本文選題：異種　切入點(diǎn)：核移植　出處：《山東大學(xué)》2006年碩士論文

【摘要】：第一部分：異種核移植來(lái)源的胚胎干細(xì)胞(rhSCNT-ESCs)分化為造血干細(xì)胞 研究目的： rhSCNT-ESCs來(lái)源于移植人體細(xì)胞核入去核的兔卵母細(xì)胞中，因而具有與供體細(xì)胞一致的基因類型。本項(xiàng)研究采用不同的誘導(dǎo)條件，通過(guò)流式分析與免疫染色比較，旨存探索rhSCNT-ESCs的定向誘導(dǎo)造血方案，為下一步的臨床應(yīng)用奠定實(shí)驗(yàn)基礎(chǔ)。 研究方法： (1) 本研究嘗試使用MEF、FL和BM來(lái)源的基質(zhì)細(xì)胞為飼養(yǎng)層，將其與rhSCNT-ES細(xì)胞共培養(yǎng)，流式檢測(cè)培養(yǎng)不同時(shí)間段CD34~+細(xì)胞百分含量。 (2) 采用分階段誘導(dǎo)造血法，將傳代的rhSCNT-ES細(xì)胞微團(tuán)接種于低粘附六孔板中，添加不同組合的生長(zhǎng)因子，，選取不同階段的細(xì)胞進(jìn)行免疫染色。 研究結(jié)果： (1) 不添加任何外源性細(xì)胞因子，rhSCNT-ESCs與基質(zhì)細(xì)胞共培養(yǎng)，在第3～4天出現(xiàn)造血細(xì)胞簇，第14天可見造血集落CFU-GEMM。體外培養(yǎng)過(guò)程中，CD34~+細(xì)胞百分比逐漸增加，三組均在第7天達(dá)峰。MEF共培養(yǎng)組ES細(xì)胞造血分化能力較弱，與另外兩組相比有顯著差異。BMSC及FLSC飼養(yǎng)層支持ES細(xì)胞向造血分化的能力相近，CD34~+細(xì)胞百分比在第7天與第14天出現(xiàn)兩個(gè)峰值。 (2) rhSCNT-ES細(xì)胞微團(tuán)在分化培養(yǎng)液中2天可觀察到簡(jiǎn)單擬胚體細(xì)胞團(tuán)生成，4天時(shí)生成的囊狀擬胚體直徑約有100-150μm，大小基本均一。將第一階段的EBs消化成單細(xì)胞后種植在甲基纖維素培養(yǎng)基中，3～4天后第一階段添加中胚層誘導(dǎo)因子BMP_4的第4，5組可觀察到明顯的呈集落狀生長(zhǎng)的細(xì)胞。此時(shí)的細(xì)胞CD34及KDR抗體免疫染色呈陽(yáng)性。 結(jié)論：
[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:
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329

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