【作者】 王心；

【导师】 尚丽新； 陈薇；

【作者基本信息】 第四军医大学 ， 妇产科学， 2009， 博士

【摘要】 邻苯二甲酸二-(2-乙基己基)酯(DEHP)作为一种增塑剂被广泛添加在各种塑料制品和化妆品中,含量最高可达30 %。目前DEHP已经形成了比较严重的环境污染,在全球几乎所有的海洋、大气、饮用水、植物及动物体内都可不同程度地被检出,它对人体的毒性作用越来越受到人们的关注。由于大量使用化妆品及洗漱用品,女性DEHP的暴露几率远高于男性,由于妊娠期对有害因素的影响更为敏感,因此有必要深入研究DEHP对女性生殖系统的不良影响。流行病学调查显示流产、子痫前期及胎儿生长受限等不良妊娠结局与邻苯二甲酸酯暴露密切相关。胎盘位于母胎界面,是胎儿对外来物质发生反应的第一线,对妊娠的发生和维持有重要的作用。我们推测胎盘滋养细胞可能是邻苯二甲酸酯重要的靶细胞,从DEHP作用后滋养细胞相关生物学功能的改变入手,就有可能抓住DEHP影响生殖健康的主要环节。本课题拟通过以下四部分实验,探讨DEHP对体外培养滋养细胞的作用及机制,并通过动物实验,探讨DEHP的雌性生殖毒性。实验一早孕绒毛外滋养细胞的分离、鉴定和培养【目的】建立分离正常早孕绒毛组织获得高纯度的绒毛外滋养细胞(EVTs)的方法,并对其进行鉴定和培养。【方法】无菌条件下获取人工流产早孕绒毛组织,经机械剪碎后通过温和的酶消化方法获得滋养细胞,经1.25-2.5 %BSA密度梯度沉降法纯化EVTs。将分离纯化后EVTs接种在Matrigel包被的细胞板上培养。用免疫细胞化学方法,进行CK-7、HLA-G和波形蛋白检测,荧光显微镜下观察鉴定EVTs。【结果】经分离纯化后的EVTs体积较大,呈不规则多边形,核大,胞浆丰富。细胞呈单片状聚集生长,但彼此并不融合。EVTs在Matrigel包被的细胞板上生长状态良好,接种于未包被Matrigel的细胞板上的细胞大多数在24h内死亡。CK-7、HLA-G在EVTs胞浆和胞膜上阳性表达,EVTs内无波形蛋白表达。成纤维细胞胞浆内可见波形蛋白表达,但CK-7和HLA-G不表达。分离的细胞中EVTs数量超过95 %。【结论】用温和的酶消化法和BSA密度梯度沉降法可获得的高纯度EVTs。Matrigel是EVTs体外培养过程必要条件。EVTs在包被Matrigel的细胞板上生长状态良好,特征性蛋白CK-7和HLA-G表达阳性。实验二DEHP对绒毛外滋养细胞浸润能力的影响及其机制【目的】探讨DEHP对原代培养人早孕绒毛外滋养细胞浸润能力的影响及其机制。【方法】以25、50、100μmol/L DEHP处理DEHP24h后,通过体外侵袭实验检测DEHP干预后EVTs浸润能力的改变,通过RT-PCT和Western Blot方法检测细胞内侵袭相关基因MMP-2和MMP-9的mRNA和蛋白表达的改变。【结果】DEHP干预后通过体外侵袭实验检测,EVTs的浸润能力具有不同程度下降。剂量为50和100μmol/L时DEHP组与对照组侵袭指数差别显著(P < 0.05)。DEHP作用后50μM和100μM剂量组MMP-2和MMP-9的mRNA和蛋白表达量较对照组明显降低(P < 0.05),而且随着DEHP剂量增加MMP-2和MMP-9 mRNA和蛋白表达量逐渐降低,呈剂量效应负相关。【结论】剂量大于50μmol/L时,DEHP可降低早孕绒毛外滋养细胞浸润能力,其机制可能与抑制MMP-2和MMP-9表达相关。实验三DEHP对绒毛外滋养细胞凋亡的影响及其机制【目的】探讨DEHP对原代培养人早孕绒毛外滋养细胞凋亡的影响及其机制。【方法】以25、50、100μmol/L DEHP处理DEHP24h后,通过原位缺口末端标记法检测DEHP干预后EVTs细胞凋亡数的改变,通过RT-PCR和Western Blot方法检测细胞内凋亡相关基因Bcl-2和Bax的mRNA和蛋白表达的改变。【结果】DEHP干预后经原位缺口末端标记法检测,不同剂量DEHP干预组EVTs细胞凋亡数均有不同程度的增加,剂量为50和100μmol/L时DEHP组与对照组凋亡数差别显著(P < 0.05)。DEHP作用后50μM和100μM剂量组Bax的mRNA和蛋白表达量较对照组明显增加,而且随着DEHP剂量增加Bax mRNA和蛋白表达量逐渐增加。DEHP各组Bcl-2的mRNA和蛋白表达量与对照组相比没有明显差异(P > 0.05)。随着DEHP剂量增加Bcl-2和Bax蛋白表达量的比值逐渐降低,呈剂量效应负相关。【结论】剂量大于50μmol/L时,DEHP可诱导EVTs细胞凋亡增加,其机制可能与诱导促凋亡基因Bax的表达相关,DEHP对Bcl-2的表达没有明显影响。实验四DEHP对雌性大鼠早期妊娠及胚胎发育的影响【目的】探讨DEHP在体内对雌性大鼠早期妊娠及胚胎发育的影响。【方法】40只大鼠分为4组,DEHP 5、25、50 mg/kg分别为低、中、高剂量组,对照组为玉米油。妊娠早期(1-6d)灌胃给予不同剂量DEHP,妊娠14d处死孕鼠剖腹检测DEHP对胚胎着床情况的影响。另外40只大鼠分组方法同上,在妊娠中晚期(7-20d)给予不同剂量DEHP,妊娠末期处死,剖腹检测胚胎畸形和胎盘发育情况。【结果】DEHP妊娠早期干预各组和妊娠中晚期干预各组大鼠体重与对照组没有显著差异(P > 0.05)。中剂量和高剂量DEHP干预后,胚胎着床数降低,胚胎数和活胎数减少,胚胎和胎盘重量均低于对照组(P < 0.05)。【结论】DEHP对母鼠没有明显影响时,在中高剂量组DEHP但仍可在妊娠早期影响胚胎着床过程,造成胚胎丢失。DEHP还可在妊娠中晚期影响胚胎及胎盘发育,导致胎儿活胎数降低,胎儿发育不良、胎儿畸形数增加。综上所述,本课题首先以完善可靠的人早孕绒毛外滋养细胞原代分离纯化方法为基础,以绒毛外滋养细胞为体外研究模型,检测了DEHP对绒毛外滋养细胞浸润的能力及细胞凋亡的影响,并初步探讨其作用机制;最后,以妊娠大鼠为体内研究模型,探讨了DEHP对早期妊娠着床及胚胎发育的影响。本课题在体内外实验中证实了DEHP对滋养细胞生物学行为有不良影响,并从基因水平阐明了其作用的分子机制,为进一步寻找有效拮抗剂及有效的防护措施提供了理论依据。更多还原

【Abstract】 DEHP is used as plasticizers for polyvinyl chloride (PVC) products and cosmetic. DEHP was detected in most of environmental samples collected from oceans, atmosphere, surface water, animals, plants and newborn baby body. With the extensive use of plastic, the adverse health of DEHP on human have arisen great concern worldwide. Because of the substantial use of cosmetics and toiletries products, women expose to the risk of DEHP is much higher than male. Women in pregnant is more sensitive to harmful factors, so it is necessary to depth study on the adverse effects of DEHP during pregnancy.Data of epidemiological investigation revealed that adverse pregnancy, such as outcomes abortion, pre-eclampsia and fetal growth restriction, is closely related to phthalate exposure. Maternal-fetal interface located in the placenta is the first barrier of the fetus to foreign substances. Placental trophoblast has an important role in the occurrence and maintenance of pregnancy. We presume that trophoblast cells are probably the target cells of DEHP. From the change in biological function of trophoblast cells after DEHP treating,we can find the effects of DEHP on health. The aim of this project is to explore the effects of DEHP on female reproductive reproductive health in vitro and in vivo. The research can be divided into four sections as following:Section I Isolation, clture and characterization of human extravillous trophoblasts from first trimester placenta[objective] To establish a method to isolate and purify extravillous cytotrophoblasts (EVTs) from the chorionic villi of first trimester human placenta.[Methods] After mild enzymatic digesttion and different density of BSA sedimentation, EVTs were collected and planted on the Matrigel-covered dishes. Immunocytochemistry revealed the various characteristics of EVTs.[Results] EVTs grow well on the Matrigel-covered dishes, but most of EVTs on no-Matrigel dishes died within 24h. EVTs exhibited positive staining for CK-7 and HLA-G, while fibroblast cells exibited positive staining for vimentin.[Conclusion] This method can be successfully used to isolate almost pure EVTs.Section II Effects of DEHP on the invasion of human first trimester EVTs[objective] To explore the effects of DEHP on the invasion in EVTs.[Methods] After intervention by different doses of DEHP for 24h, the invasion ablility was detected by Transwell. The effect of DEHP on the change of MMP-2 and MMP-9 mRNA and protein in EVTs was investigated with reverse transcription-PCR and western blot.[Results] DEHP can inhibite the invasiveness of EVTs. The EVTs incubation with 50 and 100μmol/L DEHP significant decrease the expression of MMP-2 and MMP-9.[Conclusion] DEHP can inhibite the invasiveness of EVTs, this anti-invasive activity may be the results of decrease in MMP-2 and MMP-9 expression in EVTs.Section III Effects of DEHP on the apoptosis of human first trimester EVTs[objective] To explore the effects of DEHP on the apoptosis in EVTs.[Methods] After intervention by different doses of DEHP for 24h, the apoptosis of EVTs was detected by TUNEL. The effect of DEHP on the change of Bcl-2 and Bax mRNA and protein in EVTs was investigated with reverse transcription-PCR and western blot.[Results] DEHP can induce apoptosis of EVTs. The EVTs incubation with 50 and 100μmol/L DEHP significant increase the expression of Bax, but the Bcl-2 expression does not change.[Conclusion] DEHP can induce apoptosis of EVTs, this effect may be the results of increase in Bax expression in EVTs.Section IV Effects of DEHP on female pregnancy and fetus development[objective] To investigate the effect of DEHP on female pregnancy and fetus development.[Methods] Fourty pregnant rats were radomized equally into 4 groups consisting of three treatment groups(in which DEHP was administered at 5, 25,50 mg/kg, respectively, from 1 to 6 day of pregnancy) and a control group. Anthor fourth pregnant rats were given DEHP from 7 to 20 days of pregnancy, and the grouping methods are the same as former. The pregnant rats were killed by 14 or 21 day of pregnancy. The following results in each group were recorded: embryo implantation cases, fetal malformations and developmental.[Results] DEHP has no effects on the body weight of rats. DEHP can reduce the number of embryo implantation, live births and the weight of fetal and placenta.[Conclusion] In the case of low-dose, DEHP can affect blastocyst implantation and embryo delopment.In summary, we firstly isolate and purify EVTs from the chorionic villi of placenta successfully. Then we comprehensively observed the change in biological function of EVTs after DEHP treating in vitro. We found that DEHP could suppress the activity of invisiveness of EVTs by reducing the MMP-2 and MMP-9 expression, and DEHP could suppress the activity of apotosis of EVTs by increasing the Bax expression. Finally we detected the DEHP coud affect blastocyst implantation and embryo delopment in vivo. Our research results provide a theoretical basis for further looking for an effective antagonist and effective protective measures.更多还原