| 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. |
PDF Full Text Request