胰岛素样生长因子I对人早孕滋养细胞的作用及其MAPK信号转导机制的初步研究

发布时间：2018-03-01 20:13

本文关键词： 丝裂原活化蛋白激酶胰岛素样生长因子滋养细胞信号转导细胞外信号调节蛋白激酶增殖分化侵入　出处：《华中科技大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的:研究MAPK通路信号在人早孕绒毛组织中的表达,探讨IGF-1对体外培养的人早孕滋养细胞的调节作用以及MAPK通路对这些作用的影响,阐明胚胎植入过程中滋养细胞发挥生理功能的部分分子机制。方法:以妊娠5-7周的正常和自然流产绒毛组织为标本,应用免疫组化、Western Blot、图像分析等方法,检测MAPK通路信号在人早孕绒毛组织中的表达部位、表达水平;以体外培养的人早孕滋养细胞为标本,分别用细胞ELISA法、MTT法、化学发光法以及割伤法观察IGF-1在滋养细胞增殖、分化、侵入中的调节作用以及MAPK信号转导通路对这些作用的影响。结果:(1)免疫组化结果显示:妊娠5-7周正常组绒毛组织中,磷酸化(活化型)ERK在细胞滋养细胞和合体滋养细胞呈阳性或强阳性染色,以细胞浆着色为主,而在自然流产组绒毛组织中,呈弱阳性或阴性染色,以合体滋养细胞胞浆着色为主;磷酸化p-38和磷酸化JNK在正常组绒毛细胞滋养细胞和合体滋养细胞中均呈阳性染色;而在自然流产组中,二者在细胞滋养细胞呈阳性染色,在合体滋养细胞则呈阴性染色。(2)Western Blot结果显示:磷酸化ERK在妊娠5-7周正常组和自然流产组绒毛均有表达,但在正常早孕组中表达水平明显高于自然流产组。(3)细胞ELISA法检测滋养细胞中ERK的相对激酶活性,结果发现,不同浓度IGF-1均能激活ERK,且在0.1-100nM范围内,随IGF-1浓度增高,激酶活性增强;ERK通路的特异性阻断剂U0126可抑制滋养细胞内ERK激活。(4)MTT法测定滋养细胞的增殖活性,结果发现,IGF-1可促进滋养细胞增殖,且在0.1-100nM范围内,随IGF-1浓度增高,滋养细胞增殖活性增强;U0126可抑制IGF-1对滋养细胞的促增殖作用。(5)化学发光法检测滋养细胞HCG含量,结果表明,IGF-1可促进滋养细胞分泌HCG,且在0.1-100nM范围内,随IGF-1浓度增高,滋养细胞分泌HCG增加;U0126可抑制滋养细胞中HCG分泌。(6)割伤法测定滋养细胞的迁移活性,结果表明,IGF-1对滋养细胞的迁移无明显促进作用;而ERK通路阻断剂U0126可显著抑制滋养细胞的迁移。结论: (1) MAPK信号转导通路激活可能是滋养层细胞增殖、分化及侵入过程中的重要分子机制。 (2) IGF-1在体外培养的人早孕滋养细胞中具有促进细胞增殖、分化的作用。 (3) IGF-1促进滋养层细胞增殖、分化的作用可能是通过激活ERK/MAPK信号通路实现的。
[Abstract]:Aim: to investigate the expression of MAPK pathway signal in human chorionic villi, and to investigate the regulatory effect of IGF-1 on human trophoblastic cells cultured in vitro and the effect of MAPK pathway on these effects. To elucidate some molecular mechanisms of the physiological function of trophoblast during embryo implantation. Methods: using normal and spontaneous abortion chorionic villi from 5-7 weeks of gestation as specimens, the expression of MAPK pathway signal in human early pregnancy villi was detected by immunohistochemistry and image analysis. The effects of IGF-1 on the proliferation, differentiation and invasion of trophoblastic cells were observed by ELISA assay, chemiluminescence assay and cut wound method. The effects of MAPK signal transduction pathway on these effects were observed. Results Immunohistochemical results showed that phosphorylated ERK was positive or strongly positive in cytotrophoblast and syncytiotrophoblast in normal villi of 5-7 weeks gestation, mainly cytoplasm. In the villi of spontaneous abortion group, weak positive or negative staining, mainly cytoplasmic staining of syncytiotrophoblast, phosphorylated p-38 and phosphorylated JNK were positive staining in trophoblast and syncytiotrophoblast of normal group. In spontaneous abortion group, both of them were positive in cytotrophoblast and negative staining in syncytiotrophoblast. Western Blot results showed that phosphorylated ERK was expressed in villi of normal group and spontaneous abortion group at 5-7 weeks of gestation. But the expression level in normal early pregnancy group was significantly higher than that in spontaneous abortion group. The activity of ERK relative kinase in trophoblastic cells was detected by ELISA method. The results showed that IGF-1 at different concentrations could activate ERKs and increase with the concentration of IGF-1 in the range of 0.1-100nM. U0126, a specific inhibitor of kinase-enhanced ERK pathway, inhibited ERK activation in trophoblastic cells. The proliferative activity of trophoblastic cells was determined by MTT assay. The results showed that IGF-1 could promote the proliferation of trophoblastic cells and increase with the concentration of IGF-1 in the range of 0.1 ~ 100nM. The enhancement of trophoblast proliferation activity (U0126) inhibited the proliferation of trophoblastic cells by IGF-1. The chemiluminescence method was used to detect the content of HCG in trophoblast. The results showed that the HCG level of trophoblastic cells increased with the concentration of IGF-1 in the range of 0.1 ~ 100nM. The increased secretion of HCG by trophoblastic cells inhibited the secretion of HCG in trophoblastic cells by slit method. The results showed that HCG 1 did not promote the migration of trophoblastic cells. ERK pathway blocker U0126 significantly inhibited trophoblast migration. Conclusion:. 1) Activation of MAPK signal transduction pathway may be an important molecular mechanism of trophoblast cell proliferation, differentiation and invasion. 2) IGF-1 can promote the proliferation and differentiation of human early pregnancy trophoblastic cells in vitro. 3) IGF-1 promotes the proliferation of trophoblast cells, and the differentiation of trophoblast cells may be mediated by activation of ERK/MAPK signaling pathway.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R321

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