【作者】 孟涛；

【导师】 尚涛；

【作者基本信息】 中国医科大学 ， 妇产科学， 2007， 博士

【摘要】 oxLDL和其受体LOX-1诱导滋养细胞凋亡及与子痫前期发病关系的研究目的子痫前期是孕产期严重的并发症，是导致孕产妇和新生儿病死率的重要原因，关于子痫前期的病因和发病机理研究一直是产科的重要课题。目前普遍认为，胎盘浅着床和血管重铸障碍是子痫前期发病的中心环节，而这两者都与细胞滋养细胞浸润能力降低有关。妊娠期若滋养细胞凋亡过度，就可导致这部分细胞不能向子宫蜕膜浸润，或浸润细胞数目减少及对螺旋小动脉内皮细胞替代减少，导致“浅着床”和血管重铸障碍，继而导致子痫前期的发生。大量心血管病方面的研究证实，氧化修饰低密度脂蛋白(oxidized low densitylipoprotein，oxLDL)oxLDL是引起血管内皮细胞、血管平滑肌细胞、巨噬细胞损伤和凋亡的重要因子，并且oxLDL可通过多种途径诱导细胞的凋亡。而oxLDL要通过其受体介导而发挥作用。血凝素样氧化低密度脂蛋白受体-1(Lectin likeoxidized low density lipoprotein receptor-1，LOX-1)是近年发现的oxLDL受体，LOX-1介导内皮细胞对oxLDL的摄取和代谢，在血管平滑肌细胞、巨噬细胞、成纤维细胞和血小板均有表达，在胎盘中也有表达。子痫前期常不同程度的存在代谢综合症的表现，其中血脂代谢紊乱在子痫前期中表现比较明显，之前虽已有一些研究报道，但均局限于血浆脂蛋白水平的检测，更深入的研究报道极少。在子痫前期oxLDL是否为疾病发生发展的重要因子，oxLDL是否通过诱导LOX-1的表达而在滋养细胞凋亡和子痫前期发病中发挥作用，目前尚无研究报道。研究方法(1)以ELISA方法检测正常孕妇和子痫前期患者血浆oxLDL的变化并进行差异比较。(2)以免疫组织化学和Westen-blot、RT-PCR方法检测不同孕周正常孕妇和子痫前期患者胎盘滋养细胞LOX-1表达的时空变化；Westen-blot、RT-PCR方法检测不同孕周和子痫前期患者胎盘滋养细胞凋亡相关基因Bax、Bcl-2及Caspase-3表达的变化。(3)应用具有滋养细胞特性的绒癌细胞株(JEG-3)在体外进行细胞培养实验，用细胞免疫化学、Westen-blot、RT-PCR方法检测oxLDL对滋养细胞LOX-1和凋亡相关基因表达的调控，以LOX-1阻断剂角叉菜胶来进一步检测阻断前后的变化。结果一、血浆氧化修饰低密度脂蛋白水平在子痫前期患者较正常孕妇有显著升高，两组比较有显著差异。二、正常妊娠与子癎前期胎盘组织中LOX-1和凋亡相关基因表达的时空变化1．LOX-1在正常妊娠与子癎前期胎盘组织中的表达。(1)免疫组织化学显示LOX-1在早期妊娠绒毛组织中表达于合体滋养细胞及细胞滋养细胞；在中，晚期正常妊娠与子癎前期胎盘组织中均表达在合体滋养细胞、细胞滋养细胞及血管内皮细胞。随着妊娠周数增加，LOX-1表达逐渐增强，不同孕周子癎前期胎盘组织LOX-1表达均强于同孕周正常妊娠胎盘组织。(2)RT-PCR结果显示，随着孕周增加，胎盘组织LOX-1mRNA表达逐渐增强，不同孕周子癎前期胎盘组织中LOX-1mRNA的表达较同孕周正常妊娠胎盘组织上调。(3)Westen-blot结果显示，随着孕周增加，胎盘组织LOX-1蛋白表达逐渐增强，不同孕周子癎前期胎盘组织中LOX-1表达较同孕周正常妊娠胎盘组织表达上调。2．凋亡相关基因Caspase-3、Bax、Bcl-2在正常妊娠与子癎前期胎盘组织中的表达(1)免疫组织化学显示Caspase-3在早期妊娠绒毛组织中表达于合体滋养细胞及细胞滋养细胞；在中、晚期正常妊娠和子癎前期胎盘组织中均表达在合体滋养细胞、细胞滋养细胞及血管内皮细胞。随着妊娠周数增加，Caspase-3表达逐渐增强，不同孕周子癎前期胎盘组织Caspase-3表达均强于同孕周正常妊娠胎盘组织。(2)RT-PCR结果显示，随着孕周增加，Caspase-3、Bax mRNA表达逐渐增强，而Bcl-2 mRNA表达逐渐减弱，Bax／Bcl-2逐渐增大。子癎前期胎盘组织Caspase-3、Bax mRNA表达较同孕周正常妊娠胎盘表达增强，而Bcl-2 mRNA表达减弱。(3)Westen-blot结果也显示，随着孕周增加，Caspase-3、Bax蛋白表达逐渐增强，而Bcl-2蛋白表达逐渐减弱，Bax／Bcl-2逐渐增大。子癎前期胎盘组织Caspase-3、Bax蛋白表达较同孕周正常妊娠胎盘组织表达增强，Bcl-2蛋白表达减弱。三、oxLDL对JEG-3细胞系LOX-1和凋亡相关基因表达的调控1．LOX-1和Caspase-3在JEG-3细胞中的表达通过细胞免疫化学技术检测到LOX-1和Caspase-3在JEG-3细胞浆及细胞膜中均有表达，为体外调控研究提供实验基础。2．oxLDL和角叉菜胶对JEG-3细胞LOX-1表达的调控(1)细胞免疫化学显示，JEG-3细胞未经oxLDL处理时，仅有微弱的LOX-1表达，经oxLDL处理24小时和48小时后，细胞外观未见明显变化，但细胞的LOX-1表达显著增强。同时加入LOX-1抑制剂角叉菜胶后，又逆转上述作用。(2)RT-PCR进一步显示，oxLDL可剂量依赖性和时间依赖性的诱导JEG-3细胞LOX-1 mRNA表达。当加入LOX-1抑制剂角叉菜胶后，可逆转上述作用。(3)Westen-blot也显示，oxLDL可剂量依赖性和时间依赖性诱导JEG-3细胞LOX-1蛋白表达，加入LOX-1抑制剂角叉菜胶则LOX-1表达又下调。3．oxLDL和LOX-1抑制剂角叉菜胶对JEG-3细胞凋亡相关基因Caspase-3、Bax、Bcl-2表达的调控(1)细胞免疫化学显示，JEG-3细胞未经oxLDL处理时，Caspase-3表达较微弱，经oxLDL处理24小时和48小时后，细胞外观未见明显变化，但细胞Caspase-3表达显著增强。在oxLDL作用基础上加入LOX-1抑制剂角叉菜胶继续培养后，又可逆转上述作用。(2)RT-PCR显示，oxLDL可剂量依赖性和时间依赖性地诱导JEG-3细胞Caspase-3、Bax mRNA的表达，抑制Bcl-2 mRNA的表达，随着oxLDL作用浓度和作用时间的增加，Bax／Bcl-2增大。在oxLDL作用基础上同时加入LOX-1抑制剂角叉菜胶后，可逆转上述作用。(3)Westen-bolt也同样显示oxLDL可剂量依赖性和时间依赖性地诱导JEG-3细胞Caspase-3、Bax蛋白的表达，下调Bcl-2蛋白的表达，随着oxLDL作用浓度和作用时间的增加，Bax／Bcl-2增大。在oxLDL作用基础上同时加入LOX-1抑制剂角叉菜胶后，又逆转上述作用。结论(1)子痫前期患者血浆oxLDL水平显著高于正常孕妇，提示oxLDL可能参与了子痫前期的病理生理过程。(2)在体研究显示，LOX-1在早期妊娠绒毛组织中表达于合体滋养细胞及细胞滋养细胞；在中、晚期正常妊娠与子痫前期胎盘组织中均表达在合体滋养细胞、细胞滋养细胞及血管内皮细胞。随着妊娠周数增加，LOX-1表达逐渐增强，子瘸前期胎盘LOX-1表达强于相同孕周正常妊娠胎盘组织。同时随着孕周增加，胎盘组织中凋亡相关基因Caspase-3、Bax表达逐渐增强，而Bcl-2表达逐渐减弱，Bax／Bcl-2逐渐增大。子癎前期胎盘组织Caspase-3、Bax表达均比同孕周正常妊娠胎盘组织表达增强，Bcl-2表达减弱，Bax／Bcl-2增大。提示oxLDL受体LOX-1可能参与了子痫前期的发病，并与滋养细胞凋亡有关。(3)以具有早期滋养细胞特性的绒癌细胞株JEG-3细胞进行体外培养研究显示，oxLDL可剂量依赖性和时间依赖性的诱导JEG-3细胞LOX-1的表达。同时加入LOX-1抑制剂角叉菜胶后，又可逆转上述作用。结果还显示oxLDL可剂量依赖性和时间依赖性地诱导JEG-3细胞Caspase-3、Bax的表达，抑制Bcl-2的表达，随着oxLDL作用浓度和作用时间的增加，Bax／Bcl-2升高；同时加入LOX-1抑制剂角叉菜胶后，又可逆转上述作用。提示oxLDL可诱导滋养细胞LOX-1的表达，并可能导致滋养细胞凋亡增加，为进一步解释子痫前期的发病机制提供了实验基础。更多还原

【Abstract】 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.更多还原