hBMP-2基因转染对兔骨髓间充质干细胞增殖分化能力影响的实验研究

发布时间：2018-03-29 18:36

本文选题：骨髓间充质干细胞　切入点：人骨形成蛋白2　出处：《广西医科大学》2012年硕士论文

【摘要】：目的 1.为大量获得骨组织工程所需种子细胞,于体外分离、培养、扩增、纯化兔骨髓间充质干细胞(Bone Mesenchymal Stem Cells, BMSCs). 2.为鉴定BMSCs的干细胞多分化潜能以及成骨分化的能力,利用成骨诱导培养基对其进行培养,诱导BMSCs向成骨方向分化,并鉴定其成骨分化的效果。 3.利用脂质体介导携带有hBMP-2基因pcDNA3.1质粒转染BMSCs,观察其转染效果。 4.检测转染对BMSCs的增殖分化能力的影响。方法 1.BMSCs的体外培养：取新生健康新西兰大白兔,处死后于无菌条件下取出四肢长骨,完全培养基冲洗骨髓腔得到骨髓液,用全骨髓贴壁法进行培养,原代培养6-7d后可传代培养,按照1：3的比例传代,每2-3天可传代1次。 2.BMSCs的体外成骨诱导分化：BMSCs传到第3代时用p-甘油磷酸钠,地塞米松,抗坏血酸配制而成的成骨诱导分化液进行成骨诱导14至21d,并分别用茜素红染色,ELISA以及免疫组化的方法检测其矿化结节的形成和ALP,BGP的表达。 3.脂质体介导的hBMP-2转染兔BMSCs:取第3代BMSCs,用脂质体介导携带hBMP-2基因的pcDNA3.1质粒转染入BMSCs中,观察GFP表达的情况,通过RT-PCR检测瞬时转染后hBMP-2mRNA的表达。 4.MTT法、流式细胞法检测转染后BMSCs的增殖能力,BMP-2免疫组化检测细胞的分化能力。结果 1.BMSCs体外培养的结果：BMSCs培养6-7d后即可长满瓶底,并可传代,传代后细胞增殖快,生长状态良好,可一直维持BMSCs的典型形态以及生长状态,细胞呈现长梭形形状,整体呈旋涡式的排列。 2.BMSCs的体外成骨诱导分化的结果：BMSCs可成功向成骨方向诱导,其中钙结节茜素红染色见阳性结节染色；ALP含量逐渐升高,在诱导2周内增高趋势明显,之后趋势减缓,各时间点与未诱导组有显著差异(p0.05)；BGP免疫组化也呈阳性染色。 3.脂质体介导的hBMP-2转染兔BMSCs的结果：利用脂质体介导携带hBMP-2的pcDNA3.1质粒成功转染BMSCs,转染效率约30%；转染24h能观察到弱荧光表达,48h后荧光强度增强；RT-PCR也检测到了hBMP-2mRNA的表达。 4.MTT法、流式细胞周期检测结果证实转染对BMSCs的增殖能力无明显影响,免疫组化结果显示转染hBMP-2基因后的BMSCs中BMP-2为阳性表达。结论 1.全骨髓贴壁分离培养法简便、易行,可大量扩增、纯化兔骨髓间充质干细胞。 2.本实验所获的细胞可于体外成功诱导后向成骨细胞分化。 3.通过脂质体介导的方法能成功将外源hBMP-2基因导入BMSCs。 4.转染hBMP-2对BMSCs的增殖无明显影响,但使BMSCs向成骨方向分化。
[Abstract]:Purpose. 1. In order to obtain a large number of seed cells for bone tissue engineering, bone Mesenchymal Stem cells (BMSCs) were isolated, cultured, amplified and purified from rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro. 2. In order to identify the multi-differentiation potential of BMSCs stem cells and the ability of osteogenic differentiation, BMSCs was cultured in osteogenic medium to induce the differentiation of BMSCs into osteogenesis, and the effect of osteogenic differentiation was evaluated. 3. HBMP-2 gene pcDNA3.1 plasmid was transfected into BMSCs by liposome, and the transfection effect was observed. 4. The effect of transfection on the proliferation and differentiation of BMSCs was detected. Method. In vitro culture of 1.BMSCs: after being killed, the long bones of the limbs were removed under aseptic conditions. Bone marrow fluid was obtained by washing the marrow cavity in a complete culture medium, and cultured with the whole bone marrow adherent method. After 6 to 7 days of primary culture, the bone marrow fluid could be cultured by subculture. According to the 1:3 ratio of passage, every 2-3 days can be subcultured once. In vitro osteogenic differentiation of 2.BMSCs was induced by sodium pglycerophosphate and dexamethasone. The osteogenic differentiation fluid prepared by ascorbic acid was induced by osteogenesis for 14 to 21 days. The formation of mineralized nodules and the expression of ALP BGP were detected by alizarin red staining Elisa and immunohistochemistry respectively. 3. Liposome-mediated hBMP-2 transfection into rabbit BMSCs: the pcDNA3.1 plasmid carrying hBMP-2 gene was transfected into BMSCs by liposome. The expression of GFP was observed and the expression of hBMP-2mRNA after transient transfection was detected by RT-PCR. The proliferative ability of BMSCs after transfection and the differentiation ability of BMP-2 were detected by 4.MTT and flow cytometry. Results. The results of 1.BMSCs culture in vitro were as follows: after 6-7 days of culture, 1.BMSCs could grow to the bottom of the bottle and could be subcultured. After passage, the cells could proliferate quickly and grow in good condition. They could maintain the typical morphology and growth state of BMSCs, and the cells showed a long spindle shape. The whole is arranged in the form of a vortex. The results of osteogenic induction and differentiation of 2.BMSCs in vitro showed that BMSCs could be successfully induced to osteogenesis, in which calcium nodule alizarin red staining showed that the content of 2.BMSCs increased gradually, and increased significantly within 2 weeks of induction, and then slowed down. There was significant difference between the different time points and the uninduced group, and the immunohistochemical staining of BGP was also positive. 3. The results of liposome-mediated hBMP-2 transfection into rabbit BMSCs: the pcDNA3.1 plasmid carrying hBMP-2 was successfully transfected into BMSCs by liposome, the transfection efficiency was about 30%, and the expression of hBMP-2mRNA was detected by RT-PCR after 48 h of weak fluorescence expression was observed after transfection. The results of 4.MTT assay and flow cytometry showed that transfection had no significant effect on the proliferation of BMSCs. The results of immunohistochemistry showed that BMP-2 was positive in BMSCs after transfection of hBMP-2 gene. Conclusion. 1. The whole bone marrow adherent culture method is simple and easy, and can be expanded and purified from rabbit bone marrow mesenchymal stem cells. 2. The cells obtained in this experiment can differentiate into osteoblasts after successful induction in vitro. 3. Exogenous hBMP-2 gene was successfully introduced into BMSCs by liposome mediated method. 4. Transfection of hBMP-2 had no effect on the proliferation of BMSCs, but BMSCs differentiated to osteogenesis.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】