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The effects of crude extract from the flowers of Trollius chinensis on expressions of mRNA and proteins related to vital genes (TLR 3, TBK 1, IRF 3 and IFN β) in TLR 3 signaling pathway were investigated in the presence/absence of Polyinosinic acid-polycytidylic acid (PolyI: C) to ascertain the antiviral mechanism of these flowers. Real-time PCR and western blot were applied to determine the expressions of mRNA and proteins, respectively, and immunofluorescence assay was employed to study the effect on IRF 3 distribution between nuclei and cytoplasma. In the absence of PolyI:C, the crude extract reduced the mRNA expression of TLR 3, IRF 3 and IFN β and the protein expression of TLR 3, and increased the protein expression of IRF 3 and the distribution of IRF 3 in nuclei. In the presence of PolyI:C, the extract reduced the mRNA and protein expressions of TLR 3 and the mRNA expression of IFN β, meanwhile inhibited the translocation of IRF 3 into nuclei. The antiviral mechanism of the crude extract from the flowers of T. chinensis is to protect the host from inflammatory damage through intervening the TLR 3 signaling pathway and reducing the secretion of inflammatory factors.

Mineralised collagen fibrils constitute the basic building blocks of bone, dentin and cementum. Noncollagenous proteins (NCPs) that are indispensable for collagen biomineralisation are not commercially available, and the mechanism of intrafibrillar mineralisation remains debatable. Herein, synthetic biomimetic molecules are regarded as alternative candidates for NCPs, and more convenient in revealing the mechanism of intrafibrillar mineralisation in vitro. Here, we fabricated a novel amphiphilic oligopeptide imitating a natural NCP. We aimed to investigate the effectiveness of the oligopeptide in intrafibrillar mineralisation and partially reveal the corresponding mechanism in vitro. The effectiveness of the oligopeptide in intrafibrillar mineralisation was characterised from the following aspects: (1) mineral interaction, (2) collagen binding and (3) induction of intrafibrillar mineralisation. Results indicated that the self-assembled oligopeptide could attract calcium ions inducing the formation of amorphous precursors; and bind onto the surface of collagen fibrils. These processes were mainly driven by the electrostatic attraction and hydrogen bonds. The self-assembled oligopeptide induced the intrafibrillar mineralisation of reconstituted collagen fibrils, in which the c-axis of apatite crystallites was roughly parallel to the long axis of the fibrils. The collagen mineralisation was achieved by binding with the self-assembled oligopeptide to increase the pool of mineralization precursors available for intrafibrillar mineralisation. In addition, the self-assembled oligopeptide induced dentin collagen remineralisation and formed a 30 μm-thick remineralised layer within 96 h. Our work sheds light on the fabrication of a novel biomimetic molecule for collagen mineralisation. The results should serve as a reference for understanding the mechanism of intrafibrillar mineralisation.

Background Innate immune dysfunction contributes to the development and progression of nonalcoholic fatty liver disease (NAFLD), however, its pathogenesis is still incompletely understood. Identifying the key innate immune component responsible for the pathogenesis of NAFLD and clarifying the underlying mechanisms may provide therapeutic targets for NAFLD. Recently, F-box- and WD repeat domain-containing 7 (FBXW7) exhibits a regulatory role in hepatic glucose and lipid metabolism. This study aims to investigate whether FBXW7 controls high-mobility group box 1 protein (HMGB1)-mediated innate immune signaling to improve NAFLD and the mechanism underlying this action. Methods Mice were fed a high-fat diet (HFD) for 12 or 20 weeks to establish NAFLD model. Hepatic overexpression or knockdown of FBXW7 was induced by tail-vein injection of recombinant adenovirus. Some Ad-FBXW7-injected mice fed a HFD were injected intraperitoneally with recombinant mouse HMGB1 to confirm the protective role of FBXW7 in NAFLD via inhibition of HMGB1. Results FBXW7 improves NAFLD and related metabolic parameters without remarkable influence of body weight and food intake. Moreover, FBXW7 markedly ameliorated hepatic inflammation and insulin resistance in the HFD-fed mice. Furthermore, FBXW7 dramatically attenuated the expression and release of HMGB1 in the livers of HFD-fed mice, which is associated with inhibition of protein kinase R (PKR) signaling. Thereby, FBXW7 restrains Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) signaling in HFD-fed mouse livers. In addition, exogenous HMGB1 treatment abolished FBXW7-mediated inhibition of hepatic inflammation and insulin resistance in HFD-fed mouse livers. Conclusions Our results demonstrate a protective role of FBXW7 in NAFLD by abating HMGB1-mediated innate immune signaling to suppress inflammation and consequent insulin resistance, suggesting that FBXW7 is a potential target for therapeutic intervention in NAFLD development. Electronic supplementary material The online version of this article (10.1186/s10020-019-0099-9) contains supplementary material, which is available to authorized users.

The safety and efficacy of intravenous insulin injection coupled with subsequent milk consumption was evaluated for high-quality cardiac viability F-18-fluorodeoxyglucose (A total of 328 patients with known/suspected coronary artery disease received intravenous insulin injection with or without subsequent milk consumption for cardiacIntravenous insulin injection supplemented with subsequent milk consumption is a safer formulation for cardiac viability

An improved, reliable and comprehensive method for assessing the quality of the ethyl acetate extract from persimmon leaves (EAPL) and its commercial preparation, Naoxinqing (Brain and Heart Clear capsules), has been developed and validated. Based on HPLC-DAD-ESI-Q-TOF-MS analysis, myricetin-3-O-β-D-galactoside (1), myricetin-3-O-glucoside (2), quercetin-3-O-β-D-galactoside (3), quercetin-3-O-β-D-glucoside (4), quercetin-3-O-(2″-O-galloyl-β-D-galactoside) (5), quercetin-3-O-(2″-O-galloyl-β-D-glucoside) (6), kaempferol-3-O-β-D-galactoside (7), kaempferol-3-O-β-D-glucoside (8), kaempferol-3-O-(2″-O-galloyl-β-D-galactoside) (9), kaempferol-3-O-(2″-O-galloyl-β-D-glucoside) (10), quercetin (11) and kaempferol (12) were identified from 15 batch samples. A HPLC fingerprint analytical method was established. All compounds, with the exception of compound 2, were simultaneously quantified by the single standard to determine multi-components (SSDMC) method, using kaempferol-3-O-β-D-glucoside as the internal standard. The rate of analysis was found to be faster with the SSDMC method than with current acid hydrolysis method (Pharmacopoeia of the People's Republic of China 2015 edition) and the results were more intuitive and reliable. Three-dimensional principal component analysis revealed that there were similar characteristics in persimmon leaf from same district. Analysis of the myocardial cell protection activity of 11 monomeric compounds showed that compounds 12, 11 and 10 were the main active ingredients that produce pharmacologic functions in EAPL. Among these compounds, the bioactive constituent of myricetin-3-O-β-D-galactoside was determined for the first time in Diospyros khaki. Thus, we have established an effective assessment method that can be applied to the comprehensive quality evaluation of EAPL extract and Naoxinqing capsule.

Introduction Cardiac-cerebral vascular diseases (CCVDs) are global health problems due to the characteristic of high mortality. It is found that atherosclerosis (AS), a main cause of CCVDs, is significantly relevant to the change of intimal and media thickness. Neutrophil count (NEU) and neutrophil-lymphocyte ratio (N/L) are recognized possible risk factors for atherosclerosis (AS). However, there are few studies on the separate relationship between carotid intimal thickness, media thickness and NEU, N/L. This study explored the respective effects of NEU and N/L on AS and intimal, media thickness. Materials and methods The χ2, Spearman's rho test, and multiple linear regression were implemented to analyze the relevance between blood parameters and intimal-media thickness. The potential factors, affecting non-depression time (NDT), is identified by univariate Cox regression. ROC curve was performed to determine the ability of blood parameters to predict intimal-media thickness. Immunohistochemistry was implemented. Results and conclusion Based on χ2, Spearman's rho test and multiple linear regression, NEU is related with intimal thickness (P

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is associated with various adverse human health effects; however, the knowledge of its toxicity is still very limited. Mitochondrial injury has been observed in liver cells exposed to BDE-47 in vitro. Mitophagy impairment causes the accumulation of dysfunctional mitochondria, contributing to the pathological mechanisms of liver injury. The aim of this study was to investigate whether BDE-47 impairs mitophagy to trigger mitochondrial dysfunction-related liver injury and the underlying mechanisms. This study revealed that BDE-47 elicited mitochondrial dysfunction and related oxidative liver injury by impairing mitophagy. Moreover, our results showed that NAD+ insufficiency is responsible for BDE-47-mediated mitophagy defect and mitochondrial dysfunction in mouse livers, which was associated with suppression of Sirt3/FoxO3a/PINK1 signaling. Furthermore, our results indicated a potential role of miR-34a-5p in the hepatotoxicity of BDE-47. Mechanistically, BDE-47 dramatically upregulated miR-34a-5p expression in mouse livers. The data from AAV-sponge-mediated miR-34a-5p inhibition suggested that miR-34a-5p diminished NAD+ level by directly targeting NAMPT expression in BDE-47-treated mouse livers, which was confirmed by luciferase reporter assay. Consequently, miR-34a-5p markedly abated Sirt3/FoxO3a/PINK1 signaling-mediated mitophagy to promote mitochondrial dysfunction in BDE-47-treated mouse livers. The present study provided in vivo evidence to reveal a potential mechanism for BDE-47-induced mitochondrial dysfunction and related liver injury and indicated that miR-34a-5p-mediated mitophagy impairment might be a therapeutic target for BDE-47 toxicity.

Purpose: To establish a new rat model, the pathogenesis of which is closer to the clinical occurrence of chronic obstructive jaundice with liver fibrosis. Methods: 90 SD rats were randomly divided into 3 groups. Group A common bile duct ligation, group B common bile duct injection compont and group C injection saline. The serum of three groups was extracted, and the liver function was detected by ELISA. HE staining, Masson staining and immunohistochemistry were used to detect liver pathology. Results: Group B showed a fluctuant development of jaundice, obstructive degree reached a peak at 2 weeks, and decreased from 3 weeks. HA, LA and PCIII were significantly higher than control group. 3 weeks after surgery, liver tissue fibrosis occurred in group B, and a wide range of fiber spacing was formed at 5 weeks. Immunohistochemistry showed that hepatic stellate cells were more active than the control group. Conclusion: Intra-biliary injection of Compont gel is different from the classic obstructive jaundice animal model caused by classic bile duct ligation, which can provide an ideal rat model of chronic obstructive jaundice with liver fibrosis.

Background The aim of this study was to optimize the preparation method for self-assembled glyceryl monoolein-based cubosomes containing paeonol and to characterize the properties of this transdermal delivery system to improve the drug penetration ability in the skin. Material/Methods In this study, the cubic liquid crystalline nanoparticles loaded with paeonol were prepared by fragmentation of glyceryl monoolein (GMO)/poloxamer 407 bulk cubic gel by high-pressure homogenization. We evaluated the Zeta potential of these promising skin-targeting drug-delivery systems using the Malvern Zeta sizer examination, and various microscopies and differential scanning calorimetry were also used for property investigation. Stimulating studies were evaluated based on the skin irritation reaction score standard and the skin stimulus intensity evaluation standard for paeonol cubosomes when compared with commercial paeonol ointment. In vitro tests were performed on excised rat skins in an improved Franz diffusion apparatus. The amount of paeonol over time in the in vitro penetration and retention experiments both was determined quantitatively by HPLC. Results Stimulating studies were compared with the commercial ointment which indicated that the paeonol cubic liquid crystalline nanoparticles could reduce the irritation in the skin stimulating test. Thus, based on the attractive characteristics of the cubic crystal system of paeonol, we will further exploit the cosmetic features in the future studies. Conclusions The transdermal delivery system of paeonol with low-irritation based on the self-assembled cubic liquid crystalline nanoparticles prepared in this study might be a promising system of good tropical preparation for skin application.

Objective: To use microarray chip technology for screening of stem cell radiation related miRNAs in laryngeal squamous cell carcinoma; study and explore the relationship of miRNAs with radiosensitivity of laryngeal squamous cells. Method: After conventional culture and amplification of the laryngeal squamous carcinoma cell line Hep-2, CD 133+ cells were screened out with combination of isolated culture of stem cell microspheres and FACS for preparation of laryngeal cancer stem cells. After radiation treatment, miRNAs of laryngeal squamous carcinoma stem cells before and after radiation were enriched and purified. After microarray hybridization with mammalian miRNA and scanning of fluorescence signal, the miRNAs of laryngeal squamous carcinoma stem cells before and after radiation was subject to differential screening and clustering analysis. Real-time quantitative RT-PCR was used to verify part of the differentially expressed miRNAs. Results: 70 miRNAs related to laryngeal cancer stem cell radiation with 2-fold difference in expression were screened out, in which 62 were down-regulated and 8 were up-regulated. Fluorescent quantitative RT-PCR results were consistent with miRNAs chip results. Conclusion: Some miRNAs may be involved in self-regulation with laryngeal squamous carcinoma stem cell radiation.

To explore the signalling pathways involved in aldosterone-induced inflammation and fibrosis in rat vascular smooth muscle cells (VSMCs). Using Western blotting and real-time RT-PCR, we investigated the effects of aldosterone on the expression of cyclooxygenase-2 (Cox-2) and IL-6, two important proinflammatory factors, and TGFβ1, a critical profibrotic factor, in VSMCs. Aldosterone treatment significantly increased the expression of Cox-2 and IL-6 and activation of p38MAPK and NF-κB. The expression of both Cox-2 and IL-6 could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone and the p38MAPK inhibitor SB203580. Also, the rapid phosphorylation of p38MAPK could be suppressed by SB203580 but not by spironolactone, implicating in nongenomic effects of aldosterone. Similar to SB203580 and spironolactone, the NF-κB inhibitor α-p-tosyl-L-lysine chloromethyl ketone (TLCK) markedly attenuated expression of Cox-2, indicating that MR, p38MAPK and NF-κB are associated with aldosterone-induced inflammatory responses. Furthermore, aldosterone enhanced expression of TGFβ1 in rat VSMCs. This result may be related to activation of the MR/ERK-Sp1 signalling pathway because PD98059, an ERK1/2 inhibitor, significantly blocked the rapid phosphorylation of ERK1/2 and function of Sp1 and led to reduced expression of TGFβ1. Spironolactone was also shown to significantly inhibit TGFβ1 and Sp1 expression but not ERK1/2 phosphorylation. These results suggest that aldosterone-induced inflammatory responses and fibrotic responses may be mediated by the MR/p38MAPK-NF-κB pathways and the MR/ERK-Sp1 pathways in VSMCs, respectively.

High concentrations of arsenic, which can be occasionally found in drinking water, have been recognized as a global health problem. Exposure to arsenic can disrupt spatial memory; however, the underlying mechanism remains unclear. In the present study, we tested whether exercise could interfere with the effect of arsenic exposure on the long-term memory (LTM) of object recognition in mice. Arsenic (0, 1, 3, and 10 mg/ kg, i.g.) was administered daily for 12 weeks. We found that arsenic at dosages of 1, 3, and 10 mg/kg decreased body weight and increased the arsenic content in the brain. The object recognition LTM (tested 24 h after training) was disrupted by 3 mg/ kg and 10 mg/ kg, but not 1 mg/ kg arsenic exposure. Swimming exercise also prevented LTM impairment induced by 3 mg/ kg, but not with 10 mg/ kg, of arsenic exposure. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-response element binding protein (pCREB) in the CA1 and dentate gyrus areas (DG) of the dorsal hippocampus were decreased by 3 mg/ kg and 10 mg/ kg, but not by 1 mg/ kg, of arsenic exposure. The decrease in BDNF and pCREB in the CA1 and DG induced by 3 mg/ kg, but not 10 mg/ kg, of arsenic exposure were prevented by swimming exercise. Arsenic exposure did not affect the total CREB expression in the CA1 or DG. Taken together, these results indicated that swimming exercise prevented the impairment of object recognition LTM induced by arsenic exposure, which may be mediated by BDNF and CREB in the dorsal hippocampus.

Neural tube defects (NTDs) are severe birth malformations that affect one in 1,000 live births. Recently, mutations in the planar cell polarity (PCP) pathway genes had been implicated in the pathogenesis of NTDs in both the mouse model and in human cohorts. Mouse models indicate that the homozygous disruption of Sec24b, which mediates the ER-to-Golgi transportation of the core PCP gene Vangl2 as a component of the COPII vesicle, will result in craniorachischisis. In this study, we found four rare missense heterozygous SEC24B mutations (p.Phe227Ser, p.Phe682Leu, p.Arg1248Gln, and p.Ala1251Gly) in NTDs cases that were absent in all controls. Among them, p.Phe227Ser and p.Phe682Leu affected its protein stability and physical interaction with VANGL2. Three variants (p.Phe227Ser, p.Arg1248Gln, and p.Ala1251Gly) were demonstrated to affect VANGL2 subcellular localization in cultured cells. Further functional analysis in the zebrafish including overexpression and dosage-dependent rescue study suggested that these four mutations all displayed loss-of-function effects compared with wild-type SEC24B. Our study demonstrated that functional mutations in SEC24B might contribute to the etiology of a subset of human NTDs and further expanded our knowledge of the role of PCP pathway-related genes in the pathogenesis of human NTDs.

To study the pharmacokinetics of cantide, an antisense oligonucleotide, and its metabolites after iv gtt administration in rhesus monkeys, a dual solid phase extraction pretreatment method coupling with non-gel sieving capillary electrophoresis analysis method was used for determination of cantide and its metabolites in plasma and their pharmacokinetic parameters were calculated. The pharmacokinetic behavior of cantide and its metabolites (M1 and M2) after iv gtt administration (8, 16 and 24 mg kg(-1)) in rhesus monkeys were investigated. After iv gtt administration of cantide to rhesus monkeys, cantide in plasma was eliminated rapidly and the terminal elimination half-life (t1/2) was 57.91-77.97 min, the correlation coefficients (r) to the dose of Cmax AUC(o-inf) and AUC(0-t) of the prototype was 0.9918, 0.9568 and 0.9773, respectively. The metabolites of cantide reached the Cmax following cantide immediately and the Cmax of metabolites were lower than that of the prototype. The CL(S) of cantide and its metabolites (M1 and M2) were 1.60-2.19, 5.92-8.58 and 6.07-8.78 mL min(-1) kg(-1), respectively. So, it is concluded that the Cmax of cantide and its metabolites increased with the dose, which is the same as their AUC(0-inf) and AUC(0-t). The CL(S) of metabolites were higher than that of the prototype. The MRT and t1/2 of metabolites in the high dose group increased obviously.

Polyethylenimine-cyclodextrin-tegafur (PEI-CyD-tegafur) conjugate was synthesized as a novel multifunctional prodrug of tegafur for co-delivery of chemotherapeutic agent tegafur and enhanced green fluorescent protein (EGFP) reporter plasmid DNA. Conjugation of tegafur to PEI-CyD via chemical linkage was characterized by (1)H NMR spectrometry and ultraviolet (UV) spectrometry. PEI-CyD-tegafur was able to condense plasmid DNA into complexes of around 150 nm with positive charge at the N/P ratio of 25, in accordance with electron microscopy observation of compact and monodisperse nanoparticles. The results of in vitro experiments showed enhanced cytotoxicity and considerable transfection efficiency in B16F10 cell line. Therefore, PEI-CyD-tegafur may have great potential as a co-delivery system with anti-cancer activity and potential for gene delivery.

Aims Innate and adaptive immune responses are associated with the development of hypertension-induced myocardial hypertrophy and fibrosis. As a result, we investigated whether heat shock protein (HSP) 70, which is a molecule of damage-associated molecular patterns, could induce inflammation in the myocardium and promote the development of hypertension-induced cardiac hypertrophy and fibrosis. Methods and results We found that HSP70 serum levels, as well as the amount of HSP70 translocation to the cardiomyocyte membranes and the interstitial space, were elevated in the hypertensive mice caused by abdominal aortic constriction (AAC). Transcriptional inhibition of HSP70 expression by a specific heat shock transcript factor inhibitor, KNK437, reduced the serum level, and the re-distribution of HSP70. It promoted myocardial hypertrophy and cardiac dysfunctions although it protected animals from AAC-induced cardiac fibrosis. On the other hand, the functional antagonism of HSP70 by an anti-HSP70 antibody attenuated AAC-induced cardiac hypertrophy and fibrosis without adverse haemodynamic effects. The cardioprotective effect of the anti-HSP70 antibody was largely attributed to its ability to block AAC-activated immune response in the heart, as was indicated by suppressing the hypertension-enhanced conjugation of HSP70 with toll-like receptor 4, reducing heart-infiltrating macrophages, decreasing the expression of pro-inflammatory factor monocyte chemoattractant protein-1 and profibrotic factor transforming growth factor beta 1, and attenuating pro-hypertrophy signal MAPK P38 and ERK. Conclusion These results indicate that intracellular and extracellular HSP70 have different roles in the regulation of cardiac remodelling and function in response to hypertension. Extracellular HSP70 is a potential therapeutic target against cardiac hypertrophy and fibrosis.

To prepare the anticolon carcinoma vaccine with rich chaperone peptide and to examine its anticancer immunological efficacy.CT26 colon carcinoma cells were cultured in 1 mg/L Trichosanthin 1640 medium at different temperatures to induce the chaperone expression and promote the synthesis of antigen peptides. Groups of these cells treated under the different condition were lysed by the sonic disintegration, and the lysates were centrifuged. The rawpurified proteins were obtained from the supernatants by precipitating with saturated ammonium sulfate and removing the molecules below 50,000 and above 300,000 in molecular weight via dialysis. Furthermore, the proteins with the molecular weights in 70,000, 90,000, 95,000, 110,000 and 170,000 were collected through gel filtration and SDS-PAGE. The purified proteins were analysed by Western blotting, and inspected on the anticancer immunological effects including lymphocyte proliferation and the activities of NK and CTL.Major of the chaperone peptides of anticancer effects in CT26 cells, including antigen peptides joining with HSP70, HSP90, gp96, HSP110 and HSP170, was satisfactorily extracted and condensed, and rich chaperone peptide composites were successfully obtained. The composites prepared under various condition could all enhance lymphocyte proliferation and the activities of CTL and NK(P0.01).The rich chaperone peptide composites are successfully prepared via dialysis, salt fractionation and gel filtration combined with SDS-PAGE. Both the heat stress and Trichosanthin can increase the composites, which treated by 42 centi-degree heat stress and Trichosanthin are found to possess the strongest anticancer efficacy.

Altered peptide ligand (APL), a short peptide with immune regulatory activity and substitutions of a single or multiple amino acids in an antigenic peptide, has shown potential therapeutic effect on autoimmune disease, tumor and virus infection. APL regulates immune responses by interfering the interaction between the major histocompatibility complex (MHC), antigenic peptide and T cell receptor (TCR), or by regulating the intracellular signaling of antigen presenting cells, bystander suppression and inducing heterogenous immune responses. High-specific and high-affinity APL screened from peptide laboratory by phage display, has a potential to be a new resource for drug with antigen specificity.

To develop a novel dual-modified vaccine, the superantigen-linked intestine-carcinoma cells expressing membrane-bound heat shock protein 70 (HSP70), and further examine its anticancer therapeutic effect.The pre-established intestine carcinoma CT26 line expressing membrane-bound heat shock protein 70 (HSP70) was amplified and incubated with superantigen fusion protein, staphylococcal enterotoxin A (SEA) fused with transmembrane sequence (SEA-TM), thereby the dual-modified vaccine was prepared after inactivation. The anticancer efficacy of the vaccine was examined.The laser confocal microscopy and flow cytometry showed that there co-existed much HSP70 and SEA on the vaccine membrane surface. Both of the single-modified vaccines, the SEA-linked vaccine and membrane-bound-HSP70-expressing one, displayed marked tumor suppression, a prolonged survival period, augmented lymphocyte proliferation and higher NK and CTL activity in the vaccinated mice when compared with its counterpart. Furthermore, the dually modified vaccine induced lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest tumor rejection in the vaccinated mice. The survival period of the mice was further prolonged.A new vaccine, SEA-linked and membrane-bound-HSP70-expressing intestine-carcinoma cells can induce more potent anticancer immunity and produce better therapeutic efficacy.

To develop a new vaccine expressing membrane-bound heat shock protein 70 (mbHSP70) and further study its antitumor therapeutic effect.The pre- established vector expressing mbHSP70 was transfected into CT26 cells of colorectal cancer. After the CT26 cells were incubated with 900 microg/ml G418, the sub-clones resistant to G418 were harvested and the HSP70 positive clones were selected by limiting dilution. The clones were amplified and inactivated, thereby the vaccine expressing mbHSP70 was prepared. Lymphocyte proliferation stimulated by the vaccines, NK and CTL activity was observed. The antitumor efficacy of vaccine was observed in BALB/c mice model with colorectal cancer.The laser confocal microscopy and flow cytometry showed that there existed much HSP70 on the vaccine membrane surface. The HSP70 gene-modified vaccine displayed augmented lymphocyte proliferation and higher NK and CTL activity in vitro,and marked tumor suppression and prolonged survival time of the vaccinated micein vivo, when compared with its counterpart. Furthermore, mbHSP70-expression vaccine elicited lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest antitumor effect, and prolonged survival time of the vaccinated mice.A new vaccine expressing mbHSP70 has more potent antitumor immunity and better therapeutic efficacy than HSP70 gene-modified vaccine did.

To prepare the SEA-TM and mB7.1-GPI dual-anchored EL-4 cell vaccine and to investigate its antitumor effects.mB7.1-GPI-anchored EL-4 cell vaccine, SEA-TM-anchored EL-4 cell vaccine, SEA-TM and mB7.1-GPI dual-anchored EL-4 cell vaccine were prepared. In vitro the biological activities of these vaccines were measured using a lymphocyte proliferation assay and cytokine release assay on splenocytes derived from C57BL/6 mice. The splenocytes were co-cultured with EL-4 or EL-4/mB7.1-GPI or EL-4/SEA-TM or EL-4/SEA-TM + mB7.1-GPI (treated with Mitomycin C). Lymphocyte proliferation was determined with MTT assay, the concentrations of cytokines (IL-2 and IFN-gamma) were measured using a ELISA technique. Forty C57BL/6 mice were inoculated with EL-4 cells, after 3 days the mice were randomly divided into 5 groups with 8 in each and were treated with PBS, EL-4 cell vaccine, EL-4/mB7.1-GPI cell vaccine, EL-4/SEA-TM cell vaccine and EL-4/SEA-TM + mB7.1-GPI cell vaccine respectively, vaccines were injected three time with two-day interval. Animals were observed daily, tumor sizes were measured every third day. Twenty-five days after tumor challenge, 3 mice in each group were sacrificed and splenic lymphocytes were isolated to examine the activity of natural killer cells (NK) and cytolytic T lymphocytes (CTL). The survival of the remaining 5 mice in each group was observed till the 90th day.mB7.1-GPI or/and TM-SEA fusion protein was stably anchored onto the surface of EL-4 tumor cells. EL-4/mB7.1-GPI or EL-4/SEA-TM had a stronger ability to stimulate lymphocyte proliferation and IL-2 and IFN-gamma production than EL-4 (P0.05); while EL-4/SEA-TM + mB7.1-GPI showed a further increased ability than EL-4/mB7.1-GPI and EL-4/SEA-TM in stimulating lymphocyte proliferation and cytokine production in vitro (P0.05). Volume of tumor was smaller and survival time of mice was longer in EL-4/mB7.1-GPI vaccine group, EL-4/SEA-TM vaccine group and EL-4/SEA-TM + mB7.1-GPI vaccine group, comparing with PBS group and EL-4 cell vaccine group (P0.05). Tumor volume was much smaller and survival time of mice was much longer in EL-4/mB7.1-GPI + mB7.1-GPI vaccine group, comparing with EL-4/SEA-TM vaccine group and EL-4/mB7.1-GPI vaccine group (P0.05). Lymphocytes derived from the mice treated with EL-4/SEA-TM + mB7.1-GPI showed much higher NK activity and CTL activity than those derived from EL-4/mB7.1-GPI vaccine group and EL-4/SEA-TM vaccine group (P0.05), meanwhile the NK activity and CTL activity of EL-4/mB7.1-GPI vaccine group and EL-4/SEA-TM vaccine group was higher than EL-4 vaccine group (P0.05).mB7.1-GPI or/and SEA-TM fusion protein was stably anchored onto the surface of EL-4 tumor cells. The tumor cell vaccines prepared from these cells exhibited antitumor effect. The mB7.1-GPI and SEA-TM dual-anchored tumor cell vaccine had much stronger antitumor effect than the single-anchored tumor cell vaccine.

To investigate the influencing factors of polyethylenimine (PEI) in gene transfer in vitro.Cytotoxic effects of PEI on in vitro cultured NIH 3T3 cells were quantified by MTT assay. The interaction between PEI and DNA at different charge ratios was analyzed by agarose gel electrophoresis retardation assay. The expression of gene transfer was monitored in Cos-7 cells using pEGFP and pSV beta plasmids as the reporter gene systems. Influences of chloroquine, albumin, serum, salt ion strength, and Mg(2+) ion and other factors on PEI/DNA transfer efficiency were evaluated.The survival rate of NIH3T3 cells at 6 mg/L of PEI was 64.2% and at 7 mg/L of PEI was 54.4%. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 PEI nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of PEI. Albumin and serum in the culture medium decreased the transfection efficiency. HBS(HEPES buffered solution) or 150 mmol/L NaCl as the dilution solution of PEI/DNA was superior over 278 mmol/L glucose solution in the transfection efficiency. Mg(2+) in the dilution solution decreased the transfer efficiency of PEI/DNA.PEI is efficient gene transfer agent of eukaryotes in vitro, and can be possibly used in vivo.

To study the local hemorrhagic activity of a fibrinolytic enzyme (FII(a)) from Agkistrodon acutus venom and its mechanism.The local hemorrhagic activity was determined by subcutaneous injection on the back of mouse. The effects of FIIa on factor X, prothrombin, gelatin, and collagen were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Platelet aggregation assays were performed in rat platelet-rich plasma (PRP). Human umbilical vein endothelial cells (HUVEC) were cultured and passaged in complete M199 medium. Cell viability and nuclear morphology change were determined by fluorescein diacetate (FDA) staining and Hoechst 33258 staining, respectively.The minimum hemorrhagic dose (MHD) of FIIa was 89 microg. In vitro, FII(a) (0.25 g/L) degraded factor X, prothrombin, collagen, and gelatin, and dose-dependently (0.25, 0.50, 0.75, and 1.00 g/L) inhibited the platelet aggregation induced by ADP in rat PRP. When HUVEC in culture treated with FII(a), HUVEC showed detachment in a dose-dependent manner, but no apoptosis sign was observed.FIIa had local hemorrhagic activity, and the mechanism was related to the degradation of factor X, prothrombin, gelatin, and collagen, the inhibition of ADP-induced platelet aggregation, and inducement of HUVEC detachment.

Phagocytosis and subsequent phagosome maturation by professional phagocytes are essential in the clearance of infectious microbial pathogens. The molecular regulation of phagosome maturation is largely unknown. We show that integrin β1 plays critical roles in the phagocytosis of microbial pathogens and phagosome maturation. Macrophages lacking integrin β1 expression exhibit reduced phagocytosis of bacteria, including group B streptococcus and Staphylococcus aureus. Furthermore, phagosomes from macrophages lacking integrin β1 show lowered maturation rate, defective acquisition of lysosome membrane markers, and reduced F-actin accumulation in the periphagosomal region. Integrin β1-deficient macrophages exhibit impaired bactericidal activity. We found that the expression of the Rho family GTPases Rac1, Rac2, and Cdc42 was reduced in integrin β1-deficient macrophages. Ectopic expression of Rac1, but not Cdc42, in integrin β1-deficient macrophages restored defective phagosome maturation and F-actin accumulation in the periphagosomal region. Importantly, macrophages lacking Rac1/2 also exhibit defective maturation of phagosomes derived from opsonized Escherichia coli or IgG beads. Taken together, these results suggest that integrin β1 regulates phagosome maturation in macrophages through Rac expression.