| Background:hypertensive disorder complicating pregnancy is a pregnancy- specific syndrome characterized by hypertension, edema and proteinuria, resolves spontaneously on placental delivery., is the one of important reasons of perinatal mortality. In the situation that the pathogenesis is not fully understood, which brings difficulty to the prevention and treatment of the disease.From the beginning of the embryo implantation to the successful delivery,it depend on the normal placental function. Trophoblast invasion is shallow in endometrial and uterine spiral arteries has a barrier on remodeling,which are placental pathological characteristic of hypertensive disorders.and the function of trophoblast invasion is descending during early pregnancy may the important reason result in implanting shallowly. The Shallow trophoblast invasion of endometrial,uterine spiral artery remodeling disorders, placental insufficiency, ischemia and hypoxia, leading to trophoblast cells produce a series of virulence factors into the maternal circulation, causing widespread endothelial cell damage and, ultimately, as pregnancy induced hypertension[12].Hypoxia inducible factor -1 (hypoxia inducible factor 1, HIF-1) is found that it is the only factor playing a specific transcription activity in anoxic environment. It is a mediator of hypoxia response genes transducting hypoxia signal,and activateing some hypoxia-related genes, make the cells adapt to hypoxia, but also can cause a variety of pathological changes.Matrix metalloproteinase-9 ( MMP-9) is an important member in matrix metalloproteinase system, and some physiological functions (such as embryonic implantation, tissue, wound healing) and pathologic processes (tumor invasion Run and transfer) are closely related. It needs to attachment, degradation and crossing the ECM interspace to complete embryo implantation.MMP-9 plays a direct function of enzymatic degradation in the decidual stroma, basement membrane and attachment of vascular cavity surface, as the trophoblast cell invasion removed the physical barrier,is the rate-limiting steps in trophoblast invasion, and it took a lot of well-established in vivo experiments mechanism.In this study, by detecting the differences of HIF-1αand MMP-9 in preeclampsia and normal pregnancy, and the expression of HIF-1αand MMP-9mRNA of trophoblast cells in vitro in different oxygen concentration, further confirmed HIF-1αin the pathogenesis of pregnancy hypertension plays an important role,and through the negative correlational relationship of HIF-1αand MMP-9, indicating that abnormal hypoxia response could affect placenta accreta by the expression of MMP-9, Providing new ideas for the research of etiology and pathogenesis of PIH. Objective:1. Detecting expression on the oxygen regulator of HIF-1αand its downstream regulatory factors ET-1 in patients with preeclampsia and normal pregnancy in the placenta by collected clinical specimens. indicating that the correlation between HIF-1αand ET-1, to provide a theoretical basis for the role that HIF-1αand ET-1 in hypertensive disorder complicating pregnancy play.2. Through isolating villous trophoblast cells during early pregnancy, explore the simple and efficient training methods to trophoblast villi , as to provide effective physical infrastructure for the study of trophoblastic disease in vitro.3. By detecting the expression of HIF-1αand MMP-9 in vitro trophoblast cells under different oxygen concentrations, indicating that changes in oxygen concentration on maternal-fetal interface have expression on the development of the placenta during early pregnancy,revealing the relationship between HIF-1αand MMP-9 in the formation of the placenta, providing new ideas to the pathogenesis of hypertensive disorder complicating pregnancy.Methods:1. Collection of clinical preeclampsia placenta 30 cases, (12 patients with mild and severe in 18 cases) and normal pregnancy placenta 20 cases, with SP immunohistochemical method did detect the locations and expression of the protein in the placental tissue; Western-blotting method to detect the HIF-1αand ET-1 protein expression of placenta.2. Tissue-culture method was used to get the primary human t rophoblastic cells of first rimester pregnancy. Clean repeatedly to remove blood clots, strip decidua Organization, isolated chorionic villi,and cut into pieces; paste fragments of tissue to spread evenly with spaced 5mm at the bottom of wet flask, with the amount of DMEM containing 15% FBS medium cultured; medium was changed every other day, digestion and passage after 2 weeks. Digestion and different speed adherence were used to purify the cells , immunocytochemistry with specific CK7 and Vimeintin monoclonal antibodies were used to identified the cells.3. Using chemical hypoxia inducer CoCl2 induced in different oxygen concentration environments, isolated and cultured in vitro trophoblast cells were divided into hypoxia group 1 (100μl / L 24h), hypoxic group 2 (50μl / L 24h), reoxygenation (50μl / L 6h ~ 18h), normoxia (24h),collected cells were extracted RNA, using semi-quantitative PCR detected the expression of HIF-1αand MMP-9mRNA every group.Results:1. Preeclampsia compared with normal group, HIF-1αexpression was significantly increased, the difference was significant (t = -5.710, P <0.05), HIF-1αexpression of severe preeclampsia was significantly higher than that of mild preeclampsia, the difference was significant (t =-6.945, P<0.05). Similarly, compared with the control group, ET-1 expression on preeclampsia was significantly increased, the difference was significant (t = -3.659, P <0.05), ET-1 expression on severe preeclampsia was significantly higher than the mild preeclampsia, the difference was statistically significant (t =- 7.966, P <0.05). The correlation analysis of HIF-1αand ET-1 in preeclampsia group showed obviously positive correlation in placenta tissue (γ= 0.659, P <0.05), with the deeper levels of hypoxia, the expression of HIF-1αand MMP- 9 was negatively correlated in villi.2. It was 24 hours after tissue pieces adheret, the cells began to spreadly grow around organization, and significantly increased after a week, mostly multilateral irregular shape or long spindle. After two weeks, the border of mass organizations was blurred and the surrounding cell fusion, Part mass of the cells connected as the growth of flaky tile. covered 60% of the culture flask bottom in three weeks. By the passage of adherence repeatedly and digestion elimination, epithelial cell-like cells mostly tile sheet growth as irregular polygonal shape, form almost the same, the individual was fiber-like cells. Nuclear is large and round, near the central, cell cytoplasm is rich. By immunohistochemistry staining, CK7 positive rate was 75%, vimentin positive rate of less than 10%, indicating the purity of trophoblast cells at 75% or more.3. The expression of HIF-1αmRNA the most obvious on hypoxia group 1, with the CoCl2 concentration decreased, the oxygen concentration of the cell growth conditions increased, the expression of HIF-1αmRNA showed a decreasing trend (Figure 7); the same time, the expression of MMP-9mRNA in normoxia group was the most obvious, with the CoCl2 concentration increased, the oxygen concentration of the cell growth conditions increased decreased, the expression of MMP-9mRNA showed a decreasing trend (Figure 8), showing negative correlation between them..Conclusion:1. HIF-1αexpression in the disease group was significantly higher than the normal placenta group of pregnant women, and is closely associated with vasoconstriction of ET-1 was positively correlated, HIF-1αcan cause abnormal expression of the abnormal transcription of downstream target genes and affect the placenta development. The degradation and transcription of HIF-1 is relevant with the happen of hypertensive disorder complicating pregnancy2. The incidence of hypertensive disorder complicating pregnancy is probably due to abnormal HIF-1 regulation, affecting the expression of MMP-9 which closely related to invasion of trophoblast, leading to reduced invasiveness of trophoblast cell, indicating that the process of hypoxia response with abnormal regulation of HIF-1 in early pregnancy can affect the activity of placenta accreta.3. Though cells purity would be affected by this original tissue adherent methods substituting cultured human trophoblast, which we could make available to digest and purify by the time-lag of adherence between the fibroblast and epithelioid cells, the cells were relatively easy to grow. |
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