【作者】 李维；

【导师】 杨菁；

【作者基本信息】 武汉大学 ， 妇产科学， 2017， 博士

【摘要】 研究目的本研究目的为检测PCOS及PCOS伴2型糖尿病(PCOS-2DM)大鼠胰岛内ATPsyn-β的表达改变,并通过上调PCOS-2DM大鼠胰岛细胞ATPsyn-β的表达,了解受损的胰岛β细胞功能是否能够得到改善,进一步探索ATPsyn-β在PCOS-2DM大鼠胰岛β细胞功能障碍中的作用及机制。具体的研究目的为:1.成功构建PCOS-2DM大鼠的模型,并在造模后成功分离纯化大鼠胰岛,并进行原代培养,为ATPsyn-β的上调提供基础;2.成功构建Atp5b重组慢病毒载体,为ATPsyn-β的上调提供干预手段;3.验证ATPsyn-0表达改变介入了 PCOS-2DM大鼠胰岛β细胞功能障碍的形成;4.阐明上调ATPsyn-β以后,功能受损的胰岛β细胞胰岛分泌功能有所改善。研究方法1.构建PCOS-2DM大鼠模型:通过持续DHEA(硫酸普拉睾酮钠)联合高脂高糖饮食诱导PCOS大鼠模型,在高脂高糖饮食基础上,单次大剂量STZ注射诱导2DM模型。通过观察模型大鼠一般状态、监测体重、阴道涂片了解动情周期改变、HE染色了解卵巢形态学改变、血糖仪检测血糖、ELISA法检测血清E2、T、INS水平、计算HOMA-IR指数,综合评价PCOS-2DM造模成功与否;2.大鼠胰岛细胞原代培养:各组大鼠胰腺在位给予胶原酶V灌注消化、Ficoll梯度离心纯化,DTZ染色鉴定活性,原代培养并行葡萄糖刺激下的胰岛素分泌试验,ELISA法检测胰岛素水平,评估胰岛分泌功能改变;3.构建Atp5b慢病毒载体:GV208载体进行单酶切,Atp5b基因通过RT-PCR进行扩增后交换入线性化GV208载体,转化质粒DNA进入感受态大肠杆菌,阳性克隆鉴定与测序比对,以RT-PCR及Western-Blotting方法验证构建的重组质粒,重组病毒质粒及两种辅助包装原件载体质粒共转染293T细胞,完成重组慢病毒的包装,荧光显微镜下观察转染效果,最后以Real-Time PCR法检测并计算病毒滴度;4.检测PCOS-2DM大鼠胰岛细胞内ATPsyn-β表达:以免疫组织化学法及Western-Blotting法检测大鼠胰腺组织胰岛内ATPsyn-β表达的变化;5.以构建好的Atp5慢病毒载体分别转染正常大鼠胰岛及PCOS-2DM大鼠胰岛,Western-Blotting方法检测胰岛细胞ATPsyn-β的表达变化,行葡萄糖刺激后胰岛素释放实验,ELISA法检测胰岛素分泌情况变化,ATP检测试剂盒检测胰岛ATP含量改变。实验结果1.成功构建了 PCOS-2DM大鼠模型:①PCOS组体重较正常组增加,而PCOS-2DM组体重明显减轻;②对照组约在50日龄左右开始出现动情前期-动情期-动情后期-动情间期的动情周期模式。而PCOS组及PCOS-2DM组大鼠则未建立稳定的动情周期。③PCOS-2DM组大鼠有多饮、多食、多尿症状及体重减轻;④PCOS以及PCOS-2DM组卵巢重量显著高于正常组,囊性卵泡数、囊性卵泡比例均较正常组显著增加,而颗粒细胞层数显著减少;⑤与对照组相比,PCOS组及PCOS-2DM组T、E2水平均显著增高,但PCOS组与PCOS-2DM间无明显差异。PCOS伴-2DM组大鼠FBG比正常组及PCOS组均显著增高,达到糖尿病诊断标准。PCOS组及PCOS-2DM组大鼠与正常组相比,HOMA-IR均显著增高;2.胰岛细胞的原代培养:DTZ染色胰岛为猩红色。随着培养时间的延长,胰岛细胞的外形及生长环境发生改变,培养后24小时的胰岛状态最佳;3.正常组大鼠胰岛高浓度葡萄糖刺激胰岛素分泌明显高于低浓度葡萄糖刺激,PCOS-2DM组大鼠胰岛两种浓度葡萄糖刺激下胰岛素分泌无显著差异。与对照组相比,PCOS-2DM组无论在何种浓度葡萄糖刺激下,胰岛素分泌均显著下降,PCOS组胰岛在高浓度葡萄糖刺激下,分泌胰岛素较正常大鼠胰岛显著增高。与PCOS-2DM组相比,PCOS大鼠胰岛素分泌显著增高;4.Atp5b慢病毒载体构建,病毒的滴度为:2×109TU/ml;5.免疫组织化学结果显示,与对照组大鼠胰岛相比,PCOS组大鼠胰岛略小,与对照组形态上并差别,PCOS-2DM组大鼠胰岛明显萎缩且形态不规则,胰岛细胞排列疏松且紊乱。对照组及PCOS组内棕黄色颗粒表达量肉眼观无差异,均有较明显的ATPsyn-β表达,而PCOS-2DM组大鼠胰岛内却仅见少许阳性表达颗粒,与PCOS组及对照组相差明显。以Western-Blotting的法检测大鼠胰岛内ATPsyn-β蛋白表达,结果表明PCOS-2DM组较对照组显著下降,与PCOS组相比也显著表达下降。而PCOS组与对照组相比,其表达浓度显著下降。6.PCOS-2DM大鼠胰岛内ATPsyn-β表达较正常功能胰岛显著下降。重组Atp5b慢病毒转染功能受损的胰岛后,ATPsyn-β表达较未转染组及空载体组显著增加。但转染组与正常组相比,胰岛内ATPsyn-β的表达量仍显著不及。PCOS-2DM组大鼠胰岛内ATP含量显著下降。Atp5b慢病毒转染后,ATP含量有所上升,与PCOS-2DM及空载体转染组相比均显著增加,但与正常组相比,仍显著减少。正常组大鼠胰岛25mM葡萄糖刺激时其胰岛素分泌量显著高于5mM葡萄糖刺激。而PCOS-2DM及Atp5b慢病毒转染组在低糖与高糖刺激时胰岛素分泌并无显著差异。Lenti-Atp5b转染PCOS-2DM胰岛后,其胰岛素分泌水平显著提高。在5mM浓度葡萄糖刺激时,Lenti-Atp5b转染后PCOS-2DM胰岛的胰岛素分泌与正常胰岛并无差异,而25mM浓度葡萄糖刺激时,前者胰岛素分泌仍显著低于后者。结论硫酸普拉睾酮钠联合高脂高糖饮食及STZ可以成功构建PCOS伴2型糖尿病大鼠模型。ATPsyn-β在PCOS及PCOS-2DM大鼠胰岛中表达降低,通过Atp5b慢病毒载体转染上调PCOS-2DM大鼠胰岛ATPsyn-β表达,可以改善其胰岛素分泌功能。更多还原

【Abstract】 Objectives:The aims of this study were to investigate the changes of ATPsyn-β expression in pancreas islets of PCOS with type 2 diabetes mellitus,and to investigate the changes of the distructed islet β-cell function after the up-regulation of ATPSyn-βby Lentivirus Atp5b.The purpose of the study is:1.To constract an rat model of PCOS with type 2 diabetes mellitus,and isolating and purifying of the rat pancreatic islets for primary culture was carried out to provide the matrix for up-regulation of ATPsyn-β.2.To construct Atp5b recombinant lentiviral vector,to provide intervention for ATPsyn-β up-regulation;3.To confirm the changes of ATPsyn-β expression in the formation of pancreatic islet β-cell dysfunction in PCOS with type 2 diabetes mellitus;4.To clarify after up-regulation of ATPsyn-β,the function of impaired pancreaticβ-cell islet secretion function would be improved and protected.Methods:1.Construction of PCOS with 2DM rat model:The PCOS model was induced by continuous DHEA and high fat and high glucose diet.And a single high-dose STZ injection induced type 2DM model on the basis of high fat and high glucose diet.Through the observation of the general state of the model rats,the body weight,the changes of estrous cycles by vaginal smears,the ovarian morphological changes by HE staining,the fastin blood glucose,the levels of E2,T and INS by ELISA,the HOMA-IR index and so on,to evaluate the construcion of PCOS and type 2 diabetes;2.The primary culture of pancreas islets of rat:the pancreas was digested with collagenase V,and purified with gradient centrifugation of Ficoll.DTZ staining was used to identify the activity of pancreas islets,which were cultivated with 20%FBS RPMI1640 + double anti-culture medium.Glucose stimulated insulin secretion was carried out to detect the insunlin scecretion funcion of islets;3.The construction of lentivirus Atp5b:GV208 vector was subjected to single digestion,and Atp5b gene was amplified by RT-PCR and then exchanged into a linearized GV208 vector.After the transformaion of plasmid DNA into E.coli,the identification of positive clones and sequencing was carried out.The recombinant plasmids were identified by RT-PCR and Western-Blotting.The recombinant virus plasmids and two auxiliary packaging vector plasmids were co-transfected into 293T cells to complete the packaging of the recombinant lentivirus.Fluorescence microscope was used to observe the transfection effect.Finally,the virus titer was detected and calculated by Real-Time PCR.4.Detection of ATPsyn-β expression in pancreatic cells in rat with PCOS and 2DM:expression of ATPsyn-β in pancreas islets was detected by immunohistochemical and Western-Blotting.5.The Atp5b lentiviral vector were transfected into PCOS-2DM rat islets.And then the glucose stimulated insulin secretion was carried out,with the detection of insulin secretion changes by ELISA,and the ATP content was detected with the detection kit of ATP.Results:1.PCOS with 2-DM rat model was successfully constructed:① The rats in PCOS group and PCOS-2DM group were increased when compared with control group before STZ administration.In the PCOS-2DM group,the body weight of the rats was significantly lower than that of the control group and PCOS group at the end.② The control group began to appear the estrus cycle mode of preoestus-esturs-metaoestrus-diesturs at the age of 50 days.Meanwhile,the PCOS group and the PCOS-2DM group did not establish a stable estrous cycle at the same time.③ PCOS-2DM rats got the symptoms of polydipsia,polyphagia,polyuria and weight loss,which were similar to the clinical symptoms of type 2 diabetes.④ When compared with the control group,the volume and weight of ovaries in PCOS and PCOS-2DM group increased significantly with more cystic follicules on the surface.The number of cystic follicles、the proportion of cystic follicles the in PCOS and PCOS-2DM gruoups were significantly lower than the control group.⑤ Compared with the control group,the levels of T and E2 in PCOS group and PCOS-2DM group were significantly increased,but there was no significant difference between PCOS group and PCOS-2DM group.The FBG of PCOS-2DM group was significantly higher than that of normal group and PCOS group,and the mean value was 26.59mmol/L,which reached the diagnostic criteria of diabetes mellitus.HOMA-IR were significantly increased in PCOS group and PCOS-2DM group,suggesting the existance of insulin resistance;2.Primary culture of pancreas islets:pancreatic islets was isolated and purified successfully for the primary culture,and the islets were scarlet after DTZ staining.With the prolongation of culture time,the shape and growth environment of islet cells changed,and the isle had the best status at about 24 hours after culture;3.The insulin secretion in the normal group was significantly higher when stimulated with high concentration of glucose.There was no significant difference in insulin secretion between different glucose concentration in PCOS-2DM group.Compared with the control group,insulin secretion was significantly decreased in PCOS-2DM group with different glucose stimulation,meanwhile,the insulin secretion of PCOS group was similar to the normal rats at the stimulation of low concentration of glucose.Under the stimulation of high concentration of glucose,the insulin secretion of PCOS was significantly higher than that of the control.4.The lentivirus Atp5b was constructed at titer of 2 × 109 TU/ml.5.Immunohistochemical results showed that the pancreas islets in control group were larger and plumper,and the islet cells were closely arranged.Meanwhile,the islets in the PCOS group were slightly smaller than those in the control group without a significant difference.But islets in PCOS-2DM rats were atrophic and irregularly shaped,and islet cells were loose and disordery.There was no significant difference in the expression of brown granules between the control group and PCOS group,but only a few positive expression of granules was presented in the PCOS-2DM group,with a significant difference when controlled with anoter two groups.The expression of ATPsyn-β in pancreas islets was detected with Western-Blotting,and The results showed that expression of ATPsyn-β was significantly lower in the PCOS-2DM group than in the control and PCOS group.And expression of ATPsyn-(3 was significantly lower in the PCOS group than control group.6.The expression of ATPsyn-β was significantly increased in the Atp5b lentivirus transfection group than the untransfected group and empty vector group,but still significantly lower than that in the normal group.The expression of ATP in islets of PCOS-2DM group was significantly lower than that of normal group.After transfection of Atp5b lentivirus,the ATP content increased,which was significantly increased compared with PCOS-2DM group and empty vector transfection group,but still significantly lower compared with normal group.In normal group,insulin secretion was significantly higher after 25mM glucose stimulation than 5mM.There was no significant difference in insulin secretion in both PCOS-2DM and Atp5b lentivirus transfection group between the different stimulating concentration of glucose.After the transfection of Lenti-Atp5b to the PCOS-2DM islets,the insulin secretion was significantly increased,and the difference was statistically significant.The insulin secretion of transfected PCOS-2DM islets was simlar to the normal islets at the stimulation of 5 mM glucose,whereas significantly lower than normal after the stimulation 25 mM glucose.Conclusion:The rat model of PCOS and 2DM can be successfully constructed by DHEA combined with high fat and high glucose diet and STZ.The expression of ATPsyn-βin PCOS and PCOS-2DM rats was decreased,and the expression of PCOS-2DM ATPsyn-β was up-regulated by Atp5b lentiviral vector,which could improve the insulin secretion of pancreatic β cells and protect the function of pancreatic β cells.更多还原