The Research On The Effect Of OxLDL/LOX-1 To Human Cytotrophoblasts Apoptosis And Its Role In The Etiology Of Preeclampsia

The Research of Effect of oxLDL/LOX-1 on Apoptosis in Human Cytotrophoblasts and the Relationship between this Effect and PreeclampsiaObjectivesPreeclampsia is a severe complication during the pregnant and circumnatal period, it is the reason to lead to the CFR of the parturients and neonates. The studay about etihological factor and etiopathogenesis of the preeclampsia is always an important topic of OB. Now it generally presume that abnormal placental implantation and vascular remaking disorder are pathogenetic center porints of preeclampsia, and they are both concerned with degrading infiltrating ability of langhans cells. If trephocyte apoptosis excessly duinggestational period, these cells will can't infiltrate into deciduas or the reduation of the infiltration cells population and the substitution of helix arteriole endothelial cell., inducing inadequate nidation and vascular remaking disorder, then preeclampsia happens.Lots of researches about CD confirm that oxidized low density lipoprotein, oxLDL is one of important factors that cause vascular endothelial injury, vascular smooth muscle cell and macrophage apoptosis. The studay discovers that oxLDL can induce cell apoptosis through many ways. But oxLDL only can make function by its receptor. Lectin like oxidized low density lipoprotein receptor-1,LOX-1 identified as a receptor of oxLDL in recent years, LOX-1 induces absorbing and metabolism of oxLDL expressing on endothelial cells, oxLDL express on vascular smooth muscle cell,macrophagus,mechanocyte and platelet, also on placenta. Different levels of complic metabolism complications ofen happen in preeclampsia, and blood fat metabolic is more obviously, there was already some rearch reporting. Whether oxLDL make an important factor in disease development and develing,whether oxLDL make function in trophoblast apoptosis and preeclampsia morbidity by inducing LOX-1 that yet haven't been reported until now.Methods(1) Normal pregnant woman and preeclampsia patients' oxLDL in blood plasma in different gestational weeks are tested by ELISA way, and to compare their diversifies.(2) Normal pregnant woman and preeclampsia patient's LOX-1 and apoptosis related gene Caspase-3 express in placenta trophocyte whose space time changes in differet gestational weeks are tested by Westen-blot,Immunohistochemistry and RT-PCR; Normal and preeclampsia pregnant woman's apoptosis related gene Bax and Bcl-2 expressing in placenta trophocyte in different gestational weeks are tested by Westen-blot,RT-PCR.(3) Chorioepithelioma cloned strain which prossess the characteristic of trephocyte are cultured in vitro to test oxLDL's regulation of expressing of LOX-1 and apoptosis gene in trephocyte by cell immunochemistry, RT-PCR and Westen-blot. We tested the changes anteroposterior blocking by the blocking agent of LOX-1.Results一,The level of ox-LDL in blood preeclampsia patient's blood plasma is significant higher, and the two sets have significant deviation.二,The space-time changes of the expression of LOX-1and apoptosis related genes in normal and preeclampsia pregnant woman's placenta1 The expression of LOX-1 in normal and preeclampsia pregnancy woman's placenta. (1) Immunohistochemistry displays that LOX-1 is expressed in syneytiotrophoblast and langhans cell of villus in early pregnancy, and it is expressed in syneytiotrophoblast, langhans cell and vascular endothelial cell of placenta of normal and preeclampsia in middle and advanced stage .With gestational weeks increasing the expression of LOX-1 becomes stronger and stronger, and the expression of LOX-1 in the placenta of preeclampsia patient is better than it in the placenta of normal pregnancy woman who is in the same gestational week in different gestational weeks.(2) The result of RT-PCR displays that with gestational weeks increasing the expression of LOX-lmRNA on placenta becomes stronger and stronger, and the expression in preeclampsia woman's placenta is up-regulated to compare to it in normal pregnancy woman's placenta who is in the same gestational week to the preeclampsia one in different gestationa weeks.(3) The result of Westen-bolt shows that with gestational weeks increasing the expression of LOX-1 protein becomes stronger, and the expression of LOX-1 in preeclampsia pregnancy patient's placenta is up-regulated to compare to the expression in normal pregnancy woman who is in the same gestationa week in different gestational weeks.2. The expression of apoptosis related gene Caspase-3,Bax,Bcl-2 in normal and preeclarnpsia pregnancy woman's placenta.(1) Immunohistochemistry shows that Caspase-3 is expressed in syneytiotrophoblast and langhans cell of carly pregnancy villus, and it is expressed in synetiotrophoblast, langhans cell and vascular endotheliocyte cell of placenta in middle and advanced stage of normal and preeclampsia pregnancy. With gestational weeks increasing the expression of Caspase-3 becomes stronger, and the expression of Caspase-3 in preeclampsia pregnancy woman's placenta is better than the expression in normal pregnancy woman's placenta which is in the same gestational week in different gestational weeks. (2) The result of RT-PCR displays that with gestational weeks increasing the expression of Caspase-3, Bax mRNA is gradually reinforced,but the expression of Bcl-2 mRNA becomes weaker and Bax/Bcl-2 increases gradually.The expression of Caspase-3,Bax mRNA in preeclampsia pregnancy woman's placenta is stronger than in normal pregnancy woman's placenta in the same gestational weeks but the expression of Bcl-2 mRNA becomes weaker and Bax/Bcl-2 increases.(3) The result of Westen-blot shows that with gestational weeks increasing the expressions of Caspase-3 and Bax protein become stronger gradually,but the expression of Bcl-2 weaker and weaker,Bax/Bcl-2 increases gradually.The expression of Caspase-3 and Bax protein in the preeclampsia placenta is stronger than in the same gestational weeks normal placenta,and Bcl-2 protein'expression becomes weaker and Bax/Bcl-2 increase三,The regulation of oxLDL to the expressions of LOX-1of JEG-3 cell line and apoptosis related gene1. The expression of LOX-1 and Caspase-3 in JEG-3 cellsLOX-1 and Caspase-3 can both be tested the expression in cytolymph and cellular membrane of JEG-3 cells by cell immunity chemistry technology, and this provides the foundation of experiment for the study of regulation in vitro.2. oxLDL and CAR regulate the expression of LOX-1 in JEG-3 cells(1) Cell immunity chemistry shows that when JEG-3 cells are not been handled,the expression of LOX-1 is very slight. After 24 and 48 hours treatment of oxLDL the expression of LOX-1 in cells is phenomently enhanced though the appearance of cells does not marked change. In the same time we add CAR the inhibitor of LOX-1 in them,and the above affection is reversed..(2) Futhermore RT-PCR shows that adding oxLDL whose final concentration is 25 —100μg/ml can dose dependent and time dependent induce the expression of LOX-1mRNA. When the affecting concentratiom of oxLDL is 100μg／ml, we continue to raise after adding in 125μg/ml CAR the depressantia of LOX-1, the above mentioned affection is reversed, and it gets close to control group.(3) Westen-bolt also displays that oxLDL can dose depending and time depending induce the expression of LOX-1 protein, then adding in CAR the downer of LOX-1 can reverse the above affection.3. CAR the downer of oxLDL and LOX-1 regulates the expressions of apoptosis related genes Caspase-3, Bax and Bcl-2 in JEG-3 cells(1) Cell immunity chemistry shows that when JEG-3 has not been handled, the expression of Caspase-3 is very slight, then to continue cultivate after 24 and 48 hours adding in oxLDL, the expression of Caspase-3 is phenomently enhanced though the shape of cells dosen't change. At the basement of the affection of oxLDL we continue to raising after adding in CAR the downer of LOX-1, the expression of Caspase-3 is down regulated again, getting close to control group.(2) RT-PCR shows that oxLDL can dose-dependly and time-dependly induce expressions of Caspase-3 and Bax mRNA in JEG-3 cells. It also can restrain the expression of Bcl-2 mRNA. With increasing of the effectional concentration of oxLDL, Bax/Bcl-2 accretes. At the basement of the effection of oxLDL, adding in 125μg/ml CAR can reverse above effections.(3) Westen-bolt also shows that oxLDL can dose-dependy and time-dependy induce the expression of Caspase-3 and Bax protein, and down regulate the expression of Bcl-2 protein. At the basement of the effections of oxLDL adding in 125μg/ml CAR can reverse above mentioned affections.Conclusions(1) The level of oxLDL in eclampsis patient's blood plasma is phenomently higher than in normal. This prompts that oxLDL may participate in patho-physio-process of preeclampsia.(2)The study in vivo shows that LOX-1 is expressed in syneytiotrophoblast and langhans cell in carly pregnancy villus, and expressed in syneytiotrophoblast, langhans cell and vascular endothelial cells of normal and preeclampsia pregnancy woman's placenta in middle and advance stage. With gestational weeks increasing, the expression of LOX-1 becomes stronger, and the expression of LOX-1 in placenta of preeclampsia is stronger than in placenta of normal in the same gestational weeks. At the same time with gestational weeks increasing, the expressions of apoptosis related genes Caspase-3 and Bax in placenta are reinforced gradually,but the expression of Bcl-2 becomes taper,and Bax/Bcl-2 increase gradually. The expressions of Caspase-3 and Bax in preeclampsia pregnant woman's placenta is stronger than in normal in the same gestational weeks, but the expression of Bcl-2 becomes taper, and Bax/Bcl-2 increases. This prompts that LOX-1 the receptor of oxLDL may participate in invasion of preeclampsia, and it is related to the apoptosis of trophoblasts.(3)The research of chorioepithelioma cell line JEG-3 in vitro which has the characteristic of trophoblasts in early stage shows that oxLDL can dose- dependly and time-dependly induce the expression of LOX-1. At the same time adding in CAR the downer of LOX-1 can reverse above effections. Meanwhile oxLDL can dose-dependly and time-dependly induce the expressions of Caspase-3 and Bax, and restrain the expression of Bcl-2, and with the affectional concentration of oxLDL increasing, Bax/Bcl-2 steps up. Meanwhile adding in CAR the downer of LOX-1 can reverse above affections. This prompts that oxLDL can induce the expression of LOX-1 of trophoblasts, and may induce apoptosis of trophoblasts to raise. This supply the experimental basements for the explaination of pathogenesis of preeclampsia furthermore.