【作者】 许聿新；

【导师】 陈丽；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 第一部分骨髓间充质干细胞旁分泌效应对胰腺β细胞系的影响目的:研究胰腺提取物对骨髓间充质干细胞分泌细胞因子的影响,并分析胰腺提取物处理后的骨髓间充质干细胞条件培养液对体外培养的胰腺β细胞增殖、凋亡及胰岛素分泌的影响方法:(1)制备正常大鼠胰腺提取物(normal rat pancreatic extracts,N-RPE),再生性大鼠胰腺提取物(regenerative rat pancreatic extracts,R-RPE),分离培养鉴定大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs),分别应用N-RPE和R-RPE孵育MSCs获得条件培养液(MSC conditioned media,MSC-CM),分别为N-RPE-CM,R-RPE-CM,并设立无RPE处理的对照组(CTR-CM),ELISA法分析三组CM中IGF-1,HGF,b FGF及VEGF的含量,应用RT-PCR分析RPE对MSCs中细胞因子IGF-1,HGF,b FGF及VEGF mRNA的改变。(2)应用RPE处理后的MSC-CM孵育体外培养的大鼠胰腺β细胞系INS-1细胞,设立单纯RPMI 1640培养基作为对照组(RPMI1640组)。应用MTT分析N-RPE-CM,R-RPE-CM及CTR-CM对INS-1细胞增殖的影响;应用IL-1β,TNF-α和IFN-γ诱导INS-1细胞凋亡,AnnexinⅤ/PI双染分析MSC-CM对INS-1细胞凋亡的影响。应用RIA分析MSC-CM对INS-1细胞分泌胰岛素影响,应用RT-PCR分析对INS-1细胞胰岛素mRNA的影响。结果:(1)骨髓间充质干细胞形态及细胞表型鉴定大鼠骨髓细胞原代培养10～14天细胞可达80%～90%融合,有规律的排列成放射状或旋涡状,经过3代后MSCs基本达到形态均一,流式细胞仪分析第三代MSCs细胞表型,细胞高表达CD44和CD90,而CD34、CD45低表达或表达阴性。(2) MSCs条件培养液IGF-1,HGF,b FGF及VEGF的测定四种细胞因子在三组中均可以检测到,CTR-CM,N-RPE-CM和R-RPE-CM组的IGF-1水平分别为:(123.5±15.3)pg/ml,(356.8±35.6)pg/ml和(852.3±53.9)pg/ml,P＜0.01;三组中HGF的水平分别为:(110.5±11.3)pg/ml,(206.5±23.6)pg/ml和(358.5±42.8)pg/ml,P＜0.01;三组中VEGF的水平分别为(78.6±12.6)pg/ml,(206.8±21.6)pg/ml和(702.3±43.6)pg/ml,P＜0.01;三组中bFGF的水平分别为(89.6±11.6)pg/ml,(285.3±25.6)pg/ml和(683.6±42.6)pg/ml,P＜0.01。N-RPE-CM和CTR-CM相比,三种细胞因子的水平也有显著性差异,P＜0.01。(3)胰腺提取物对MSCs中IGF-1,HGF,VEGF及b FGF mRNA的影响RT-PCR结果显示,与对照组(CTR组)相比,经过N-RPE和R-RPE处理MSCs后,IGF-1,HGF,VEGF和bFGF电泳条带均变深,MSCs的IGF-1,HGF,VEGF及bFGF的mRNA相对表达量均明显升高(P＜0.01)。R-RPE组与N-RPE组比较,IGF-1,HGF,VEGF及bFGF的mRNA相对表达量也均明显升高(P＜0.01)。(4)大鼠MSCs条件培养液对INS-1细胞增殖及凋亡的影响应用MTT检测细胞存活及增殖。RPMI 1640组,CTR-CM组,N-RPE-CM组及R-RPE-CM吸光度值分别为0.382±0.016,0.403±0.028,0.519±0.035及0.626±0.048,有显著性差异(P＜0.01),N-RPE-CM及R-RPE-CM组吸光度值升高更明显。应用AnnexinⅤ/PI双染分析INS-1细胞凋亡,荧光显微镜及流式细胞仪观察:凋亡细胞及坏死细胞数在RPE-CM干预组明显减少,以R-RPE-CM组凋亡细胞及坏死细胞最少。(4)大鼠MSCs条件培养液对INS-1细胞胰岛素分泌及胰岛素mRNA的影响5.6 mmol/L葡萄糖浓度刺激下各组胰岛素分泌分别为(ng/ml):0.225±0.019(RPMI1640组)vs 0.242±0.035(CTR-CM组)vs 0.351±0.052(N-RPE-CM组)vs 0.486±0.063(R-RPE-CM组),P＜0.01;20mmol/L葡萄糖浓度刺激下各组胰岛素分泌分别为(ng/ml):0.318±0.021(RPMI1640组)vs 0.346±0.025(CTR-CM组)vs 0.662±0.052(N-RPE-CM组)vs 0.801±0.077(R-RPE-CM组),P＜0.01。分析MSC-CM在葡萄糖浓度11.1mmol/L下对INS-1细胞胰岛素mRNA的影响。与RPMI 1640组相比,经过N-RPE-CM和R-RPE-CM处理INS-1细胞后,胰岛素电泳条带均变深,INS-1细胞胰岛素的mRNA相对表达量均明显升高(P＜0.01)。结论:(1)胰腺提取物可以促进骨髓间充质干细胞分泌IGF-1,HGF,VEGF及b FGF,mRNA表达增加,再生性胰腺提取物促进作用更强。(2)胰腺提取物处理后的骨髓间充质干细胞条件培养液促进体外培养的INS-1细胞增殖,抑制其凋亡,促进INS-1细胞胰岛素分泌及mRNA表达(3) MSCs条件培养液的胰腺保护作用提示MSCs的旁分泌效应可能在MSCs移植治疗糖尿病中发挥重要的作用。第二部分骨髓间充质干细胞条件培养液对糖尿病大鼠胰岛细胞的作用目的:探讨应用骨髓间充质干细胞条件培养液对糖尿病大鼠模型的血糖控制、胰岛细胞增殖及凋亡的影响。方法:1%STZ 60mg/kg单次腹腔注射制备糖尿病大鼠模型,分为四组:糖尿病对照组(DM-CTR组,腹腔注射生理盐水),n=6;CTR-CM组(腹腔注射CTR-CM),n=8;N-RPE-CM组(腹腔注射N-RPE-CM),n=8;R-RPE-CM组(腹腔注射R-RPE-CM),n=8。分析条件培养液对糖尿病大鼠体重,血糖的影响;胰腺HE染色分析对胰岛形态的改变,应用免疫组织化学胰岛素及Ki67染色,分析对胰岛细胞增殖的影响;应用TUNEL分析条件培养液对胰岛细胞凋亡的影响。结果:(1)各组体重及血糖的变化,R-RPE-CM组腹腔注射糖耐量试验(IPGTT)情况:DM-CTR,CTR-CM组体重降低,到实验结束时分别降至(141.5±10.3)g及(149.2±11.2)g;N-RPE-CM组和R-RPE-CM组体重变化不明显,分别为(223.7±18.6)g和(231.8±16.9)g,N-RPE-CM组,R-RPE-CM组体重与DM-CTR及CTR-CM组之间有显著性差异,P＜0.01。在治疗之前,四组血糖水平没有显著性差异,P＞0.05;治疗一周后,N-RPE-CM组和R-RPE-CM组血糖开始下降,与DM-CTR组及CTR-CM组相比有显著性差异,P＜0.01。在治疗后的第2,3,4周,N-RPE-CM组和R-RPE-CM组血糖继续下降,而DM-CTR及CTR-CM组则有升高趋势,在第四周DM-CTR组,CTR-CM组,N-RPE-CM组和R-RPE-CM组血糖分别为(25.5±2.0)mmol/L,(24.5±2.1)mmol/L,(13.6±1.7)mmol/L,(7.8±1.8)mmol/L,四组有显著性差异,P＜0.01,其中R-RPE-CM组血糖比N-RPE-CM组更低,有显著性差异,P＜0.01。IPGTT显示治疗后2周及4周与治疗前相比,R-RPE-CM组各时点的血糖均有明显降低,均P＜0.01。(2)胰腺组织学检查HE染色结果:DM-CTR组及CTR-CM组动物胰岛数量明显减少,残存胰岛萎缩,变性坏死,细胞核固缩;N-RPE-CM组胰岛数目少、形态欠完整,但与DM-CTR组及CTR-CM组相比,胰岛细胞坏死明显减轻;R-RPE-CM组胰岛数目明显较DM-CTR组及CTR-CM组增多,体积增大,坏死减轻,水样变性较多。胰岛素染色结果:R-RPE-CM组及N-RPE-CM组胰岛素阳性表达面积与视野中总面积的比例,胰岛素阳性表达面积和胰岛素染色的平均光密度与DM-CTR组及CTR-CM组相比均有显著性差异,P＜0.01。胰岛β细胞增殖情况:DM-CTR组及CTR-CM组Ki67阳性细胞非常少见,两组没有显著性差异,P＞0.05,而N-RPE-CM组及R-RPE-CM组胰岛中Ki67阳性细胞较DM-CTR组及CTR-CM组常见,具有显著性差异,P＜0.01;而R-RPE-CM组Ki67阳性细胞比N-RPE-CM组多见,P＜0.01。胰岛β细胞凋亡情况:在DM-CTR组及CTR-CM组凋亡细胞多见,而经过N-RPE-CM治疗后,与DM-CTR组及CTR-CM组相比明显减少,有显著性差异,P＜0.01;而经过R-RPE-CM治疗后凋亡细胞数与N-RPE-CM组相比明显减少,有显著性差异,P＜0.01。结论:骨髓间充质干细胞条件培养液可以降低糖尿病大鼠的血糖,促进大鼠胰岛细胞的增殖,抑制其凋亡。更多还原

【Abstract】 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.更多还原