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Cognitive dysfunction caused by chronic cerebral hypoperfusion is a common underlying cause of many cognition-related neurodegenerative diseases. The mechanisms of cognitive dysfunction caused by CCH are not clear. Long non-coding RNA is involved in synaptic plasticity and cognitive function, but whether lncRNA is involved in cognitive dysfunction caused by CCH has not yet been reported. In the present study, we identified the altered lncRNAs and mRNAs by deep RNA sequencing. A total of 128 mRNAs and 91 lncRNAs were up-regulated, and 108 mRNAs and 98 lncRNAs were down-regulated. Real-time reverse transcription-polymerase chain reaction verified the reliability of the lncRNA and mRNA sequencing. Gene Ontology and KEGG pathway analyses showed that differentially-expressed mRNAs were related to peptide antigen binding, the extracellular space, the monocarboxylic acid transport, and tryptophan metabolism. The co-expression analysis showed that 161 differentially expressed lncRNAs were correlated with DE mRNAs. By predicting the miRNA in which both DE lncRNAs and DE mRNAs bind together, we constructed a competitive endogenous RNA network. In this lncRNAs-miRNAs-mRNAs network, 559 lncRNA-miRNA-mRNA targeted pairs were identified, including 83 lncRNAs, 67 miRNAs, and 108 mRNAs. Through GO and KEGG pathway analysis, we further analyzed and predicted the regulatory function and potential mechanism of ceRNA network regulation. Our results are helpful for understanding the pathogenesis of cognitive dysfunction caused by CCH and provide direction for further research.

Chronic cerebral hypoperfusion (CCH) induces cognitive deficits. Although CCH can be improved, cognitive impairment is not improved accordingly. To date, many studies have focused on investigating the pathophysiological mechanisms of CCH; however, the treatment of the induced cognitive impairment remains ineffective. Thus, the mechanisms underlying cognitive impairment after CCH and potential agents for treating this impairment need to be explored further. Oxiracetam is a nootropic drug that improves clinical outcomes for some central nervous system (CNS) disorders. Whether it can improve cognitive impairment after CCH is unknown. In this study, we used behavioural methods, electrophysiology, biochemistry, histopathological staining and transmission electron microscope to investigate rat's cognitive impairment by CCH, and found that Oxiracetam could improve CCH-induced cognitive impairment and prevent deficits of neural plasticity, white matter lesions, and synaptic ultrastructure. These results suggest that Oxiracetam may be effective as a potential agent against CCH-induced cognitive impairment.

Chronic cerebral hypoperfusion (CCH) affects the aging population and especially patients with neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. CCH is closely related to the cognitive dysfunction in these diseases. Glucagon-like peptide-2 receptor (GLP2R) mRNA and protein are highly expressed in the gut and in hippocampal neurons. This receptor is involved in the regulation of food intake and the control of energy balance and glucose homeostasis. The present study employed behavioral techniques, electrophysiology, western blotting, immunohistochemistry, quantitative real time polymerase chain reaction (qRT-PCR), and Golgi staining to investigate whether the expression of GLP2R changes after CCH and whether GLP2R is involved in cognitive impairment caused by CCH. Our findings show that CCH significantly decreased hippocampal GLP2R mRNA and protein levels. GLP2R upregulation could prevent CCH-induced cognitive impairment. It also improved the CCH-induced impairment of long-term potentiation and long-term depression. Additionally, GLP2R modulated after CCH the AKT-mTOR-p70S6K pathway in the hippocampus. Moreover, an upregulation of the GLP2R increased the neurogenesis in the dentate gyrus, neuronal activity, and density of dendritic spines and mushroom spines in hippocampal neurons. Our findings reveal the involvement of GLP2R via a modulation of the AKT-mTOR-p70S6K pathway in the mechanisms underlying CCH-induced impairments of spatial learning and memory. We suggest that the GLP2R and the AKT-mTOR-p70S6K pathway in the hippocampus are promising targets to treat cognition deficits in CCH.

Autoclaved aerated concrete (AAC) has widely been utilized as a lightweight, porous insulation material for energy-efficient buildings. The knowledge on the thermal conductivity of AAC is required for thermal design of building envelopes. The effective thermal conductivity of AAC is strongly dependent on the moisture content. Such dependence, however, is not well documented in available literature. In this work, AAC bricks with three different bulk densities of 415, 520, and 630 kg/m3, were obtained as the raw materials, and the samples were prepared by humidification to a set of moisture content levels up to 100% by mass. The effective thermal conductivity of the moisturized samples was measured by means of the transient plane source technique. Meanwhile, fractal models for predicting the effective thermal conductivity were proposed based on construction of the porous structure of AAC by self-similar Sierpinski carpet. A two-phase fractal model was first proposed for dry AAC samples, and then an extension to a three-phase model was developed by considering the presence of water phase in the pores for unsaturated, moist samples. It was shown that the thermal conductivity increases with increasing the moisture content, by a factor up to 3.8 over the studied range of moisture content, following a two-section piecewise linear variation. A high-to-low slope change was found to be around a moisture content of 15% for all the AAC samples. A correlation was proposed for the measured thermal conductivity as a function of both moisture content and porosity. Appropriate parameters for the two-phase model were determined by comparing the predicted results to the measured data at dry state. The three-phase fractal model was exhibited to be able to predict the hygric dependence of thermal conductivity. The discrepancy among the predictions by the three-phase model with different geometric parameters was discussed in relation to the constructed pore structures. The predicted results by the two configurations of the three-phase model, i.e., with and without considering the presence of connected water bridges in the pores, were also presented. A reasonable elimination of the presence of connected bridges was shown to lead to better predictions in the low moisture content regime.

Non-isothermal crystallization of aqueous nanofluids in the presence of two types of high aspect-ratio carbon nano-additives, i.e., carbon nanotubes (CNTs) and graphene nanoplatelets (GNPs), was characterized by means of differential scanning calorimetry (DSC). A parametric investigation was performed on various concentrations of the nanofluids as well as various cooling rates during the DSC tests. In addition to the supercooling degree and latent heat of crystallization, the crystallization kinetics was also analyzed by both the Ozawa method and a modified Ozawa-based method. It was shown that dilute loading of CNTs or GNPs leads to reduction of the supercooling degree up to 5 °C due to the nucleating effect. The planarly-shaped GNPs featuring large contact area perform better than CNTs in facilitating heterogeneous nucleation, which greatly suppress crystal growth during the late phase of non-isothermal crystallization. In contrast to the case of GNPs, CNTs may be able to accelerate crystallization up to nearly 37% at relatively dilute loadings, especially when the cooling rate is low. The CNT-based aqueous nanofluids exhibit more balanced performance in supercooling degree, latent heat of crystallization, and crystallization kinetics, which may be utilized as an emerging candidate of phase change materials for cold thermal energy storage.

Neuroinflammation and oxidative stress play an important role in cognition deficit following chronic cerebral hypoperfusion (CCH). Luteolin, a natural flavonoid found in many plants, is known for a variety of pharmacological activities, such as its anti-inflammatory, anti-allergy, urate, anti-tumor, antibacterial, and antiviral effects. To assess whether luteolin could prevent CCH-induced cognitive dysfunction, through its anti-inflammatory and anti-oxidative-stress effects, we used enzyme-linked immunosorbent assays, enzyme activity assays, behavioral methods, immunohistochemistry, and electrophysiology to detect neuroinflammation and oxidative stress, cognition alterations, and long-term potential (LTP), in a bilateral common carotid arteries ligation (2VO) rat model. We demonstrated that CCH increased tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and malondialdehyde (MDA), and decreased superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels. Further, it caused microglia over-activation and astrogliosis, learning and short-term memory dysfunction, and an LTP deficit. Luteolin treatment reversed CCH-induced changes. Specifically, luteolin prevented the increase of TNF-α and IL-1β, IL-6, and MDA, improved the activity of SOD and GPx, inhibited microglia over-activation and astrogliosis (particularly in the hippocampus and cortex), and ameliorated learning and short-term memory dysfunction, and LTP deficit. Thus, our study suggested that luteolin could be a preferable anti-inflammatory agent to protect cognitive function and synaptic plasticity following CCH. Luteolin could also be putative therapeutic candidate for other inflammation-related brain diseases.

Background Chronic Cerebral Hypoperfusion (CCH) is an important vascular risk factor for vascular-related dementia cognitive impairment and there are no effective measures for the prevention and treatment of cognitive deficit by CCH and the underlying mechanisms are still poorly understood. Methyl cytidine-phosphate-guanosine (CpG) binding protein 2 (MeCP2), regulated by microRNA 132 (miR-132), is as a transcriptional repressor in high concentrations in the brain, which regulates the expression of synaptic proteins and neuroplasticity, and may be involved in the cognitive deficit after CCH. But no relevant studies have been reported. The aim of this study is to investigate the status of MeCP2 expression after CCH and explore whether MeCP2 changes is associated with cognitive deficits after CCH. Methods We investigated MeCP2 expression after CCH using Western blotting, quantitative Real- Time Polymerase Chain Reaction (qRT-PCR) analysis and immunofluorescence technique in a rat model of permanent bilateral common carotid artery occlusion (2VO) to mimic CCH. We determined the effect of MeCP2 expression on cognitive deficits and neuroplasticity after CCH through lenti-virus stereotaxic injection, the Morris water maze and electrophysiology. Results CCH contributed to the down-regulation of MeCP2 and mecp2 expressions in the hippocampus and cortex. miR-132 up-regulated by 2VO was distinctly negatively correlated with MeCP2 down-regulation by miR-132 inhibitors. MeCP2 over-expression improved learning and memory impairment, as well as neuroplasticity after 2VO. Brain-Derived Neurotrophic Factor (BDNF) and the activities of its downstream pathways moleculars, tropomyosin receptor kinase B (TrkB) and the cAMP Response Element Binding Protein (CREB) were down-regulated by 2VO and rescued by MeCP2 over-expression. Conclusion Our study found that miR-132 may participate in the down-regulation of MeCP2 after CCH and MeCP2 down-regulation was possibly involved in the cognitive deficit through regulation of BDNF and its downstream pathways after 2VO. Our findings expounded the underlying mechanisms of cognition deficit after CCH, which contributes to understanding the mechanisms of vascular dementia.

Gastric cancer is the fourth most common malignancy and the third leading cause of cancer‑associated deaths worldwide. It has previously been demonstrated that microRNAs (miRNAs) are actively involved in the pathogenesis of gastric cancer. Therefore, miRNAs have been proposed as promising therapeutic targets in gastric cancer patients. MiR‑744 is aberrantly expressed in different types of human cancer. However, the expression pattern and biological roles of miR‑744 in gastric cancer remain unknown. The present study demonstrated that miR‑744 expression was low in gastric cancer tissues and cell lines. Low expression levels of miR‑744 was significantly correlated with lymph node metastasis, invasive depth and TNM staging in gastric cancer patients. The restoration of miR‑744 expression inhibited cell proliferation and invasion in vitro. Bioinformatic prediction, luciferase reporter assay, reverse transcription‑quantitative polymerase chain reaction and western blot analysis verified that brain‑derived neurotrophic factor (BDNF) is a direct target of miR‑744 in gastric cancer cells. Furthermore, BDNF was upregulated in gastric cancer tissues and inversely correlated with miR‑744 expression. Furthermore, enforced BDNF expression reversed the tumor‑suppressing effects of miR‑744 on the proliferation and invasion of gastric cancer cells, indicating that BDNF is a functional mediator of miR‑744 in gastric cancer. The present study suggests that miR‑744 is a potential prognostic biomarker and treatment target in gastric cancer patients.

An experimental assessment was performed on the thermal energy storage performance of a novel family of paraffin-based composite phase change materials (PCMs) filled with hexagonal boron nitride (h-BN) nanosheets. A series of composite PCM samples were prepared at different loadings (0, 1, 2, 5, and 10 wt.%) of h-BN nanosheets in the absence of any surfactants. The composite PCMs were subjected to a variety of characterization techniques. It was shown that the thermal conductivity of the composite PCMs, in both solid and liquid phases, increases by a factor up to 60% with raising the loading of h-BN nanosheets. The melting/solidification points of the composite PCMs were found to be nearly unvaried upon adding h-BN nanosheets, whereas their latent heat of fusion slightly decreases with the loading. In addition, acceleration of both melting and solidification heat transfer rates, afforded by the increased thermal conductivity of the composite PCMs, was clearly exhibited. The observations suggested that h-BN nanosheets may serve as promising fillers for preparing custom high-conductivity composite PCMs toward thermal energy storage applications.

Alkanes and their mixtures (paraffins) have widely been used as phase change materials (PCMs) for low-to-medium temperature thermal energy storage. Among the various alkanes, eicosane, with a nominal melting temperature of 37 °C, has emerged in energy-storage-based passive thermal management technologies, for electronics for example. In an effort to increase the thermal conductivity of eicosane, the effect of adding graphene nanoplatelets (GNPs) as thermally conductive nanofillers was investigated experimentally. The composite PCM samples were prepared by dispersing GNPs in liquid eicosane at various loadings (0, 1, 2, 5, and 10 wt.%) without any surfactants. Thermal conductivity of the composite PCM samples in their solid phase was then measured by means of the transient plane source technique at elevated temperatures from 10 to 35 °C. Latent heat of fusion and melting point of the samples were also characterized using a differential scanning calorimeter. It was shown that for the highest loading examine...

The effects of the spin coating process parameters on the thickness of the SiO x layer of the BOPP/SiO x composite film were investigated. When the concentration of tetraethoxysilane (TEOS) increased from 12.5 vol% to 55% vol%, the SiO x thickness increased from about 80 nm to 470 nm. In the sol time range of 1.5 h to 5 h the SiO x layer thickness reached a maximum at about 4 h and the change of the thickness roughly matched the change of the silica colloidal sphere sizes in sol. When the spin-coating speed of the dispensing stage increased from 450 r/min to 500 r/min, the SiO x layer thickness drastically decreased from about 1.67 μm to 400 nm. While the spin-coating speed of the thinning and drying stage went up to 1200 r/min, the SiO x layer thickness was in the range of 330 nm to 390 nm. It was also found that the SiO x layer thickness was almost increased linearly from about 500 nm to 1.02 μm with the ratio of the commercial silica colloidal to the TEOS from 0.2 to 1.0. The water contact angles decreased to about 23.0° for the BOPP/Si-Sol composite film with 1.67 μm SiO x layer and about 4.0° for the BOPP/mixing Si-Sol composite film with 1.02 μm SiO x layer. Compared to BOPP, the light transparency of the BOPP/Si-Sol composite films decreased by about 5.5% with the SiO x layer from about 80 nm to 1.67 μm and by 7.0% for the BOPP/mixing Si-Sol composite film with the SiO x layer from about 350 nm to 1.02 μm respectively.

A series of titanium complexes of the general formula [N-(3,5-di-tert-butylsalicylidene)-2-alkyl-sulfanylanilinato]Ti(IV)Cl3 (5a, b, e and f) exhibited very high catalytic activities in ethylene homo- and copolymerization with various α-olefins in the presence of modified methylaluminoxane (MMAO). The results showed that steric hindrance of the alkylthio groups in 5a–f strongly influenced the polymerization behavior. An increase in the steric hindrance of these alkyl units led to the decrease in both catalytic activity and comonomer incorporation ratio. On the other hand, the amount of α-olefin incorporated into the copolymer chain depended on the molar ratio of the monomers in the polymerization reaction and was slightly influenced by the chain length of α-olefin. Comparisons between alkylthio and arylthio substituents on catalyst performance were also discussed in this article.

A family of titanium complexes of the general formula [N-(3,5-di-tertbutylsalicylidene)-2-alkylsulfanylanilinato]Ti(IV)Cl-3 5a-f was prepared from the reaction of TiCl4 with the potassium salts of the corresponding ligands. These complexes were fully characterized by various spectroscopic techniques and elemental analyses. The molecular structures of 5b and 5e were further confirmed by single-crystal X-ray analyses. Complexes 5a-f (except for 5c) exhibited good to high catalytic activities in ethylene copolymerization with cycloolefins such as norbornene, cyclopentene, dicyclopentadiene in the presence of modified methylaluminoxane. The reaction conditions and the steric hindrance of the alkyl substituents on sulfur atom in the precatalysts influenced strongly the copolymerization behaviors and the structures of the resultant copolymers. Complex 5c with bulky tert-butylthio sidearm showed both low catalytic activity and comonomer incorporation ratio. The n-alkylthio complexes 5a, 5d-f all exhibited good ethylene copolymerization capabilities with cycloolefins, which is superior to the corresponding phenylthio complex 5g. (c) 2008 Wiley Periodicals, Inc.

BackgroundThe value of transplantation of BM stem cells in aged (12-month-old) mdx was evaluated because it is thought to be a more ideal model for studying the praxiology of Duchenne muscular dystrophy (DMD). The possible mechanisms of stem cell differentiation were then discussed.MethodsBM was isolated from 8–10-week-old male C57 BL/10 mice. After injecting BM cells into 12-month-old female mdx mice through the tail vein, the expression of dystrophin and MyoD was detected at different time points by immunofluorescence staining, RT-PCR and Western blot.ResultsThe C57 male mice donor-specific and Y-chromosome-specific sequence could be detected in all female aged mdx mice, implying the success of the transplantation. Expression of dystrophin and MyoD was detected and increased over time.DiscussionBM cells were recruited to the muscle and partially restored specific pathophysiologic features of the dystrophic muscle in aged mdx mice. Muscle differentiation of BM cells recapitulated embryonic myogenesis.

Novel titanium complexes containing monoanionic [NOP] ligands based on a phenoxy−imine ligand and their derivatives were synthesized and characterized. Their performance as ethylene polymerization and copolymerization catalysts were studied. Upon treatment with MMAO, the [NOP]TiCl3 complexes were robust and highly active for the polymerization of ethylene, even at very low cocatalyst/catalyst ratios. These catalysts also have good copolymerization capabilities.

Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay. It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plant DNA, the plasmid rescue technique was used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it. For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6% homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA. Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.

To investigate the neuroprotective effects of Lycium barbarum extract against MPP(+) -induced neurotoxicity in Caenorhabditis elegans and PC12 cells and its mechanism.Pretreated MPP(+) -induced nearotoxicity in C. elegans and PC12 cells with Lycium barbarum at different dosages. The viability and DA neurodegeneration was assessed in C. elegans selectively expressing green fluorescent protein (GFP) in DA neurons. PC12 cell damage was measured using MTT and nuclear morphology. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential and total GSH were assessed.Lycium barbarum extract protected against MPP(+) -induced loss of viability and DA neurodegeneration in C. elegans in a dose-dependent manner. Similar neuroprotection was replicated in MPP + PC12 cell model. Lycium barbarum extract attenuated MPP(+) -induced intracellular ROS accumulation, loss of mitochondrial membrane potential and restored total GSH levels in PCl2 cells.Lycium barbarum extract protects against MPP(+) -induced neurotoxicity in C. elegans and PC12 cells and its machanism may be related to its antioxidative property and restoration of total GSH level.

To investigate the effect of suppression of CCL5 ligand gene on the proliferation of human breast cancer cells.A lentiviral vector carrying a short interfering RNA (siRNA) targeting CCL5 was transfected into human breast cancer cell line MCF-7 and MDA-MB-231 cells. The expression of CCL5 mRNA in the cells was detected by real-time PCR. The proliferation of MCF-7 and MDA-MB-231 cells was assessed by MTT assay and FACS assay, and the colony formation ability of both cell lines were measured, respectively.Real time PCR showed a good knockdown effect of CCL5 in both cell-lines. Colony-forming assay showed that the ability of colony formation of MCF-7/CCL5-siRNA and MDA-MB-231/CCL5-siRNA was decreased markedly. The colony number of MCF-7/CCL5-siRNA group was (0.34 ± 0.08), significantly lower than 0.81 ± 0.12 in the MCF-7/CCL5-N group and 0.92 ± 0.12 in the MCF-7 group (P0.05). The colony number of MDA-MB-231/CCL5-siRNA group was 0.33 ± 0.10, significantly lower than 0.97 ± 0.09 in the MDA-MB-231/CCL5-N group and 1.04 ± 0.07 in the MDA-MB-231 group (P0.05). However, MTT assay revealed that the proliferation of MCF-7/CCL5-siRNA cells was not significantly different from that of MCF-7/CCL5-N or MCF-7 cells, respectively (P0.05), and the same result was found in MDA-MB-231 cells. FACS assay showed that the proliferation index (PI) of groups MCF-7/CCL5-siRNA, MCF-7/CCL5-N and MCF-7 were 0.48 ± 0.03, 0.43 ± 0.01 and 0.45 ± 0.02. The PI of groups MDA-MB-231/CCL5-siRNA, MDA-MB-231/CCL5-N and MDA-MB-231 cells were 0.48 ± 0.02, 0.44 ± 0.05 and 0.47 ± 0.02. There was no statistical difference among them (P0.05).The down-regulation of CCL5 gene in human breast cancer cells may significantly suppress their colony formation ability, rather than affecting their population doubling time to some extent.

To study the impact of organized stroke ward on the therapeutic effect in stroke patients.A total of 2637 patients with acute stroke were randomly assigned to organized stroke ward or the general ward for treatment, and the rates of mortality, nonrecovery, improvement, and recovery were compared between the two groups.The rates of mortality, nonrecovery, improvement, and recovery in 5 years were 2.00%, 0.90%, 74.94% and 22.16% respectively in the organized stroke ward group, as compared to 3.26%, 1.02%, 74.01% and 21.71% in the general ward group, respectively. The mortality rate was significantly lower in organized stroke ward (P0.05), but no significant difference was found in the rates of nonrecovery, improvement, or recovery between the two groups (P0.05).Admission of the stroke patients in organized stroke ward for treatment can be associated with lowered mortality rate.

To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice.BrdU-labeled fifth-passage rat MSCs were transplanted in mdx mice with previous total body gamma irradiation (7 Gy). At 4, 8, 12 and 16 weeks after the transplantation, the mice were sacrificed to detect dystrophin/BrdU and utrophin expressions in the gastrocnemius muscle using immunofluorescence assay, RT-PCR and Western blotting. Five normal C57 BL/6 mice and 5 mdx mice served as the positive and negative controls, respectively.Four weeks after MSC transplantation, less than 1% of the muscle fibers of the mdx mice expressed dystrophin, which increased to 15% at 16 weeks. Donor-derived nuclei were detected in both single and clusters of dystrophin-positive fibers. Some BrdU-positive nuclei were centrally located, and some peripherally within myofibers. Utrophin expression decreased over time after transplantation.The myofibers of mdx mice with MSC transplantation express dystrophin, which is derived partially from the transplanted MSCs. Dystrophin expression from the transplanted MSCs partially inhibits the upregulation of utrophin in mdx mouse muscle, showing a complementary relation between them.

To study the effects of BMP-2 and FGF-2 on osteoblast differentiation of murine MSCs in vitro.The bone marrow cells were collected from 3-18 month old C57BL/6J mice (50 mice), and they were isolated, enriched and expanded using bone marrow adherent culture, and then purified by immunomagnetic microbeads. At last they were identified as mesenchymal stem cells (MSCs). After the MSCs were cultured adherently for 24 hours, 100 microg/L BMP-2 and 0.5 nmol/L FGF-2 were added into osteogenic media separately for 7, 14 and 21 days, alkaline phosphatase (ALP) staining, alkaline phosphatase activities, Vonkossa staining, and Alizarin red S staining were performed step by step. Osteoblast differentiation marker genes including Runx2/cbfa1, ALP, collagen-1, and osteocalcin were investigated by RT real time PCR.ALP activities, calcium deposition and the osteoblast differentiation marker genes of BMP-2 group were markedly higher than those of osteogenic group. The mRNA of Runx2/cbfa1, ALP, collagen-1, and osteocalcin of BMP-2 group was highly expressed than that of osteogenic group. It was the same with FGF-2 group, but less evident than BMP-2 group.BMP-2 and FGF-2 can induce murine MSCs to differentiate into osteoblast at different degrees.

To investigate the dynamic distribution of human bone marrow mesenchymal stem cells (hBM-MSCs) in mdx mice.Twenty-four 8-10-week-old immunocompromised mdx mice were transplanted with 1 x 10(7) passage 5 hBM-MSCs labeled with bromodeoxyuridine (BrdU) by means of injection into the tail vein. The mice were euthanized 48 hours and 2, 4, 8, 12, 16, 20, and 24 weeks after transplantation. BrdU-positive cells in tissue and organs of the mice were detected by immunofluorescence analysis. Skeletal muscle was stained for anti-human nuclei mouse monoclonal antibody (anti-Hu) and analyzed for human dystrophin (Dys) expression by immunohistochemistry and reverse transcription-polymerase chain reaction.After transplantation, BrdU-positive cells were found in most organs (especially in bone marrow, liver, and lung) within 4 weeks, and these cells in liver and lung decreased gradually after 4 weeks. At 48 hours after transplantation, BrdU-positive cells were found in bone marrow, which reached a peak level after 2 weeks and were still detectable after 16 weeks. BrdU-positive cells in skeletal muscle increased gradually over time of transplantation. A small number of anti-Hu positive cells were detected in skeletal muscle 2 weeks after transplantation. A small number of Dys positive cell were seldom found at 4 weeks and small Dys mRNA expression detected 4 weeks after transplantation. The proportion of anti-Hu in parallel with Dys positive cells and Dys mRNA in skeletal muscle of mdx mice increased gradually over time of transplantation.After being transplanted into mdx mice, hBM-MSCs are mainly distributed in bone marrow, liver, and lung during the early time (2-4 weeks) , and then in bone marrow and skeletal muscle (after 4 weeks).

Amniotic fluid-derived stem cells have attracted considerable attention in the field of regenerative medicine. Approach of genetic modification probably enhances their regenerative potential. In this work, we wanted to determine whether baculovirus as a new gene vector could efficiently and safely transduce mouse amniotic fluid-derived stem cells (mAFSs). Cells were isolated from mouse amniotic fluid and cultured in vitro. These cells were analyzed by examining phenotypes and differentiation potential. They were further transduced with baculovirus. Baculovirus-transduced mAFSs were induced to differentiate into adipogenic, osteogenic, myogenic, and neurogenic lineages. Mouse amniotic fluid-derived stem cells were successfully isolated and cultured in vitro. They were positive for CD29 and Sca-1, but negative for CD34, CD45, or CD11b. Furthermore, they could differentiate into adipocytes, osteocytes, myocytes, and neurocytes in vitro. Baculovirus could efficiently transduced mAFSs. More importantly, baculovirus-transduced mAFSs retained differentiation potential. Thus, baculovirus vector effective and safe transduction is an attractive promise for genetic modification of mAFSs. Baculovirus genetically modified mAFSs will probably be more suitable as vehicles for regenerative medicine.

In this paper, a thermal tomography method, which can deduce the temperature distribution of the inner heat source from the surface thermal map, is applied to the clinical breast diagnosis. With the comparison to the conventional mammography and ultrasound imaging, the clinical value of thermal tomography on breast diagnosis is discussed. Moreover, with the help of the infrared thermal imaging, the different rules of the temperature response on normal or cancerous mammary tissue by activating the sympathetic nervous system with a cold stimulus on hands is studied preliminarily.

To investigate the effect on bone marrow stem cell transplantition (BMST) combinated with Bushen fang therapy in mdx mice.Cultured the bone marrow cells of C56BL/6 in vitro and tranplante these cells to mdx mice after irradiation. Bushen fang was used to cure the mdx mice after BMST. The survivel rate, symptom of Graft Versus Host Disease (GVHD) and motor function of model mice following irradiation and transplantation were observed 4 or 8 months after BMST, prepared the freeze-splice of muscles of each group, and made HE staining. The dystrophin (DYS) was detected by SABC immunofluorescene.Bushen fang could decrease the rate of centrally nucleated fibers (CNF), enhance expressional rate of DYS in different level and relieve the symptom of GVHD in transplanted mice.Bushen fang can improve the therapeutic efficacy of BMST.

To detect the female carriers from the intron and/or exon-deletion Duchenne/Becker musclular dystrophy (DMD) familial members for prenatal or preimplantation genetic diagnosis.Using method of PCR to five microsatellite markers (located in 5' terminus and intron 44, 45, 49, 50), analysing of the short tandem repeat sequence polymorphism with the genescan and binding with the quantitative polymerase chain reaction, we detected the DMD carriers from 1 intron and exon -deletion family and 1 intron-deletion family.The STR-50 genotype of II 2 in family 5 was 245/245, so II3 is DMD gene carrier. The STR-45 genotype of II6 and II8 were del/172, III19 was del/178, so they were all DMD gene carriers.The STR haploid linkage analysis combined with quantitative polymerase chain reaction is accurate and efficient to detect the female carriers from the intron and/or exon-deletion DMD familial members.

To determine the effects of wild-type mouse bone marrow stem cells transplants on survival time and motor functions in the human mutant SOD1-G93A mouse model of familial amyotrophic lateral sclerosis (ALS).Bone marrow stem cells derived from the wild-type male mice were delivered intravenously into 25 ALS transgenic female mice (carrying the human SOD1 gene with Gly 93 Ala mutation) that had been pretreated with 5.5-6.5 Gy gamma-ray 5-7 days before. The onset time of limbs paralysis, lifespan and the graft versus host disease (GVHD) symptoms were observed in the treated group and statistically compared with control group of 15 media-injected ALS transgenic mice. The Sry gene (sex determine region on the Y chromosome) were detected by polymerase chain reaction technique in the blood sample of treated female ALS mice after 8 weeks of transplantation. A series of animal motor tests including rotating rods, rotated wheel and extension reflex were performed in both two groups at the same age of 16-17 weeks to assess the mice survival motor functions. Results A few treated mice (7/25) had different clinical presentations of GVHD. The semi-quantity evaluation score of average GVHD among the treated ALS mice was not over 1-2. The detection of Sry gene on these treated female ALS group was positive. The average onsets of limb paralysis and survival time were prolonged for about 5 weeks. At the age of 16-17 weeks, the motor function in the treated group was significantly better than in the ALS control group (P0.01).Transplantation of wild-type mice bone marrow stem cells can prolong survival in the recipient mice and ameliorate motor dysfunction. Intravenous administration of normal bone marrow stem cells may have therapeutic values for ALS.

To construct the retroviral vector containing human micro-dystrophin gene and detect the expression of human micro-dystrophin in mdx mice bone marrow-derived mesenchymal stem cells (MSCs) after retrovirus infection.Retroviral vector for micro-dystrophin gene was constructed and transferred into the packing cell PA317 mediated by Lipofectamine 2000. The retroviral supernatant containing the target genes were subsequently used to infect mdx mice MSCs. Micro-dystrophin expression was examined by methods of immunofluorescence staining and reverse transcriptase-polymerase chain reaction.Micro-dystrophin retroviral vector was successfully constructed and transferred into PA317 cells, and 48 h after infection with the recombinant retrovirus in mdx mice MSCs, 319 bp fragment could be detected by electrophoresis in the RT-PCR products. The red particles could be detected in some infected mdx mice MSCs with immunofluorescence staining. CONCLUSION mdx mice MSCs infected with retrovirus containing micro-dystrophin gene can express micro-dystrophin protein.

To detect the disease-causing point mutations in the dystrophin gene of Duchenne muscular dystrophy (DMD) patients.The approach of denaturing high performance liquid chromatography (DHPLC) coupling with sequencing was used to screen the point mutations of 79 exons and the untranslated regions of dystrophin gene without large deletions/duplications, which was in 6 unrelated DMD probands from 6 DMD families.Five disease-causing mutations, 697-698insGT, C616T, G1255T, C4279T, and C2302T, were ides created the new stop codons in downstream sites of mutations, respectively. In addition to the disease-causing point mutations, a point mutation T5586+61A in intron 39 was also found at patient 3, and a missense mutation A694T in exon 8 was detected at patient 5. Four point mutations, C2168+13T, 5740-13dupG, G5234A and C5280T, were also detected at patient 6 whose causative point mutation was unavailable. Seven point mutations have not been reported previously. Bi-directional PCR amplification of specific alleles (Bi-PASA) method was established to distinguish the haplotypes of heterozygote or homozygote in a single PCR reaction.Via automated DHPLC screening or detecting the subexonic mutations in dystrophin gene is feasible to clinical laboratories, and also is a superior method in terms of sensitivity and efficiency.

To investigate the dynamic changes of dystrophin expression in mdx mice after bone marrow stem cells transplantation.The bone marrow stem cells of C57 BL/6 mice (aged 6 to 8 weeks) were injected intravenously into the mdx mice (aged 7 to 9 weeks), which were preconditioned with 7Gy gamma ray. The amount of dystrophin;expression in gastrocnemius was detected by immunofluorescence, reverse transcription-polymerase chain reaction and Western blot at week 5, 8, 12 and 16 after transplantation.At week 5 after bone marrow stem cells transplantation, the dystrophin expression detected in mdx mice were very low; however, its expression increased along with time. At week 16 week, about 12% muscle cells of all transplanted mice expressed dystrophin. There were less centrally placed myonuclei than the control mdx mice, whereas peripheral myonuclei increased.After having been injected into mdx mice, the allogenic bone marrow stem cells have a trend to reach the injured muscle tissues and differentiate to fibers that can express dystrophin and the expression increased with time. The bone marrow stem cells participates in the repair and regeneration of the injured tissues permanently and constantly.

To establish transgenic mouse models of familial amyotrophic lateral sclerosis (FALS) and identify the genotype of the first filial generation.Six male B6SJL SOD1G93A/+ hemizygote mice were mated with 6 female B6SJLF1/J+/+ mice to produce the filial generation. The genomic DNA was extracted from the tail vein blood of the first filial generation mice and PCR was performed to amplify the hmSOD1 gene fragment. The genotype of the mice was determined by electrophoresis, and the PCR product was purified for further gene sequence analysis and detection of mutation loci.Fifty-three progeny mice were born and the survival rate before ALS onset was 98% (52/53), and among the survived mice, the positivity rate for hmSOD1 gene was 44.2% (23/52). Electrophoresis result showed that the PCR product of 236 bp was consistent with the hmSOD1 gene fragment, and the sequence of the PCR product was identical with hmSOD1 gene sequence of G93A mutant.Transgenic mouse models of ALS can be established in the first filial generation of male B6SJL SOD1G93A/+ mice mated with female B6SJLF1/J+/+. PCR technique can precisely identify the genotype of the filial generation.

To investigate the effect of shenqi fuzheng injection (SFI) in inducing differentiation of human mesenchymal stem cells (hMSCs) in brain stem and its effect on nervous function in model rats of cerebral infarction.Middle cerebral artery occlusion model rats were made, and hMSCs was injected into their brain after being amplified in vitro and incubated with SFI for 0.5 h, then the survival, migration and differentiation of hMSCs in brain stem as well as the change of nervous function in model rats were observed.The post-transplantation reject reaction to hMSCs was low, it could survive as long as 6 weeks or more. No difference in area of infarction was shown before and after transplantation. Immunohistochemical staining showed that hMSCs expressed human neuron specific enolase (NSE), neurofilament (NF) and glial fibrillary acid protein (GFAP). The limb-kinetic function and tactile perception were improved in the model rats.SFI can induce hMSCs differentiate into neurons in vivo, and hMSCs may be the ideal germinal cells for treating cerebral infarction.

To analyze a Duchenne muscular dystrophy(DMD) patient's muscular regeneration, dystrophin expression and locomotive variation before and after he underwent umbilical cord blood stem cell transplantation in order to assess the therapeutic effect.A 12-year-old DMD boy who could not walk for 3 years was confirmed by gene analysis and dystrophin protein immune test on his muscle. He had no other chronic disease. By HLA matching, a piece of umbilical cord blood stem cell with 6 HLA sites matching to the boy was found in Guangdong Umbilical Cord Blood Bank. The number of the nucleated cells of the umbilical cord blood stem cell was 24.08x 10(8). After pretreatment for the DMD boy with busulfan, cyclophosphamide and rabbit anti-human thymocyte globulin, the allergenic cord blood stem cells were transplanted into him by intravenous injection. Cyclosporin A, methylprednisolone, MMF, prostaglandin E1 and ganciclovir were given after the transplantation. At the same time, Gran, the granulocytic cell stimulating factor, and gamma globulin were administered. The biochemistry profile including serum creatine kinase (CK), the reconstruction of blood making, the deletion exon of DMD gene, the regenerating muscles, the dystrophin protein expression, and the locomotive function of the DMD boy were tested regularly.(1) The white blood cells (WBC) of peripheral blood decreased gradually to zero after pretreatment. In a period of 15 days after transplantation, the neutrophil increased to 0.5x 10(9)/L; at 25 days, WBC increased to normal level. Blood platelet was more than 20x 10(9)/L at 22 days. The hemoglobin rose to 85-100 g/L. At 140 days, sternal puncture revealed the rapid growth of neutrophil, blood platelet and hemoglobin. (2)At 140 days, the blood type of the DMD boy transformed from type O to type AB (the donor's blood type being AB). There was no grafe versus host reaction. (3) At 18, 30, 43, 55, 74 and 233 days after transplantation, the PCR-short tandem repeat test of the boy's peripheral blood DNA showed that his genotype was completely the same as the donor's. The results of PCR-short tandem repeat tests of the bone marrow cells DNA by sternal puncture at 140, 183 and 235 days were the same as those of the blood DNA. (4) At 60 days, DMD gene analysis by PCR showed that the defected DMD gene (exon 19 deletion) had been corrected by the umbilical cord stem cells transplantation. (5) At 75 days, the biopsy of calf muscle showed there were myoblast cells and muscular tubes growing. The dystrophin expressions were weak, but a few of them were strong. DNA analysis showed that the donor's gene DNA accounted for 1%-13%. At 126 days, obviously increased dystrophin positive muscular fibers of the boy were found. The donor's fibers rose to 2.5%-25%. (6) The serum CK of the boy declined from 5735 U/L to 274 U/L. (7) At 100 days, physical examination revealed improvement in his arms and legs.The therapy of Duchenne muscular dystrophy with allogeneic umbilical cord blood hematopoietic stem cell transplantation may reset up the blood-making function, decrease the serum CK level, restore the dystrophin in muscles, and improve the locomotive function of the DMD boy. These data suggest that the allogeneic umbilical cord blood hematopoietic stem cell transplantation may benefit the DMD boys.

To observe gene reversion during differentiation of human adult bone marrow-derived mesenchymal stem cells (MSCs) into neuron-like cells induced by Shenqiye.The MSCs were separated, cultured and expanded in the culture medium and induced to differentiate into neuron-like cells with Shenqiye. The expressions of neuron-specific enolase (NSE), neurofilament (NF), and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method, and the changes of 10 genes of the cells after differentiation were detected by reverse transcriptional PCR.The MSCs exhibited neuronal phenotype when treated with Shenqiye and the neuron-like cells were positive for expressions of NSE and NF but not for glial astrocyte marker GFAP. After withdrawal of Shenqiye from the medium, the neuron-like cells were reversed to MSC, flat or spindal in morphology. The gene expression profiles of the redifferentiated cells were similar to those of undifferentiated MSCs.Shenqiye can induce MSC differentiation into neuron-like cells during and after which gene reversion may occur.

To investigate the feasibility of using human bone marrow-derived mesenchymal stem cells (hBM- MSCs) for repairing the skeletal muscle sarcolemma lesions in mdx mice and characterize the distribution of the transplanted hBM-MSCs.Eighteen 8- to 10-week-old immunosuppressed mdx mice received transplantation with 1x10(7) of hBM-MSCs (the fifth passage) with 3H-thymidine (3H-TdR) labeling by injection of the cells into the tail vein. The mice were killed at 24 h, 48 h, 2 weeks, and 1, 2 and 4 months after the transplantation, respectively, to measure the radioactivity in the tissues and organs. Dystrophin expression on the sarcolemma was detected by immunofluorescence analysis.One month after transplantation, the mice with cell transplantation showed greater radioactivity in most of the tissues and organs than the control mice, especially in the bone marrow, liver and spleen. The radioactivity was then gradually lowered but in the skeletal muscle, the radioactivity increased progressively since 2 weeks after transplantation, reaching the peak of 27.65+/-3.53 Bq/mg at 1 month. Compared with that in the control mice, the radioactivity in the bone marrow and skeletal muscle was persistently higher in mice with cell transplantation 1 month after transplantation. No dystrophin-positive cells were found in the mdx mice at 2 weeks but detected at 1 month. The percentage of dystrophin-positive fibers in each section ranged from a 6.6% (1 month) to 8.9% (4 months).hBM-MSCs engrafted in immunosuppressed mdx mice may differentiate into skeletal muscle cells to repair the pathological lesion of the skeletal muscle sarcolemma. The hBM-MSCs reside mainly in the bone marrow, liver and spleen in the early stage following transplantation, homing into the bone marrow and skeletal muscle later.

To study the therapeutic effects of granulocyte colony-stimulating factor (G-CSF) on focal cerebral ischemia-reperfusion injury in rats.Adult male SD rats were subjected to transient (2 h) middle cerebral artery occlusion (MCAO) and scored 48 h later for such neurological functions as spontaneous movement, paresis, forelimb motor function, climbing ability, pain sensation, and position sense. Twenty rats with the total score less than 12 were divided equally into two groups to receive daily subcutaneous injection of 20 microg/kg of G-CSF for 19 days or the same dose of saline injection (control group). The rats in the two groups were also given intraperitoneal injection of Brdu10 mg/kg for 19 days. The neurological functions of the rats in both groups were examined on a weekly basis after MCAO and on day 21, the rats were killed to prepare frozen sections of the brain tissues for double immunohistochemical staining with Brdu and glial fibrillary acidic protein antibody to identify the neural cells newly evolved from the stem cells.The rats in G-CSF group showed better neurological function recovery than the control rats 3 weeks after MCAO (P0.05). Obvious regeneration of the neural cells was observed around the infarction area in the rats receiving G-CSF treatment.G-CSF promotes neurological function recovery and neural cell regeneration after cerebral infarction in rats and can be effective for intervention of focal cerebral ischemia-reperfusion injury.

To probe the conditions for inducing human cord blood monocytes to differentiate into neuron-like cells.The mononuclear cells were isolated from human umbilical cord blood samples and plated in 25-mm culture flasks containing DMEM/F12 medium. The fifth passage of mesenchymal stem cells (MSCs) were induced by beta-mercaptoe- thanol (beta-ME), dimethyl sulfoxide (DMSO) and conditioned medium for neuron induction, respectively, to differentiate into neuron-like cells. The expression of nestin, neuron-specific enolase (NSE) and neurofilament (NF) on the treated cells were detected by immunocytochemical method.Nestin expression were found in 6.7% of the primary, 12.4% of the second passage, 20.8% of the fifth passage of MSCs, respectively. Thirty-six percent of cell cultures treated with the conditioned medium for neuron induction were immunoreactive for NSE and NF, and 33% of the cells induced by beta-ME and 25% of the cells by DMSO also expressed NSE and NF.Cord blood MSCs possess some features of neural stem cells, and have the capacity to differentiate into neuron-like cells under proper conditions.