利用生物素—链霉亲和素进行骨髓间充质干细胞表面标记的实验研究

发布时间：2018-04-04 01:29

  本文选题：骨髓间充质干细胞　切入点：急性肾损伤　出处：《南昌大学》2015年硕士论文

【摘要】：目的:1、通过生物素-链霉亲和素反应体系建立一种简单可行的细胞表面化学修改方法。2、评价生物素和链霉亲和素进行骨髓间充质干细胞表面标记的效率。3、探讨此种化学共价结合方法对骨髓间充质干细胞生物学功能的影响,为这项技术在促进干细胞在急性肾损伤中归巢中的研究奠定实验基础。方法:1、通过全骨髓培养法得到第三代骨髓间充质干细胞备用,并采用流式细胞仪鉴定BMSCs;2、以磺化生物素-N-羟基琥珀酰亚胺、生物素、链霉亲和素为材料,通过共价结合的化学方法将粘附分子配体SLeX装备到BMSCs表面;3、通过荧光显微镜评估化学修改方法进行BMSCs表面标记的效率;4、采用台盼蓝染色法检测化学修改方法对BMSCs细胞活性的影响;5、CCK-8比色法检测化学修改方法对BMSCs的细胞增殖功能影响;6、成脂、成骨诱导检测化学修改方法对BMSCs多分化功能的影响。结果:1、骨髓间充质干细胞表面标志物鉴定:通过全骨髓培养法培养2周,可得到第三代BMSCs,采用流式细胞仪进行BMSCs细胞表型鉴定,结果如下:CD9099.9%;CD29 99.8%,CD34 0.1%,CD45 0%,符合BMSCs表型2、化学修改方法效率的评估:通过生物素及链霉亲和素进行BMSCs表面标记,细胞表面修改成功率达(87.8±3.1%),明显比对照组B+S(33.2±1.3)%(P0.001)和P+S组(6.4±1.3%)(P0.001)高;3、化学修改方法对BMSCs生物学特性的影响:BMSCs细胞活性在BNHS化学修饰细胞后24h和48h细胞活性为83%和76%,CCK-8检测细胞的增殖功能发现,BNHS修饰后72h细胞增殖功能受一定影响。BNHS修饰的细胞和PBS处理后BMSCs均能被成功能诱导分化,成脂诱导23后进行油红染色均可见大量脂质沉淀,成骨诱导23天后茜素红染成骨分化细胞均可见明显深红色矿化结节。结论:1、本研究通过生物素-链霉亲和素反应体系成功将粘附分子配体装备到骨髓间充质干细胞表面,本方法进行细胞表面标记,操作简单、反应条件温和。2、本标记方法效率高,而且对细胞的毒性小,对细胞增殖、分化功能影响不大。3、这项技术对于针对特定组织的细胞靶向治疗具有广泛的运用前景,可能有助于促进干细胞在急性肾损伤中的归巢。
[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.缁嗚優琛ㄩ潰淇�鏀规垚鍔熺巼杈�(87.8卤3.1%),鏄庢樉姣斿�圭収缁凚 S(33.2卤1.3)%(P0.001)鍜孭 S缁，

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