Regulation Of Invision By Leptin And Its Mechanism In First Trimester Cytotrophoblastic Cells

Pregnancy is a complicated and delicate process,and the maintenance of pregnancy depends on the normal functions of trophoblastic invasion.The invasion of trophoblastic has a strict time-space,and its invasion increased during early pregnancy, and reduced or disappeared during intermediate or evening pregnancy.The depth of invasion is limited to decidual and nearly 1/3 myometrium.Studies showed that the balance of MMPs/TIMPs played a major role in human embryonic implantation,but its regulatory mechanism is not clear now.Recent studies showed that trophoblast cells expressed leptin.Leptin receptor was detected at the eighth weeks of pregnancy in implantation sites.So we supposed that leptin and its receptor system may be involved in the of expression trophoblastic MMPs/TIMPs or the process regulation of blastocyst implantation.Objective:To Observe the regulation of MMPs/TIMPs by leptin in first-trimester cytotrophoblastic cells under the conditions of the micro-environment simulation of early pregnancy(17-β-Estradiol and Progesterone) in vitro,and futher explore its regulation mechanism and significance.Methods:According to conventional methods,We isolated first trimester villi(6-9 weeks) of normal pregnant woman,and used immunohistochemical staining(Cytokeratin 7 and vimentin) to identify extravillous trophoblast.After cultured for 24h in 1640 medium containing 10%FBS in the environment of 37℃,5%CO₂,we continued to culture in serum-free medium.Then we divided cytotrophoblastic cells into 2 groups: control group or 17-β-Estradiol and progesterone group.After 24h,we collected the supernatant or cell.We detected soluble leptin receptor contained in supernatant with ELISA.We applied FCM to detect leptin receptor expression of the cell surface.Base on the above findings.After cultured for 24h in 1640 medium containing 10%FBS in the environment of 37℃,5%CO₂,we continued to culture in serum-free medium. Then we divided cytotrophoblastic cells into 4 groups:17-β-Estradiol and progesterone,leptin,17-β-Estradiol and progesterone and leptin,the control group. After 24h,Collected cells and culture medium after cultured 24h in the environment of 37℃,5%CO₂.MMP-2,MMP-9,TIMP-1,TIMP-2 mRNA were detected by the semi-quantitative RT-PCR method.MMP-2 or MMP-9 level of supernatant were determined by zymography.Results:1,The purity of human extravillous trophoblast cells was more than 90%after immunohistochemical staining of cytokeratin 7 or vimentin.The viability of human extravillous trophoblast cells was more than 90%by counting living cells of trypan blue exclusion.2,FCM results showed that leptin receptor level were significantly upregulated by 17-β-Estradiol and progesterone combined treatment(P＜0.05).3,Soluble leptin receptor was undetected in the 17-β-Estradiol and progesterone combined treatment group or the control group.4,the results of RT-PCR detection showed that compared with the control group,the transcript level of MMP-2,MMP-9 mRNA were significantly up-regulated in the three groups(17-β-Estradiol and progesterone treated group,leptin treatment group, 17-β-Estradiol and progesterone and leptin treated group).TIMP-2 were down-regulated(P＜0.05).Compared with 17-β-Estradiol+progesterone treated group or leptin treatment group,the transcript level of MMP-2,MMP-9 mRNA of 17-β-Estradiol and progesterone and leptin combined treatment group was significantly up-regulated,TIMP-2 expression was down-regulated(P＜0.05). However,compared with the control group,the transcript level of TIMP-1 of 17-β-Estradiol and progestin treatment group or leptin treatment group was no significant difference.It is interesting that the transcript level of TIMP-1 of 17-β-Estradiol and progesterone and leptin treatment group was down-regulated.And compared with 17-β-Estradiol and progesterone treatment group or leptin treatment group,the transcript level of TIMP-1 was up-regulated in the 17-β-Estradiol and progesterone and leptin combined treatment group(P＜0.05).5,the results of zymography showed that compared with the control group,the protein level of MMP-2 or MMP-9 was significantly up-regulated in the three groups (17-β-Estradiol and progesterone treated group,leptin treatment group,17-β-Estradiol and progesterone and leptin treated group)(P＜0.05).Compared with 17-β-Estradiol+ progesterone treated group or leptin treatment group,the protein level of MMP-2 or MMP-9 of 17-β-Estradiol and progesterone and leptin combined treatment group was significantly up-regulated(P＜0.05).Conclusions:The human extravillous trophoblast cell we isolated and cultured expressed strongly leptin recptor,but did not express the soluble leptin recptor.There was a pathway of leptin-leptin recptor,leptin up-regulated the expression of MMP-2 or MMP-9 of human extravillous trophoblast cell through the pathway of leptin-leptin receptor,and involved the regulation and control of the human extravillous trophoblast cell invasion.Leptin-leptin receptor pathway to participate in the up-regulation of MMP-2 or MMP-9 in the environment of 17-β-Estradiol and progesterone,and the up-regulation was enhanced by leptin.Meanwhile,the expression of TIMP-1or TIMP-2 was down-regulated,leptin play a similar role in the regulation and control of invasion.This pathway might regulate the balance of MMPs/TIMPs expression,thereby regulating invasion of cytotrophoblastic cells.