The Effect Of Morphine On The Expression Of LHR MRNA Of Human Luteinizing Granulose Cells In Vitro

Objective:To study the effect of morphine on the expression of LHR mRNA of human luteinizing granulose cells in vitro,to probe into the direct influence of morphine on ovary,so as to put forward theoretic basis for negative effect of reducing or obstructing morphine dependence on ovary.Materials and Methods:Human granulosa cells were obtained from patients undergoing in vitro fertilization-embryo transfer(IVF-ET)by ultrasound-guided follicular aspiration.30 dysgenesia patients were chose,mean age was 30,menstrual cycle was normal,the reasons of dysgenesia were both from oviduct or male.The patients with genital system organic disease and other endocrine system disease all over the body were removed.They accepted in vitro fertilization -transplantation of embryowere(IVF-ET)treatments in the reproduction medical center,the First Hospital of shanxi medical university in April-september in 2006.All patients adopted superovulation program,monitoring follicular development per vagina B ultrasound,human chorionic gonadotropin(HCG)was administered by intramuscular when more than one ovarian follicle developed to diameter reaching 16mm.In 34～36h after injecting HCG,ovum was obtained per vagina B ultrasound,and emanent follicular fluid was placed in bioclean and dried centrifuge tube,they were as soon as possible delivered to the laboratory by heat preservation, then did isolating culture.Most of the granulosa cells adhered after 24 hours of culture in vitro,then identified them by the appearance of cells.All cells were randomly divided into 3 groups,one group was control group that nothing was added,other group was added morphine(including 10^-6,10^-5,10^-4Mol/L),the other group was added morphine and naloxone(both final concentration were 10^-5Mol/L).We observed the morphologic change of granulosa cells by inverted microscope and after 24 hours,we collected all cells and analyzed the expression of LHR mRNA by RT-PCR method.Statistical calculations were done by SPSS 12.0 for windows.Result:1,After 24 hours of human granulosa cells culture of in vitro,cell adherence had been observed under inverted microscope,it grows explanately,rich in granula,and there were also cell junction between them.Culturing continuously for 2-3 days,granula cells keeped on growing,with the apophysis grows longer,and cell junction increased.After 6-7 days' culture, cells were out of their shape,star shape and filaceous apophysis disappeared,and at about the end of the 10th day,they were shedding.2,PCR showed that the expression of LHR mRNA of human granulosa cells is decreased significantly in contrast with that of control(P＜0.05).While there was no significant difference in LHR mRNA between morphine Narcon combined group and controlling group(P＞0.05). The expression of LHR mRNA had negative correlation with the concentration of morphine (r=-0.932,P＜0.01).Conclusion:1,Human granulose ceils of ovary were cultured successfully in vitro.2,Morphine could reduce the expression of LHR of human granulosa cells directly with dose dependent,while Nareon could work against morphine's negative effect.