本文選題：蛻膜基質(zhì)細(xì)胞 + 傳代次數(shù)��； 參考：《山東大學(xué)》2010年碩士論文

【摘要】： 從免疫學(xué)角度講,妊娠相當(dāng)于成功的半同種異體移植,妊娠建立與維持取決于胚胎植入及發(fā)育過程中母體蛻膜局部乃至全身免疫系統(tǒng)的狀態(tài)。母體與胎兒之間維持著精妙的免疫平衡,一方面耐受胎兒使其得以植入并在宮內(nèi)正常發(fā)育,另一方面適度活化抵抗病原體侵襲,以免感染后蛻膜局部免疫系統(tǒng)發(fā)生變化,打破母胎間的免疫平衡,使母體排斥胎兒,進(jìn)而引起流產(chǎn)、胎兒畸形、胎兒宮內(nèi)生長受限、子癇前期子癇等病理妊娠的發(fā)生。母胎之間的免疫平衡有賴于多種免疫細(xì)胞和細(xì)胞因子的協(xié)調(diào)作用,研究發(fā)現(xiàn)單核細(xì)胞趨化蛋白-1(MCP-1)具有抗感染及介導(dǎo)耐受的雙向作用,妊娠早期蛻膜基質(zhì)細(xì)胞高表達(dá)MCP-1及其受體CCR2,多種炎性因子可刺激蛻膜基質(zhì)細(xì)胞(DSC)分泌MCP-1,提示其在感染過程中蛻膜局部免疫平衡的維持方面具有重要作用,因此推測感染可影響其在DSC的表達(dá)。目前尚無DSC培養(yǎng)的公認(rèn)方法,為建立一種科學(xué)的DSC體外培證明本實(shí)驗(yàn)結(jié)果的可靠性,本實(shí)驗(yàn)同時(shí)進(jìn)行了DSC體外培養(yǎng)的方法學(xué)探討。 目的： 觀察體外模擬早孕微環(huán)境(雌激素、孕激素)條件下革蘭氏陰性細(xì)菌內(nèi)毒素脂多糖(LPS)對(duì)人蛻膜基質(zhì)細(xì)胞MCP-1表達(dá)的影響,同時(shí)探討蛻膜基質(zhì)細(xì)胞體外培養(yǎng)時(shí)傳代純化法對(duì)該表達(dá)的影響。 方法： 選取正常妊娠婦女人工流產(chǎn)蛻膜組織(6-8周),按常規(guī)方法分離蛻膜基質(zhì)細(xì)胞,在含有或不含有雌激素、孕激素培養(yǎng)基中傳代培養(yǎng),普通光鏡下分別觀察第一代(P1)與第三代細(xì)胞(P3)的形態(tài)；采用免疫細(xì)胞化學(xué)法(細(xì)胞角蛋白7和波形蛋白)分別鑒定兩代細(xì)胞的純度,采用流式細(xì)胞技術(shù)檢測兩代細(xì)胞表面Toll樣受體-4(TLR4)、CCR2分子的表達(dá)水平；當(dāng)細(xì)胞鋪板率為80%時(shí),將第一代細(xì)胞和第三代細(xì)胞分別分為空白對(duì)照組、LPS (1μg/mL)刺激組。另外,細(xì)胞分別在含與不含雌激素+孕激素的培養(yǎng)基中培養(yǎng)傳代三次后各分為空白對(duì)照組、LPS刺激組。37℃、5%CO2培養(yǎng)24h后收集上清,Trizol裂解后按常規(guī)方法提取總RNA,用RT-PCR與實(shí)時(shí)定量PCR技術(shù)檢測MCP-1轉(zhuǎn)錄水平的表達(dá),ELISA法檢測上清中MCP-1的表達(dá)。 結(jié)果： 1、DSC體外培養(yǎng)的方法學(xué)探討： ①與傳代一次的細(xì)胞相比,傳代三次的細(xì)胞形態(tài)變規(guī)則,細(xì)胞突起減少,胞漿內(nèi)顆粒明顯減少,透明度增加,極性減弱或者消失。 ②免疫細(xì)胞化學(xué)染色顯示,傳代三次細(xì)胞較傳代一次細(xì)胞DSC純度增加(P0.05)。 ③流式細(xì)胞儀檢測結(jié)果顯示,與傳代一次細(xì)胞相比,傳代三次細(xì)胞表面TLR4、CCR2分子表達(dá)下調(diào)(P0.05)。 2、LPS對(duì)DSC MCP-1表達(dá)水平的影響： ①RT-PCR、ELISA顯示,,傳代一次細(xì)胞LPS刺激前即高表達(dá)MCP-1,刺激后變化無統(tǒng)計(jì)學(xué)差異(P□0.05)；與傳代一次細(xì)胞空白對(duì)照組相比,傳代三次細(xì)胞空白對(duì)照組MCP-1表達(dá)水平顯著降低(P0.05),但經(jīng)LPS刺激后,其LPS刺激組較對(duì)照組顯著上調(diào)(P0.05)；傳代三次細(xì)胞LPS刺激組MCP-1表達(dá)水平與傳代一次細(xì)胞LPS刺激組無明顯差異(P□0.05)。 ②RT-PCR、實(shí)時(shí)定量PCR、ELISA顯示,與空白對(duì)照組相比,傳代三次細(xì)胞LPS刺激組、雌激素+孕激素處理組、雌激素+孕激素+LPS處理組在轉(zhuǎn)錄水平與分泌水平上MCP-1的表達(dá)顯著上調(diào)(P0.05)；與雌激素+孕激素處理組和單純LPS刺激組相比,雌激素+孕激素+LPS處理組MCP-1表達(dá)水平顯著上調(diào)(P0.05)。 結(jié)論： 1、傳代可純化分離培養(yǎng)的DSC,但也使其胞膜表面分子TLR4、CCR2表達(dá)水平下降。 2、LPS通過與DSC表面TLR4作用,直接或間接地在轉(zhuǎn)錄水平與分泌水平上調(diào)DSC的MCP-1表達(dá),影響蛻膜局部免疫狀態(tài)。 3、雌、孕激素聯(lián)合作用可上調(diào)早孕期DSC MCP-1的表達(dá),提示MCP-1可能對(duì)于正常妊娠的發(fā)生和維持具有不可忽視的作用。 4、雌、孕激素聯(lián)合作用還可增強(qiáng)LPS對(duì)MCP-1的上調(diào)作用,說明MCP-1在妊娠早期蛻膜抗感染方面有重要意義。 創(chuàng)新點(diǎn)： 1、取材新：目前國內(nèi)外研究妊娠蛻膜微環(huán)境的實(shí)驗(yàn)室及相關(guān)文獻(xiàn)報(bào)道較少。 2、本實(shí)驗(yàn)首次發(fā)現(xiàn)LPS可上調(diào)蛻膜基質(zhì)細(xì)胞MCP-1表達(dá)水平。 3、本實(shí)驗(yàn)首次對(duì)傳代培養(yǎng)DSC的方法進(jìn)行了較完善的研究,發(fā)現(xiàn)傳代可下調(diào)DSC表面TLR4表達(dá)水平,但不影響本實(shí)驗(yàn)過程中TLR4的活性。
[Abstract]:From an immunological point of view, pregnancy is equivalent to a successful semi allograft. The establishment and maintenance of pregnancy depends on the state of the part of the mother's decidua and the whole body immune system during the implantation and development of the embryo. The mother and the fetus maintain an excellent immune balance between the fetus and the fetus to be implanted in the uterus and develop normally in the uterus. On the one hand, it is moderately activated to resist the invasion of the pathogen, so as to avoid the changes in the local immune system of the decidua, break the immune balance between the mother fetus, make the mother repel the fetus, cause abortion, fetal malformation, fetal intrauterine growth and eclampsia and other pathological pregnancies. The immune balance between the mother's fetus depends on a variety of immune fines. In the coordination of cell and cytokine, it is found that monocyte chemoattractant protein -1 (MCP-1) has a two-way effect of anti infection and mediated tolerance. Early decidual stromal cells express MCP-1 and its receptor CCR2 in early pregnancy, and many inflammatory factors can stimulate decidual stromal cells (DSC) to secrete MCP-1, suggesting that the decidual immune balance in the process of infection is partial to the partial immune balance of decidua. It is important to maintain that infection can affect the expression of DSC. At present, there is no recognized method of DSC culture to establish a scientific DSC in vitro culture to prove the reliability of the results of the experiment. This experiment has also carried out a methodological approach to the culture of DSC in vitro.
Objective:
To observe the effect of gram-negative bacterial lipopolysaccharide (LPS) on the expression of MCP-1 in human decidua stromal cells under the conditions of early pregnancy microenvironment (estrogen and progesterone), and to explore the effect of the purification of the decidual stromal cells on the expression of the decidual stromal cells in vitro.
Method錛，

本文編號(hào)：2000057