The Effect Of Insulin、Testosterone、AngⅡ、Ang-(1-7) On Renin-angiotensin Receptors In Endometrial Cell And Endometrial Cell Development

Objective: To establish a primary endometrial glandular epithelial andstromal cells model in vitro from the method of isolation、puanifying、andculturing human endometrial specimensï¼›Detecting the changes of angiotensinreceptors in endometrial cells after stimulating by insulin、testosterone andcell proliferation and apoptosis of endometrial cells after stimulating by AngII、Ang-（1-7）.Discussing the relationship between changes of RAS inpolycystic ovarian syndrome （PCOS） and endometrial cells development, toprovide a new thinking for pathomechanism and therapeutic approaches ofPCOS.Methods:1Human normal endometrial specimens were separated and purificated byenzymolysis,two filter and pasted wall,then identifed the purify of two typescells by immunoocytochemistry,observed the cell morphology by H-E sta-ining,watched the cell growth state and the cell growth environment by micr-oscope.2Endometrial stromal cells were stimulated with insulin at differentconcen-tration （physiological dose of10mIu/l,as the dose of the pcos of30mIu/l） and testosterone (physiological dose of10^-9mmol/ml,as the dose ofthe pcos of10^-5mmol/ml) for24h、48h、72h, and the growth of theproliferative stromal cells were detected by MTT, and The mRNA expressionof angiotensin II （Ang II） type1receptor （AT₁R）, type2receptor （AT₂R）,angiotensin1-7（Ang-（1-7）） receptor （mas） on the proliferative andsecretory endometrium stromal cells and glandular epithelium weredetermination by Reverse transcription polymerase chain reaction （RT-PCR）. 3The same growth stage of proliferative and secretory endometriumstromacells and glandular epithelium were stimulated with insulin at differentconcentration of AngⅡ（10-12mol/L¹0-5mol/L）and Ang-（1-7）(10^-12mol/L¹0^-5mol/L)for24h,48h,72h, Method of MTT was used to detect the OD andeffect on cell proliferation.The proliferation of stromal cells were stimulatedwith10-6mol/L Ang II and Ang-（1-7） for48hours and detected the effect onproliferation and apoptosis of cells by flow cytometry.Results:1The culture success rate of primary endometrial cell was97%.The purityof stromal cells was95%, limited passage, survived one month. The purity ofglandular epithelium was90%, not easily passage,olny surviving for10da-ys.Stromal cells were proliferating with time, the fastest proliferation time was48hours. Glandular epithelial cells had no obvious change in24,48,72hours.2The changs of endometrium stromal cells and glandular epithelium afterstimulating by INS and T2.1Statisticing the OD values: physiological dose, high dose of INS canpromote the proliferation of stromal cells proliferation period, with theincrease of concentration of insulin, the growth of cells nhancing,, comparedwith the control group were significantly different, and are time dependenting,48hours was strongest function.Different concentrations of testosterone couldinhibit the proliferation of stromal cells, compared with the control group weresignificantly different, and have the time dependence, the strongest functionwas48hours.2.2The expression of AT₁R、AT₂R、MASmRNA.2.2.1The level of AT1R、AT2RmRNA in the secretory was obviously higherthan those in the proliferative, glandular epithelium was obviously higher thanthose stromal cells. The level of MAS mRNA in glandular epithelium wasobviously higher than those stromal cells. The indifference of MAS mRNAbetweenSecretory and proliferation was not statistically significant.2.2.2The difference in the expression of AT₁R、AT₂R、MASmRNA betweenthe stimulation group of the physiological doses of insulin、testosterone and the control group had no statistical significance，The difference in theexpression of AT₁R、AT₂R、MASmRNA between the stimulation group of thehigh doses of insulin、testosterone and the control group had statisticalsignificance3The proliferation and apoptosis of endometrium stromal cells and glandularepithelium after stimulate AngⅡ、Ang-（1-7）3.1Statisticing the OD values: less than physiological dose of Ang II did notplay a role on the proliferation of endometrial cells, physiological dose of Angâ…¡ promoted the growth of Secretory and proliferation of endometrial cells,when large dose Ang â…¡inhibited the proliferative of Secretory andproliferation endometrial cells,with the increasing of concentration, theinhibition was more strong,10-6mol/L had the strongest effect. With increasingof time, the inhibition was more strong,48hours had the strongest inhibitioneffect, there were statistical significance. Less than physiological dose ofAng-（1-7） did not play a role in the proliferation of endometrial cells,physiological dose and large dose of Ang-（1-7） inhibited the proliferative andsecretory phase endometrium stromal and glandular epithelial cell sgrowth,the effect of inhibitory enhanced with the increasing of concentration,10-6mol/L had the strongest effect. With increasing of time, inhibition also increased,48hours had the strongest inhibition effect, there were statistical signifi-cance.3.2The statistical results of flow cytometry show: percentage of S phase cellsdecreased after stimulating the proliferation of stromal cells48h by10^-6mol/Lconcentration of Ang â…¡a nd Ang-（1-7）, control group was8.586±1.420, Angâ…¡group was5.146±1.339, Ang-（1-7） group was3.850±0.9702. Sphase cellsIn the DNA active synthesis phase decreased, cell cycle transition fromS/G2-M to G0/G1phase, Inhibiting effect of Ang-（1-7） were stronger than thatof Angâ…¡, with statistical significance. the apoptosis index of10^-6mol/Lconcentration of Ang II, Ang-（1-7） compared with the control group wereincreased, the control group was0.4880±0.0747, Ang â…¡group was0.6980±0.0907, Ang-（1-7） group was1.282±0.3539, had statistical significance. 10^-6mol/L concentration of Ang II, Ang-（1-7） inhibited the proliferation ofstromal cells proliferation and promoted apoptosis,Ang-（1-7） were morestronger than Ang â…¡.Conclusions:1Enzymolysis, two filter and pasted wall is the time-saving,effciencymethod of culturing endometrial cell. Stromal cells can be limited passag-e,cells of2-3generation is the best period for experimental study. Glandularepithelium cannot growth permanently, the cells of3-4days is the best periodfor experimental study.2INS can promote and Testosterone could inhibit the proliferation ofstromal cells.3Physiological doses of insulin and testosterone does not change theexpreesion of AT₁R、AT₂R、MASmRNA in endometrium cells.The level ofAT₁R、AT₂R、MASmRNA in endometria cell of PCOS dose was obviouslyhigher than those in the control group, with statistically significantdifference.The changs of reninangiotensin receptors of pcos in endometrialcell may be associated with insulin andtestosterone.4Physiological dose of Angâ…¡ promote cell growth, Ang-（1-7） inhibitecell growth, The balance of RAS may be benifete of human endometral cellsdevelopment. Large doses of Ang Ⅱ、Ang-（1-7） can inhibit proliferation andpromote apoptosis of endometrial cells,It is may be involved in the path-ophysiology of PCOS and normal endometium.