BMP-2慢病毒载体的构建及转染后骨髓间充质干细胞骨软骨分化的研究

发布时间：2018-03-18 00:04

  本文选题：慢病毒　切入点：基因转染　出处：《福建医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：【目的】 1.构建骨形态发生蛋白-2（BMP-2）重组慢病毒表达载体； 2.TGF-β1、BMP-2重组慢病毒转染BMSCs后向成骨、软骨细胞的分化,为探索体外组织工程化骨软骨的构建提供新的思路。 【方法】 1.构建携带BMP-2基因的重组慢病毒表达载体：将提取的BMP-2cDNA包装至慢病毒表达载体，在脂质体Lipofectamine2000作用下转染293T细胞，通过酶切电泳分析和及DNA测序的方法对重组DNA进行鉴定，RT-PCR测定病毒滴度；Westernblot法检测所构建质粒能在293T细胞中表达活性。 2.以BMP-2重组慢病毒载体转染BMSCs，通过荧光表达法判定感染复数（Multiplicitiesofinfection,MOI），免疫组化、Real-timePCR、Westernblot和酶联免疫吸附试验(Enzymelinkedimmunosorbentassay,ELISA)判定感染后的BMSCs中BMP-2的表达情况；MTT法检测其转染后的增殖活性；ALP活性检测、碱性磷酸酶染色和茜素红染色观察矿化结节形成能力对其成骨分化能力进行分析；以TGF-β1重组慢病毒载体转染BMSCs，经一段时间的培养后，II型胶原免疫组织化学染色及RT-PCR法检测经TGF-β1转染后BMSCs的成软骨分化能力。 【结果】 1.成功构建BMP-2重组慢病毒表达载体，经酶切及测序结果完全正确，转染293T细胞后获得滴度为4.07×108/ml慢病毒浓缩液。 2.BMP-2重组慢病毒可高效转染BMSCs，最佳MOI值100，转染后BMSCs可高效稳定表达BMP-2RAN及蛋白；MTT法检测显示转染后BMSCs的生长增殖明显增强；碱性磷酸酶染色、碱性磷酸酶活性检测及钙结节染色发现转染两周后碱性磷酸酶和钙结节表达阳性；免疫组织化学染色及RT-PCR法发现TGF-β1转染后的BMSCs可表达II型胶原蛋白及II型胶原RNA。 【结论】 成功构建BMP-2重组慢病毒表达载体，转染BMSCs后可持续高效表达BMP-2RAN及蛋白；BMSCs经BMP-2、TGF-β1慢病毒转染后，，经一定时间的培养后可诱导其向成骨、软骨细胞分化。
[Abstract]:[purpose]. 1.Recombinant lentivirus expression vector of bone morphogenetic protein-2BMP-2 was constructed. 2. The recombinant lentivirus TGF- 尾 1 and BMP-2 were transfected into BMSCs and differentiated into osteoblasts and chondrocytes, which provided a new idea for the construction of tissue engineered bone cartilage in vitro. [methods]. 1. Construction of recombinant lentivirus expression vector carrying BMP-2 gene: the extracted BMP-2cDNA was packaged into the lentivirus expression vector and transfected into 293T cells under the action of liposome Lipofectamine2000. The recombinant DNA was identified by restriction endonuclease electrophoresis and DNA sequencing. RT-PCR was used to detect the expression activity of the constructed plasmid in 293T cells by Western blot. 2. The BMP-2 recombinant lentivirus vector was used to transfect BMSCs. The expression of BMP-2 in infected BMSCs was determined by fluorescence expression method. The expression of BMP-2 in infected BMSCs was determined by Western blot and enzyme-linked immunosorbent assay (Elisa). Alkaline phosphatase staining and alizarin red staining were used to observe the ability of mineralized nodule formation to analyze its osteogenic differentiation ability. BMSCs were transfected with TGF- 尾 1 recombinant lentivirus vector. After a period of culture, the ability of cartilage differentiation of BMSCs transfected with TGF- 尾 1 was detected by immunohistochemical staining of type II collagen and RT-PCR method. [results]. 1.Recombinant lentivirus expression vector of BMP-2 was successfully constructed. The results of restriction endonuclease digestion and sequencing were correct. After transfection into 293T cells, the titer of lentivirus concentrate was 4.07 脳 10 8 / ml. 2. BMP-2 recombinant lentivirus could efficiently transfect BMSCs, the best MOI value was 100. After transfection, BMSCs could express BMP-2RAN efficiently and stably. Alkaline phosphatase activity and calcium nodule staining showed that the expression of alkaline phosphatase and calcium nodule was positive after two weeks of transfection, and the BMSCs transfected with TGF- 尾 1 could express type II collagen and type II collagen RNAA by immunohistochemical staining and RT-PCR method. [conclusion]. The recombinant lentivirus expression vector of BMP-2 was successfully constructed. After transfection of BMSCs, BMP-2RAN and BMSCs could be induced to differentiate into osteoblasts and chondrocytes after transfection with BMP-2TGF- 尾 1 lentivirus.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329


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