The Influence Of Klotho Gene Transfer Into Diabetic Rats On The Concentration Of HDL、LDL、iNOS And P-selectin

ObjectiveKlotho gene is known to be associated with aging and metabolic,andaging-related diseases such as coronary heart disease, also have a certain relationshipwith hypertension. Studies have shown that the incidence of diabetes has significantlycorrelated with Klotho signal weakens and there is positive correlation between theseverity and decreased Klotho signal. Klotho gene plays an important role in theregulation of insulin and IGF-1signaling pathway， moreover diabetes andcardiovascular disease is significantly correlated. It is found that in mice with Klothogene expression deficient will occur aging-related human disease phenotypes,such asatherosclerosis, osteoporosis,glucose and energy metabolism change,suggesting thatKlotho gene regulation may occur in the human metabolic syndrome happen anddevelopment. Overseas studies have confirmed that inlet the Klotho gene to themouse model of atherosclerosis, endothelial function abnormalities be improved、lowed the hypertension、alleviate the peripheral vascular fibrosis and middle arterialcalcification and inhibit the further development of target organ damage andsecondary effects of hypertension,indicate that Klotho gene can protect the coronaryartery. This study was designed to observe and compare blood iNOS, P-selectin,low-density lipoprotein, high density lipoprotein levels between Klotho gene and non-Klotho gene rats throug import gene carriers，to prepare for the further study thatKlotho gene ‘s protective effect on coronary heart disease.MethodsFirst, gene cloning and construction of Klotho gene carrier: according to Klothogene sequence timing primer， constructing Klotho carrier by applying PCRtechnology to amplify the target gene.Then establish animals model of diabetic ratsand transfer the Klotho gene into rats. Measuring mRNA and protein expressionlevels definite time of Klotho gene between experimental group and control group,byusing PT-PCR,Westen blot technology and so on.Kill rats after a successful modelingafter twelve weeks respectively, measuring Klotho mRNA and protein expression,testing blood concentration of low-density lipoprotein、 high density lipoprotein、nitric oxide synthase (Nos) expression and P-selectin.ResultsThe concentration of high-density lipoprotein in gene therapy diabetic ratsgroup is (0.61±0.06) mmol/L、negative vector control group is (0.48±0.11) mmol/L、normal rats group is (0.67±0.06) mmol/L.After two independent samples t-test，the result of therapy group compared with control group show that P <0.05,statistically significant. Compared with the normal group, the results of diabetic ratswere significantly decreased; The concentration of low therapy group compaired withcontrol group show that P-density lipoprotein in gene therapy group is (0.44±0.09)mmol/L, negative vector control group is (0.45±0.10) mmol/L, normal non-diabeticrats group is (0.10±0.01) mmol/L, therapy group compared with control group showthat P〉0.05, no statistically significant; Compared with the normal group, low-densitylipoprotein in diabetic groups are significantly elevated; The concentration of iNOSin gene therapy diabetic rats group is（8.53±2.22）u/ml、negative vector control groupis （11.24±4.22） u/ml、normal rats group is （2.51±1.02）u/ml, therapy groupcompared with control group show that P 〈0.05, statistically significant; The concentration of P-selectin in gene therapy diabetic rats group is （3.07±0.67）ng/mL、negative vector control group is （5.21±1.12）ng/mL、normal rats group is（2.71±0.46）ng/mL, therapy group compared with control group show that P <0.05,statistically significant.ConclusionsThrough the experiment, Klotho gene can make high-density lipoprotein indiabetic rats increased, and slow nitric oxide synthase increase level,but for theinfluence of low LDL is uncertain,need further study. Klotho gene also reduceP-reactive protein level influenced by diabetic, in general, Klotho gene can inhibitionrisk factors for coronary heart disease and protects the arterial intima.It is certainthat,Klotho gene therapy in human to protect coronary artery will soon come true.