【摘要】：胎盤滋養(yǎng)細胞在人類妊娠早期胚泡植入過程中發(fā)揮著重要作用,滋養(yǎng)細胞增殖、凋亡、侵襲和遷移等功能適度是生理妊娠建立、維持和適時終止的關鍵。功能過強可能發(fā)生滋養(yǎng)細胞腫瘤(如葡萄胎、絨毛膜癌等)；過弱則可導致妊娠失敗(如自然流產、早產等)和病理妊娠(如子癇前期、胎兒宮內生長受限等)。滋養(yǎng)細胞功能異常的分子機制一直被廣泛研究,然而通過何種關鍵性靶分子,經過何種細胞途徑作用于滋養(yǎng)細胞,從而引起增殖、侵襲等生物學行為改變的分子生物學機制尚不明確。S100P是鈣結合蛋白S100家族的一員,在人類胎盤組織中首次被檢測到,其發(fā)現即預示著與妊娠關系密切。然而,目前尚沒有文獻報道s100P在人類妊娠不同階段胎盤組織的表達和分布——也沒有報道S100P與滋養(yǎng)細胞功能之間的關系。本研究首先檢測了Sl00P在正常妊娠不同階段胎盤組織的表達模式,并比較了自然流產及妊娠滋養(yǎng)細胞腫瘤中S100P表達的差異；其次,通過體外細胞實驗及體內動物實驗來研究S100P對滋養(yǎng)細胞生物學功能的影響及可能的分子作用機制。本研究旨在探討S100P表達與胎盤滋養(yǎng)細胞功能的相關性,第一部分S100P在胎盤組織中的表達目的：探討S100P在正常妊娠不同階段絨毛或者胎盤組織以及早孕期自然流產絨毛組織以及妊娠滋養(yǎng)細胞腫瘤中的表達。材料與方法：利用RT-PCR、定量實時PCR、western-blot以及免疫組織化學染色法檢測S100P在胎盤組織中的表達。(1)收集正常妊娠不同階段絨毛或胎盤組織,包括12個早孕期絨毛組織,10個中孕期胎盤組織,和12個足月妊娠胎盤組織,檢測Sl00P在絨毛和胎盤組織的表達與定位。(2)比較16個早孕期自然流產與12個早孕期正常妊娠絨毛組織,比較兩組S100P表達量的差異；(3)收集了23例妊娠滋養(yǎng)細胞腫瘤的組織樣本,10例葡萄胎、3例侵襲性葡萄胎、10例絨毛膜癌,其中5例同時具有原發(fā)灶組織和轉移灶組織；對葡萄胎、侵蝕性葡萄胎、絨毛膜癌原發(fā)灶及轉移灶組織的Sl00P蛋白表達進行免疫組化表達分析。結果：(1)S100P在整個妊娠階段早孕期、中孕期以及足月妊娠絨毛或胎盤組織均呈高表達,相比較而言,早孕期絨毛組織的S100P mRNA和蛋白表達水平更高一些。免疫組化法檢測到S100P蛋白在絨毛合體滋養(yǎng)細胞強陽性表達,細胞滋養(yǎng)細胞發(fā)現中等強度表達。S100P在合體滋養(yǎng)細胞的細胞核及細胞漿均呈強陽性表達,而在細胞滋養(yǎng)細胞及細胞滋養(yǎng)細胞柱只表達于細胞漿。在中孕期與足月妊娠胎盤,S100P在合體滋養(yǎng)細胞的細胞核和細胞漿表達。(2)與早孕期正常妊娠絨毛組織相比,自然流產絨毛組織的S100P蛋白表達水平明顯降低(P0.05)。(3)在妊娠滋養(yǎng)細胞腫瘤標本中,10例葡萄胎組織的免疫組化結果顯示,部分病人(3/10)S100P蛋白在合體滋養(yǎng)細胞為強陽性,在細胞滋養(yǎng)細胞為中等強度陽性或強陽性表達；而在3例侵蝕性葡萄胎(3/3)、6例絨毛膜癌原發(fā)灶(6/10)和5例肺轉移灶組織(5/5)中,細胞滋養(yǎng)細胞S100P均為強陽性表達。結論：S100P在正常妊娠不同階段胎盤滋養(yǎng)細胞的特異性表達和分布顯示其在滋養(yǎng)細胞的功能可能隨著定位不同發(fā)生改變。S100P在早孕期自然流產絨毛組織中表達量下降且在良惡性滋養(yǎng)細胞腫瘤表達存在差異,提示S100P與胎盤滋養(yǎng)細胞的功能可能存在一定的關系,但具體作用與可能的分子機制還有待進一步驗證。第二部分s100P對滋養(yǎng)細胞生物學功能的影響及分子機制探討目的：探討S100P對滋養(yǎng)細胞生物學行為的影響及可能的分子機制。材料與方法：體外細胞實驗部分：培養(yǎng)JAR和JEG-3滋養(yǎng)細胞系,構建真核表達載體和小干擾RNA分別轉染JAR細胞和JEG-3細胞。不同分組滋養(yǎng)細胞分別用XTT細胞生長實驗、細胞凋亡實驗、平板克隆形成實驗及transwell細胞遷移實驗分析S100P對滋養(yǎng)細胞生長、凋亡、克隆形成及遷移能力的影響。體內動物實驗部分：應用裸鼠體內成瘤實驗和尾靜脈注射轉移瘤實驗觀察S100P對滋養(yǎng)細胞體內成瘤能力和細胞轉移能力的影響。為了進一步探討S100P的作用機制,我們用western-blot檢測P38信號通路相關分子在JAR細胞中的作用。結果：(1)構建S100P真核表達載體和S100P小干擾RNA (siRNA1、siRNA2),轉染滋養(yǎng)細胞系JAR和JEG-3。轉染質粒過表達S100P的JAR和JEG-3細胞均比對照組(空載體轉染組)細胞生長能力增快,細胞克隆形成能力增加,且細胞體外遷移能力亦明顯增加(P0.05)。與之相反,轉染小干擾RNA的滋養(yǎng)細胞生長能力要明顯慢于對照組,且細胞克隆形成能力降低,體外遷移能力也降低(P0.05)。(2)我們接著檢測上調S100P的表達水平對裸鼠體內成瘤能力和尾靜脈注射轉移瘤形成的影響,結果顯示,與轉染空載體的對照組細胞相比,S100P過表達滋養(yǎng)細胞形成的腫瘤體積明顯大于對照組,差異有統(tǒng)計學意義(P0.05)。同時,轉移瘤實驗顯示S100P過表達組JAR細胞在裸鼠體內轉移能力顯著增強(P0.05)。(3)進一步分子機制實驗結果顯示S100P過表達后滋養(yǎng)細胞JAR的P-P38和P-ERK表達升高；利用抑制劑SB203580阻斷P38 MAPK,上調S100P的表達不能促進JAR細胞的增殖作用,而ERK通路抑制劑PD98059阻斷后對S100P引起的細胞增殖沒有作用。結論：s100P可以促進滋養(yǎng)細胞的體外增殖、克隆、遷移能力以及體內成瘤和轉移能力,且S100P可能通過P38 MAPK信號途徑促進滋養(yǎng)細胞的增殖。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R714

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