钙结合蛋白S100P在胎盘滋养细胞的表达与功能研究

发布时间：2018-08-18 17:50

【摘要】：胎盘滋养细胞在人类妊娠早期胚泡植入过程中发挥着重要作用,滋养细胞增殖、凋亡、侵袭和迁移等功能适度是生理妊娠建立、维持和适时终止的关键。功能过强可能发生滋养细胞肿瘤(如葡萄胎、绒毛膜癌等)；过弱则可导致妊娠失败(如自然流产、早产等)和病理妊娠(如子痫前期、胎儿宫内生长受限等)。滋养细胞功能异常的分子机制一直被广泛研究,然而通过何种关键性靶分子,经过何种细胞途径作用于滋养细胞,从而引起增殖、侵袭等生物学行为改变的分子生物学机制尚不明确。S100P是钙结合蛋白S100家族的一员,在人类胎盘组织中首次被检测到,其发现即预示着与妊娠关系密切。然而,目前尚没有文献报道s100P在人类妊娠不同阶段胎盘组织的表达和分布——也没有报道S100P与滋养细胞功能之间的关系。本研究首先检测了Sl00P在正常妊娠不同阶段胎盘组织的表达模式,并比较了自然流产及妊娠滋养细胞肿瘤中S100P表达的差异；其次,通过体外细胞实验及体内动物实验来研究S100P对滋养细胞生物学功能的影响及可能的分子作用机制。本研究旨在探讨S100P表达与胎盘滋养细胞功能的相关性,第一部分S100P在胎盘组织中的表达目的：探讨S100P在正常妊娠不同阶段绒毛或者胎盘组织以及早孕期自然流产绒毛组织以及妊娠滋养细胞肿瘤中的表达。材料与方法：利用RT-PCR、定量实时PCR、western-blot以及免疫组织化学染色法检测S100P在胎盘组织中的表达。(1)收集正常妊娠不同阶段绒毛或胎盘组织,包括12个早孕期绒毛组织,10个中孕期胎盘组织,和12个足月妊娠胎盘组织,检测Sl00P在绒毛和胎盘组织的表达与定位。(2)比较16个早孕期自然流产与12个早孕期正常妊娠绒毛组织,比较两组S100P表达量的差异；(3)收集了23例妊娠滋养细胞肿瘤的组织样本,10例葡萄胎、3例侵袭性葡萄胎、10例绒毛膜癌,其中5例同时具有原发灶组织和转移灶组织；对葡萄胎、侵蚀性葡萄胎、绒毛膜癌原发灶及转移灶组织的Sl00P蛋白表达进行免疫组化表达分析。结果：(1)S100P在整个妊娠阶段早孕期、中孕期以及足月妊娠绒毛或胎盘组织均呈高表达,相比较而言,早孕期绒毛组织的S100P mRNA和蛋白表达水平更高一些。免疫组化法检测到S100P蛋白在绒毛合体滋养细胞强阳性表达,细胞滋养细胞发现中等强度表达。S100P在合体滋养细胞的细胞核及细胞浆均呈强阳性表达,而在细胞滋养细胞及细胞滋养细胞柱只表达于细胞浆。在中孕期与足月妊娠胎盘,S100P在合体滋养细胞的细胞核和细胞浆表达。(2)与早孕期正常妊娠绒毛组织相比,自然流产绒毛组织的S100P蛋白表达水平明显降低(P0.05)。(3)在妊娠滋养细胞肿瘤标本中,10例葡萄胎组织的免疫组化结果显示,部分病人(3/10)S100P蛋白在合体滋养细胞为强阳性,在细胞滋养细胞为中等强度阳性或强阳性表达；而在3例侵蚀性葡萄胎(3/3)、6例绒毛膜癌原发灶(6/10)和5例肺转移灶组织(5/5)中,细胞滋养细胞S100P均为强阳性表达。结论：S100P在正常妊娠不同阶段胎盘滋养细胞的特异性表达和分布显示其在滋养细胞的功能可能随着定位不同发生改变。S100P在早孕期自然流产绒毛组织中表达量下降且在良恶性滋养细胞肿瘤表达存在差异,提示S100P与胎盘滋养细胞的功能可能存在一定的关系,但具体作用与可能的分子机制还有待进一步验证。第二部分s100P对滋养细胞生物学功能的影响及分子机制探讨目的：探讨S100P对滋养细胞生物学行为的影响及可能的分子机制。材料与方法：体外细胞实验部分：培养JAR和JEG-3滋养细胞系,构建真核表达载体和小干扰RNA分别转染JAR细胞和JEG-3细胞。不同分组滋养细胞分别用XTT细胞生长实验、细胞凋亡实验、平板克隆形成实验及transwell细胞迁移实验分析S100P对滋养细胞生长、凋亡、克隆形成及迁移能力的影响。体内动物实验部分：应用裸鼠体内成瘤实验和尾静脉注射转移瘤实验观察S100P对滋养细胞体内成瘤能力和细胞转移能力的影响。为了进一步探讨S100P的作用机制,我们用western-blot检测P38信号通路相关分子在JAR细胞中的作用。结果：(1)构建S100P真核表达载体和S100P小干扰RNA (siRNA1、siRNA2),转染滋养细胞系JAR和JEG-3。转染质粒过表达S100P的JAR和JEG-3细胞均比对照组(空载体转染组)细胞生长能力增快,细胞克隆形成能力增加,且细胞体外迁移能力亦明显增加(P0.05)。与之相反,转染小干扰RNA的滋养细胞生长能力要明显慢于对照组,且细胞克隆形成能力降低,体外迁移能力也降低(P0.05)。(2)我们接着检测上调S100P的表达水平对裸鼠体内成瘤能力和尾静脉注射转移瘤形成的影响,结果显示,与转染空载体的对照组细胞相比,S100P过表达滋养细胞形成的肿瘤体积明显大于对照组,差异有统计学意义(P0.05)。同时,转移瘤实验显示S100P过表达组JAR细胞在裸鼠体内转移能力显著增强(P0.05)。(3)进一步分子机制实验结果显示S100P过表达后滋养细胞JAR的P-P38和P-ERK表达升高；利用抑制剂SB203580阻断P38 MAPK,上调S100P的表达不能促进JAR细胞的增殖作用,而ERK通路抑制剂PD98059阻断后对S100P引起的细胞增殖没有作用。结论：s100P可以促进滋养细胞的体外增殖、克隆、迁移能力以及体内成瘤和转移能力,且S100P可能通过P38 MAPK信号途径促进滋养细胞的增殖。
[Abstract]:Placental trophoblasts play an important role in the process of human embryo implantation in early pregnancy. Moderate trophoblast proliferation, apoptosis, invasion and migration are the key to the establishment, maintenance and timely termination of physiological pregnancy. Such as spontaneous abortion, premature delivery, and pathological pregnancy (such as preeclampsia, fetal intrauterine growth restriction, etc.). The molecular mechanism of trophoblast dysfunction has been extensively studied. However, the key target molecule, the cellular pathway through which the trophoblast acts, leading to proliferation, invasion and other biological behavior changes in molecular biology. S100P, a member of the calcium-binding protein S100 family, is first detected in human placenta, indicating a close relationship with pregnancy. This study first examined the expression patterns of Sl00P in placenta of different stages of normal pregnancy, and compared the differences of S100P expression between spontaneous abortion and gestational trophoblastic tumors; secondly, we studied the effects of S100P on the biological function of trophoblastic cells in vitro and in vivo through animal experiments. The purpose of this study was to investigate the correlation between the expression of S100P and the function of placental trophoblasts. Part I: Expression of S100P in placental tissues. Objective: To investigate the expression of S100P in villi or placental tissues of different stages of normal pregnancy, villi of spontaneous abortion in early pregnancy and gestational trophoblastic tumors. The expression of S100P in placenta was detected by RT-PCR, quantitative real-time PCR, Western-blot and immunohistochemical staining. Expression and localization of S100P were compared between 16 spontaneous abortions and 12 normal villous tissues of early pregnancy. Results: (1) S100P protein was highly expressed in villi and placenta tissues of early pregnancy, middle pregnancy and full-term pregnancy. Strong positive expression of S100P was detected in chorionic syncytiotrophoblasts by immunohistochemistry, and moderate expression was found in cytotrophoblasts. Strong positive expression of S100P was found in both nucleus and cytoplasm of syncytiotrophoblasts, but only in cytoplasm of cytotrophoblasts and cytotrophoblasts during the second trimester. The expression of S100P in the nucleus and cytoplasm of syncytiotrophoblast was significantly lower in spontaneous abortion villi than in normal early pregnancy villi (P 0.05). 0) S100P protein was strongly positive in syncytiotrophoblast, moderately or strongly positive in cytotrophoblast, and strongly positive in 3 invasive hydatidiform moles (3/3), 6 primary choriocarcinoma (6/10) and 5 lung metastases (5/5). The specific expression and distribution of placental trophoblasts indicated that the function of placental trophoblasts might change with the localization of placental trophoblasts. The expression of S100P decreased in the villi of spontaneous abortion in early pregnancy and was different in benign and malignant trophoblastic tumors, suggesting that S100P might be related to the function of placental trophoblasts, but there was a relationship between S100P and placental trophoblasts. The effects of s100P on the biological functions of trophoblasts and the possible molecular mechanisms were investigated. Materials and Methods: In vitro cell experiments: JAR and JEG-3 trophoblasts were cultured. JAR cells and JEG-3 cells were transfected with eukaryotic expression vector and small interfering RNA respectively. The effects of S100P on the growth, apoptosis, clone formation and migration of trophoblasts were analyzed by XTT cell growth assay, cell apoptosis assay, plate clone formation assay and Transwell cell migration assay. Part I: The effects of S100P on the tumorigenesis and metastasis of trophoblasts in nude mice and tail vein injection metastasis were observed. In order to further explore the mechanism of S100P, we detected the role of P38 signaling pathway related molecules in JAR cells by Western blot. JAR and JEG-3 cells overexpressing S100P by expression vector and S100P small interfering RNA (siRNA1, siRNA2), transfected with JAR and JEG-3, grew faster than the control group (empty vector transfection group), and the ability of cell cloning and migration in vitro were also increased significantly (P 0.05). (2) We then examined the effect of up-regulation of S100P expression on tumorigenesis in nude mice and the formation of metastatic tumor by tail vein injection. The results showed that compared with the control cells transfected with empty vectors, S100 had a lower ability of cell cloning and migration in vitro (P 0.05). At the same time, the metastasis ability of JAR cells in the S100P overexpression group was significantly enhanced in nude mice (P 0.05). (3) Further molecular mechanism experiment showed that the expression of P-P38 and P-ERK in JAR cells increased after S100P overexpression. Conclusion: s100P can promote the proliferation, cloning, migration, tumorigenesis and metastasis of trophoblasts in vitro, and S100P can promote the proliferation of JAR cells. It can promote the proliferation of trophoblast cells through P38 MAPK signaling pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714

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