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。
PartⅠStudy on the expression of leptin in the placenta and serum of normalpregnancy and preeclampsia
Objective 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-α
3.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 oftrophoblast
Objective 1.To investigate the effect of CoCl_2 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 CoCl_2 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 CoCl_2.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 CoCl_2,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 CoCl_2 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 oftrophoblast
Objective To investigate the effects of CoCl_2 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 CoCl_2, 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 CoCl_2 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 cell
Objective 1.To investigate the effect of CoCl_2 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 CoCl_2 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 CoCl_2.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 CoCl_2, 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 CoCl_2 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.
引文
[1] 林其德．妊娠期高血压综合征病因学研究进展与展望．中华妇产科杂志，2003，38(8)：471-473．
[2] Cunningham FG, Gant NF, Leveno KJ, et al. Willianms Obstetrics, 21th ed. New York: McGraw Hill, 2001: 585-588.
[3] Aizawa-Abe M, Ogawa Y, Masuzaki H, et al. Pathophysiological role of leptin in obesity-related hypertension. J Clin Invest, 2000, 105(9): 1243-1252.
[4] Shek EW, Brands MV, Hall JE. Chronic leptin infusion increases arterral pressure. Hypertension, 1998, 31: 409-414.
[5] Dotsch J, Nusken KD, Knerr I, et al. Leptin and neuropeptids Y gene expression in human placenta: ontogeny and evidence for similarities to hypothalamic regulation. J Clin Endocrinol Metab, 1999, 84: 2755-2758.
[6] Solomon CG, Seely EW. Brief review: hypertension in pregnancy: a manifestation of insulin resistance syndrome. Hypertension, 2001, 37(2): 232-239.
[7] Laivuori H, Kaaja R, Koistinen H, et al. Leptin during and after preeclampsia or normal pregnangcy: its relation to serum insulin and insulin sentivity. Metabolism, 2000, 49(2): 259-263.
[8] Fujita Y, Murakami M, Ogawa Y, et al. Leptininhibits stress-induced apoptosis of T lymphocytes. Clin Exp Immunol, 2002, 128(1): 21-26.
[9] Bajoria R, Sovranna SR, Ward BS, et al. Prospective function of placental leptin at maternal-fetal interface. Placenta, 2002, 23(2-3):103-115.
[10] Gonzalez RR, Devoto L, Campana A, et al. Effects of leptin, interleukin-1 alpha,interleukin-6, and transforming growth factor-beta on markers of trophoblast invasive phenotype:integrins and metalloproteinases. Endocrine, 2001,15(2): 157-64.
[11] Gabay C, Dreyer M, Pellegrinelli N, et al. Leptin directly induces the secretion of interleukin in 1 receptor antagonist in human monocytes. J Clin Endocrinol Metab, 2001,86(2): 783-791. .
[12] Grunfeld C, Zhao C, Fuller J, et al. Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. J Clin Invest, 1996, 97: 2152-2157.
[13] Jose L, Bartha, Raquel RC, et al. The relationship between leptin and inflammatory cytokines in women with preeclampsia. BJOB, 2001,108: 1272-1276.
[14]Bmun J M, Lihn A S, Ventich C, et al. Regulation of adiponectin by adipose tissue-derived cytekines: in vivo and in vitro investigations in humans. Am J Physiol Endocrinal Metab, 2003, 285(3): E527-E533.
[15] Zhang Y, Proence R, Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue. Nature, 1994, 372: 425.
[16]屈会起,邱明才,村珊.瘦素与肾脏.国外医学内科学分册, 2000, 27(3):111.
[17] Myers MG J.Leptin receptor signaling and the regulation of mammalian physiology.Recent Prog Horm Res,2004,59:287-304.
[18]Cauzac M,Czuba D,Girard J,et al.Transduction of leptin growth signals in placental cells is independent of JAK-STAT activation. Placenta, 2003 ,24(4):78-384.
[19] Cohen P, Zhao C, Cai X, et al. Selective deletion of leptin receptor in neurons leads to obesity. J Clin Invest, 2001,108(8):1113-1121.
[20] Gabay C, Dreyer M, Pellegrinelli N, et al. Leptin directly induces the secretion of interleukin in 1 receptor antagonist in human monocytes. J Clin Endocrinol Metab,2001, 86(2): 783-791.
[21] Grunfeld C, Zhao C, Fuller J, et al. Endotoxin and cytokines induce expression of leptin , the ob gene product, in hamsters. J Clin Invest, 1996, 97: 2152-2157.
[22]Arnalich F, Lopez J, Codoceo R, et al. Relationship of plasma leptin to plasma cytokines and human survivalin sepsis and septic shoch. J Infect Dis, 1999, 180:908-911.
[23]Zumbach MS, Boehme MW, Wahl P, et al. Tumor necrosis factor increases leptin levels in humans. J Clin Endocrinol Metab, 1997, 82: 4080-4082.
[24] Clapp JF, Kiess W. Effects of pregnancy and exercise on concentrations of the metabolic markers tumor necrosis factor and leptin. Am J Obstet Gynecol, 2000, 182:300-306.
[25]Melczer Z, Banhidv F, Csomor S, et al. Acta Obstet Gynecol Scand, 2003 May, 82(5):432-438.
[26]Pajvani UB, Du X, Combs TP, et al. Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin, Implications for metabolic regulation and bioactivity. J Biol Chem, 2003, 278: 9073-9085.
[27]Matsuda M, Shimonura I, Sata M, et al. Role of adiponectin in preventing vascular stenosis: the missing link of adipo-vascular axis. J Biol Chem, 2002, 277:37487-37491.
[28] Braun JM, Lihn AS, Ventich C,et al. Regulation of adiponectin by adipose tissue-derived cytekines:in vivo and in vitro investigations in humans. Am J Physiol Endocrinal Metab, 2003, 285(3): E527-E533.
[29] D'Anna R, Baviera G, Corrado F, et al. Plasma adiponectin concentration in early pregnancy and subsequent risk of hypertensive disorders. Obstet Gynecol, 2005 Aug,106 (2): 340-4.
[30] Kern PA, DiGregotio GB, Lu T, et al. Adiponectin expression from human adipose tissue relation to obesity,insulin resistance, and tumor necrosis factor-α expression.Diabetes, 2003, 52:1779-1785,
[31]Maeda N, Shimomura I, Kishida K, et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med, 2002, 8: 731-737.
[32] Koket F, Adamezak M, Wiecek A, et al. Does leptin play a role in the pathogenesis of essential hypertension? Kindney Blood Press Res, 1999,22(3): 154.
[33] Suther PM, Locher R, Vetter W, et al. Is there a role for the ob gene product leptin in essential hypertension. Am J Hypertens, 1998, 11(11pt1): 1305.
[34] Chan TF, Su JH, Chung YF, et al. Amniotic fluid and maternal serum leptin levels in pregnant women who subsequently develop preeclampsia. Eu J of Obstet & Gynecol and trprod bio, 2003,108: 50-53.
[35] McCarthy JF, Misra DN, Roberts JM. Maternal plasma leptin is increased in preeclampsia and positively correlates with fetal cord concentration. Am J Obstet Gynecol, 1999,180(3 pt 1): 731.
[36] Reimer T, Koczan D, Gerber B. Microarray analysis of differentially expressed genes in placental tissue of preeclampsia: Up-regulation of obesity-related genes. Mol Hum Reprod, 2002, 8: 674-680.
[37] Odegard RA, Vatten ST, Nilsen ST. Umbilical cord plasma leptin is increased in preeclampsia. Am J Obstet Gynecol, 2002,186(3): 427-435.
[38] Clapp JF, Kiess W. Effects of pregnancy and exercise on concentrations of the metabolic markers tumor necrosis factor and leptin. Am J Obstet Gynecol, 2000, 182:300- 306.
[39] Melczer Z, Banhidv F, Csomor S, et al. Acta Obstet Gynecol Scand, 2003 May, 82(5):432-438.
[40] Jose L, Bartha, Raquel RC, et al. The relationship between leptin and inflammatory cytokines in women with preeclampsia. BJOB, 2001,108: 1272-1276.
[41] Bmun JM, Lihn AS, Ventich C,et al. Regulation of adiponectin by adipose tissue-derived cytekines: in vivo and in vitro investigations in humans. Am J Physiol Endocrinal Metab, 2003, 285(3): E527-E533.
[42] Naruse K, Yamasaki M, Umekage H, et al. Peripheral blood concentrations of adiponectin, an adipocyte-specific plasma a protein, in normal pregnancy and preeclampsia. J Reprod Immunol, 2005, 65: 65-75.
[43] Shimabukuro M, Higa N, Asahi T, et al. Hypoadiponectinemia is closely linked to endothelial dysfunction in human. J Clin Endocrinol Metab, 2003, 88: 3236-3240.
[44] Ouchi N, Ohishi M, Kihara S, et al.Association of hypoadiponectinemia with impaired vasoreactivity. Hypertension, 2003, 42: 231-234.
[45] Maeda N, Shimomura I, Kishida K, et al. Diet-induced insulin resistance in mice lacking adiponectin/Acrp30. Nat Med, 2002, 8: 731-737.
[46] Seely EW, Solomon CG. Insulin resistance and its potential role in pregnancy-induced hypertension. J Clin Endocrinol Metab, 2003, 88: 2393-2398.
[47] Meissner U, Ostreicher I, Allabauer I. Synergistic effects of hypoxia and insulin are regulated by different transerpitional elements of the human leptin promotor. Biochem Biophys Res Commun, 2003, 303: 707-712.
[48] Kaaja R, Laivuori H, Pulkki P, et al. Is there any link between insulin resistance and inflammation in established preeclampsia. Metabolism, 2004, 53: 1433-1435.
[49] Sibai BM. Preeclampsia: an inflammatory syndrome. Am J Obstet Gynecol, 2004,191:1061-1062.
[50] Shimabukuro M, Higa N, Asahi T, et al. Hypoadiponectinemia is closely linked to endothelial dysfunction in human. J Clin Endocrinol Metab, 2003, 88: 3236-3240.
[51] Camions JE, Nogueiras R, Gallego R, et al. Expression and regulation of adiponectin and receptor in human and rat placenta. J Clin Endocrinol Metab, 2005, 90(7):4276-4286.
[1] Feng Q, Liu Y, Liu K, et al. Expression of urokinase, plasminogen activator inhibitors and urokinase receptor in pregnancy rhesus monkey uterus during early placentation.. Placenta, 2000, 21(2-3): 184-193.
[2] Haigh T, Chen C, Jones CJ, Aplin JD. Studies of mesenchymal cells from 1st trimester human placenta: Expression of cytokeratin outside the trophoblast lineage. Placenta 1999, 20: 615-25.
[3] Maldonado-Estrada J, Menu E, Roques P, Barre-Sinoussi F, Chaouat G. Evaluation of cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry. J Immunol Methods. 2004, 286: 21-34.
[4] Proll J, Blaschitz A, Hutter H, Dohr G. First trimester human endovascular trophoblast cells express both HLA-C and HLA-G. Am J Reprod Immunol 1999, 42: 30-6.
[5] McMaster MT, Librach CL, Zhou Y, et al. Human placentalHLA-G expression is restricted to differentiated cytotrophoblasts. J Immunol 1995, 154: 3771-8.
[6] Hirano T, Higuchi T, Ueda M, et al. CD9 is expressed in extravillous trophoblasts in association with integrin alpha3 and integrin alpha5. Mol Hum Reprod 1999, 5: 162-7.
[7] De Marco CS, Caniggia I. Mechanisms of oxygen sensing in human trophoblast cells. Placenta, 2002, 23: 58.
[8] Zhu H, Bunn HE. Oxygen sensing and signaling impact on the regulation of physiologically important genes. Res physiol, 1999, 115: 239-247.
[9] 吴维光，陈亚琼，王玉萍等．不同缺氧方法诱导BeWo细胞缺氧诱导因子1表达的实验研究．武警医学院学报．2005，14(2)：86-89．
[10] O'Neil JS, Green AE, Edwards DE, et al. Regulation of leptin receptor in baboon pregnancy: effect of advancing gestation and fetectomy. J Clin Endocrinol Metab, 2001, 86(6): 579-583.
[11] Linnemann K, Malek A, Schneider H, et al. Physiological and pathological regulation of feto/placento/maternal leptin expression. Biochem Soc Tran, 2001,29(pt2): 86-90.
[12] Sattar N, Greer IA, Pirwani I, et al. Leptin levels in pregnancy: marker for fat accumulation and mobilization. Acta Obstet Gynecol Scand, 1998, 77: 278-283.
[13] Grosfeld A, Turban S, Andre J, et al. Transcriptional effect of hypoxia on placental leptin. FEBS Lett, 2001, 502(3): 122-126.
[14] Grosfels A, Andre J, Mouzon SH, et al. Hypoxia-inducible factor-1 transactivates the human leptin gene promoter. J B tol Chem, 2002, 277(45): 42953-42957.
[15] Sagawa N, Mori T, Masuzaki H, et al. leptin production by hydatidiform mole. Lancet, 1997, 350: 1518-1523.
[16] Camions JE, Nogueiras R, Gallego R, et al. Expression and regulation of adiponectin and receptor in human and rat placenta. J Clin Endocrinol Metab, 2005, 90(7): 4276-4286.
[17] Macara L, Kingdom JC, Kaufmann P, et al. Structural analysis of placental terminal villi from growth restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta, 1996, 17(1): 37-48.
[18] Genbacev O, Zhou Y, Ludlow JW, et al. Regulation of human placental development by oxygen tension. Science, 1997, 277(5332): 1669-1672.
[19] Kilburn BA, Wang J, Duniec Dmuchkowski ZM, et al. Extracellularmatrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line. Biol Reprod, 2000, 62(3): 739-747.
[20] Esterman A, Greco MA, Mitani Y, et al. The effect of hypoxia on human trophoblast in culture: morphology, glucose transport and metabolism. Placenta, 1997, 18(2-3): 129-136.
[21] Genbacev O,DiFederico E,Mc MasterM,et al.Invasive cytotrophoblast apoptosis in preeclampsia.Hum Reprod,1999,14[Supp 12]: 59-66.
[22] 胡蓉，李笑天，左极等．缺氧促进妊娠早期细胞滋养细胞凋亡机理研究．中华围产医学杂志，2006，9(4)：225-228．
[23] Aschkenazi S, St raszewski S, Verwer KM, et al. Differential regulation and function of the Fas/ Fas ligand system in human trophoblast cells. Biol Reprod, 2002, 66: 1853-1861.
[24] Nechushtan A, Smith CL, Lamensdorf I, et al. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol, 2001, 153: 1265-1276.
[25] Morin D, Pires F, Plin C, et al. Role of the permeability transition pore in cytochrome C release from mitochondria during ischemia reperfusion in rat liver.Biochem Pharmacol, 2004, 68: 2065-2073.
[26] Finck BN, Johnson RW. Tumor necrosis factor (TNF)-alpha induces leptin production through the p55 TNF receptor. Am J Physiol Regul Integr Comp Physiol, 2000, 278(2): 537-543.
[1] Bischof P, Meisser A, Campana A. Paracrine and autocrine regulators of trophoblast invasion-a review. Placenta, 2000, 60: S55-S60.
[2] Bischof P, Meisser A, Campana A. Mechanisms of endometrial control of trophoblast invasion. J Reprod Fertil, 2000, (Suppl55): 65-71.
[3] 郭丽娜，崔澄，刘复权．影响滋养细胞侵蚀能力的因素及其与妊高征的关系．国外医学妇幼保健分册，2003，14(1)：34-36．
[4] Bischof P. Endocrine, paracrine and autocrine regulation of trophoblastic metalloproteinases. Early Pregnancy, 2001, 5(1):30-31.
[5] Caniggla I, Mostachfi H, Winter J, et al. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFβ3. J Clin Invest, 2000, 105: 577-587.
[6] Morrish DW, Dakour J, Li H. Functional regulation of human trophoblast differentiation. J Reprod Immunol, 1998, 39(1-2): 179-195.
[7] Staff AC, Ranheim T, Henriksen T, et al. 8-Iso-prostaglandin F (2α) reduces trophoblast invasion and matrix metalloproteinase activity. Hypertension,2000,35(6): 1307-1313.
[8] Gonzalez RR, Devoto L, Campana A, et al. Effects of leptin, interleukin-1 alpha, interleukin-6, and transforming growth factor-beta on markers of trophoblast invasive phenotype: integrins and metalloproteinases. Endocrine, 2001, 15(2): 157-164.
[9] Henson MC, Castracane VD. Leptin: roles and regulation in primate pregnancy. Semin Reprod Med, 2002, 20(2): 113-122.
[10] Bjorbaek C, KahnB B. Leptin signaling in the central nervous system and the periphery. Recent Prog Horm Res, 2004, 59: 305-336.
[11] Myers MG J. Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog Horm Res, 2004, 59: 287-304.
[12] Cauzac M, Czuba D, Girard J, et al. Transduction of leptin growth signals in placental cells is independent of JAK-STAT activation. Placenta, 2003, 24(4): 378-384.
[13] Hardwick J, Brink VDG, Oerhaus G, et al. Leptin is a growth factor for colonic epithelial cells. Gastroenterology, 2001, 121(79-90): 2355-60.
[14] Tang M, Potter JJ, Mezey E. Leptin enhances the effects of transforming growth factor beta in increasing type 1 collagen formation. Biochem Biophys Res Commun, 2002, 297(4): 906-911.
[15] Domnguez F, Pellicer A, Smon C. Paracrine dialogue in implanation. Mol Cell Endocrinol, 2002, 186(2): 175-181.
[16] Kupferminc MJ, Peaceman AM, Wigton TR, et al. Tumor necrosis factor α is elevated in plasma and amniotic fluid of patients with severe preeclampsia. Am J Obstet Gynecol, 1994, 170: 1752-1759.
[17] Reister F, Frank HG; Kingdom JC, et al.Macrophage-induced apoptosis limits endovascular trophoblast invasion in the uterine wall of preeclamptic women. Lab Invest, 2001, 81(8): 1143-1152.
[18] Nagamatsu T, Fujii T, Kusumi M, et al. Cytotrophoblasts up-regulate soluble fms-like tyrosine kinase-1 expression ender reduced oxygen: an implication for the placental vascular development and the pathophysiology of preeclampsia. Endocrinology, 2004,145(11):4838-4845.
[19] 李云，胡继芬．可溶性血管内皮生长因子受体1与子痫前期研究进展．国外医学妇产科学分册，2006，33(5)：346-349．
[1] Crocker IP, Kenny LC, Thornton WA, et al. Excessive stimulation of poly (ADP-ribosyl) ation contributes to endothelial dysfunction in preeclampsia. Br J Phamacol, 2005, 144(6): 772-780.
[2] Zhao S, Zhang Y, Gu Y, et al. Heme oxygenase-1 mediates up-regulation of adhesion molecule expression induced by peroxynitrite in endothelial cells. J Soc Gynecol Investig, 2004, 11(7): 465-471.
[3] Zhang Y, Zhao S, Gu Y, et al. Effects of peroxynitrite and superoxide radicals on endothelial monolayer permeability: potential role of peroxynitrite in preeclampsia. J Soc Gynecol Investig, 2005, 12(8): 586-592.
[4] Cooke CL, Davidge ST. Peroxynitrite increases iNOS through NF-kappaB and decreases prostacyclin synthase in endothelial cell. Am J Physiol Cell Physiol, 2002, 282(2): 395-402.
[5] Narumiya H, Zhang Y, Fernandez-Patron C, et al. Matrix metalloproteinase-2 is elevated in the plasma of women with preeclampsia. Hypertens Pregnancy, 2001, 20(2): 185-194.
[6] Merchant SJ, Narumiya H, Zhang Y, et al. The effects of preeclampsia and oxygen environment on endothelial release of matrix metalloproteinase-2. Hypertens Pregnancy, 2004, 23(1): 47-60.
[7] Maynard SE, Min JY, Merchan J,et al. Excess placental soluble fms-like tyrosine kinase 1(sFLT-1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest, 2003, 111(5): 649-658.
[8] Wang Y, David FL, Gu Y, et al. Placental trophoblast-derived factors diminish endothelial barrier function. J Endocri Metab, 2004, 89(5): 2421-2428.
[9] 李东至，林其德，林建华．胎盘绒毛缺氧与血管内皮细胞损伤关系的实验研究．中国病理生理杂志，2004，20(3)：411、416．
[10] 郭钰珍，陈汉平．二氯化钴化学缺氧对人脐静脉内皮细胞增殖和血红素氧合酶表达的影响及其临床意义．现代妇产科进展，2006，15(3)：212-214．
[11] Teppa RJ, Ness RB, Crombleholme WR, et al. Free leptin is increased in normal pregnancy and further increased in preeclampsia. Metabolism, 2000, 49(6): 1043-1048.
[12] Wu G, Brouckaert P, Olivecrona T. Rapid downregulation of adipose tissue lipoprotein lipase activity on food deprivation: evidence that TNF-is involved. Am J Physiol Endocrinol Metab, 2004, 286(5): 711 -717.
[13] Ouchi N, Kihara S, Arita Y, et al. Adiponectin, anadipocyte derived plasma protein, inhibits endothelial NF kappa B signaling through a cAMP dependent pathway. Circulation, 2000, 102(3): 1296-1301.
[14] Yokota T, Oritani K, Takahashi I, et al. Adiponectin, anewmember of the flamily of soluble defense collagens, negatively regulates the growth of myekomonocytic progenitors and the functions of macrophages. Blood, 2000, 96(5): 1723-1732.
[15] Matsuda M, Shmomura I, Sata M, et al. Role of adiponection in preventing vascular stenosis, The missing link of adipo-vascular axis. J Biol Chen, 2002, 277(4): 37487-37491.
[16] Ouchin, Kihara S, Arita Y, et al. Adipocyte derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression human monocyte derived macrophages. Circulation, 2001, 103(3): 1057-1063.
[1] Yang YS,Song HD,Li RY, et al.The gene expression profiling of human visceral adipose tissue and its secretory function.Biochem Biophys Res Commun,2003,300:839-846.
[2] Grosfeld A,Andre J,Hauguel-De Morzon S,et al.Hypoxia-inducible factor 1 (HIF-1) transactivates the human leptin gene promoter.J Biol Chem,2002,4.
[3] Iwagaki S, Yokoyama Y, Tang L, et al.Augrnentation of leptin and hypoxia-inducible factor 1alpha mRNAs in the pre-eclamptic placenta.Gynecol Endocrinol,2004, 18(5): 263-8.
[4] Melczer Z,Banhidy F, Csomor S,et al.Influence of leptin and the TNF system on insulin resistance in pregnancy and their effect on anthropometric parameters of newboms.Acta Obstet Gynecol Scand,2003,82:432-438.
[5] D'Anna R,Baviera G, Corrado F,et al.Plasma adiponectin concentration in early pregnancy and subsequent risk of hypertensive disorders.Obstet Gynecol,2005 Aug, 106 (2):340-4.
[6] Hendler I,Blackwell SC,Mehta SH, et al.The levels of leptin,adiponectin,and resistin in normal weight,overweight,and obese pregnancy women with and without preeclampsia.Am J Obstet Gynecol,2005 Sep,193(3):979-83.
[7] Camions JE,Nogueiras R,Gallego R,et al.Expression and regulation of adiponectin and receptor in human and rat placenta.J Clin Endocrinol Metab,2005,Jul,90(7):4276-86.
[8] Haugen F,Ranheim T,Harsem NK,et al.Increased plasma levels of adipokines in preeclampsia:relationship to placenta and adipose tissue gene expression.Am J Physiol Endocrinol Metab,2005 sep 6.
[9] Park KG,Park KS,Kim MJ,et al.Relationship between serum adiponectin and leptin concentrations and body fat distribution.Diabetes Res Clin Pract,2004,63(2):135-142.
[10]Bacha F,Saad R,Gungor N,et al.Adiponectin in youth:relationship to visceral adiposity,insulin sensitivity,and beta-cell function.Diabetes Care,2004,27:547-552.
[11]Naoto K,Yasuo T,Toshimasa Y,et al.Disruption of adiponectin causes insulin resistance and neoinimal formation.J Biol Chem, 2002,277:25863-25866.
[12]Bellowski J.Adiponectin and resistin梟ew hormones of white adipose tissue.Med Sci Monil,2003,9(2):RA55-RA61.
[13] Yang Z,Zhang M,Xu L,et al.Effect of tumor necrosis factor-alpha on resistin expression in 3T3-L1 adipocytes and its mechanism.J Huazhong Univ Sci Technolog Med Sci,2005,25(2): 121-30.
[14] Chen D,Dong M,Fang Q,et al.Alterations of serum resistin in normal pregnancy and pre-eclampsia.Clin Sci,2005 Jan,108(l):8l-4.
[15]Silswal N,Singh AK,Aruna B,et al.Human resistin stimulates the pro-inflammatory cytokines TNF-alpha and IL-12 in macrophages by NF-kappaB-dependent pathway.Biochem Biophys Res Commun,2005 Sep 9,334(4): 1092-101.
[16]Ishihara N,Matsuo H,Murakoshi H,et al.Increased apoptosis in the syncytotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation.Am J Obstet Gynecol,2002,186(l):158-166.
[17] Kirwan JP,Hauguel-De Mouzon S,et al. TNF-alpha is a predictor of insulin resistance in human pregnancy.Diabetes,2002 Jul,51(7):2207-13.
[1] Gonzalez RR, Devoto L, Campana A, et al. Effects of leptin, interleukin-lalpha, interleukin-6, and transforming growth factor-beta on markers of trophoblast invasive phenotype:integrins and metalloproteinases. Endocrine, 2001, 15(2):157-64.
[2] Hui Chen Feng,Mei Yee Choy,Wen Deng,et al.Establishment and Characterization of a Human First-Trimester Extravillous Trophoblast Cell Line(TEV-1).J Soc Gynecol Investig,2005,12(4):22-32.
[3] Sato, Y, H. Fujiwara,T. Higuchi, S. Yoshioka,K. Ta～umi, M. Maeda& S. Fujii,2002, Involvement of dipeptidyl peptidase Ⅳ in extravilous trophoblast invasion an diferentiafion. Clim Endocrinol. Metab. 87: 4287-4296.
[4] Castellucci M, Matteis DR, Meisser A, et al. Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion. Mol Hum Reprod, 2000, 6(10):951- 8.
[5] Dominguez F, Pellicer A & Simon C. Paracrine dialogue in implantation.Mol Cell Endocrinol, 2002, 186(2): 175-81.
[6] Aschkenazi S , St raszewski S , Verwer KM , et al. Differential regulation and function of the Fas/Fas ligand system in human trophoblast cells. Biol Reprod ,2002 , 66 : 1853-1861.
[7] Nechushtan A ,Smith CL ,Lamensdorf I ,et al.Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol ,2001,153 :1265-1276.
[8] Morin D, Pires F, Plin C,et al.Role of the permeability transition pore in cytochrome C release from mitochondria during ischemia-reperfusion in rat liver.Biochem Pharmacol,2004,68:2065-2073.
[9] Bajoriaa RD, Soorannac SR & Warda BS.Prospective function of placental leptin at maternal-fetal interface. Placenta,2002, 23:103-15.
[10] Bjorbaek C,KahnB B.Leptin signaling in the central nervous system and the periphery.Recent Prog Horm Res,2004,59:305-336.
[11] Myers MG J.Leptin receptor signaling and the regulation of mammalian physiology.Recent Prog Horm Res,2004,59:287-304.
[12] Cauzac M,Czuba D,Girard J,et al.Transduction of leptin growth signals in placental cells is independent of JAK-STAT activation. Placenta , 2003 ,24 (4):378-384.
[13] Hardwick J, Brink VDG, Oerhaus G,et al.Leptin is a growth factor for colonic epithelial cells.Gastroenterology,2001,121(79-90): 2355-60.