Objective To study the survival and melanogenic potential of human melanocytes reversibly immortalized via SV40T antigen gene and Cre/loxP system in Guinea pigs. Methods The supernatants of retrovirus vector Cre-ERT2 were used to infect melanocytes which had been successfully transfected by SV40TAg gene (MCT), then the expression of Cre recombinase was induced with tamoxifen in infected cells; subsequently, the surviving cells, which were named as MCTC, were subjected to expansion culture. Guinea pigs were utilized to establish animal models of vitiligo, then MCTC and primary melanocytes were transplanted respectively into the animal models. The repigmentation at the transplanted area was observed with naked eyes successively until 3 months after the transplantation when tissue samples were obtained from implanted area and nonimplanted area of guinea pigs and subjected to Masson-Fontana silver stain and Hematoxylin-eosin stain for the analysis of melanocyte distribution and melanin deposition in epidermis. Results Repigmentation started 4 weeks after the transplantation, and dark or brown patches, which ranged in size from 0.5 to 1 cm, were observed in the implanted area 3 months after the transplantation. The repigmentation rate was of no significant difference between pigs transplanted with MCTC and those with primary melanocytes (82.5% vs 76.7%, P > 0.05). Pathological examination revealed melanin deposition in the basal layer of epidermis and some hair follicles in transplanted area. Conclusions SV40T antigen gene combined with Cre/loxP site-specific recombinase system can induce the reversible immortalization of human melanocytes, and the immortalized melanocytes have a favorable profile of biological safety and similarity in survival rate and melanogenic potential to primary melanocytes.

China ; Humans ; Laparoscopy ; education ; Mentors ; Urology ; education

Objective To perform the epidemiological analysis of the developmental dysplasia hip(DDH) in Shigatse of Tibet in order to explore the correlation between the onset of DDH with some risk factors such as altitude and take targeted intervention measures to reduce the disability rate of DDH.Methods A total of 5900 children aged 0 to 14 year-old from different altitudes in Shigatse of Tibet were surveyed randomly from June 2013 to June 2014.SPSS 18.0 statistical software was used to analyze the correlation of the onset of DDH with residence altitude,production and living ways,binding method of the lower limbs,delivery situation and genetic factor.Results One hundred and ninety-one DDH cases were screened out,and the average incidence of DDH in Shigatse of Tibet was about 32.4‰,and unilateral was higher than bilateral,fight side(33 cases)∶left side(68 cases) =1.00∶ 2.06.The result of Pearson correlation analysis of the onset of DDH with residence altitude showed that there was significant correlation (r=0.820,P=0.046).The result of Spearman correlation analysis of the onset of DDH with production and living ways showed that there was significant correlation(r=0.530,P =0.048).The result of x2 test analysis of the onset of DDH with binding method of the double lower limbs and breech delivery showed that there was statistical significance (P=0.0017,0.0082).Conclusions The DDH incidence of children living in high altitude areas in Tibet has certain regional characteristics indicating that it has clear correlation with some environmental risk factors such as altitude,methods of the lower limbs binding,production and living ways and breech delivery,especially the bundled package of lower limbs is the main environmental risk factor of DDH.

OBJECTIVE To design and construct eukaryocyte expression vector of SV40 virus large T antigen and induce its targeted expression in eukaryocyte.METHODS SV40 large T gene which excised intron was cloned by SOE(splicing by overlapping extension) and digested with restricted enzymes EcoR Ⅰ and BamH Ⅰ.By the same methods,we got the digested product of pEGFP-N1.After that,the two fragments were ligated to form SV40(TEGFP) by Ligation Kit,and sequenced by TaKaRa ABI Prism Terminator Cycle Sequence Kit.The reconstructed vector was transfected into primary cultured human fibroblast using a Lipofectin transfection method.At 48 h(after) transfection,the expression of SV40T was detected with PCR and RT-PCR using specific primer of T gene.(RESULTS) The restricted enzymes digested and sequencing results showed that SV40 large T gene had cloned into pEGFP-N1 vector successfully.The genome DNA and total RNA were isolated from the positive cells.With these samples,the specific 288 bp fragment was amplified using PCR and RT-PCR.CONCLUSIONS The recombinant plasmid SV40TEGFP will be a stable and valuable molecular tool for human eukaryocyte study.

Objective To isolate melanocytes from epidermis and identify their biological characters. Methods Taking TPA/bFGF/IBMX as basic supplements in medium DMEM/F12(1∶1), the cultured cells from the resected foreskin were purified with trypsin digestion and G418 selection, and the cellular structure and function were observed by Dopa-staining, melanin content assay, tyrosinase activity assay and immunohistochemical staining. Results Dopa-staining showed that the cultured melanocytes had melanin synthesis, melanin content assay and tyrosinase activity assay showed the tyrosinase function of the cells was normal, immunohistochemical staining demonstrated the cell differentiation was normal. Conclusion Melanocytes could be cultured by TPA/bFGF/IBMX and would maintain normal structure and biological function under such conditions.

ObjectiveTo evaluate prognostic factors affecting local tumor progression after radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC).MethodsA total of 246 HCC patients (343 lesions) underwent RFA treatment in our department and were enrolled into this study.The average tumor size was 3.7 cm ( range 0.9 ～ 3.7 cm).Regular follow-up with enhanced CT was performed to evalutate the treatment results.Kaplan-Meier model and log-rank test were used in univariate analysis and COX regression model was used in multivariate analysis to identify risk factors for local tumor progression.ResultsThe local tumor progression rate was 11.4％ (39/343 lesions),and the average time from initial RFA to local tumor progression was 12.0 months.Univariate analysis indicated tumor size ( P ＜0.001 ),close to intrahepatic vessels ( P ＜0.001),tumor boundary ( P =0.020),pathological grade( P =0.010) and CEUS before RFA ( P =0.001) as risk factors for local progression.The following factors were identified as independent prognostic factors for local tumor progression by multivariate model:tumor size (P ＜ 0.001),isolated or close to intrahepatic vessels( P ＜0.001) and CEUS before RFA(P =0.018).ConclusionsTumor size,CEUS before RFA and close to intrahepatic vessels are the most important factors for local progression after RFA.Being awaring of possible risk factors for local tumor progression may increase the treatment efficacy and help to promote the use of RFA technique.

Objective To investigate the expressions of heparanase(Hpa) and basic fibroblast growth factor(bFGF) as well as their relationship with angiogenesis in human ovarian carcinoma.MethodsForty-one ovarian carcinoma specimens resected from the patients diagnosed with ovarian carcinoma from January 2003 to November 2007 were examined by immunohistochemistry for the expressions of Hpa and bFGF and microvessel density(MVD).ResultsHpa highly expressed in ovarian carcinoma,and its expression was positively correlated with MVD and bFGF expression(r=0.351,P

The specific fragment of Pneumococcal surface protein A(PspA)and Pneumococcal Surface Adhesin A(PsaA)gene was amplified by PCR from Streptococcus pneumonia 5 and Streptococcus pneumonia 19.The amplified fragnent of PspA and PsaA gene was ligated into pET-27b(+)vector and transformed into BL 21 E.coli for expression and obtain the expressive production of PspA and PsaA.Induced by IPTG,the expression level was as high as 75 % of the total disolube protein.The result showed that the recombinant plasmid could express a specific 75 kDa and 37 kDa fusion protein in E.coli BL 21,which showed the good immunogenicity and a broadly cross reactivity with the other serotypes.

G protein-coupled receptors (GPCRs) are the largest cell surface receptor family, which mediates activities of almost all known cellular response to ligands, including hormones release, neurotransmitters and sensory input.GPCRs can promote development and progression of gastric cancer, colorectal cancer, lung cancer and breast cancer and other tumors.Tyrosine kinase receptors (RTKs) are another important family of membrane receptors, which can regulate cell proliferation, differentiation, migration and survival.Overexpression of RTKs has been found in many cancer cells.Therefore, GPCRs and RTKs are equally important in the clinical treatment of cancer therapeutic.However, GPCRs and RTKs are not independent, and they can use common signal transduction.The present study show that crosstalk between GPCRs and RTKs can facilitate migration of lung epithelial cells, increasing survival of nerve cells and promoting tumor occurrence and development.This article mainly focuses on crosstalk between GPCRs and RTKs and their roles in tumorigenesis and oncotherapy.

Aim To study the effect of Baoxinbao film on endothelin(ET) and nitric oxide(NO) secretion in patients with stable angina pectoris(SAP).Methods 76 patients with SAP were randomly divided into two groups, with 40 cases in the baoxinbao group plastered with baoxinbao film and 36 cases in the isosorbide dinitrate group receiving isosorbide dinitrate. The levels of plasma ET and NO before and after treatment were observed. Results The concentrations of plasma ET were increased and plasma NO reduced significantly in the SAP patients respectively, as compared with those in the control group(all P