Purpose: Isocitrate dehydrogenase 1 (IDH1) mutations are the most common genetic abnormalities in low-grade gliomas and secondary glioblastomas. Glioma patients with these mutations had better clinical outcomes. However, the effect of IDH1 mutation on drug sensitivity is still under debate. Materials and Methods: IDH1-R132H mutant cells were established by lentivirus. IDH1-R132H protein expression was confirmed by western blot. The expression of ataxia telangiectasia mutated (ATM) signaling pathway and apoptosis-related proteins were detected by immunofluorescence and western blot. Temozolomide (TMZ) induced cell apoptosis was detected by flow cytometry. Tumor cell proliferation was detected by Cell Counting Kit-8. In vivo nude mice were used to confirm the in vitro roles of IDH1 mutation. Results: We established glioma cell lines that expressed IDH1-R132H mutation stably. We found that TMZ inhibited glioma cells proliferation more significantly in IDH1 mutant cells compared to wild type. The IC50 of TMZ in IDH1-R132H mutant group was less than half that of wild-type group (p < 0.01). TMZ significantly induced more DNA damage (quantification of γH2AX expression in IDH1 mutation vs. wild type, p < 0.05) and apoptosis (quantification of AnnexinV+propidium iodide–cells in IDH1 mutation versus wild type, p < 0.01) in IDH1 mutant gliomas compared to wild-type gliomas. The ATM-associated DNA repair signal was impaired in IDH1 mutant cells. Inhibiting the ATM/checkpoint kinase 2DNA repair pathway further sensitized IDH1 mutant glioma cells to chemotherapy. We found that IDH1 mutation significantly inhibited tumor growth in vivo (the tumor size was analyzed statistically, p < 0.05). Moreover, we confirmed that gliomas with IDH1 mutation were more sensitive to TMZ in vivo compared to wild type significantly and the results were consistent with the in vitro experiment. Conclusion These results provide evidence that combination of TMZ and ATM inhibitor enhances the antitumor effect in IDH1 mutant gliomas.

Purpose: Isocitrate dehydrogenase 1 (IDH1) mutations are the most common genetic abnormalities in low-grade gliomas and secondary glioblastomas. Glioma patients with these mutations had better clinical outcomes. However, the effect of IDH1 mutation on drug sensitivity is still under debate. Materials and Methods: IDH1-R132H mutant cells were established by lentivirus. IDH1-R132H protein expression was confirmed by western blot. The expression of ataxia telangiectasia mutated (ATM) signaling pathway and apoptosis-related proteins were detected by immunofluorescence and western blot. Temozolomide (TMZ) induced cell apoptosis was detected by flow cytometry. Tumor cell proliferation was detected by Cell Counting Kit-8. In vivo nude mice were used to confirm the in vitro roles of IDH1 mutation. Results: We established glioma cell lines that expressed IDH1-R132H mutation stably. We found that TMZ inhibited glioma cells proliferation more significantly in IDH1 mutant cells compared to wild type. The IC50 of TMZ in IDH1-R132H mutant group was less than half that of wild-type group (p < 0.01). TMZ significantly induced more DNA damage (quantification of γH2AX expression in IDH1 mutation vs. wild type, p < 0.05) and apoptosis (quantification of AnnexinV+propidium iodide–cells in IDH1 mutation versus wild type, p < 0.01) in IDH1 mutant gliomas compared to wild-type gliomas. The ATM-associated DNA repair signal was impaired in IDH1 mutant cells. Inhibiting the ATM/checkpoint kinase 2DNA repair pathway further sensitized IDH1 mutant glioma cells to chemotherapy. We found that IDH1 mutation significantly inhibited tumor growth in vivo (the tumor size was analyzed statistically, p < 0.05). Moreover, we confirmed that gliomas with IDH1 mutation were more sensitive to TMZ in vivo compared to wild type significantly and the results were consistent with the in vitro experiment. Conclusion These results provide evidence that combination of TMZ and ATM inhibitor enhances the antitumor effect in IDH1 mutant gliomas.

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection

Objective To investigate the efficacy and safety of mesenchymalstem cells (MSCs)in improving the renal transplantation functional recovery of donation after citizens death (DCD)derived renal donors.Methods A retrospective analysis of 97 cases of DCD renal transplantation was performed in our center from July 2011 to December 2016.Among them,50 cases were treated with MSCs (the treatment group) and 47 cases without MSCs (the control group).In the treatment group,the umbilical cord stem cell suspension (50 mL,approximately 1 106/kg weight) was infused before vascular patency and 1 week,2 weeks,and 3 weeks after renal transplantation.Postoperative renal function within 3 months of recovery,delayed graft function (DGF) incidence and duration,the incidence of pulmonary infection,acute rejection and surgical complications were analyzed.Resuts The incidence rate of DGF and duration in the treatment group were 16.0％ and (14.42 ± 3.95) days,and those in the control group were 27.7％ and (17.85 ± 6.25) days respectively,and there were statistically significant difference.The incidence of acute rejection in the treatment group and the control group was 12.0 and 21.3％ respectively with the difference being statistically significant.The incidence rate of pulmonary infection in the treatment group and control group was 10％ and 12.7％respectively with the different being not statistically significant.Comparison of eGFR at 1st,1st month and 3rd month after operation showed that the eGFR in the control group was statistically significantly lower than that in the treatment group at 1st and 3rd month,but there was no significant difference in eGFR at 1st week between the two groups.There was no significant difference in the surgical complications between the two groups.Conclusion In the treatment group,the incidence rate of DGF was lower,and DGF duration was shorter than in the control group.The acute rejection rate in the control group increased significantly,but there was no significant difference in pulmonary infection and surgical complications between two groups.The recovery trend of renal function in the treatment group was better than that in the control group.

BACKGROUND:Bone mesenchymal stem cells have immunological regulation function both in vitro and in vivo, while the effect of bone marrow mesenchymal stem cells on CD4+T cellimmune function in patients receiving kidney transplantation remains unclear. OBJECTIVE:To explore the monitoring significance of CD4+T-cellimmune function by ImmuKnow assay and to determine the effect of induction therapy with bone marrow mesenchymal stem cells on cellimmune function in patients receiving kidney transplantation. METHODS:From January 2011 to June 2013, 24 patients receiving al ograft renal transplantation with autologous bone marrow mesenchymal stem cells were included and another 48 patients receiving al ograft renal transplantation and Simulect induction therapy with various matched preoperative characters served as controls. In both groups, adenosine triphosphate levels in CD4+T cells in the peripheral blood were determined by the ImmuKnow assay preoperatively and at 14, 30, 60, 90, 180 days postoperatively, as wel as during acute rejection and infection episodes. RESULTS AND CONCLUSION:During the 180 days postoperatively, fewer patients in the bone marrow mesenchymal stem cellgroup had acute rejection and injection than the Simulect group, but no significant differences were observed. Postoperative adenosine triphosphate levels in CD4+T cells were significantly lower than those determined preoperatively in both groups (P<0.05), while no significant differences were observed between the two groups. A total of 12 patients in the bone marrow mesenchymal stem cellgroup and 26 patients in the Simulect group had infection episodes, and the adenosine triphosphate levels in CD4+T cells during the infection episodes were lower than clinical stable patients in both groups (P<0.01). For patients receiving renal transplantation, induction therapy with bone marrow mesenchymal stem cells can effectively decrease the cellimmune function, which can be reflected by the adenosine triphosphate levels in CD4+T cells in the peripheral blood determined by the ImmuKnow assay.

Objective To evaluate the values of cell-mediated immune function in elderly renal allograft recipients.Method The levels of immuknowTM ATP was sequentially monitored by means of Cylex immuknowTM assay in 52 elderly renal allograft recipients including 11 with infection and 8 with acute rejection.Results No statistically significant difference was found between stable allograt function and uremia (P＞0.05).The levels of immuknowTM ATP during infection was significantly lower than those with stable allograft function with acute rejection (P ＜ 0.01).The levels of immuknowTM ATP during acute rejection was significantly higher than those with stable allograft function with infection (P＜0.01).Conclusion Sequential monitoring of immuknowTM ATP is helpful for elderly renal allograft recipients in individualized immunosuppression therapy.Cylex immuknowTM assay can be used as a potent tool for assessment of high risk in infection and rejection.

Objective To analyze the clinical application of donor specific antibodies (DSAs) detected by a single antigen Luminex virtual crossmatch,and to discuss the treatment of DSA and the impact of DSA on renal function.Methods Serum from living-relative renal recipients before and after transplantation was investigated using a Luminex single antigen assay.The relation between DSA and renal acute rejection as well as renal function was analyzed.Results A total of 30 patients and 173 serum samples were tested,including 47 serum samples before transplantation,and 126 after transplantation.DSA was positive in one patient before transplantation,and 8 patients after transplantation.Three of the patients positive for DSA were treated by Bortezomib,3 by addition of MMF,2 by addition of CNI,1 by addition of Sirolimus.The MFI of DSA in one of the patients treated by Bortezomib was decreased to below 1000,while that in the other two decreased by more than 50 ％.The renal eGFR at the time with and without DSA was (1.50 ± 0.59) and (1.23 ± 0.38)ml/s respectively (P＜0.05).Conclusion Dynamic monitoring of single bead antigen antibody DSA conduces to direct the adjustment of immunosuppressant.The appearance of DSA contributes to the declination of renal function.Application of Bortezomib decreased the MFI of DSA.

Objective To evaluate the efficacy and safety of mesenchymal stem cells(MSCs)in preventing early acute rejection after renal transplantation.Methods Eighty-eight primary cadaveric renal allograft recipients in our department were randomized into two groups treated with bone marrow MSCs (BMSCs group,n =43) or not (control group,n =45).Main immunosuppressive therapy regimen consisted of steroids,tacrolimus or cyclosporine and mycophenolate mofetil in all recipients.Estimated glomerular filtration rate (eGFR) of transplant kidney,incidence of acute reaction (AR),graft survival and incidence of adverse events were recorded within 24 months.Results In BMSCs group,the incidence of AR was 4.7 ％ and 9.3 ％ at 3rd month and 6th month respectively,significantly lower than 20.0 ％ and 26.7 ％ (P＜0.05) in the control group.The eGFR at day 7,14and 30 post-transplantation was significantly higher in the BMSCs group than in the control group (P＜0.01,P＜0.01,P＜0.05 respectively).The incidence of adverse events in the BMSCs group and the control group was 44.2 ％ (19/43) and 66.7 ％ (30/45,P ＜ 0.05) respectively and the rate of infection was 37.2 ％ (16/43) and 33.3 ％ ( 15/46,P ＞ 0.05) respectively within 24 months.Conclusion Induction therapy with autogenous BMSCs appeared to be more effective in the prevention of AR following cadaveric kidney transplantation and was associated with better clinical outcomes as far as early renal graft function without compromising patient safety.

Objective To evaluate the life quality in front and at the back of the cure of the floating population tuberculosis patients.Methods The SF-36 scale was applied to the new smear-positive pulmonary tuberculosis patient in floating population using face to face interview in Shenzhen 6 districts.The contrast was tuberculosis patient in the population of the household register,the people stayed temporarily in the same term.Results The score of general health(43.36 ± 15.67),vitality(57.96 ± 16.54) and mental health(61.46 ± 14.26) in the floating population was lower than the appraisal result with the population of the household register(49.28 ± 17.85,66.38 ± 17.03 and 66.70 ± 15.12),the people stayed temporarily(45.00 ± 17.22,60.99 ± 16.59 and 63.13 ±13.71 ) (P ＜ 0.05 ).It was still lower than the other two groups after 2 (3) months and 6 (7) months (P ＜ 0.05 ).But the score of social functioning in floating population ( 88.92 ± 14.88 ) was higher than the other two groups (82.62 ± 14.44,68.02 ± 27.30).The change rate of general health ( (76.05 ± 132.82) ％,(69.32 ± 111.98 ) ％and (48.36 ± 101.31 ) ％ ) and social functioning scores( (46.49 ± 85.18 ) ％,( 26.33 ± 50.25 ) ％ and ( 51.09±73.44)％ ) were significant difference in the three groups(x2 =12.088,P=0.002;x2 =11.611,P=0.003).The change rate of the general health score in floating population was higher than the household register population and people stayed temporarily.Conclusion It is necessary to implement the management in whole course to the floating population tuberculosis patients.

Objective To establish a new technique of isolating pancreatic islet of langerhans and glueoeortieoid-free immunosuppressive regimen and to evaluate the clinical efficacy and safety of simultaneous adult islet-kidney transplantation in the treatment of type 1 diabetes mellitus with endstage renal failure.Methods Pancreases were stored using the"2-layer method"of the oxygenated perfluoroehemieal and UW solution.The pancreases were digested by Liberase collagenase enzyme and purified using continuous gradients of Ficoll-diatrizoic acid on a refrigerated COBE 2991 centrifuge to separate the islets.Cadaver kidney was transplanted by conventional method and cultured islets were infused by surgical approach to the liver via portal vaseulature using glucocorticoid-free immunosuppressive regimen.Clinical metabolic data such as blood glucose,dose of insulin,C-peptide,HbAlc,liver function and renal function,were determined and compared with the pre-transplant data.ResuitsIslets of langerhans were isolated successfully in 23 pancreases.The average islet yield was 300000 islet equivalents(IEQ).Islet purity and viability were 91.6%,94.6%,respectively.The stimulation index as assessing function of human islet was 3.16 and etiology results in vivo were negative.Twelve islet transplant infusions were carried out in 7 patients after kidney transplantation.Three recipients received 2 islet infusions,1 patient had 3 transplants,and 3 patients received 1 transplant only.The average islet mass for infusion was 1 1 820 IEQ/kg.The immunosuppressive regimen glucocorticoid.During 18 months to 3 yearg follow-up,4 recipients had insulin independence,the dosage of insulin decreased by 70%in 3 patients.The level of blood glucose and H bAlc,liver and renal function were normal throughout follow-up period.C-peptide of all patients was positive after islet transplantation.No adverse effects and complications related to islet infusion procedure were found.Conclusions New technique has proved tO be suitable for isolating pancreatic islet of langerhans.Simuhaneous adult islet-kidney transplantation could be used as an effective and safe way for treating type 1 diabetes mellitus with end-stage renal failure.