The Influence Of Mesenchymal Stem Cells On The Proliferation And Apoptosis Of Isle T Cells Through Paracrine Effects

PartⅠTHE INFLUENCE OF PARACRINE EFFECTS OF MESENCHYMAL STEM CELLS ON THE PROLIFERATION AND APOPTOSIS OF PANCREATICβCELL LINEObjective:To investigate the influence of rat pancreatic extracts on the secretion of cytokines of mesenchymal stem cells and to analyze the effects of conditioned media of mesenchymal stem cells treated with rat pancreatic extractss on the proliferation, apoptosis and insulin secretion of pancreaticβcell line INS-1 cells.Methods:1.The normal rat pancreatic extracts(N-RPE) and regenerative rat pancreatic extracts (R-RPE) were prepared respectively.Mesenchymal stem cells(MSCs) were isolated, cultured and identified.N-RPE conditioned media(N-RPE-CM) and R-RPE conditioned media(R-RPE-CM) were acquired by incubating the MSCs with N-RPE and R-RPE.CTR-CM was acquired by incubation of MSCs without RPE.The levels of IGF-1,HGF,VEGF and b FGF in MSC-CM were analyzed by ELISA and the mRNA expression of IGF-1,HGF,VEGF and b FGF in MSCs was assessed by RT-PCR.2.The pancreaticβcell line INS-1 cells were incubated with conditioned media of MSCs treated with RPE,with the standard RPMI 1640 medium as the control group (RPMI 1640 group).The effects of N-RPE-CM,R-RPE-CM and CTR-CM on proliferation of the INS-1 cells were analyzed by MTT.The influence of MSC-CM on the INS-1 cell apoptosis induced by the mixture of IL-1β,TNF-αand IFN-γwas measured by Annexin V/PI double staining.The insulin secretion and mRNA expression of INS-1 cells were analyzed by RIA and RT-PCR,respectively.Results:1.The cell phenotype identification of MSCsThe bone marrow cells reached 80%-90%confluence in 10-14 days after primary culture,arranged regularly with clear boundary and showed spindle-like or radiation morphology.The morphology of MSCs was about uniform at passage 3. Flow cytometric analysis of the MSCs at passage 3 showed that these cells were negative for CD34 and CD45 and expressed high levels of CD44 and CD90.2.Assay of IGF-1,HGF,VEGF and b FGF in MSC conditioned mediaThe four cytokines were all measured in three groups.The IGF-1 levels of CTR-CM group,N-RPE-CM group and R-RPE-CM group were(123.5±15.3) pg/ml,(356.8±35.6) pg/ml and(852.3±53.9) pg/ml,respectively,P＜0.01. The HGF levels of CTR-CM group,N-RPE-CM group and R-RPE-CM group were(110.5±11.3) pg/ml,(206.5±23.6) pg/ml and(358.5±42.8) pg/ml, respectively,P＜0.01.The VEGF levels in three groups were(78.6±12.6) pg/ml, (206.8±21.6) pg/ml and(702.3±43.6) pg/ml,respectively,P＜0.01.The b FGF levels were(89.6±11.6) pg/ml,(285.3±25.6) pg/ml and(683.6±42.6) pg/ml, respectively,P＜0.01.There was significant difference in the levels of the four cytokines between N-RPE-CR and CTR-CM groups(P＜0.01).3.The influence of RPE on the IGF-1,HGF,VEGF and b FGF mRNA expression of MSCsThe RT-PCR electrophoresis results indicated that electrophoresis strip density of IGF-1,HGF,VEGF and bFGF in MSCs treated with N-RPE and R-RPE was higher in comparison to CTR,that is,the relative mRNA expression of IGF-1,HGF,VEGF and bFGF was significantly increased(P＜0.01).The relative mRNA expression of IGF-1,HGF,VEGF and bFGF was significantly increased in the R-RPE treated group in comparison to N-RPE treated group.4.The effects of MSC-CM on the proliferation and apoptosis of INS-1 cellsThe viability and proliferation of INS-1 cells was assayed by MTT.The optical density of RPMI 1640 group,CTR-CM group,N-RPE-CM group and R-RPE-CM group was 0.382±0.016,0.403±0.028,0.519±0.035 and 0.626±0.048,respectively,P＜0.01.The N-RPE-CM group and R-RPE-CM group have higher optical density.The apoptotic cells were analyzed by Annexin V/PI double staining.The fluorescence microscope and flow cytometer results indicated that the apoptotic cells and necrotic ceils in the RPE-CM groups were decreased compared with RPMI1640 control group and CTR-CM group.The R-RPE-CM group had the least number of apoptotic ceils and necrotic cells.4.The effects of MSC-CM on the insulin secretion and insulin mRNA expression of INS-1 cellsThe insulin levels secreted by INS-1 cells in the presence of 5.6mmol/L glucose were 0.225±0.019(RPMI 1640 group) vs 0.242±0.035(CTR-CM group) vs 0.351±0.052(N-RPE-CM group) vs 0.486±0.063(R-RPE-CM group),P＜0.01. The insulin levels secreted by INS-1 cells in the presence of 20mmol/L glucose were 0.318±0.021(RPMI 1640 group) vs 0.346±0.025(CTR-CM group ) vs 0.662±0.052(N-RPE-CM group) vs 0.801±0.077(R-RPE-CM group),P＜0.01.The insulin mRNA expression of INS-1 cells in the presence of 11.1 mmol/L glucose was assessed by RT-PCR.The electrophoresis strip density of insulin in INS-1 cells treated with N-RPE-CM,R-RPE-CM and CTR-CM was higher in comparison to RPMI 1640.Conclusion:1.The rat pancreatic extracts can promote the MSCs to secret IGF-1,HGF, VEGF and b FGF and increase the mRNA expression.The R-RPE has the strongest effect of promotion.2.The conditioned media of MSCs treated with rat pancreatic extracts can stimulate the proliferation,inhibit the apoptosis,and promote the insulin secretion and insulin mRNA expression of INS-1 cells.3.The panpreaprotective effects of MSC conditioned media indicate that paracrine action of MSCs may play an important role in the cell therapy of diabetes PartⅡTHE EFFECTS OF CONDITIONED MEDIA OF MESENCHYMAL STEM CELLS ON THE ISLET CELLS OF DIABETIC RATSObjective:To investigate the effects of conditioned media ofmesenchymal stem cells on the glycemia control,islet cell apoptosis and proliferation in diabetic rats. Methods:Diabetic rats induced by single intraperitoneal injection of 1%STZ at 60mg/kg body weight were randomly divided into four groups:diabetic control group (DM-CTR,only injecting saline,n=6),CTR-CM group(injecting CTR-CM,n=8), N-RPE-CM group(injecting N-RPE-CM,n=8) and R-RPE-CM group(injecting R-RPE-CM,n=8).The effects of conditioned media were analyzed on the body weight and blood glucose of diabetic rats.The histopathology and immunohistochemistry were essayed with H.E staining assessing the morphology of islets and insulin and Ki67 staining analyzing the proliferation of islet cells.The apoptosis of islet cells were evaluated by TUNEL assay.Result:1.The body weight and blood glucose in four groups and the IPGTT results in R-RPE-CM groupThe body weight of DM-CTR and CTR-CM groups decreased into(141.5±10.3) g and(149.2±11.2) g respectively at the end of the research.There was no obvious change in the body weight of N-RPE-CM(223.7 g±18.6 g) and R-RPE-CM(231.8 g±16.9 g) groups during the experiment.There was significant difference in the body weight among the four groups,P＜0.01.There was no significant difference in the fasting blood glucose among the fours groups at the beginning of experiment,P＞0.05.Blood glucose levels in N-RPE-CM and R-RPE-CM groups began to decline from 1 week after injection CM and continue at least four weeks.Blood glucose levels in DM-CM and CTR-CM groups did not decrease 1 week after treatment,and on the contrary,had the trend to increase from 2 weeks after treatment.There was significant difference in the blood glucose concentrations among four groups since 1 week.The blood glucose levels in the DM-CTR,CTR-CM,N-RPE-CM and R-RPE-CM groups 4 week after treatment were (25.5±2.0) mmol/L,(24.5±2.1)mmol/L,(13.6±1.7) mmol/L and(7.8±1.8)mmol/L, respectively,P＜0.01.The levels of blood glucose in R-RPE-CM group were lower in comparison to N-RPE-CM group,P＜0.01.However,there was no significant difference of blood glucose levels between CRT-CM and DM-CTR groups.IPGTT results showed that the blood glucose at 30min,60 min,120rain and 180min significantly declined at 2 and 4 weeks after treatment compared with the level before treatment.2.Histopathology,immunohistochemistry and TUNEL assaysIn diabetic rats with CTR-CM and saline,the most consistent findings were the degenerative and necrotic changes,and shrinking of the islets of Langerhans.In diabetic rats with the treatment of N-RPE-CM for seven days,the severity of degenerative and necrotic changes in the islet of Langerhans parenchyma was less than those with saline or CTR-CM.In diabetic rats with R-RPE-CM,the majority of cells showed significantly light hydropic degeneration as compared to islet cells of diabetic rats of the other three groups,and the islets of Langerhans were distinctly increased in size.The number of islet was significantly decreased in the DM-CTR and R-RPE-CM group in comparison to N-RPE-CM and R-RPE-CM groups.In immunohistochemical staining of the pancreatic tissues of diabetic rats with CTR-CM or saline treatment,the cells were essentially negative for insulin -immunoreactivity.In diabetic rats with N-RPE-CM treatment,a fewβcells in some islets displayed insulin immunopositivity in small granules.In diabetic rats with R-RPE-CM,both the number of insulin immunoreactiveβcells and their granules increased and insulin immunoreactiveβ-cells increased distinctly in number and displayed intense immunostaining when compared to diabetic rats with N-RPE-CM treatment.Treatment with R-RPE-CM induced a marked increase in the size of the islets.The average optical of insulin staining was obviously different among the four groups,P＜0.01.The Ki67 positive cells in the DM-CTR and CTR-CM groups seldom appeared. There was no significant difference between DM-CTR and CTR-CM groups, P＞0.05.The Ki67 positive cells were obviously increased in the N-RPE-CM and R-RPE-CM groups in comparison to DM-CTR and CTR-CM groups,P＜0.01.There were more Ki67 positive cells in R-RPE-CM group compared with N-RPE-CM group,P＜0.01.There were more apoptotic pancreatic cells in DM-CTR and CTR-CM groups. The apoptotic cells significantly declined after treatment of N-RPE-CM,P＜0.01. There were significant decline in the apoptotic cells in R-RPE-CM group,compared with N-RPE-CM group,P＜0.01.Conclusion:The conditioned media of MSCs treated with rat pancreatic extracts can lower the blood glucose levels of diabetic rats,promote the proliferation and inhibit the apoptosis of the pancreatic cell.