本文選題：骨髓間充質(zhì)干細(xì)胞　切入點：急性腎損傷　出處：《南昌大學(xué)》2015年碩士論文

【摘要】：目的:1、通過生物素-鏈霉親和素反應(yīng)體系建立一種簡單可行的細(xì)胞表面化學(xué)修改方法。2、評價生物素和鏈霉親和素進(jìn)行骨髓間充質(zhì)干細(xì)胞表面標(biāo)記的效率。3、探討此種化學(xué)共價結(jié)合方法對骨髓間充質(zhì)干細(xì)胞生物學(xué)功能的影響,為這項技術(shù)在促進(jìn)干細(xì)胞在急性腎損傷中歸巢中的研究奠定實驗基礎(chǔ)。方法:1、通過全骨髓培養(yǎng)法得到第三代骨髓間充質(zhì)干細(xì)胞備用,并采用流式細(xì)胞儀鑒定BMSCs;2、以磺化生物素-N-羥基琥珀酰亞胺、生物素、鏈霉親和素為材料,通過共價結(jié)合的化學(xué)方法將粘附分子配體SLeX裝備到BMSCs表面;3、通過熒光顯微鏡評估化學(xué)修改方法進(jìn)行BMSCs表面標(biāo)記的效率;4、采用臺盼藍(lán)染色法檢測化學(xué)修改方法對BMSCs細(xì)胞活性的影響;5、CCK-8比色法檢測化學(xué)修改方法對BMSCs的細(xì)胞增殖功能影響;6、成脂、成骨誘導(dǎo)檢測化學(xué)修改方法對BMSCs多分化功能的影響。結(jié)果:1、骨髓間充質(zhì)干細(xì)胞表面標(biāo)志物鑒定:通過全骨髓培養(yǎng)法培養(yǎng)2周,可得到第三代BMSCs,采用流式細(xì)胞儀進(jìn)行BMSCs細(xì)胞表型鑒定,結(jié)果如下:CD9099.9%;CD29 99.8%,CD34 0.1%,CD45 0%,符合BMSCs表型2、化學(xué)修改方法效率的評估:通過生物素及鏈霉親和素進(jìn)行BMSCs表面標(biāo)記,細(xì)胞表面修改成功率達(dá)(87.8±3.1%),明顯比對照組B+S(33.2±1.3)%(P0.001)和P+S組(6.4±1.3%)(P0.001)高;3、化學(xué)修改方法對BMSCs生物學(xué)特性的影響:BMSCs細(xì)胞活性在BNHS化學(xué)修飾細(xì)胞后24h和48h細(xì)胞活性為83%和76%,CCK-8檢測細(xì)胞的增殖功能發(fā)現(xiàn),BNHS修飾后72h細(xì)胞增殖功能受一定影響。BNHS修飾的細(xì)胞和PBS處理后BMSCs均能被成功能誘導(dǎo)分化,成脂誘導(dǎo)23后進(jìn)行油紅染色均可見大量脂質(zhì)沉淀,成骨誘導(dǎo)23天后茜素紅染成骨分化細(xì)胞均可見明顯深紅色礦化結(jié)節(jié)。結(jié)論:1、本研究通過生物素-鏈霉親和素反應(yīng)體系成功將粘附分子配體裝備到骨髓間充質(zhì)干細(xì)胞表面,本方法進(jìn)行細(xì)胞表面標(biāo)記,操作簡單、反應(yīng)條件溫和。2、本標(biāo)記方法效率高,而且對細(xì)胞的毒性小,對細(xì)胞增殖、分化功能影響不大。3、這項技術(shù)對于針對特定組織的細(xì)胞靶向治療具有廣泛的運用前景,可能有助于促進(jìn)干細(xì)胞在急性腎損傷中的歸巢。
[Abstract]:Objective to establish a simple and feasible method of cell surface chemical modification by using biotin-streptavidin reaction system to evaluate the efficiency of biotin and streptavidin on the surface labeling of bone marrow mesenchymal stem cells (BMSCs), and to explore the effect of biotin and streptavidin on the surface labeling of bone marrow mesenchymal stem cells.Effects of chemical covalent binding methods on the biological function of bone marrow mesenchymal stem cellsIt provides experimental basis for the study of stem cells homing in acute renal injury.Methods the third generation of bone marrow mesenchymal stem cells were obtained by whole bone marrow culture. BMSCs 2 was identified by flow cytometry. Sulfonated biotin-N-hydroxysuccinimide, biotin and streptavidin were used as materials.The adhesion molecular ligand SLeX was equipped on the surface of BMSCs by covalent chemical method. The efficiency of BMSCs surface labeling by chemical modification method was evaluated by fluorescence microscope. The trypan blue staining method was used to detect the chemically modified BMSCs.The effect of CCK-8 colorimetric assay on the proliferation of BMSCs cellsEffects of chemical modification of osteogenic induction on the multidifferentiation of BMSCs.Results: the surface markers of bone marrow mesenchymal stem cells (BMSCs) were identified. The third generation of BMSCs was obtained by whole bone marrow culture for 2 weeks. The phenotype of BMSCs was identified by flow cytometry.The results were as follows: 1 / CD9099.9 / CD29 / 99.8 / CD34 / 0.1 / CD 450, according to the phenotype of BMSCs, and the evaluation of the efficiency of chemical modification method: biotin and streptavidin were used to mark the surface of BMSCs.緇嗚優(yōu)琛ㄩ潰淇敼鎴愬姛鐜囪揪(87.8鹵3.1%),鏄庢樉姣斿鐓х粍B S(33.2鹵1.3)%(P0.001)鍜孭 S緇，

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