本文選題：臍帶 + 基質(zhì)干細胞　； 參考：《第三軍醫(yī)大學(xué)》2008年碩士論文

【摘要】： 目的:1.建立分離培養(yǎng)人臍帶基質(zhì)干細胞(umbilical cord matrix stem cells , UC- MSCs)的方法,了解其生物學(xué)特性及分化潛能。2.探討UC-MSCs在體外向生殖細胞分化的潛能。3.觀察UC-MSCs移植后在小鼠早衰卵巢中的定位及生長。 方法:1.無菌條件下取正常足月順產(chǎn)新生兒的臍帶,用Ⅰ型膠原酶消化,分離單個核細胞,DMEM培養(yǎng),獲得貼壁細胞,監(jiān)測細胞生長曲線、流式細胞儀檢測其免疫表型。采用不同條件培養(yǎng)基誘導(dǎo)細胞向成脂、成骨、成軟骨方向分化。2.通過RT-PCR檢測UC-MSCs中生殖系特異性標記物的表達,采用卵泡液作為條件培養(yǎng)基,體外誘導(dǎo)UC-MSCs向生殖細胞分化。3.通過化療藥物處理建立卵巢早衰(Premature ovarian failure , POF)動物模型,腺病毒介導(dǎo)Ad-GFP轉(zhuǎn)染UC-MSCs,經(jīng)尾靜脈移植于POF小鼠體內(nèi),在不同時間點取材進行冰凍切片,通過激光共聚焦檢查移植細胞在卵巢中的定位和生長情況。 結(jié)果: 1.分離的臍帶單核細胞培養(yǎng)后呈紡錘體樣,其倍增時間為31.11±2.45 h,體外增殖達10代以上,P3代細胞中約85.84%處于G0/ G1期, 14.16%的細胞處于S + G2 + M期。其分子免疫表型為:CD29、CD44、CD73(SH3)、CD90、CD105 (SH2)陽性;SSEA-4弱陽性;CD31、CD34、CD45、HLA-DR陰性,與BM-MSCs的免疫表型相似。在不同的誘導(dǎo)條件下,UC-MSCs可形成脂滴、骨結(jié)節(jié)、軟骨結(jié)節(jié)等結(jié)構(gòu),并表達脂肪、骨及軟骨細胞相關(guān)的特異性基因。2. UC-MSCs表達OCT4、Stella、Ifitm3等生殖系標記物,在含5%、10%、20%等不同濃度卵泡液的誘導(dǎo)條件下細胞可聚集分化形成卵母細胞樣結(jié)構(gòu)。3.采用環(huán)磷酰胺(cyclophosphamide , CTX)和白消安(busulfan , BUS)處理可成功建立小鼠卵巢早衰模型;腺病毒介導(dǎo)Ad-GFP轉(zhuǎn)染UC-MSCs的效率達95%左右;UC-MSCs經(jīng)尾靜脈移植后,在受損小鼠卵巢中可發(fā)現(xiàn)大量GFP陽性細胞存活生長,但在大腦、肝臟、腎臟、胰腺等組織中未發(fā)現(xiàn)GFP陽性細胞存在。 結(jié)論:1.人UC-MSCs可在體外培養(yǎng)、擴增,細胞免疫表型與BM-MSCs相似,具有成脂、成骨、成軟骨等多向分化潛能,是一種理想的成體干細胞來源。2.UC-MSCs表達生殖系特異性標記物,體外誘導(dǎo)可形成卵母細胞樣結(jié)構(gòu),初步表明UC-MSCs具有分化為生殖細胞的潛能。3.UC-MSCs移植后能夠遷移至功能早衰卵巢并存活生長,提示UC-MSCs可能具有卵巢損傷修復(fù)的作用。
[Abstract]:Purpose 1. To establish a method of isolation and culture of human umbilical cord stromal stem cells (UC- MSC) and to understand its biological characteristics and differentiation potential. 2. To investigate the potential of UC-MSCs to differentiate into germ cells in vitro. Objective: to observe the localization and growth of UC-MSCs in mouse ovarian premature failure after transplantation. Umbilical cord of normal full-term homogenetic newborns was collected under aseptic condition, digested with type I collagenase, mononuclear cells were isolated and cultured with DMEM, adherent cells were obtained, cell growth curve was monitored, and immunophenotype was detected by flow cytometry. Different conditions were used to induce adipogenesis, osteogenesis and cartilage differentiation. The expression of reproductive lineage specific markers in UC-MSCs was detected by RT-PCR. Follicular fluid was used as a conditioned medium to induce UC-MSCs to differentiate into germ cells in vitro. The animal model of premature ovarian failure, POF was established by chemotherapeutic drug treatment. Ad-GFP was transfected into UC-MSCs by adenovirus, then transplanted into mice via tail vein, and frozen sections were obtained at different time points. The localization and growth of transplanted cells in ovary were examined by confocal laser. Results: 1. The isolated umbilical cord monocytes were spindle-like after culture, and the doubling time was 31.11 鹵2.45 h. About 85.84% of the cultured cells were in G _ 0 / G _ 1 phase and 14.16% were in S _ 2M phase. Its molecular immunophenotype was CD29, CD44, CD73, SH3, CD90, CD105, SH2), SSEA-4 weak positive, CD31, CD34, CD45, CD45, HLA-DR negative, similar to the immunophenotype of BM-MSCs. Under different induction conditions, UC-MSCs could form lipid droplets, bone nodules and cartilage nodules, and express specific genes of fat, bone and chondrocytes. UC-MSCs expressed OCT4Stella-Ifitm3 and other reproductive markers. Under the conditions of different concentrations of follicular fluid, such as 10% or 20%, the cells could aggregate and differentiate to form oocyte like structure. Cyclophosphamide (CTX) and busulfan (BuUS) were used to establish mouse ovarian premature failure model, and adenovirus-mediated Ad-GFP transfection efficiency of UC-MSCs was about 95% after tail vein transplantation, and UC-MSCs were successfully transfected into UC-MSCs by adenovirus-mediated transfection of UC-MSCs. A large number of GFP positive cells were found in the ovary of damaged mice, but no GFP positive cells were found in brain, liver, kidney and pancreas. Conclusion: 1. Human UC-MSCs can be cultured and amplified in vitro. The immunophenotype of UC-MSCs is similar to that of BM-MSCs, and has the potential to differentiate into lipid, osteogenesis and cartilage, which is an ideal source of adult stem cells. 2. UC-MSCs express reproductive lineage specific markers. UC-MSCs could be induced to form oocyte like structure in vitro, which indicated that UC-MSCs had the potential to differentiate into germ cells. 3. UC-MSCs could migrate to premature ovarian failure and survive and grow after transplantation, suggesting that UC-MSCs might play a role in ovarian damage and repair.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

【參考文獻】

相關(guān)期刊論文 前4條

1 鄧黎;梁志清;李俊男;李宇迪;徐惠成;史常旭;;人臍血間充質(zhì)干細胞體外分離、培養(yǎng)與鑒定[J];重慶醫(yī)學(xué);2007年08期

2 姜曉兵,趙洪洋,周鳳,劉如恩,周偉,張建國;EGFP在EGFP/GDNF融合基因修飾骨髓基質(zhì)干細胞中的示蹤作用[J];中華神經(jīng)外科疾病研究雜志;2004年06期

3 郭連瑞,谷涌泉,張建,張淑文,吳英鋒,崔葉青,郭德玉,汪忠鎬;不同途徑移植骨髓單個核細胞治療大鼠后肢缺血[J];中國臨床康復(fù);2005年10期

4 李東,崔磊,舒朝鋒,柳向東,張英,柴崗,劉偉,曹誼林;熒光活性染料DiI標記觀察人骨髓基質(zhì)干細胞與部分脫鈣骨粘附的實驗研究[J];中華創(chuàng)傷骨科雜志;2004年03期

，

本文編號：2007528