本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：角膜緣干細(xì)胞　出處：《南昌大學(xué)》2009年碩士論文

【摘要】： 目的:探討體外誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞(HBMSCs,human bone mesenchymal stem cells)分化為角膜緣干細(xì)胞(LSCs,limbal stem cells)的可行性。 方法:用密度梯度離心法體外分離HBMSCs,在含10%胎牛血清(FBS,fetal bovine serum)的LG-DMEM/F12培養(yǎng)基中擴(kuò)增培養(yǎng),流式細(xì)胞儀檢測(cè)細(xì)胞的表面抗原;用消化后組織塊培養(yǎng)法分離培養(yǎng)LSCs,在含20%FBS的HG-DMEM/F12培養(yǎng)基中擴(kuò)增培養(yǎng),流式細(xì)胞儀檢測(cè)細(xì)胞的核心抗原P63。將第四次傳代的HBMSCs按5 X 104/ml的細(xì)胞密度接種于六孔Transwell培養(yǎng)板底層,分成兩組:實(shí)驗(yàn)組將第二次傳代的LSCs按5 X 104/ml接種于Transwell培養(yǎng)板上層,用一種特殊的培養(yǎng)基(含90% LG-DMEM/F12,10%FBS,10μg/ml EGF, 10ng/ml bFGF,1μg/mLPS)共同培養(yǎng)10天。對(duì)照組不加任何誘導(dǎo)劑,以原LG-DMEM/F12培養(yǎng)基培養(yǎng)。在相差顯微鏡下進(jìn)行形態(tài)學(xué)觀察。 結(jié)果:密度梯度離心法能分離出純度較高的HBMSCs。典型的HBMSCs貼壁生長(zhǎng),呈長(zhǎng)梭形,漩渦狀盤(pán)旋排列。流式細(xì)胞分析結(jié)果顯示第四代HBMSCs表達(dá)相關(guān)的抗原標(biāo)記CD29(96.78%), CD44(96.23%), CD105(91.45%), CD166(81.70%),不表達(dá)造血細(xì)胞系的表面標(biāo)志CD34(2.34%), CD45(3.92%)及主要組織相容性抗原HLA-DR(1.11%)。在體外成功培養(yǎng)出LSCs,細(xì)胞呈圓形、橢圓形、多角形,胞體透亮,約2周后細(xì)胞融合呈鑲嵌狀排列。第二代LSCs表達(dá)P63(83.35%)。實(shí)驗(yàn)組共同培養(yǎng)72小時(shí)后,貼壁HBMSCs部分呈橢圓形、圓形改變,在體外微環(huán)境中誘導(dǎo)10天后大部分細(xì)胞呈多邊形、圓形、橢圓形,少量細(xì)胞呈梭形。對(duì)照組細(xì)胞呈典型HBMSCs貼壁生長(zhǎng),以長(zhǎng)梭形為主。試驗(yàn)組P63弱陽(yáng)性(7.16±0.56%),對(duì)照組P63陰性(0.74±0.49%)。 結(jié)論:與受炎性刺激的LSCs共培養(yǎng)后,HBMSCs有橫向分化為類LSCs細(xì)胞的能力。
[Abstract]:Aim: to investigate the feasibility of inducing the differentiation of human bone mesenchymal stem cells from human bone marrow mesenchymal stem cells into limbal stem cells in vitro. Methods: HBMSCs were isolated by density gradient centrifugation in vitro and cultured in LG-DMEM/F12 medium containing 10% fetal bovine serum (FBS). Flow cytometry was used to detect the surface antigen of the cells. LSCs were isolated and cultured by digested tissue mass culture method, and amplified in HG-DMEM/F12 medium containing 20s. The core antigen P63of the cells was detected by flow cytometry. The fourth passage of HBMSCs was inoculated at the bottom of the six-well Transwell culture plate according to the cell density of 5 X 104/ml. The experimental group was divided into two groups: the second passage of LSCs was inoculated on the top of the Transwell culture plate according to 5X 104/ml, and co-cultured on a special medium (90% LG-DMEM / F12, 10s 10 渭 g/ml EGF, 10ng/ml bFGF-1 渭 g / mLPS-1 渭 g / mLPS1 渭 g / mL PSS) for 10 days, the control group was not treated with any inducer. The morphology was observed under phase contrast microscope in the culture medium of LG-DMEM/F12. Results: high purity HBMSCs could be isolated by density gradient centrifugation. Typical HBMSCs adherent growth was long fusiform. The results of flow cytometry showed that the antigen markers associated with the expression of HBMSCs in the fourth generation were CD2996.78, CD44C96.23K, CD105, 91.4545, CD166, 81.70T, the surface markers of hematopoietic cell line CD34C ~ 2.34, CD453.922), and the main histocompatibility antigen HLA-DR1.11T. The cells were round, and the cells were round and round. Oval, polygonal and bright cell bodies. After about 2 weeks, the cells were arranged in mosaics. The second generation of LSCs expressed P63O83.35. After 72 hours of co-culture, the HBMSCs parts of the experimental group were oval and round. After 10 days of induction in microenvironment in vitro, most of the cells were polygonal, round, oval, and a few cells were fusiform. The cells of the control group were typical HBMSCs adherent growth, mainly long fusiform. In the test group, the P63 weak positive cells were 7.16 鹵0.56 and those in the control group were 0.74 鹵0.49m. Conclusion: hBMSCs co-cultured with inflammatory LSCs have the ability to differentiate into LSCs like cells.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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