The Biological Foundation And Effectiveness Of "Phlegm Accumulation In The Uterus"(6)

一,Objective: Based on observing gene expression of key point signal protein: Glut₄ ahd MAPK in insulin signal transduction of IR,which induced by wortmannin,the PI-3K catastaltica,we approached the molecule mechanism of IR signal transduction. The aim of our in vitro studies was to examined the mechanism of action of IGF-I to determine its potential role in the influence of sexual Hormone secretion and steroidogenic enzyme , which are P450arom,P450scc and StAR, by IR and normal porcine granulosa cells treating with IGF-I. And we researched the influence of the euglycemic agent to IR,which including of the traditional Chinese medicine and western medicine: troglitazone,dimethyl biguani,berberine and sal viol.二,Methods :The model of IR granulosa cell was established in vitro culture by treating with wortmannin, the PI-3K catastal- tica, and assessing the amount of glucose in cells by [3-³H] glucose. The corpuscular consumption of glucose was assessed by glucose oxidase method. Culture medium samples were subjected to chemoluminescence test for the level of E₂,P,and T. The proteinum qualitative assay of Glut4,MAPK and PPARγwere did by Immumofluorescence method. And then semiquanti- tative analyzing the expression of Glut₄,MAPK,PPARγ,P450arom,P450scc and StAR were used RT-PCR.三,Results: Results: (1)The corpuscular intake of glucose was significantly decreased by treating with 1.5μmol/L wortmannin (P<0.05) , and tne amount of leftover sugar was increased by 13.76% or so. (2)The immune of luorescence was shown there was the expression of Glut₄ and MAPK with the chromatosis mainly on cytolymph, as well as that of PPARγmainly on cytolymph and cellular membrane. (3)It was found that IGF-I elevated the secretion of E₂ in both IR and normal granulosa cells(P<0. 05)and moreover, the difference between the normal group and the group of normal cells treated with IGF-1 was bigger than that between the IR group and the group of IR cells treated with IGF-1. Compared with the normal group,the secretion of T in the group of normal cells treated with IGF-1 was decreased (P<0.05), increasedly in the IR group(P<0.05), and increasedly in the group of IR cells treated with IGF-1 withnot statistical significance (P> 0.05). Moreover, the difference between the normal group and the group of normal cells treated with IGF-1 was smaller than that between the IR group and the group of IR cells treated with IGF-1 on the secretion of T. (4)The expression of P450arom mRNA in the group of normal cells treated with IGF-1 was increased compared with the normal group, and inversely, P450arom in the group of IR cells treated with IGF-1 was decreased compared with the other groups. The expression of P450scc and StAR mRNA in the group of normal cells treated with IGF-1 was increased compared with the normal group, and similaly, P450scc and StAR in the group of IR cells treated with IGF-1 was increased compared with the other groups. (5)When granulosa cells were IR, the expression of Glut₄ was hyp-expression , and inversely, that of MAPK and PPARγwere hypsi-expression. (6)After IR granulosa cells were treated with euglycemic agent (troglitazone,dimethyl biguani,berberine and salviol),the expression of Glut₄ was increased, but lower than that of the normal group; and inversely, the expression of MAPK and PPARγwere decreased, but higher than that of the normal group.å››,Conclusions: (l)Wortmannin can blockade the signal transduction of PI-3K pathway and lead to IR .The decreased expression of Glut₄ influenced the signal transduction pathway of metabolism. However, the increased expression of MAPK influenced the signal transduc- tion pathway of caryocinesia. In short, there are alternating insulinum signal transduction pathways in ovaries. (2)For first time, we produced the method of making model of IR granulosa cell by utilizing the PI-3K catastaltica(wortmannin), authenticated with the glucose oxidase method and assessing the amount of glucose in cells by[3-³H]glucose.(3)These observations demonstrate that IGF-I stimulates expression of P450arom,StAR,P450scc mRNA and brings about increased estrogen secretion in granular cell;and in IR granulosa cells, confirm the effection of IGF-I on androgen by increasing the volume of both secretory and synthesis, as well as obviously elevated StAR,P450scc expression.(4)These observations suggest an inter-relationship among effecttion of IGF-I on follicular development , the intracellular signaling system, as well as hormone and growth factors in microenvironment of ovaries.(5)On effect of droping blood glucose, the feature of berberine and salviol is similar to troglitazone,and better then dimethyl biguani.(6)We enter to the molecular level of ovaries cell to approach the molecule mechanism of IR signal transduction and researched the influence of the euglycemic agent of the TCM and western medicine to IR. This study conduce to interpret nosogenesis of IR, especially on molecule target and intracellular mechanism of signal transduction; provide theoretical evidence to prevent and control PC0S,as well as to improve IR;and offer new method on clinical application of TCM.