The Effects Of Leptin On Invasiveness Of Cytotrophoblasts And Injury Of Endothelial Cell And The Relationship Between These Effects And Pre-eclampia

Partâ… Study 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.Partâ…¡Study 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. Partâ…¢Study 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.Partâ…£Study 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.