【作者】 黄伟；

【导师】 谢琪璇；

【作者基本信息】 暨南大学 ， 发育生物学， 2011， 硕士

【摘要】 目的：探索小鼠MSC具有多分化潜能的原因以及MSCs对卵巢GC的影响。方法：1、从小鼠骨髓中分离MSCs,并进行原代培养,分别于0、2、4、6、8、10天提取总RNA,RT-PCR检测多潜能标记基因和相关因子、三个胚层标记基因的表达情况。2、利用分离、纯化的MSCs与GC,建立共培养体系。实验分3组：单独培养MSCs组、单独培养GC组及MSCs和GC共培养组。分别在4天和6天提取总RNA,RT-PCR检测卵巢内标记基因在GC和MSCs中的表达情况,以及共培养MSCs多潜能标记基因和相关因子及三个胚层基因的表达情况。并在3天和5天通过MTT法检测各组细胞的增殖情况。结果：小鼠MSCs表达多潜能性标记基因Oct-4、nanog和相关因子Klf4和c-Myc.三个胚层基因nestin、SM22α、CYP51均有表达。在共培养实验中,单独培养和共培养GC均有ZP1.ZP2.ZP3和FSHR的表达,但ZP3.FSHR和BMP-15表达量不同。MSCs可促进GC增殖。不同天数单独培养和共培养MSCs均未检测到卵巢内标记基因的表达,但多能性基因和相关因子表达量不同,三个胚层基因表达无变化。此外,GC可促进MSCs增殖。结论：贴壁筛选得到的小鼠MSCs表达多能性标记基因以及三个胚层标记基因。在共培养体系中,GC与MSCs之间可以相互促进增殖并影响基因的表达。更多还原

【Abstract】 Objective:To explore the cause of multi-differentiation potential of mouse bone marrow mesenchymal stem cell(MSCs) and the effect of MSCs on mouse ovarian granulosa cells (GC) in vitro co-cultured.Methods:MSCs were isolated from the bone of the adult mouse and cultured in vitro.Total RNA were extracted from the mouse bone marrow (Day 0) and cultured mouse MSCs at 2,4,6,8 and10 days. The pluripotency marker genes Oct-4 and nanog, pluripotency related transcription factors Klf4 and c-Myc and the marker genes of three embryonal layers, such as CYP51, SM22αand nestin, which are typical genes specific for endoderm, mesoderm and ectoderm respectively, were detected by RT-PCR.MSCs and GC from mouse were cultured and purified in vitro using their adherent characteristics. A co-culture system was established by culturing MSCs in the Transwell insert and GC on the out plastic plates (6 wells). The cells were divided into 3 groups:alone cultured MSCs, alone cultured GC and MSCs co-cultured with GC. Total RNA were extracted from the cells of these 3 groups at the 4th day and the 6th day respectively. The ovarian marker genes mRNA were detected by RT-PCR. The pluripotency related genes and the three embryonal layers genes were also detected at the 4th day and the 6th days respectively. Cell proliferation of these 3 groups were detected by MTT assay after 3 days and 5 days respectively.Results:The mRNA of Oct-4, nanog, Klf4 and c-Myc were detected in mouse MSCs. Furthermore, in addition to SM22a, RT-PCR revealed the presence of mRNA for CYP51 and nestin during the primary culture of mouse MSCs. the mRNA of ZP1, ZP2 and ZP3, FSHR were detected in both alone cultured and co-cultured GC at the 4th day and the 6th day. But the mRNA of ZP3, FSHR and BMP-15 were different between co-cultured GC and alone cultured GC.The mouse MSCs promoted the proliferation of the mouse ovarian GC. No mRNA of ovarian characteristics genes were detected in alone cultured or co-cultured mouse MSCs. The mRNA of pluripotency marker genes and pluripotency related transcription factors were different between co-cultured MSCs and alone cultured MSCs, while the mRNA of genes of the three embryonal layers did not significantly change. In addition, the mouse ovarian GC promoted the proliferation of mouse MSCs.Conclusion:Mouse MSCs, which were isolated by adhesion purification, expressed the mRNA of the pluripotency marker genes and the marker genes of three embryonal layers. In vitro co-culture, the proliferation of the mouse ovarian GC and the mouse MSCs were promoted mutually and affected the quantity of mRNA for some genes.更多还原