[ ABSTRACT] Diabetes is characterized by an absolute or relative deficiency inβ-cell mass, which cannot be re-versed with existing therapeutic strategies.The restoration of the endogenous islet β-cells can stabilize the level of blood glucose.The isletβ-cells can be obtained from the directional differentiation of stem cells, but the process is complex and has the risk of teratomas generation.Cell direct reprogramming, one terminal differentiated cell can transdifferentiate into another kind of terminal differentiated cell, which is other than directional differentiation from stem cells.Direct reprogram-ming gives rise to the generation of isletβ-cells from one terminal differentiated cell, may be preferable for diabetes therapy because of its unique advantage.

Objective To study the effect of environmental estrogen, bisphenol A (BPA), on proliferation of human uterine leiomyoma cells in culture. Methods The primary culture and subculture of the human uterine leiomyoma cells which were identified at each passage by means immunocytochemical staining with a monoclonal anti-?-smooth muscle actin antibody, were performed. The cells (passage 3-5) were incubated with BPA (including three dosimetric systems, 1?10-7 mol/L, 50 ?10-7 mol/L and 100?10-7 mol/L) for 24, 48 and 72 h, and the control cells received only the vehicle (ethanol, 0.1 %, V/V). The proliferation of cultured human uterine leiomyoma cells was analyzed by MTT assay and flow cytometry. Results The limited human uterine leiomyoma cell lines were successfully established. After 24 h, 48 h and 72 h of treatment in 100?10-7 mol/L and 48 h, 72 h, at 50?10-7 mol/L , BPA could enhance the cells proliferation. After 72 h of treatment in 50?10-7 mol/L and 100?10-7 mol/L, BPA promoted cells into S cell cycle phase and elevated mitotic index (P

ObjectiveTo investigate the effect of individual donation-nucleic acid amplification test (ID-NAT) and minipool of 16 donations-NAT (P16-NAT) on the results of NAT of blood donors.Methods From February 2009 to June 2009,samples randomly collected from voluntary blood donors in Beijing were tested individually or in pooling of 16 donations by the PROCLEIX ULTRIO assay.For ID-NAT reactive samples with HBsAg,anti-HCV,or anti-HIV serologically unqualified,ID-NAT repeat reactive samples with serologically qualified,and P16-NAT reactive and followed resolution ID-NAT reactive samples,were performed for further discriminatory assays for HIV-1,samples and followed resolution ID-NAT reactive samples,were performed for further discriminatory assays for HBV,HCV and HIV-1discriminatory reagents.Samples which were HBV NAT + alone with serologically qualified were further quantified and confirmed of HBV DNA by Roche HBV quantitative PCR,analyzed by HBV serology and were diluted to simulate if they could be detected in P16-NAT.Results ( 1 ) Among 7613 samples tested by ID-NAT,26 were NAT positive,i.e.the ID-NAT positive rate was 0.34％ ( 26/7613 ). ( 2 ) Among 1004 P16 samples from 16 064 blood donations,27 were NAT positive,i.e.the P16-NAT positive rate was 0.17％ (27/16 064).(3)In serological qualified donations,ID-NAT yield rate (1 in 826,9/7438 ) was much higher than P16-NAT ( 1 in 7875,2/15 750) (x2 =11.880,P ＜ 0.05 ).All these 9 ID-NAT positive and 2 P16-NAT positive donations were discriminated as HBV NAT positive.There were no HCV NAT yield or HIV NAT yield samples. (4) Dilution assay showed only 2 of the 9 (22.22％ ) ID-NAT HBV yields were detected by P16-NAT.(5)Eight ID-NAT and 2 P16-NAT positive samples were quantified for HBV DNA and confirmed as HBV NAT yield,although the virus loads were very low:2 samples had HBV viral loads of 15 IU/ml and 472 IU/ml,6 samples ＜ 12 IU/ml,and 2 could not be detected in the original samples while had ＜ 12 IU/ml and 14.3 IU/ml in the 10 times concentrated samples.(6)Among 11 HBV NAT yield cases,3 (27.3％ ) were possible HBV window-period donors with all HBV seromarkers negative,the other 8 (72.7％ ) had occult HBV infections with anti-HBc or anti-HBe positive,however anti-HBc IgM negative.(7) The rate of initial P16-NAT reactive pools needed to be further tested by ID-NAT was 2.49％(25/1004).Initial P16-NAT reactive pools which caused by serologically qualified donations was 0.20％(2/1004).ConclusionsHBV NAT yield cases are detected at a higher frequency with ID-NAT than P16-NAT.In order to avoid samples with low viral loads would be undetected,NAT assay with high sensitivity should be selected and tested in minimized minipool donations or even with individual donation.

BACKGROUND:In recent years a large number of studies have suggested that bone marrow mesenchymal stem cells can ease hyperglycemia of diabetic rats, but the related mechanism is unclear and controversial. OBJECTIVE:To investigate the relevant mechanism of bone marrow mesenchymal stem cells on pancreas microenvironment in vivo in diabetic rats. METHODS:Bone marrow mesenchymal stem cells were transfected with enhanced green fluorescent protein (EGFP) and administered to diabetic rats via the subcapsular pancreas. Blood glucose levels were monitored. The expressions of the key genes in islet development in these EGFP positive pancreatic cells were analyzed by Real-time quantitative PCR at different times. EGFP and insulin double-positive cells were detected by immunofluorescence. Flow cytometry was performed to analyze cellcycle and DNA ploidy. RESULTS AND CONCLUSION:Blood glucose levels were effectively reduced after transplantation. The expressions of the key genes in islet development reached their own peak values at different times after transplantation:Nestin at week 1, Nkx 2.2 at week 3, Pax 4 and Ngn 3 at week 4, insulin and glucagon at week 12, PDX-1 at week 8 until week 12. The cells double-positive for EGFP and insulin cells were observed. In the pancreas, EGFP positive cells at S+G 2/M phase were significantly increased, and there were no polyploid and aneuploid cells. In pancreas microenvironment, the bone marrow mesenchymal stem cells transplanted into the diabetic pancreas can differentiate into isletβ-like cells under gene control, but not through the fusion with tissue cells.

AIM: To determine the expression level of Fas molecules on the T cells isolated from systemic lupus erythematosus (SLE) patients. METHODS: The expression of Fas on T cells and the apoptotic rate of 36 patients with SLE in active phase and 18 normal people were determined with flow cytometry. The patients were treated with impact therapy of prednisone, methylprednisolone or cyclophosphamide, respectively, according to their conditions. Within 3 days after admission, immediately before discharging (the average days in hospital is 22.6 d), and 4 weeks after discharging, the amount of Fas molecules expressed on T cell surface was determined respectively with flow cytometry in each patient. RESULTS: There was a positive correlation between SLEDAI, apoptotic rate of T cells and Fas molecules on T cells in SLE patients of active phase (P

AIM:To investigate the role of B cells in CD45RB antibody-induced transplantation immune toler-ance.METHODS:Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibod-y, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice.The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process .The skin graft model was set up with B 6.μMT-/-mice as re-ceptors and BALB/c mice as donors.CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3+CD45RBhi cells were detected by flow cytometry .In another mixed lymphocyte culture , CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT-/-mice through the tail vein.The heart transplanta-tion model was established with B 6.μMT-/-mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed .RESULTS:When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody (mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control .In vivo without B lympho-cytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes.The reduction was faster and the percentage of CD3 +CD45RBhi T cells was not altered as compared with the control .The B lymphocytes treated with an-ti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete toler -ance.After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation , B cells migrated to the thymus in B6.μMT-/-mice.CONCLUSION:B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance .However, the induction of immune tolerance can not only rely on B cells .

Objective To investigate the immune regulatory effects of allogeneic bone marrowderived mesenchyrnal stem cells (MSCs) co-transplanted with islets. Methods The 18 diabetic mice were randomly divided into 3 groups: Diabetic group, without any transplantation; Islet transplantation group, in a sterile operation, with 10 μl purified islets (about 200 islets)transplantation to the left renal subcapsule of recipients; Islet + MSCs transplantation group, in addition to transplantation as the former group, 1 × 106 MSCs were given to the recipients via tail vein on 3, 2 and 0 days before islet transplantation. Blood glucose in recipient mice was monitored for 30consecutive days after transplantation. Pathological characteristics of left kidney were analyzed on the day 14 and 28 after transplantation. Th1/Th2, Tc1/Tc2, naive and memory T cells from peripheral blood and bone marrow-derived dendritic cells (DCs) were analyzed by multi-color flow cytometry.Results As compared with the islet transplantation group, blood glucose was significantly reduced,inflammatory cell infiltration decreased in the place of transplantation, graft survival prolonged, the number of Th1 and Tc1 cells was obviously reduced, the number of Th2 and Tc2 cells increased, the ratio of Th1/Th2 and Tc1/Tc2 cells was significantly decreased, the naive and memory T cells were significantly inhibited, the maturity of DCs and the secretion of interleukin-12 decreased in the islet transplantation group. Conclusion Through the immunomodulation on T-cell and DCs function,MSCs can alleviate graft verse host disease and prolong allograft survival.

Objective:To investigate the effect of piggyBac transpon,as a carrier of four defined transcription factors Oct4,Sox2,Klf4 and c-Myc,in the reprogramming of mouse embryonic fibroblasts (MEFs)to induced pluripotent stem cells (iPSCs).Methods:The MEFs were isolated from Oct4-GFP fetal mice and transfected by piggyBac transposon with four factors (Oct4,Sox2,Klf4 and c-Myc).The morphological changes of clones were traced with microscope during the process of induction.The chromosomes were analyzed to evaluate the karyotypic variation of iPSCs.The mRNA expressions of Oct4, Nanog and FGF4 associated with embryonic stem cells (ESCs)in the iPSCs of mice were tested by RT-PCR;the protein expressions of SSEA-1,Nanog and Alkaline phosphatase in the iPSCs of mice were determined by flow cytometry,immunofluorescence and AP staining.The iPSCs were transplanted into the NOD-SCID mouse groin,4 weeks later,the teratomas were removed for HE staining and the differentiation of tissue was observed.Results:The iPSCs were successfully obtained from MEFs by piggyBac carrying Oct4,Sox2,Klf4,and c-Myc.The round or oval iPSCs clones were similar to ESCs with clear boundry and large dense nuleus.The iPSCs showed the normal karyotypic and expressed the marker genes (Oct4,Nanog and FGF4)and proteins (SSEA-1,Nanog and AP)of ESCs.Teratomas containing three germ layers were formed in NOD-SCID mice after tanspalantation of iPSCs.Conclusion:The iPSCs are reprogrammed from MEFs by piggyBac transposon with four transcription factors-Oct4,Sox2,Klf4 and c-Myc,and the iPSCs with normal karyotype possess the characteristics of ESCs.

Objective To investigate smoking status,knowledge of smoking hazards,attitude of tobacco control and skill of assisting smoking cessation of the staff in a teaching hospital in Chongqing and to provide references for the further construction of‘smoking-free hospital’. Methods General investi-gation was taken on the staff in a teaching hospital in Chongqing with a self-designed questionnaire. Main contents of questionnaire include:social demographic information,smoking status,awareness of tobacco hazard,willingness and methods of tobacco control,etc. All the data were inputted with software Epidata 3.1 and were analyzed with SPSS 13.0. Results The total smoking rate was 9.65%,with 30.49%for male, 2.75%for female and 12.50%for clinician. The age distribution of smoking staff was described as follow-ing:91.97% being under 50 year-old and more than 50.00% being 20-35 year-old. 52.43% of the surveyed did not know Framework Convention of Tobacco Control of WHO . Relatively ,most of the surveyed only knew well the relationship between respiratory diseases and tobacco use and the relation-ship between fetal abnormalities and tobacco use. 84.99%of the surveyed agreed with outdoor-smoking policy;83.56%of the surveyed claimed that they had discouraged smoking behaviors in public at various extents,14.20%of the surveyed agreed that assisting the public in smoking cessation was one of the aims of constructing‘smoking-free hospital’;70.00%clinicians claimed routinely inquiring and noting smok-ing status of patients, which was better than nurses and medical technicians;almost 30.00%clinic staff did not know quitting smoking drugs at all,approximately 70.00%clinic staff claimed a lack of confidence in smoking control and approximately 70.00% clinicians and nurses did not recommend pro-fessional methods of smoking cessation in practice. Conclusions Smoking staff in the teaching hospital are almost younger people,which is an alarm of the urgent need for tobacco control education. Most staff reach a consensus on keeping smoking-free environment in hospital,but they do not sufficiently acknowledge their social responsibility for tobacco control,and also there is a distance before they can serve as a smok-ing cessation assistant. Tobacco control must be incorporated in long-term mechanism of hospital con-struction. There are three steps in the construction of smoking-free hospital:①creating a smoking-free en-vironment in hospital;②encouraging patients to quit smoking and providing professional service of smoking cessation;③making a positive effort on social tobacco control and advocating smoking cessation in public.

Objective: To understand cognition and willingness of EV71 vaccination among parents of children, so as to provide scientific basis for vaccination. Methods: A cross-sectional survey (census) was adopted. A questionnaire survey was conducted among parents of 2 783 preschool children from 12 kindergartens to analyze the parents’ cognition towards EV71 vaccine and willingness of vaccination of EV71 vaccine. Results: Among the 2 783 parents surveyed, 1 843 heard of EV71 vaccine. The parents’ awareness rate of EV71 vaccine was 51.9%. The main source of EV71 vaccinerelated information was obtained through hospitals(n=887), followed by schools(n=752). 1 014 received EV71 vaccine. The main reason of EV71 vaccination was that vaccine was believed to be effective in preventing hand,foot and mouth disease(HFMD) (76.1%), while the main reason for unwillingness for vaccination was lack of knowledge(31.7%). And 2 478 considered that EV71 vaccine should be included in planned immunization. Multivariate Logistic regression analysis showed that children in public schools(OR=0.72, 95%CI=0.59-0.89), mothers from urban residents(OR=0.76, 95%CI=0.64-0.90), parents lack of EV71 vaccine awareness(OR=0.42,95%CI=0.35-0.49), and perceived high price of EV71 vaccine (OR=0.66, 95%CI=0.47-0.92) had a low vaccination rate. Higher EV71 vaccination rate was related to children’ status as baby class (OR=2.55,95%CI=1.18-5.52),bottom class (OR=2.24,95%CI=1.54-3.24) and middle class(OR=1.51, 95%CI=1.05-2.15)(P<0.05). Conclusion EV71 vaccination in preschool children in our jurisdiction was relatively high. School type, mother’s household registration, children’s grade and the parental percption of EV71 vaccine price are the main factors influencing EV71 vaccine vaccination in preschool children.