| Background and ObjectiveThe medical profession has paid more and more attention to diabetes,so far diabetes do harm to the worldwide people about 415 million,it is predicted that there will be 642 million diabetics in the year of 2046,most of the diabetics are suffered from type 2 diabetes mellitus(T2DM).The symptoms of T2 DM are hypeglycemia and insulin resistance(IR).IR means:the role of insulin target organs,mainly liver,muscle and adipose tissue,effect on insulin sensitivity decreased,which promote the target cells to absorb and use glucose function below the normal level,so the body compensatory secretes more insulin,but for the patient’s blood sugar,insulin secretion is still relatively insufficient.Studies have found that diabetes has more complications of vessels than the non diabetes.some scholars predict the IR leads to vascular diseases,such as atherosclerosis.DCCT/EDIC research team found that reduction of insulin sensitiity to that of people with diabetes,the blood insulin levels significantly increased,the incidence of atherosclerosis was also significantly higher than in patients with insulin sensitivity.The mechanism of insulin promotes cardiovascular events,may mainly by regulating lipid metabolism,activate vascular endothelin,promote the migration of vascular smooth muscle cell proliferation and lead to endothelial dysfunction and chronic inflammation.Early experiments have confirmed that estrogen has cardiovascular protective effect to human body,its mechanism mainly by the regulations of blood coagulation,blood lipid,expansion of blood vessels,anti-inflammatory,smooth muscle functions etc.And the role estrogen played mainly through combination with estrogen receptor,through a membrane receptor or nuclear receptor pathways to regulate cell physiological activities,the study found that estrogen receptors expressed in atherosclerosis decreased significantly,and this may associated with estrogen receptor gene promoter region methylationCoincidentally,the study found that insulin can lead to atherosclerosis at the same time can cause gene methylation,but if it could lead to the estrogen receptor gene promoter region methylation,we do not know.So to figure out whether there is a potential link between insulin and estrogen receptor methylation,will helps us to know more mechanisms of atherosclerosis leaded by insulin.Method1、Animal feeding,experimental group:intraperitoneal injections of insulin 4IU 5 times a week.control group intraperitoneal injections of saline 5 times a week.Total time is 3 months2 、 Take a the abdomen aorta of mice,followed by HE staining and ER-α immunohistochemical experiments,respectively to observe the atherosclerosis and ER-α protein expression induced by insulin.3、The cultivation of SD rat VSMC cell lines4、Use 100-nm insulin treat VSMC 24 h,48 h,72 h,respectively.Using flow cytometry to detect VSMC cell cycle5、Use 100-nm insulin treat VSMC24 h,48 h,72 h,respectively.Then use rt-pcr and WB detection of VSMC in each group,to see ER-α,DNMT3 a gene m RNA and protein expression.6、VSMC treated together with insulin and 5-Aza,then use rt-pcr and WB detection of VSMC in each group,to see ER-α gene and protein expression7、Use virus,high(+)or no(-)express ER-α,to infect VSMC.Then use rt-pcr and WB to detect ER-α gene and protein expression in VSMC8、Use flow cytometry technique and scratch test to detect cell cycle and migration influenced by ER-αResult1、HE shows insulin can promote atherosclerosis and immunohistochemical shows that,in experiment group there is less ER-α expression in nuclei of VSMC compared with the control group(p<0.05)2、Flow cytometry technique shows that after cultivating 72 hours,the control group VSMC shows G2 blocking,while the insulin treated group can promote cell proliferation.3、RT-PCR and WB show that,ER-α expressed higher in the experimental group than in the control group(p<0.05),while DNMT3 a expressed lower in the experimental group than in the control group(p<0.05).4、RT-PCR and WB show that,VSMC treated together with insulin and 5-Aza show no difference between groups.5、Fluorescence microscope shows that the virus infection efficiency is over 90%.RT-PCR and WB show that “+”group expressed more ER-α than the “-”and control group(p<0.01).6、Flow cytometry technique and scratch test show that,in“+”group the proliferation and migration ability of VSMC is weaker than the “-”and control groupConclusion1 、 Animal experiment confirmed that insulin can induce the occurrence of atherosclerosis,and ER-α expression is declined in VSMC of atherosclerotic plaque2、Cell test confirmed that insulin can promote the expression of DNMT3 a and suppression ER-α,while 5-Aza can reverse the phenomenon3、ER-α has the function of weaken the proliferation and migration ability of VSMC... |
