诱导异种核移植来源的胚胎干细胞（rhSCNT-ESCs）向造血分化

发布时间：2018-03-28 10:28

本文选题：异种　切入点：核移植　出处：《山东大学》2006年硕士论文

【摘要】：第一部分：异种核移植来源的胚胎干细胞(rhSCNT-ESCs)分化为造血干细胞研究目的： rhSCNT-ESCs来源于移植人体细胞核入去核的兔卵母细胞中，因而具有与供体细胞一致的基因类型。本项研究采用不同的诱导条件，通过流式分析与免疫染色比较，旨存探索rhSCNT-ESCs的定向诱导造血方案，为下一步的临床应用奠定实验基础。研究方法： (1) 本研究尝试使用MEF、FL和BM来源的基质细胞为饲养层，将其与rhSCNT-ES细胞共培养，流式检测培养不同时间段CD34~+细胞百分含量。 (2) 采用分阶段诱导造血法，将传代的rhSCNT-ES细胞微团接种于低粘附六孔板中，添加不同组合的生长因子，，选取不同阶段的细胞进行免疫染色。研究结果： (1) 不添加任何外源性细胞因子，rhSCNT-ESCs与基质细胞共培养，在第3～4天出现造血细胞簇，第14天可见造血集落CFU-GEMM。体外培养过程中，CD34~+细胞百分比逐渐增加，三组均在第7天达峰。MEF共培养组ES细胞造血分化能力较弱，与另外两组相比有显著差异。BMSC及FLSC饲养层支持ES细胞向造血分化的能力相近，CD34~+细胞百分比在第7天与第14天出现两个峰值。 (2) rhSCNT-ES细胞微团在分化培养液中2天可观察到简单拟胚体细胞团生成，4天时生成的囊状拟胚体直径约有100-150μm，大小基本均一。将第一阶段的EBs消化成单细胞后种植在甲基纤维素培养基中，3～4天后第一阶段添加中胚层诱导因子BMP_4的第4，5组可观察到明显的呈集落状生长的细胞。此时的细胞CD34及KDR抗体免疫染色呈阳性。结论：
[Abstract]:Part I: differentiation of embryonic stem cells from xenotransplantation into hematopoietic stem cells. Objectives of the study:. RhSCNT-ESCs is derived from rabbit oocytes transplanted with human nuclei and thus has the same gene type as donor cells. In this study, different induction conditions were used, flow analysis and immunostaining were used to compare the results. The aim of this study was to explore the directed induction of hematopoiesis in rhSCNT-ESCs, and to lay an experimental foundation for clinical application in the next step. Research methods:. 1) in this study, the stromal cells derived from MEF FL and BM were used as feeder layer, co-cultured with rhSCNT-ES cells, and the percentage of CD34 ~ cells was detected by flow cytometry. The passage of rhSCNT-ES cells was inoculated into the low adhesion six-hole plate with different combinations of growth factors and the cells in different stages were selected for immunological staining by stepwise induction of hematopoiesis. Results of the study:. Without adding any exogenous cytokines rhSCNT-ESCs co-cultured with stromal cells, hematopoietic cell clusters appeared on day 34, and hematopoietic colony CFU-GEMMM were observed on day 14. The percentage of CD34 ~ cells increased gradually during in vitro culture. The hematopoietic differentiation ability of es cells in the three groups was weak on the 7th day. Compared with the other two groups, there were significant differences. The percentage of CD34 ~ cells in es cells in the feeder layer of BMSC and FLSC was similar to that in the other two groups. The percentage of CD34 ~ cells appeared two peaks on day 7 and day 14. (2) rhSCNT-ES cell microclusters were observed in differentiation medium for 2 days. The diameter of the vesicular embryoid bodies was about 100-150 渭 m after the formation of simple embryoid somatoid clusters for 4 days. The EBs of the first stage was digested into single cells and planted in A cells. In the first stage of the basal cellulosic medium, 4 days later, the cells with colony growth were observed in the first stage of addition of mesoderm inducible factor BMP_4. The CD34 and KDR antibody immunoreactivity of the cells were positive at this time. Conclusion:
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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