The Study Of Female Reproductive Toxicity Induced By DEHP

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.