脐带基质干细胞在不同诱导条件下向生殖细胞分化的实验研究

发布时间：2018-06-10 03:14

本文选题：脐带 + 基质干细胞　；参考：《第三军医大学》2009年硕士论文

【摘要】： 目的:1.建立分离培养脐带基质干细胞(umbilical cord matrix stem cells , UC-MSCs)的方法,了解其生物学特性及分化潜能。2.探讨体外微环境对UC-MSCs向生殖细胞分化的影响。3.观察UC-MSCs移植后在小鼠早衰卵巢中的分布及生长,探讨体内微环境对UC-MSCs分化的影响。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、成肌方向分化2.采用两种方法对UC-MSCs进行诱导分化,观察体外微环境对UC-MSCs分化的影响。①类胚体诱导:将UC-MSCs进行悬滴培养,使其形成类胚体(embryonic body,EB),进而采用将EB与人或小鼠卵巢颗粒细胞共培养、卵泡液条件培养基培养等方法,体外诱导UC-MSCs向早期生殖细胞分化。通过RT-PCR方法检测特异性基因的表达,流式细胞术和免疫组化检测其免疫表型,观察是否有生殖系特异性标记物的出现。②单层细胞诱导: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60COγ射线照射处理建立卵巢早衰(Premature ovarian failure , POF)动物模型,慢病毒介导绿色荧光蛋白(green fluorescent protein ,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,通过共聚焦显微镜和荧光原位杂交(Fluorescence in situ hybridization ,FISH)方法检测移植细胞在小鼠体内的分布和生长情况;通过计数非闭锁始基卵泡数目和测定雌激素水平等方法观察卵巢的损伤修复情况。结果:1.分离的脐带单个核细胞培养后呈纺锤体样,体外增殖达10代以上,P3代细胞中80以上处于G0/ G1期,约20%的细胞处于S + G2 +M期。其分子免疫表型为:CD44、CD73、CD90、CD105阳性,CD31、CD34、CD45、HLA-DR阴性,与骨髓间充质干细胞的免疫表型相似。在不同的诱导条件下,UC-MSCs可向成脂、成骨、成肌方向分化。 2.诱导分化培养结果①将UC-MSCs进行悬滴培养,其能够形成EB。②RT-PCR结果发现: EB形成后5天表达生殖系标记物Oct-4、Ifitm-3、stella、vasa、DAZL,作为对照的UC-MSCs仅表达Oct-4、Ifitm-3、stella。③流式细胞术检测结果:EB形成5天后,其中SSEA-1阳性细胞占15.61%。④免疫组化检测结果:形成后5天的EB与人或小鼠的卵巢颗粒细胞共培养,10天后生殖系标记物stella、vasa、SCP3、GDF-9阳性表达,而颗粒细胞及采用卵泡液培养的EB均无表达。⑤单层细胞培养诱导:UC-MSCs用含5%卵泡液培养基诱导20天后,细胞卷曲形成类组织样团块,团块周边不断有悬浮细胞生成并随之脱落,但未检测到生殖系特异性标记物的表达。3.①60COγ射线处理后病理切片提示:小鼠短期内即出现卵巢储备下降、卵泡闭锁、卵巢萎缩等POF样病变,心脏、肝脏、脾脏、肺脏、肾脏、肠道、大脑、骨髓等组织未见明显病理损伤,说明模型构建成功。②慢病毒介导GFP转染UC-MSCs的效率达80%以上;UC-MSCs经尾静脉移植后,在受损小鼠卵巢中可发现大量GFP阳性细胞存活并生长,但在心脏、肝脏、脾脏、肺脏、肾脏、肠道、大脑、骨髓等组织中均未发现GFP阳性细胞存在。③卵泡计数和雌激素水平均提示干细胞移植组小鼠卵巢损伤修复情况比对照组小鼠修复情况好。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、成肌等多向分化潜能,是一种理想的成体干细胞来源。2.UC-MSC体外悬滴培养可形成EB,与人或小鼠卵巢颗粒细胞共培养后均表达生殖系特异性标记物,提示体外微环境对UC-MSCs向生殖细胞方向分化发挥十分重要的作用,初步表明UC-MSCs具有向早期生殖细胞分化的潜能。3.UC-MSCs移植后能够迁移至功能早衰卵巢并存活生长,卵泡计数和E2水平提示UC-MSCs可能具有卵巢损伤修复作用,表明体内微环境对UC-MSCs的的分化也发挥重要作用。
[Abstract]:Objective: 1. to establish a method of isolation and culture of umbilical cord matrix stem cells (UC-MSCs), and to understand its biological characteristics and differentiation potential.2. to explore the effect of.2. on the differentiation of UC-MSCs to germ cells in vitro..3. observation of the distribution and growth of UC-MSCs after UC-MSCs transplantation in the premature ovarian failure of mice, and the study of the microenvironment in vivo to UC. The effect of -MSCs differentiation.
Methods: 1. the umbilical cord of normal full-term newborns was taken under aseptic condition. The mononuclear cells were digested with type I collagenase, and the mononuclear cells were isolated and cultured in DMEM. The adherent cells were obtained. The cell growth curve was monitored. The immunophenotype was detected by flow cytometry. Two methods were used to induce the cells to be fat, osteogenic and myogenic differentiation by different conditions. UC-MSCs was induced and differentiated, and the effect of microenvironment in vitro on UC-MSCs differentiation was observed. (1) embryoid body induction: UC-MSCs was cultured to form embryo like body (embryonic body, EB), and the methods of co culture of EB with human or mouse ovarian granulosa cells and culture medium culture of follicle liquid were used to induce early reproduction of UC-MSCs to early reproduction. Cell differentiation. The expression of specific genes was detected by RT-PCR, flow cytometry and immunohistochemistry were used to detect its immunophenotype, and the appearance of specific markers in the reproductive system was observed. 2 monolayer cells were induced: using follicular fluid as the conditioned medium, and inducing the differentiation of UC-MSCs into the germ cell differentiation.3. by 60CO gamma ray irradiation in vitro The animal model of Premature ovarian failure (POF) was established. The lentivirus mediated green fluorescent protein (green fluorescent protein, GFP) transfected UC-MSCs and transplanted into POF mice through the tail vein. The methods were obtained at different time points, through confocal microscopy and fluorescein in situ hybridization (Fluorescence in) method. The distribution and growth of the transplanted cells in the mice were detected, and the repair of the ovarian injury was observed by counting the number of non atresia base follicles and determining the level of estrogen.
Results: 1. the 1. isolated umbilical cord mononuclear cells showed spindle like, in vitro proliferation of more than 10 generations, more than 80 of P3 cells were in G0/ G1 phase, and about 20% of the cells were in the S + G2 +M phase. The immunophenotype of the cells was CD44, CD73, CD90, CD105 positive, CD31, CD34, and negative, similar to the immunophenotype of bone marrow mesenchymal stem cells. Under the same induction conditions, UC-MSCs can differentiate into adipocytes, osteoblasts and myoblasts.
2. induced differentiation and culture results (1) the suspension culture of UC-MSCs was carried out, and it could form EB. (RT-PCR). The results showed that the reproductive system markers were expressed as Oct-4, Ifitm-3, Stella, Vasa, DAZL 5 days after the formation of EB, and the UC-MSCs only expressed Oct-4, Ifitm-3, and flow cytometry: 5 days later, the positive cells accounted for 15.6. 1%. (4) immunohistochemical detection results: 5 days after formation of EB and human or mouse ovarian granulosa cells co culture, 10 days later, the reproductive markers Stella, Vasa, SCP3, GDF-9 positive expression, but the granular cells and the follicle culture of EB were not expressed. 5 monolayer culture induction: UC-MSCs with 5% follicle culture medium induction 20 days, cell volume In the form of tissue like mass, there were suspending cells in the periphery of the group. But the expression of the specific markers of the reproductive system was not detected. The pathological sections of the.3. (60CO) gamma ray treatment suggested that the mice had POF like lesions, such as ovarian reserve, follicle atresia, ovarian atrophy, and the heart, liver, spleen, lungs and kidneys in the short term. There was no obvious pathological damage in the intestine, brain, bone marrow and other tissues, indicating the success of the model construction. (2) the efficiency of GFP transfected by lentivirus was more than 80%. After the transplantation of the tail vein, UC-MSCs could find a large number of GFP positive cells in the injured mouse's ovary to survive and grow, but in the heart, liver, spleen, lungs, kidney, intestines, brain, bone marrow, and so on GFP positive cells were not found in the tissue. The follicle count and estrogen level all suggest that the repair of ovarian injury in the stem cell transplantation group is better than that of the control group.
Conclusion: 1. human UC-MSCs can be cultured and amplified in vitro, and the cell immunophenotype is similar to BM-MSCs. It has multipotential differentiation potential, such as fat formation, osteogenesis and myoblast. It is an ideal adult stem cell source.2.UC-MSC in vitro suspension culture can form EB, and the reproductive system specific markers are expressed after CO culture with human or mouse ovarian granulosa cells. The external microenvironment plays an important role in the direction differentiation of UC-MSCs to the germ cells. It is preliminarily indicated that UC-MSCs has the potential to differentiate into early germ cells..3.UC-MSCs can migrate to the function of premature ovarian failure and survive. The follicle count and the level of E2 suggest that UC-MSCs may have the repair effect of ovarian injury, indicating the microloop in the body. It also plays an important role in the differentiation of UC-MSCs.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【引证文献】