Objective To study the effects of bFGF on the proliferation of cultured human melanocytes, and to seek a quick method for in vitro culture of human melanocytes. Methods Melanocytes were isolated from human foreskin, and divided into two parts to be cultured with or without the presence of bFGF (0.3 μg/L). Second-passage melanocytes were identified with immunochemical stain. The growth of melanocytes was observed every 3 days for 12 days. Third-passage melanocytes were treated with various concentrations (0.3 - 2.1 μg/L) of bFGF for 72 hours followed by the detection of proliferation of and trosinase activity in melanocytes. Results Human melanocytes were obtained from primary culture in medium containing certain concentrations of bFGF, which were identified with immunohistochemical stain. The morphology of cultured melanocytes varied with growth stage of cells. The bFGF-treated melanocytes appeared to grow more rapidly than untreated melanocytes. Further more, a significant increase was observed in the proliferation rate of melanocytes treated with bFGF of 0.3 and 0.6 μg/L (P＜0.05 or 0.01 ) and tyrosinase activity in melanocytes treated with bFGF of 1.5 and 1.8 μg/L (P ＜ 0.05 or 0.01 ) in comparison with the untreated melanocytes.Conclusions The addition of certain concentrations of bFGF to defined medium can benefit the primary culture of melanocytes and make it possible to get large quantities of purified melanocytes with high viability in short periods. Certain concentrations of bFGF can up-regulate the proliferation of and tyrosinase activity in melanocytes.

Objective To study the in vitro culture condition for melanoblasts from human foreskin tissue. Methods The skin tissue taken from foreskin of children was treated with 0.5% dispase Ⅱ to separate epidermis from dermis, then with trypsin to obtain single cell suspension, which was cultured in modified medium for melanoblasts, i.e., MCDB254 medium supplied with several cell growth factors. Finally, melanoblasts were obtained based on the difference of adhesion speed. The morphology and proliferation of cultured melanoblasts were observed under a light microscope. DOPA staining, immunostaining with anti- S-100 and -tyrosinase related protein 2 (TRP2) antibodies, and transmission electron microscopy were per- formed to identify the cultured melanoblasts. Results The cultured human melanocytes displayed a match-like shape, scattered arrangement, syrmnetric double poles, slim cell body, highly refractive nuclei; meanwhile, the melanoblasts exhibited plentiful cytoplasm, large volume, bipolar or irregular shape and clonal growth. Additionally, the melanocytes were positive for TRP2, S-100 and Dopa staining, while the melanoblasts were positive only for TRP2. Electron microscopy revealed the presence of mature melanin granules (stage Ⅲ-Ⅳ ) in melanocytes but immature melanin granules (stage Ⅰ ) in melanoblasts. Conclu- sion Stable pure culture of melanoblasts has been realized with the reformed medium, which may lay a foundation for the investigation into the mechanism of epidermal pigmentation.

Objective Linkage analysis were performed in 2 pure Chinese paroxysmal kinesigenic dyskinesia families to localize the locus of them. Method Microsatellites markers corresponding to pericentrometric region of chromosome 16 were used in parametric and nonparametrie linkage analysis for 27 members in the 2 pedigrees, haplotypes were constructed subsequently. Result The maximum LOD score and NPL score in the 2 families were all negative, P values were significantly larger than 0.05.No haplotype segregated with PKD phenotype was found. It showed no evidence of association with known PKD loci in both pedigrees, providing evidence for a novel PKD locus. Conclusion PKD is heterogeneous, a novel PKD locus may be in pure Chinese pedigrees.

Objective To establish a large animal ( sheep) model to serve the experiments of domestic pulsatile ventricular assist device.Methods Three small-tail Han-sheep were anesthetized and the vein access and artery access were achieved.The cardiopulmonary bypass was established through left thoracotomy.Ventricular fibrillation was induced. An hole was made in the apex of left ventricle and the apex cannulation was sutured to it.The aortic cannulation was su-tured to the descending aorta.The two cannulations were connected to the domestic pulsatile ventricular assist device ( DP-VAD) and the driver was turned on.The working of DPVAD and the conditions of the animals were observed.Results The DPVAD worked well and uni-directional blood flow was driven by positive and negative pressure.The left ventricle was unloaded and the blood pressure was raised up.Conclusion The establishment of sheep model of pulsatile ventrieular as-sist device may play important role for the research and development of DPVAD in our country.

Objective:To study the effect of Shenqi Fuzheng injection combined with interferon-α (IFN-α) on the expression of STAT1 gene in hepatocellular carcinoma cells,and to elucidate its mechanism of IFN-α synergistic effect.Methords:The effect of IFN-α injection alone or combined with Shenqi Fuzheng injection on the proliferation of MHCC97-L cells was detected by MTT assay.IFN-α was detected by Real-time PCR and Western-blot respectively.The expression of STAT1 mRNA and protein in the experimental group,the negative control group (NaCl) and the blank control group were determined,and the effect of Shenqi Fuzheng injection on the transcription and expression of STAT1 was determined.The expression of STAT1 in lentiviral vector MTT assay was used to detect the effect of IFN-α alone or in combination with Shenqi Fuzheng injection on the STAT1 gene silencing cells.The expression of STAT1 was detected by RT-PCR and Western-Blot in MHCC97L cells.Strain,on cell proliferation.Results:Compared with IFN-α alone,Shenqi Fuzheng injection combined with IFN-α enhanced the inhibitory effect of IFN-α on MHCC97-L (P＜0.01) and up-regulated the expression of STAT1mRNA and protein (P＜0.05).Successfully constructed the STAT1 gene silent in the MHCC97-L cell line.There was no significant difference in the inhibition rate of MHCC97-L between Shenqi Fuzheng injection and IFN-α (P＞0.05).Conclusion Shenqi Fuzheng Injection can up-regulate the expression of STAT1 in human hepatocellular carcinoma cell line MHCC97-L,so as to increase the effect of IFN-α.

Objective To investigate the influence of early growth response gene-1 (EGR1) on the autophagy of host cells following infection with human T cell leukemia virus type 1 (HTLV-1).MethodsA HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model.Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1.Luciferase reporter gene analysis was used to detect the transcriptional activity of 5′-regulatory sequence of EGR1 at different time points after co-culturing.An effective small interfering RNA (siRNA) targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000.Then the transfected HeLa cells were co-cultured with the HTLV-1-positive cell line MT2 for 24 h.Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3.Real-time PCR was performed to detect viral load.Autophagosome was analyzed by immunofluorescence after co-culturing.Results The expression of EGR1 and the transcriptional activity of pEGR1-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h (P<0.01).The expression of EGR1 was positively correlated with host cell autophagy following HTLV-1 infection.The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2.The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was concomitant with a persistent decrease of intracellular autophagosome (P<0.01).Conclusion EGR1 is associated with host cell autophagy and viral replication in HTLV-1 infection.

Objective To explore the regulatory effects of HTLV-1 ( human T-cell leukemia virus type 1 ) Tax protein on the expression of HMGB 1 ( high mobility group box 1 ) gene in T cells .Methods Total RNA and protein were extracted from Tax +-T cells ( TaxP ) , Tax--T cells ( TaxN ) and Jurkat cells which were stably transfected with pCMV-Tax and pCMV-Neo, respectively.Then, the expression levels of HMGB1 mRNA and protein in different CD 4+T cells were analyzed by real-time PCR and Western blot (WB).By using liposome-mediated method, pGL3-HMGB1-luc reporter genes and pGL3-neo-luc were tran-siently transfected into TaxP and TaxN cells and the basal transcriptional activity was observed in different T cells.Additionally, pCMV-Tax and pGL3-HMGB1-luc reporter genes were also co-transfected into Jurkat cells and the regulatory effects of Tax protein on HMGB 1 gene was detected .The chromatin immunoprecipi-tation (ChIP) assay was used to identify HMGB1 genomic sites directly targeted by Tax .Results The ex-pression levels of HMGB1 mRNA and protein in Tax+-T cells ( TaxP) were higher than those in Tax--T cells (TaxN).The transcription regulation trends for HMGB1 gene in TaxN and TaxP cells were similar but not identical in diverse T cells.pHLuc3 (containing -504-+83 HMGB1) showed the highest transcriptional ac-tivity of HMGB1 gene in both TaxP and TaxN cells , but HMGB1 transcriptional activity of pHLuc 6 in TaxP cells was significantly stronger than that in TaxN cells .Luciferase assays also showed that Tax protein promo-ted the transcription of HMGB1 gene in a dose-dependent manner .The ChIP assay further confirmed that Tax protein enriched at the HMGB1 region of -1163--1043.Conclusion The region of nt -504--383 is essen-tial for the basal promoter activity of -1163-+83 HMGB1 gene originated from pHLuc 6 reporter plasmid , and Tax protein enriched probably at the HMGB 1 site of -1163--1043 enhances HMGB1 transcription.

Objective To explore the clinical efficacy and mechanism of Qinggan Sanjie Xiaoying Decoction in the treatment of Hashimoto's thyroiditis with syndrome of stagnation heat of liver meridian and stagnation of spleen deficiency and phlegm.Methods Totally 70 patients were divided into control group and medicine group according to their wishes,with 35 patients in each group.Both groups were restricted to an iodine diet.The medicine group was given Qinggan Sanjie Xiaoying Granules,1 sachet at a time,twice a day,orally.The treatment for both groups lasted for 4 weeks.20 healthy people were chosen as the healthy group.The clinical efficacy of both groups was observed.TCM symptom score,thyroid antibody titer levels(TPOAb,TGAb),changes in thyroid volume and isthmus of both groups before and after treatment were compared.Levels of serum IKKα,IKBα and TNF-α of the three groups were compared.Adverse reactions of patients daring the treatment period were monitored.Results The total effective rate of the medicine group was 85.71%(30/35),while the control group was 20.00%(7/35).The medicine group was superior to the control group(P<0.05).Compared with before treatment,the medication group showed significant improvement in TCM symptom scores,TPOAb and TGAb titer levels,thyroid volume,and thyroid isthmus thickness after treatment(P<0.05).After treatment,TCM symptom score,thyroid volume in the medicine group were lower than those in the control group(P<0.05),and the decrease rate of TPOAb titer was higher than that in the group(P<0.05).The levels of IKKα and TNF-α before treatment of medicine group and control group were higher than that in the healthy control group,and the level of IKBα was lower than that of the healthy control group(P<0.05);compared with before treatment,the levels of IKKα and TNF-α in the medicine group decreased,and the level of IKBα increased(P<0.05);after treatment,the levels of IKKα and TNF-α in the medicine group were lower than that in the control group,and IKBα was higher than the control group(P<0.05).No adverse events were observed during the treatment period in both groups of patients.Conclusion Qinggan Sanjie Xiaoying Decoction can reduce the antibody titer level,thyroid enlargement,isthmus thickness,and TCM syndrome score in the treatment of Hashimoto's thyroiditis.It is safe and effective in clinical practice.Qinggan Sanjie Xiaoying Decoction may play a therapeutic role by interfering with NF-κB signaling pathway.

Introducing the continuing education of the dental hygienist in eight states of the U.S.,to understand the the category of practice,professional ability,curriculum, teaching methods and teaching evaluation standard. To analyze the continuing education in eight states to provide a reference for constructing a curriculum system that is suitable for China.

Objective:To analyze the mechanism of Jindan Tablets, Xiaoyan Lidan Tablets and ursodeoxycholic acid in the treatment of gallstone and cholecystitis based on network pharmacology; To conduct a comparative analysis.Methods:The chemical components of Jindan Tablets, Xiaoyan Lidan Tablets and ursodeoxycholic acid and their drug targets were collected from Traditional Chinese Medicine Database and Analysis Platform (TCMSP). DAVID 6.8 database was used to search for the associated diseases of the drug targets. The disease targets of gallstone and cholecystitis were collected from GeneCards and other databases. The protein-protein interactions network was established based on the intersecting targets of three drugs and two diseases. KEGG enrichment analysis was performed based on the DAVID 6.8 database. Cytoscape 3.7.1 software was used to construct a complex network and topology analysis of component- target- disease between three drugs and diseases.Results:222 chemical components and 3 133 drug targets were collected for Jindan Tablets. 104 chemical components and 1 425 action targets were collected for Xiaoyan Lidan Tablets. 1 chemical component and 119 action targets were collected for ursodeoxycholic acid. The three drugs were associated with 31 diseases. 1 382 disease targets for gallstones and cholecystitis were collected. There were 237, 163 and 33 targets for gallstones and cholecystitis in the three drugs, of which 17 were shared by the three drugs and 20 were shared by Jindan Tablets and Xiaoyan Lidan Tablets. Based on the DAVID database, 113, 74 and 10 significant KEGG enrichment pathways were obtained for the three drugs respectively.Conclusions:The three drugs shared many targets and pathways in the treatment of gallstones and cholecystitis, which all had the function of regulating metabolism and inhibiting inflammatory response, while participating in apoptosis, oxidative stress and cancer pathology process. However, they had their own special effects, with Jindan Tablets favoring involving in the cancer process and inhibition of inflammation, and promoting angiogenesis. Xiaoyan Lidan Tablets and ursodeoxycholic acid focused on regulating cholesterol metabolism, and Xiaoyan Lidan Tablets also regulated steroid metabolism and inhibit inflammation, while ursodeoxycholic acid regulated bile acid metabolism.