脐带基质干细胞的分离、鉴定及其向生殖细胞分化的初步研究

发布时间：2018-06-12 01:06

本文选题：脐带 + 基质干细胞　；参考：《第三军医大学》2008年硕士论文

【摘要】： 目的:1.建立分离培养人脐带基质干细胞(umbilical cord matrix stem cells , UC- MSCs)的方法,了解其生物学特性及分化潜能。2.探讨UC-MSCs在体外向生殖细胞分化的潜能。3.观察UC-MSCs移植后在小鼠早衰卵巢中的定位及生长。方法:1.无菌条件下取正常足月顺产新生儿的脐带,用Ⅰ型胶原酶消化,分离单个核细胞,DMEM培养,获得贴壁细胞,监测细胞生长曲线、流式细胞仪检测其免疫表型。采用不同条件培养基诱导细胞向成脂、成骨、成软骨方向分化。2.通过RT-PCR检测UC-MSCs中生殖系特异性标记物的表达,采用卵泡液作为条件培养基,体外诱导UC-MSCs向生殖细胞分化。3.通过化疗药物处理建立卵巢早衰(Premature ovarian failure , POF)动物模型,腺病毒介导Ad-GFP转染UC-MSCs,经尾静脉移植于POF小鼠体内,在不同时间点取材进行冰冻切片,通过激光共聚焦检查移植细胞在卵巢中的定位和生长情况。结果: 1.分离的脐带单核细胞培养后呈纺锤体样,其倍增时间为31.11±2.45 h,体外增殖达10代以上,P3代细胞中约85.84%处于G0/ G1期, 14.16%的细胞处于S + G2 + M期。其分子免疫表型为:CD29、CD44、CD73(SH3)、CD90、CD105 (SH2)阳性;SSEA-4弱阳性;CD31、CD34、CD45、HLA-DR阴性,与BM-MSCs的免疫表型相似。在不同的诱导条件下,UC-MSCs可形成脂滴、骨结节、软骨结节等结构,并表达脂肪、骨及软骨细胞相关的特异性基因。2. UC-MSCs表达OCT4、Stella、Ifitm3等生殖系标记物,在含5%、10%、20%等不同浓度卵泡液的诱导条件下细胞可聚集分化形成卵母细胞样结构。3.采用环磷酰胺(cyclophosphamide , CTX)和白消安(busulfan , BUS)处理可成功建立小鼠卵巢早衰模型;腺病毒介导Ad-GFP转染UC-MSCs的效率达95%左右;UC-MSCs经尾静脉移植后,在受损小鼠卵巢中可发现大量GFP阳性细胞存活生长,但在大脑、肝脏、肾脏、胰腺等组织中未发现GFP阳性细胞存在。结论:1.人UC-MSCs可在体外培养、扩增,细胞免疫表型与BM-MSCs相似,具有成脂、成骨、成软骨等多向分化潜能,是一种理想的成体干细胞来源。2.UC-MSCs表达生殖系特异性标记物,体外诱导可形成卵母细胞样结构,初步表明UC-MSCs具有分化为生殖细胞的潜能。3.UC-MSCs移植后能够迁移至功能早衰卵巢并存活生长,提示UC-MSCs可能具有卵巢损伤修复的作用。
[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 卤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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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