【摘要】：胎盤(pán)滋養(yǎng)細(xì)胞在人類(lèi)妊娠早期胚泡植入過(guò)程中發(fā)揮著重要作用,滋養(yǎng)細(xì)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(shí)終止的關(guān)鍵。功能過(guò)強(qiáng)可能發(fā)生滋養(yǎng)細(xì)胞腫瘤(如葡萄胎、絨毛膜癌等)；過(guò)弱則可導(dǎo)致妊娠失敗(如自然流產(chǎn)、早產(chǎn)等)和病理妊娠(如子癇前期、胎兒宮內(nèi)生長(zhǎng)受限等)。滋養(yǎng)細(xì)胞功能異常的分子機(jī)制一直被廣泛研究,然而通過(guò)何種關(guān)鍵性靶分子,經(jīng)過(guò)何種細(xì)胞途徑作用于滋養(yǎng)細(xì)胞,從而引起增殖、侵襲等生物學(xué)行為改變的分子生物學(xué)機(jī)制尚不明確。S100P是鈣結(jié)合蛋白S100家族的一員,在人類(lèi)胎盤(pán)組織中首次被檢測(cè)到,其發(fā)現(xiàn)即預(yù)示著與妊娠關(guān)系密切。然而,目前尚沒(méi)有文獻(xiàn)報(bào)道s100P在人類(lèi)妊娠不同階段胎盤(pán)組織的表達(dá)和分布——也沒(méi)有報(bào)道S100P與滋養(yǎng)細(xì)胞功能之間的關(guān)系。本研究首先檢測(cè)了Sl00P在正常妊娠不同階段胎盤(pán)組織的表達(dá)模式,并比較了自然流產(chǎn)及妊娠滋養(yǎng)細(xì)胞腫瘤中S100P表達(dá)的差異；其次,通過(guò)體外細(xì)胞實(shí)驗(yàn)及體內(nèi)動(dòng)物實(shí)驗(yàn)來(lái)研究S100P對(duì)滋養(yǎng)細(xì)胞生物學(xué)功能的影響及可能的分子作用機(jī)制。本研究旨在探討S100P表達(dá)與胎盤(pán)滋養(yǎng)細(xì)胞功能的相關(guān)性,第一部分S100P在胎盤(pán)組織中的表達(dá)目的：探討S100P在正常妊娠不同階段絨毛或者胎盤(pán)組織以及早孕期自然流產(chǎn)絨毛組織以及妊娠滋養(yǎng)細(xì)胞腫瘤中的表達(dá)。材料與方法：利用RT-PCR、定量實(shí)時(shí)PCR、western-blot以及免疫組織化學(xué)染色法檢測(cè)S100P在胎盤(pán)組織中的表達(dá)。(1)收集正常妊娠不同階段絨毛或胎盤(pán)組織,包括12個(gè)早孕期絨毛組織,10個(gè)中孕期胎盤(pán)組織,和12個(gè)足月妊娠胎盤(pán)組織,檢測(cè)Sl00P在絨毛和胎盤(pán)組織的表達(dá)與定位。(2)比較16個(gè)早孕期自然流產(chǎn)與12個(gè)早孕期正常妊娠絨毛組織,比較兩組S100P表達(dá)量的差異；(3)收集了23例妊娠滋養(yǎng)細(xì)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(shí)具有原發(fā)灶組織和轉(zhuǎn)移灶組織；對(duì)葡萄胎、侵蝕性葡萄胎、絨毛膜癌原發(fā)灶及轉(zhuǎn)移灶組織的Sl00P蛋白表達(dá)進(jìn)行免疫組化表達(dá)分析。結(jié)果：(1)S100P在整個(gè)妊娠階段早孕期、中孕期以及足月妊娠絨毛或胎盤(pán)組織均呈高表達(dá),相比較而言,早孕期絨毛組織的S100P mRNA和蛋白表達(dá)水平更高一些。免疫組化法檢測(cè)到S100P蛋白在絨毛合體滋養(yǎng)細(xì)胞強(qiáng)陽(yáng)性表達(dá),細(xì)胞滋養(yǎng)細(xì)胞發(fā)現(xiàn)中等強(qiáng)度表達(dá)。S100P在合體滋養(yǎng)細(xì)胞的細(xì)胞核及細(xì)胞漿均呈強(qiáng)陽(yáng)性表達(dá),而在細(xì)胞滋養(yǎng)細(xì)胞及細(xì)胞滋養(yǎng)細(xì)胞柱只表達(dá)于細(xì)胞漿。在中孕期與足月妊娠胎盤(pán),S100P在合體滋養(yǎng)細(xì)胞的細(xì)胞核和細(xì)胞漿表達(dá)。(2)與早孕期正常妊娠絨毛組織相比,自然流產(chǎn)絨毛組織的S100P蛋白表達(dá)水平明顯降低(P0.05)。(3)在妊娠滋養(yǎng)細(xì)胞腫瘤標(biāo)本中,10例葡萄胎組織的免疫組化結(jié)果顯示,部分病人(3/10)S100P蛋白在合體滋養(yǎng)細(xì)胞為強(qiáng)陽(yáng)性,在細(xì)胞滋養(yǎng)細(xì)胞為中等強(qiáng)度陽(yáng)性或強(qiáng)陽(yáng)性表達(dá)；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(zhuǎn)移灶組織(5/5)中,細(xì)胞滋養(yǎng)細(xì)胞S100P均為強(qiáng)陽(yáng)性表達(dá)。結(jié)論：S100P在正常妊娠不同階段胎盤(pán)滋養(yǎng)細(xì)胞的特異性表達(dá)和分布顯示其在滋養(yǎng)細(xì)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(chǎn)絨毛組織中表達(dá)量下降且在良惡性滋養(yǎng)細(xì)胞腫瘤表達(dá)存在差異,提示S100P與胎盤(pán)滋養(yǎng)細(xì)胞的功能可能存在一定的關(guān)系,但具體作用與可能的分子機(jī)制還有待進(jìn)一步驗(yàn)證。第二部分s100P對(duì)滋養(yǎng)細(xì)胞生物學(xué)功能的影響及分子機(jī)制探討目的：探討S100P對(duì)滋養(yǎng)細(xì)胞生物學(xué)行為的影響及可能的分子機(jī)制。材料與方法：體外細(xì)胞實(shí)驗(yàn)部分：培養(yǎng)JAR和JEG-3滋養(yǎng)細(xì)胞系,構(gòu)建真核表達(dá)載體和小干擾RNA分別轉(zhuǎn)染JAR細(xì)胞和JEG-3細(xì)胞。不同分組滋養(yǎng)細(xì)胞分別用XTT細(xì)胞生長(zhǎng)實(shí)驗(yàn)、細(xì)胞凋亡實(shí)驗(yàn)、平板克隆形成實(shí)驗(yàn)及transwell細(xì)胞遷移實(shí)驗(yàn)分析S100P對(duì)滋養(yǎng)細(xì)胞生長(zhǎng)、凋亡、克隆形成及遷移能力的影響。體內(nèi)動(dòng)物實(shí)驗(yàn)部分：應(yīng)用裸鼠體內(nèi)成瘤實(shí)驗(yàn)和尾靜脈注射轉(zhuǎn)移瘤實(shí)驗(yàn)觀察S100P對(duì)滋養(yǎng)細(xì)胞體內(nèi)成瘤能力和細(xì)胞轉(zhuǎn)移能力的影響。為了進(jìn)一步探討S100P的作用機(jī)制,我們用western-blot檢測(cè)P38信號(hào)通路相關(guān)分子在JAR細(xì)胞中的作用。結(jié)果：(1)構(gòu)建S100P真核表達(dá)載體和S100P小干擾RNA (siRNA1、siRNA2),轉(zhuǎn)染滋養(yǎng)細(xì)胞系JAR和JEG-3。轉(zhuǎn)染質(zhì)粒過(guò)表達(dá)S100P的JAR和JEG-3細(xì)胞均比對(duì)照組(空載體轉(zhuǎn)染組)細(xì)胞生長(zhǎng)能力增快,細(xì)胞克隆形成能力增加,且細(xì)胞體外遷移能力亦明顯增加(P0.05)。與之相反,轉(zhuǎn)染小干擾RNA的滋養(yǎng)細(xì)胞生長(zhǎng)能力要明顯慢于對(duì)照組,且細(xì)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(cè)上調(diào)S100P的表達(dá)水平對(duì)裸鼠體內(nèi)成瘤能力和尾靜脈注射轉(zhuǎn)移瘤形成的影響,結(jié)果顯示,與轉(zhuǎn)染空載體的對(duì)照組細(xì)胞相比,S100P過(guò)表達(dá)滋養(yǎng)細(xì)胞形成的腫瘤體積明顯大于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。同時(shí),轉(zhuǎn)移瘤實(shí)驗(yàn)顯示S100P過(guò)表達(dá)組JAR細(xì)胞在裸鼠體內(nèi)轉(zhuǎn)移能力顯著增強(qiáng)(P0.05)。(3)進(jìn)一步分子機(jī)制實(shí)驗(yàn)結(jié)果顯示S100P過(guò)表達(dá)后滋養(yǎng)細(xì)胞JAR的P-P38和P-ERK表達(dá)升高；利用抑制劑SB203580阻斷P38 MAPK,上調(diào)S100P的表達(dá)不能促進(jìn)JAR細(xì)胞的增殖作用,而ERK通路抑制劑PD98059阻斷后對(duì)S100P引起的細(xì)胞增殖沒(méi)有作用。結(jié)論：s100P可以促進(jìn)滋養(yǎng)細(xì)胞的體外增殖、克隆、遷移能力以及體內(nèi)成瘤和轉(zhuǎn)移能力,且S100P可能通過(guò)P38 MAPK信號(hào)途徑促進(jìn)滋養(yǎng)細(xì)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(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R714

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5 周曉宇;劉霞;金小英;彭華;李琦偉;鐘少平;鄒麗;;血清剝奪對(duì)滋養(yǎng)細(xì)胞生物行為的影響及其機(jī)制研究[A];2011年浙江省婦產(chǎn)科學(xué)學(xué)術(shù)年會(huì)暨“婦產(chǎn)科常見(jiàn)疾病的臨床研究新進(jìn)展”學(xué)習(xí)班論文匯編[C];2011年

6 張弘;林其德;;妊娠高血壓綜合征患者滋養(yǎng)細(xì)胞浸潤(rùn)相關(guān)基因及其蛋白表達(dá)[A];澳門(mén)、香港、內(nèi)地生殖健康研討會(huì)論文集[C];2004年

7 周純芝;楊通明;唐石初;吳澤惠;劉世平;彭季蘭;程智;;彩色多普勒血流顯像對(duì)滋養(yǎng)細(xì)胞腫瘤化療前后應(yīng)用研究[A];中華醫(yī)學(xué)會(huì)第六次全國(guó)超聲醫(yī)學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2001年

8 黃煜;李大金;;趨化因子CXCL16對(duì)早孕期人滋養(yǎng)細(xì)胞生物學(xué)功能的自分泌調(diào)控作用[A];首屆滬浙婦產(chǎn)科學(xué)術(shù)論壇暨2006年浙江省婦產(chǎn)科學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2006年

9 董e，

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