Expression Of RAS Receptors In The Luteinized Granu1osa Cells Of Patients With Polycystic Ovary Syndrome And Effect Of AngⅡ、 Ang(1-7) For Human Luteinized Granulosa Cells

Objective: First, to discuss whether Renin-Angiotensin System（RAS）take part in the pathogenesis of polycystic ovarian syndrome by measuringthe expression level of each receptor of angiotensinII and angiotensin-1-7（AT1、AT2、MAS）in the luteinized granu1osa cel1s.Second, to observethe effect of RAS in proliferation and secretion function of granulosa cell. bycu1turing human luteinized granulosa cells in vitro with differentconcentrations of AngII、Ang1-7.Methods:1Granu1osa cel1s were obtained from infertile patients undergoingIVF-ET cycles in the reproductive center of the second Hospital of HeBeiMedical Univereity. from November2011to June2012.Age with24～38yearsold,mean age23±0.67years old. All patients using Iong period schemaover-ovulation stimulants,when follice>17mm, injected Human chlrionicgonadotrophin（10000U）,after for34-36h,follicular fluid was take out of ovumvia vagina-ultrasound condueted.Control group:11cases of married women were diagnosed as infertilitywith fallopian tube factors or male factors.Menstrual regularity,sex hormonesin the normal level,there is no abnormal pelvic ultrasound.And exclusion ofother endocrine diseases. without using other hormone drugs within threemonths,no heart,liver,kidney disease historyExperiment group:10cases of married women were diagnosed asPCOS.The diagnosis of PCOS refer to the2003Rotterdam diagnosticcriteria[1]:①Oligo-and/or anovulation②Clinical and/or biochemical signs ofhyperandrogenism③Polycystic ovaries （presence of12or more follicles ineach ovary measuring2±9mm in diameter,and/or increased ovarian volume （>10ml））,these three with two can diagnosis.And exclusion of other endocrinediseases from adrenal or pituitary.All the patients require nearly three monthswith no other hormone drugs application history,no heart, liver, kidneydisease history.2Granulosa cells of polycystic ovary syndrome and control group werecu1tured in vitro for72h. By retrovirus polymerase chain reaction （RT-PCR）for detection of AT1, AT2,and MASmRNA.3Granulosa cells of control group were cu1tured in vitro and interferedwith the10^-10～10^-5mmol/Ldose drug medicine of AngII、Ang1-7for24、48、72h，the viability of cells was determined by using the method of MTT.4Granulosa cells of control group were cu1tured in vitro and interferedwith the10-6mmol/L dose median1of AngII、Ang1-7for24、48、72h，Theculture medium was collected，and the estradiol（E2） and progesteron（eP）levelwere detected by chemiluminescence method.Results:1Morphological trait of granulosa cellWe observed the cell morphological trait under inverted phase contrastmicroscope, adherence rate was above90%,Granulosa cell was roundinoculated instantly，adherenece after24h，Hematoxylin&Eosin （HE）staining of cultured ovary granulosa cell was observed nucleolus big roundand morphological was fusiform regularly, nucleolus staining was deepblue,cytopalsm was light red, rich invacuole and granule.2The results of AT1, AT2, and MASmRNA by RT-PCR2.1The expression of AT1mRNABoth the human luteinized granulosa cells and the luteinized granu1osacel1s of polycystic ovary syndrome expressed AT1.The results of theAT1mRNA expression were2.895±0.9625、5.266701±1.7663.The level ofAT1mRNA in the PCOS group was obviously higher than those in the controlgroup,and the difference was statistically significant （P<0.05）.2.2The expression of AT2mRNABoth the human luteinized granulosa cells and the luteinized granu1osa cel1s of polycystic ovary syndrome expressed AT2.The results of theAT2mRNA expression were1.8662±0.669、5.9741±2.1063.The level ofAT2mRNA in the PCOS group was obviously higher than those in the controlgroup,and the difference was statistically significant （P<0.05）.2.3The expression of MASmRNABoth the human luteinized granulosa cells and the luteinized granu1osacel1s of polycystic ovary syndrome expressed MAS.The results of theMASmRNA expression were0.5337±0.1974、1.4830±0.2593.The level ofMASmRNA in the PCOS group was obviously higher than those in thecontrol group,and the difference was statistically significant （P<0.05）3MTT assay of granulosa cell3.1MTT assay of AngII interfere granulosa cellCompared with control group，Absorbance value of granulose cell of thedifferent dose drug of AngII (10^-10～10^-5mmol/L)all decreased，and thedifference was statistically significant （P<0.05）.With increase of dose(10-9～10^-6mmol/L)，there was differences betweendrug groups，the difference was statistically significant （P<0.05）,apart from10^-9、10–10and10^-6、10–5group.3.2MTT assay of Ang1-7interfere granulosa cellCompared with control group，Absorbance value of granulose cell of thedifferent dose drug of Ang1-7(10^-10～10^-5mmol/L)all decreased，and thedifference was statistically significant （P<0.05）.With increase of dose(10-9～10^-6mmol/L)，there was differences betweendrug groups，the difference was statistically significant （P<0.05）,apart from10^-9、10–10and10^-6、10–5group.3.3MTT assay of different timeAs the extension of time, Absorbance value of granulose cell of thedifferent dose drug of AngII and Ang1-7all increased，and the difference wasstatistically significant （P<0.05）。4E2and P level of granulosa cell4.1Compared with control group，the E2level scereted by human granulose cell of AngII、Ang1-7groups increased，and the difference was statisticallysignificant （P<0.05），there was no significant difference between AngII andAng1-7.4.2Compared with control group，the P level scereted by human granulosecell of AngII、Ang1-7groups nodistinct.4.3As the extension of time，the E2、P level scereted by human granulose cellwas increased，and the difference was statistically significant （P<0.05）.Conclusions:1Granulosa cells of human ovary were cu1tured in vitro adherence ratewasvary highly.2AT1、AT2、MAS express enhancement at the molecular level ingranulosa cells of PCOS patients， They may make granulosa cellsdevelopment obstruction and participate in the pathological process of PCOS.3Physiological dose（10–10mmol/L）of AngII、Ang1-7restrain granulosacells,and with increase of dose，the effect was enhance,when the dose was10^-6mmol/L, the effect was the best. As the extension of time，the granulosacells was proliferation.4The E2level secreted by human granulose of the AngII、Ang1-7increased，but the P level nodistinct; As the extension of time, the E2、P levelsecreted by human granulose all increased.