重组hTAZ慢病毒表达载体的构建及其对前成骨细胞MC3T3-E1分化的影响

发布时间：2018-07-09 15:28

  本文选题：骨质疏松 + 细胞分化　； 参考：《第四军医大学》2010年硕士论文

【摘要】：目的：构建人真核表达的hTAZ慢病毒载体pLenti6/V5-hTAZ，转染293FT细胞获得重组慢病毒颗粒，研究hTAZ对前成骨细胞MC3T3-E1分化的调控作用。方法：将已经过测序验证的，含有hTAZ基因慢病毒入门质粒pENTR221-hTAZ通过LR反应克隆到慢病毒载体pLenti6/V5-DEST中。对重组质粒进行酶切鉴定并在细胞中验证表达。将该重组载体和慢病毒包装质粒充分混合，用阳离子脂质体Lipofactamine转染293FT细胞，培养和待细胞完全裂解后收集富含hTAZ基因的慢病毒颗粒上清液，取适量上清液感染MC3T3 E1细胞，采用杀稻瘟菌素（Blastcidin）筛选，建立了稳定过量表达hTAZ蛋白的MC3T3-E1/hTAZ细胞系。用条件培养基诱导MC3T3-E1和MC3T3-E1/hTAZ细胞成骨、成脂分化，von Kossa和茜素红染色比较MC3T3-E1和MC3T3-E1/hTAZ成骨分化程度差异；油红O染色比较其成脂分化差异，，考察hTAZ基因过表达对前成骨细胞分化的影响。结果：凝胶电泳和测序结果均证明hTAZ重组慢病毒载体pLenti6/V5-hTAZ构建正确，并能在细胞中正确表达。与辅助质粒共转包装细胞获得慢病毒颗粒，并成功感染MC3T3-E1细胞。von Kossa染色和茜素红染色结果表明，MC3T3-E1/hTAZ细胞成骨分化强于未转染细胞，而其成脂分化受抑制。结论：本研究构建了人真核表达的质粒载体pLenti6/V5-hTAZ，并能在细胞中正确表达。成功包装了hTAZ病毒并获得过表达hTAZ的MC3T3－E1细胞。过表达hTAZ可促进MC3T3-E1向成骨细胞分化，抑制其成脂分化。
[Abstract]:Aim: to construct hTAZ lentivirus vector pLenti6 / V5-hTAZ and transfect 293FT cells to obtain recombinant lentivirus particles, and to study the effect of hTAZ on the differentiation of preosteoblast MC3T3-E1. Methods: the primer plasmid pENTR221-hTAZ containing hTAZ gene was cloned into lentivirus vector pLenti6 / V5-DEST by LR reaction. The recombinant plasmid was digested and expressed in cells. The recombinant vector and the lentivirus packaging plasmid were fully mixed and transfected into 293FT cells by cationic liposome Lipofactamine. The supernatants of lentivirus particles rich in hTAZ gene were collected after the complete cell lysis, and the supernatants were used to infect MC3T3 and E1 cells. A MC3T3-E1 / hTAZ cell line with stable overexpression of hTAZ protein was established by Blastcidin screening. The osteogenesis of MC3T3-E1 and MC3T3-E1 / hTAZ cells was induced by conditioned medium, and the degree of osteogenic differentiation of MC3T3-E1 and MC3T3-E1 / hTAZ cells was compared with that of MC3T3-E1 and MC3T3-E1 / hTAZ cells by lipogenic von Kossa and alizarin red staining, and the effect of overexpression of hTAZ gene on the differentiation of preosteoblast was investigated. Results: the results of gel electrophoresis and sequencing proved that hTAZ recombinant lentivirus vector pLenti6 / V5-hTAZ was constructed correctly and expressed correctly in cells. The lentivirus particles were obtained by co-transfection with the co-packaged cells and infected with MC3T3-E1 cells. Von Kossa staining and alizarin red staining showed that the osteogenic differentiation of MC3T3-E1 / hTAZ cells was stronger than that of untransfected cells, but the adipogenic differentiation of MC3T3-E1 cells was inhibited. Conclusion: the plasmid vector pLenti6 / V5-hTAZ was constructed and expressed correctly in human eukaryotic cells. The hTAZ virus was successfully packaged and MC3 T 3-E1 cells expressing hTAZ were obtained. Overexpression of hTAZ could promote the differentiation of MC3T3-E1 into osteoblasts and inhibit the adipogenic differentiation of MC3T3-E1.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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