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Objective To investigate the efficiency of different concentrations of genipin and silk fibroin (SF)∶ chitosan (CS) ratios on the package and release of SF-CS composite microspheres. Methods Selected the microspheres with a SF∶CS ratio of 1∶1 to encapsulate different concentrations of bovine serum albumin (BSA) and compared their encapsulation efficiency and sustained-release rate with those of pure chitosan microspheres. SF-CS microspheres were prepared with emulsion cross-linking technique. The microspheres were observed by scanning electron microscope. Size distribution was measured by a laser particle size analyzer. X-ray diffractometry (XRD), fourier transform infrared spectroscopy (FTIR) and thermogravimetricanalyzer (TGA) were used to analyze their structural characteristics. BCA method was used for determination of the drug entrapment, loading rate and cumulative release of the total drug. Results The five types of microspheres (m (CS): m (SF) = 1∶0.5, 0.1 g or 0.5 g genipin; m (CS)∶m (SF) = 1∶1, 0.05 g or 1 g genipin; m (CS)∶m (SF) = 1∶2, 0.5 g eniping) had a more spherical shape and smooth surface with particle size between 70-147 μm. The microspheres prepared with m (CS)∶m (SF) of 1∶1 and 0.05 g genipin in the presence of 10 mg,20 mg, or 50 mg BSA of BSA burst-released (30.79±3.43)%, (34.41±4.46)%, or (41.75±0.96)% of the entrapped BSA on the first day, and cumulatively released (75.20±2.52)%, (79.16±4.31)%, and (89.04±4.68)% in 21 d, respectively. The pure CS microspheres prepared in the presence of 10 mg BSA burst-released (39.53±1.76)% on the first day and cumulatively released (83.57±2.33)% of the total encapsulated BSA in 21 d. Conclusion The SF-CS composite microspheres had a higher sustained release rate than the pure CS microspheres and may be a better drug carrier.

Objective: To prepare and characterize a nano-scale fibrous hydrophilic poly-L-lactic acid/ Bioglass (PLLA/BG) composite membrane and evaluate its biocompatibility as a composite membrane for guiding bone regeneration (GBR)., Methods: PLLA/BG-guided bone regeneration membrane was treated by oxygen plasma to improved its hydrophilicity. The growth of MG-63 osteoblasts on the membrane was observed using Hoechst fluorescence staining, and the biocompatibility of the membrane was evaluated by calculating the cells adhesion rate and proliferation rate. Osteogenesis of MG-63 cells was assessed by detecting alkaline phosphatase (ALP), and the formation of calcified nodules and cell morphology changes were observed using scanning electron microscope (SEM)., Results: The cell adhesion rates of PLLA/BG-guided bone regeneration membrane treated with oxygen plasma were (30.570±0.96)%, (47.27±0.78)%, and (66.78±0.69)% at 1, 3, and 6 h, respectively, significantly higher than those on PLLA membrane and untreated PLLA/BG membrane (P<0.01). The cell proliferation rates on the 3 membranes increased with time, but highest on oxygen plasma-treated PLLA/BG membrane (P<0.01). Hoechst fluorescence staining revealed that oxygen plasma treatment of the PLLA/BG membrane promoted cell adhesion. The membranes with Bioglass promoted the matrix secretion of the osteoblasts. Under SEM, the formation of calcified nodules and spindle-shaped cell morphology were observed on oxygen plasma-treated PLLA/BG membrane., Conclusion: Oxygen plasma-treated PLLA/BG composite membrane has good biocompatibility and can promote adhesion, proliferation and osteogenesis of the osteoblasts.

Objective: To study the cultural method and identification of human umbilical vein endothelial cells (HUVECs), and investigate the expression of tyrosine kinase-2 with immunoglobulin-like and epidermal growth factor homology domains(Tie-2) in HUVECs., Methods: HUVECs were isolated from umbilical veins by the technique of irrigative digestion, and were cultivated in plates. The cells were identified by VIII monoclonal antibody. Tie-2 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and SABC immunocytochemistry., Results: HUVECs could adhere to the plates completely after 24 hours, and confluence a monolayer 4-5 days later. The band of Tie-2 mRNA was obviously and the expression of Tie-2 protein was strongly positive by immunocytochemistry in HUVECs. The positive rate was over 85%., Conclusion: Highly purified endothelial cells were isolated. And there were overexpression of Tie-2 in HUVECs.

Objective: To construct an eukaryotic coexpression vector containing Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and enhanced green fluorescent protein (EGFP) controlled by cytomegalovirus promoter using pIRES-EGFP vector., Methods: The mtHSP70 gene fragment was amplified by PCR from pVAX-mtHSP70-HSV2gD using specific primers. The PCR product was cloned into the vector pMD 18-T vector, and the correct clone was selected according to DNA sequence analysis. The interested mtHSP70 gene fragment was subcloned into pCMV-IRES-EGFP vector with XhoI and EcoR I digestion. The recombinant plasmid was transfected into mouse melanoma B16 cell line, and the green fluorescent cells were detected by fluorescence microscopy and mtHSP70 expression was detected by Western blotting., Results: The recombinant plasmid obtained was confirmed by enzyme digestion. The transfected mouse melanoma B16 cells exhibited green fluorescence under fluorescence microscopy and expressed mtHSP70 protein as demonstrated by Western blotting., Conclusion: The eukaryotic coexpression vector PCMV-mtHSP70-IRES-EGFP has been established to allow further investigation of the role of mtHSP70 gene in tumor immunotherapy.

Objective: To investigate the apoptosis and proliferation activity of Tca8113 cells., Methods: The vector that involves short hairpin RNA of iNOS was transfected to Tca8113 cells. The change of iNOS expression was observed using immunohistochemistry technique, the apoptosis rate examined by flow cytometry, and the proliferation Tca8113 cells examined by methyl thiazolyl tetrazolium (MTT)., Results: The expression of iNOS in Psilencer-iNOS group was lower than that in control groups (P < 0.01), the apoptosis rate was higher than that in control groups (P < 0.01); whereas the proliferation activity of Tca8113 cells in Psilencer-iNOS group was lower than that in control groups., Conclusions: Down expression of iNOS by RNAi can promotes apoptosis of Tca8113 cells and has an anti-proliferation activity effect.

Objective: To investigate the effect of inducible nitric oxide synthase (iNOS) on vascular endothelial growth factor (VEGF) expression., Methods: The vector containing short hairpin RNA of iNOS was transfected into Tca8113 cells using the RNA interference (RNAi) technique. The gene and protein expression of iNOS and VEGF was examined by RT-PCR and Western blot., Results: The expression of iNOS, VEGF gene in Tca8113 cells was significantly different between the experimental and control groups 24 h and 48 h after transfection (P < 0.05). The protein expression of iNOS was different between the two groups 36 h and 48 h after transfection, and of VEGF was also different between the two groups 48 h after transfection (P < 0.05)., Conclusions: The expression of VEGF could be down-regulated by silencing the iNOS gene in Tca8113 cells.

Background & Objective: Recently, most of studies have indicated that nitric oxide (NO) plays an important role in carcinogenesis and tumor progression. Inducible nitric oxide synthase(iNOS) has been found at significantly higher expression levels in head and neck squamous cell carcinoma (HNSCCA) specimens as compared with that in normal tissue. Furthermore, these data provided strong evidance for the role of iNOS generating NO in HNSCCA progression. However, there was no report about whether expression of iNOS mRNA in oral carcinoma is associated with its biological behavior. This study was designed to investigate the correlation between expression of inducible nitric oxide synthase mRNA (iNOS mRNA) in squamous cell carcinoma of tongue tissue and its invasive growth, neck lymph node metastasis, and prognosis., Methods: Sixty-eight cases with squamous cell carcinoma of tongue tissues were investigated. All cases with tongue carcinoma were followed up to obtain the information of prognosis. In situ hybridization assay was used to detect the expression of iNOS mRNA in tongue carcinoma tissues. The correlation between expression of iNOS mRNA in squamous cell carcinoma of tongue tissue and its invasive growth, neck lymph node metastasis, and prognosis was analyzed., Results: 1. The positive expression rate of iNOS mRNA was 86.1% and 48.0% in the group of invasive growth and no invasive growth respectively. The difference between the two groups was significant with coefficient of 0.3. 2. The positive expression rate of iNOS mRNA was 85.7% and 62.5% in the group of positive lymph node metastasis and negative lymph node metastasis respectively. The difference between the two groups was significant, which the correlation coefficient was 0.3. 3. Three-year survival rate was significantly lower in the group of positive expression of iNOS mRNA than that in the group of negative expression of iNOS mRNA in the tongue carcinoma., Conclusions: 1. A positive correlation was found between the positive expression of iNOS mRNA and invasive growth as well as neck lymph node metastatsis of squamous cell carcinoma of tongue. 2. The patient's prognosis of the positive expression of iNOS mRNA would be worse than that of the negative expression of iNOS mRNA in tongue carcinoma tissue.