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Objective To design and construct a controlled adenovirus vector in degradating by itself after induction for solving the problem of stimulating host immune and producing replication adenovirus and providing a secure exogenous gene vectors for clinical practice. Methods Based on the traditional adenovirus vector AdEasyTM system, we inserted the Cre gene which belongs to Cre-LoxP system into the downstream of Tet-On inducible expression system. Two LoxP sites were inserted into two sides of the shuttle plasmid′s right arm genome. Then, the full-length human insulin gene was inserted HindIII enzyme site. After the recombinant adenovirus infected the rat bone marrow-derived mesenchymal stem cells , fluorescent protein expression and insulin secretion were detected before or after induction by Dox. Results A new controlled recombinant adenovirus vector carrying human insulin gene was constructed successfully, and was named AdEasyN/INS. After the transfection of this new vector into QBI-293A cells and rat bone marrow mesenchymal stem cells , green fluorescent protein could be observed. After induction by Dox, both of the ratio of fluorescent cells/total cell and the levels of insulin significantly decreased. Conclusion Construction and preliminary validation of a controlledrecombinant adenovirus vector carrying human insulin gene is constructed successfully , it could infect rat bone marrow mesenchymal stem cells, and degradate by itself after Dox induction, realize the controllability of exogenous gene carrier.
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Objective To explore the diagnostic value of the concentration of plasma circulation microRNA155 (miRNA155) in ulcerative cilitis (UC) and its correlation with clinical characteristic of UC.Methods From October 2010 to August 2012,a total of 136 patients diagnosed as UC were enrolled,and at same time,170 healthy individuals were set as healthy control.The blood samples of all participants were obtained and plasma was isolated.The adsorption column was used for RNA extraction according to miRNeasy kit instruction.RNA was reverse-transcribed into cDNA with miScript reverse transcription kit.cDNA was a template and miRNA155 real-time quantitative polymerase chain reaction (PCR) was performed with miScript SYBR Green PCR kit.The relative quantity of miRNA155 expression was calculated with 2-△△Ct method.Analysis of variance were performed for comparison between groups.Receiver operating characteristic curve analysis was used for the diagnostic value of miRNA155 concentration in UC.Multiple linear regression analysis was used for the correlation between miRNA155 concentration and clinical characteristics of UC.Results The concentration of plasma circulation miRNA155 of patients with UC ((1357.43±326.15) fmol/L)was higher than that of healthy controls ((1140.70 ± 312.47) fmol/L) and the differences were statistically significant (F=35.56,P＜0.01).The area under receiver operating characteristic curve of the concentration of plasma circulation miRNA155 of patients with UC was 0.847,and the 95 ％CI was 0.806 to 0.888 (P＜0.01).When the concentration of plasma circulation miRNA155 was 1404.51 fmol/L,its specificity in the diagnosis of UC was 94.7％,and sensitivity was 40.4％.There was correlation between the concentration of plasma circulation miRNA155 and the disease activity in patients with UC (F=12.91,P＜0.05).However there was no correlation with the severity and location of the disease (both P＞0.05).Conclusion Plasma circulation miRNA155 highly expressed in patients with UC,and its concentration is correlated with the disease activity.
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Objective To compare the efficacy and safety of excimer laser 308 nm phototherapy alone and the combination of excimer laser 308 nm and topical application of vitamine D3 alanogue tacalcitol in the treatment of vitiligo. Methods Seventy-eight patients with vitiligo were enrolled in the single-blind clinical trial, treated with excimer laser 308 nm. The lesions were devided into two groups: patients in the experimental group were instructed to use tacalcitol ointment and the control group were applied with placebo ointment. The lesions were evaluated once per month and photos taken for analyses of clinical effects. Results The results in different locations were compared, the effective rates of the experimental group in cephalofacial site, trunk and limbs were 93.51%, 84.16 % and 42.35 %, respectively. The effective rates of control group in opposite and adjacent sites were 90.9 %, 77.45 % and 34.15 %, respectively (P ＜ 0.05). The comparison of results in different types of lesions indicated that the effective rate of the experimental group in vitiligo vulgaris and segmental vitiligo were 73.81% and 84.00 %, respectively. The effective rate of control group in vulgaris and segmental vitiligo were 86.8 %, 77.45 % and 34.15 %, respectively (P ＜0.05 ). The comparison of results in radiation times and doses of phototherapy showed that the radiation time and dose on the time of initial pigment regeneration were (16. 15 ± 3.22)times and (4.40 ± 5.03)J/cm2 in the experimental group, while ( 18.56 ± 3.50) times and ( 6.60 ± 1.01 ) J/cm2 ( P ＜ 0.05 ) in the control group, the time and dose on the time of apparent effect were ( 20. 36 ± 1.50 ) times and ( 7.50 ± 3.54 ) J/cm2 in the experimental group, and (21.68 ± 2.40) times and( 8.80 ± 9.24)J/cm2 (P ＜ 0.05 ) in the control group. Conclusion Application of tacalcitol ointment in combination with twice-weekly 308 nm excimer laser light phototherapy is an effective alternative treatment for patients with generalized vitiligo.
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Objective To study the effects of 8-methoxypsoralen (8-MOP) on a ctivation, proliferation and melanin synthesis of hair-follicle melanocytes. Me thods The cultured human hair follicle melanocytes were treated with various con centrations of 8-MOP(10～500 ?mol/L) in vitro. The effects of 8-MOP were obse rved on cellular morphologic changes, proliferation, tyrosinase activity and mel anin contents of cultured melanocytes. Results Seven days after treatment with 8-MOP(50 ?mol/L), striking changes were found in cellular morphology with many elongated dendrites, and deposition of brown granules in cytoplasm. Tyrosinase a ctivity and melanin contents also remarkably increased in treated cells compared to those in untreated cells(P
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Objective To construct eukaryotic expression vector of attenuated Salmonella typhimurium containing hCTLA-4Ig cDNA and identify its expression in COS-7 cells for the further study of function in SLE models. Methods Touchdown PCR was used to amplify hCTLA-4Ig cDNA.The PCR product was ligated into the multiple clone site of eukaryotic expression vector pcDNA3.1(+) by gene recombination technique.Then the recombinated plasmid pcDNA3.1(+)-CTLA-4Ig was transfected into COS-7 cells using DOTAP.The expression of interest protein in the supernatant of the cell disruption was detected with SDS-PAGE and Western blotting.Results Restriction analysis and DNA sequence analysis showed that the CTLA-4Ig cDNA had been successfully inserted into pcDNA3.1(+) eukaryotic expression vector.The interest protein could be detected in the supernatant of cell disruption 48h after the transfection of pcDNA3.1(+)-CTLA-4Ig.This protein specifically bound with human CTLA-4 monoclonal antibody.Conclusion The eukaryotic expression vector containing hCTLA-4Ig gene was successfully constructed and bioactive interest protein could be successfully expressed in mammalian cells.
