【作者】 吴媛媛；

【导师】 乔福元；

【作者基本信息】 华中科技大学 ， 妇产科学， 2009， 博士

【摘要】 第一部分瘦素在正常妊娠与子痫前期患者血清及胎盘组织中的表达的研究目的1．研究正常妊娠及子痫前期患者血清及胎盘组织中瘦素蛋白及mRNA表达差异，探讨瘦素与子痫前期的关系。2．检测TNF—α和脂联素在正常妊娠及子痫前期患者血清及胎盘组织中的表达，探讨它们与瘦素在子痫前期发病机制中的相关性。方法1．采用酶联免疫吸附法(ELISA)定量检测24例正常妊娠及44例子痫前期患者血清中瘦素、TNF—α及脂联素的表达水平。2．采用伊红—苏木素(HE)染色，光镜下观察正常妊娠胎盘与子痫前期患者胎盘结构的病理学改变，用免疫组织化学法半定量检测21例正常妊娠及55例子痫前期患者胎盘组织中瘦素、TNF—α及脂联素的蛋白表达。3．采用逆转录聚合酶链反应(reverse transcription polymerase chain reaction，RT-PCR)分别检测10例正常足月妊娠及20例子痫前期患者(其中轻度10例，重度10例)的胎盘组织中瘦素和脂联素的mRNA表达水平。结果1．轻度及重度子痫前期患者瘦素血清水平均明显高于正常孕妇，重度组脂联素水平明显低于正常组，而TNF-α水平明显高于正常组；且重度组中瘦素与TNF—α呈正相关，脂联素与TNF—α成负相关。2．妊娠时胎盘中瘦素主要分布于滋养细胞胞膜、胞浆及核膜上。子痫前期胎盘中瘦素蛋白表达较正常妊娠显著升高，而脂联素蛋白表达较正常妊娠明显降低，重度子痫前期组中瘦素与TNF—α表达呈正相关。3．正常妊娠胎盘中瘦素与脂联素mRNA的表达量分别为0.564±0.336、1.610±0.528，而子痫前期胎盘中瘦素的表达量明显增加，轻度组为0.778±0.175，重度组为1.174±0.556；与之相反地，脂联素的表达量明显下降，轻度组为1.363±0.475，重度组为0.636±0.218。且随着病情的加重，瘦素表达量的升高，脂联素表现出明显的下降趋势。结论瘦素表达增高与子痫前期密切相关，其他脂肪细胞因子如TNF—α和脂联素与瘦素之间的相互作用相互影响，可能是其参与子痫前期发病重要机制之一。第二部分瘦素在滋养细胞中的表达及对滋养细胞增殖和分泌功能的影响目的1．研究缺氧对人早孕绒毛滋养细胞株(TEV—1)瘦素及脂联素表达的影响，从细胞水平探讨瘦素及其他脂肪细胞因子与子痫前期的关系。2．研究缺氧、重组人leptin蛋白和TNF—α蛋白对滋养细胞株增殖及分泌功能的影响，深入研究其对滋养细胞生物学特性的调控作用。方法1．利用二氯化钴诱导滋养细胞化学缺氧，模拟体外缺氧环境，采用逆转录聚合酶链反应(reverse transcription polymerasechain reaction，RT-PCR)检测瘦素和脂联素的基因表达。2．利用二氯化钴、不同浓度的重组人leptin溶液和TNF—α溶液在体外分别干预滋养细胞生长，采用荧光定量PCR检测瘦素的基因表达，噻唑兰(Methl thiazolyltetrazolium，MTT)比色法测定缺氧、leptin和TNF—α对滋养细胞增殖的影响。结果1．随着缺氧时间的增加，滋养细胞瘦素mRNA表达逐渐增加，至12h达峰值，且与正常培养相比瘦素表达量显著升高，此后逐渐下降；随着缺氧时间的延长，滋养细胞脂联素mRNA表达逐渐减少，至12h时与正常培养相比脂联素表达量明显降低，此后逐渐下降。2．终浓度为5ng／ml瘦素和不同浓度的TNF—α均可显著促进滋养细胞leptinmRNA的表达，干预时间对此作用无明显影响。3．缺氧抑制了滋养细胞的增殖，且24h时抑制最为明显；瘦素干预滋养细胞24h后，且终浓度为10ng／ml的瘦素抑制作用最为明显；而TNF—α对滋养细胞增殖无明显影响。结论子痫前期中瘦素表达的增加与脂联素减少与滋养细胞缺氧是密切相关的。体外缺氧可诱导滋养细胞发生类似子痫前期的改变。另外，TNF—α可促进滋养细胞分泌瘦素，并且小剂量瘦素对滋养细胞瘦素的自分泌起到正反馈调节作用。第三部分瘦素、TNF—α和缺氧对滋养细胞侵袭能力影响的研究目的研究缺氧、重组人leptin蛋白和TNF—α蛋白对滋养细胞株侵袭功能的影响，进一步研究其对滋养细胞生物学特性的调控及其在子痫前期发病中的机制。方法利用二氯化钴、不同浓度的重组人leptin溶液和TNF—α溶液在体外分别干预滋养细胞生长，采用Transwell体外侵袭实验检测缺氧、leptin和TNF—α对滋养细胞侵袭能力的影响。结果缺氧抑制了滋养细胞的侵袭，且缺氧24h时抑制作用最显著；瘦素可促进滋养细胞的侵袭，且随着瘦素浓度的增高，滋养细胞的侵袭越显著；TNF—α也可促进滋养细胞侵袭，但其作用与浓度无关。结论体外培养时缺氧条件下滋养细胞的侵袭是受到抑制的，而瘦素及TNF—α均可促进滋养细胞侵袭，这是它们引起子痫前期发病的机制之一。第四部分瘦素在内皮细胞中的表达及对内皮细胞损伤的影响目的1．研究缺氧对人脐静脉内皮细胞株(VEC304)瘦素表达的影响，从细胞水平探讨瘦素在内皮细胞损伤方面与子痫前期的关系。2．研究缺氧、重组人leptin蛋白和TNF—α蛋白对内皮细胞株增殖及分泌功能的影响，深入研究胎盘源性毒性因子在内皮细胞损伤中的作用。方法1．利用二氯化钴诱导内皮细胞化学缺氧，模拟体外缺氧环境，采用逆转录聚合酶链反应(reverse transcription polymerasechain reaction，RT-PCR)检测瘦素基因的表达。2．利用二氯化钴、不同浓度的重组人leptin溶液和TNF—α溶液在体外分别干预内皮细胞生长，采用荧光定量PCR检测瘦素的基因表达，噻唑兰(Methl thiazolyltetrazolium，MTT)比色法测定缺氧、leptin和TNF—α对内皮细胞增殖的影响。结果1．随着缺氧时间的增加，内皮细胞瘦素mRNA表达量逐渐降低，但仍高于同期对照组，培养24h时却明显低于同期正常对照，此后逐渐下降。2．终浓度为5ng／ml瘦素和不同浓度的TNF—α均可显著促进内皮细胞leptinmRNA的表达，干预时间对此作用无明显影响。3．缺氧抑制了内皮细胞的增殖，且48h时抑制最为明显；瘦素和TNF—α对内皮细胞的增殖均无明显影响。结论瘦素和TNF—α可以作为“胎盘来源的毒性因子”参与了内皮细胞的损伤，它们是联系子痫前期胎盘灌注减少和全身血管内皮细胞广泛激活和(或)损伤这两部分病理改变的关键因素。更多还原

【Abstract】 PartⅠStudy on the expression of leptin in the placenta and serum of normalpregnancy and preeclampsiaObjective 1.To investigate the expression of leptin in placentas and sera obtained fromnormal pregnancy and preeclampsia, and the relationship between leptin and preeclampsia.2.To investigate the correlation of TNF-α,adiponectin and leptin on thepathogenic mechanism of preeclampsia by detecting the expressions of TNF-αandadiponectin in placentas and sera obtained from normal pregnancy and preeclampsia.Methods 1.In sera from 24 cases of normal pregnancy and 44 cases of preeclampsia,ELISA was used to detect the expressions of leptin, TNF-αand adiponectin.2.HE staining observed the pathology changes in placental tissues obtained fromnormal pregnancy and preeclampsia.Immuno-histochemistry was used to semiquantitativlydetect the protein expressions of leptin, TNF-αand adiponectin in placentas from 21 casesof normal pregnancy and 55 cases of preeclampsia.Result 1.The protein expression levels of leptin in sera of mild preeclampsia and severepreeclampsia were significantly higher than that of normal pregnancy.In sera of severepreeclampsia the level of adiponectin was significantly lower than that of control, but thelevel of TNF-αwas obviously higher than that of control; and there was obvious positivecorrelation between expression of leptin and TNF-α, whereas negative correlation betweenexpression of adiponectin and TNF-α.2.The positive staining of leptin were mainly located at cell membrane,cytoplasmaand nuclear membrane of trophoblast.Compared with normal group, the expression ofleptin protein in preeclampsia group was significantly higher, and the expression ofadiponectin protein in preeclampsia group was significantly lower.And in severe preeclampsia group there was obvious positive correlation between expression of leptin andTNF-α3.In placentas from normal pregnancy the mRNA expressions of leptin andadiponectin were respectively 0.564±0.336、1.610±0.528, and in placentas frompreeclampsia the mRNA expression of leptin increased obviously, that of mild group was0.778±0.175, and that of severe group was 1.174±0.556.Contrast with normal group, themRNA expression of adiponectin in placentas from preeclampsia decreased obviously, thatof mild group was 1.363±0.475, and that of severe group was 0.636±0.218.Along with thecourse of preeclampsia and the increasing of leptin expression, the expression ofadiponectin showed a obviouse downward trend.Conclusion The increase of leptin is closely correlated with preeclampsia.The influenceand interaction between leptin and other adipocytokines, such as TNF-αand adiponectin,may play an important role in the pathogenic mechanism of preeclampsia.PartⅡStudy on the expression of leptin in human first-trimester extravilloustrophoblast cell line and the effects of leptin on proliferation and secretion oftrophoblastObjective 1.To investigate the effect of CoCl₂ induced hypoxia on the expression of leptinand adiponectin of human first-trimester cxtravillous trophoblast cell line（TEV-1） in orderto evaluate the relationship on the cell level between leptin as well as other adipocytokinesand preeclampsia.2.To investigate the effects of CoCl₂ induced hypoxia, human recombinantleptin protein and human recombinant TNF-αprotein on proliferation and secretion oftrophoblast in order to further study the regulation of those on biological characteristics oftrophoblast.Methods 1.TEV-1 was cultured respectively in normal and hypoxia circumstance which induced by CoCl₂.Reverse transcription polymerase chain reaction （RT-PCR） was used todetect expressions of leptin and adiponectin mRNA of TEV-1.2.In vitro the culture of trophoblast was respectively interfere with CoCl₂,human recombinant leptin solution and TNF-αsolution in different concentrations.RealTime quantitative RT-PCR was used to detect expression of leptin mRNA of those TEV-1,and MTT assay was used to detect the effects of hypoxia, leptin and TNF-αonproliferation of trophoblast.Result 1.The levels of leptin mRNA in TEV-1 of hypoxia group increased gradually as theculturing time went on, and they went to the top at the 12-hour cultured time, and comparedwith normal culture group, the levels of leptin mRNA was significantly higher.But thelevels of adiponectin mRNA in TEV-1 of hypoxia group decreased gradually as theculturing time went on, and at the 12-hour cultured time compared with normal group, thatwas significantly lower, after 12-hour that decreased gradually.2.Leptin in 5ng/ml final concentration and TNF-αin different concentrationssignificantly promoted the expression of leptin mRNA of trophoblast, and culture time didnot influence those effects.3.Hypoxia induced by CoCl₂ inhibited the proliferation of trophoblast, and at the24-hour cultured time the inhibition was the most obviously.After the cultured oftrophoblast was interfere with leptin for 24 hours, the inhibition Of leptin in 10ng/ml finalconcentration was most apparent, and TNF-αhad no significant effect on the proliferationof trophoblast.Conclusion The increase of leptin expression and the decrease of adiponectin expression inpreeclampsia are closely correlated with hypoxia of trophoblast.Hypoxia in vitro caninduce the changes of trophoblast which are similar to the changes in preeclampsia.Inaddition, TNF-αpromote the secretion of leptin in trophoblast, and leptin in lowerconcentration play a positive feedback role on the autocrine of leptin in trophoblast. PartⅢStudy on the effects of leptin, TNF-αand hypoxia on invasiveness oftrophoblastObjective To investigate the effects of CoCl₂ induced hypoxia, human recombinant leptinprotein and human recombinant TNF-αprotein on invasiveness of trophoblast in order tofurther study the regulation of those on biological characteristics of trophoblast and the rolein pathogenic mechanism of preeclampsia.Methods In vitro the culture of trophoblast was respectively interfere with CoCl₂, humanrecombinant leptin solution and TNF-αsolution in different concentrations.The invasivecapability of trophoblast under the effects of hypoxia, leptin and TNF-αwas examined bytranswll invasive system.Result Hypoxia induced by CoCl₂ inhibited the invasion of trophoblast, and at the 24-hourcultured time the inhibition was the most obviously.Leptin promoted trophoblastinvasiveness in a concentration-dependent manner; and TNF-αpromoted also the invasionof trophoblast, but this effect had nothing to do with the concentration.Conclusion Under hypoxia condition in vitro the invasiveness of trophoblast is inhibited,whereas leptin and TNF-αmay promote trophoblast invasion, which is one of thepathogenic machanisms of preeclampsia.PartⅣStudy on the expression of leptin in human umbilical vein endothelial cellline and the effects of leptin on injury of endothelial cellObjective 1.To investigate the effect of CoCl₂ induced hypoxia on the expression of leptinof human umbilical vein endothelial cell line（VEC304） in order to evaluate the relationshipon the cell level between leptin and preeclampsia. 2.To investigate the effects of CoCl₂ induced hypoxia, human recombinantleptin protein and human recombinant TNF-αprotein on proliferation and secretion ofendothelial cell in order to further study the effects of toxia factors derived from placentaon endolial injury.Methods 1.VEC304 was cultured respectively in normal and hypoxia circumstance whichinduced by CoCl₂.Reverse transcription polymerase chain reaction （RT-PCR）was used todetect expressions of leptin mRNA of VEC304.2.In vitro the culture of VEC304 was respectively interfere with CoCl₂, humanrecombinant leptin solution and TNF-αsolution in different concentrations.Real Timequantitative RT-PCR was used to detect expression of leptin mRNA of those cells, andMTT assay was used to detect the effects of hypoxia, leptin and TNF-αon proliferation ofVEC304.Result 1.The levels of leptin mRNA in VEC304 of hypoxia group decreased gradually asthe culturing time went on, but compared with control group,the levels of leptin mRNAwere significantly higher, and at the 24-hour cultured time compared with control group,that was significantly lower, after 24-hour that decreased gradually.2.Leptin in 5ng/ml final concentration and TNF-αin different concentrationssignificantly promoted the expression of leptin mRNA of VEC304, and culture time did notinfluence those effects.3.Hypoxia induced by CoCl₂ inhibited the proliferation of VEC304, and at the48-hour cultured time the inhibition was the most obviously.Leptin and TNF-αhad nosignificant effects on the proliferation of VEC304.Conclusion Served as toxic factors derived from placenta, leptin and TNF-αinvolve inendothelial injury, and they are the key factors which are linked to the reduce of placentalperfusion and activation or iniury of systemic vascular endothelial cell in preeclampsia.更多还原