单质粒Tet-BMP2载体的构建及其对ADSCs调控作用的研究
发布时间：2018-12-20 07:01
【摘要】：目的:Tet-on系统的优势在于其调控的严谨、可控、背景表达低,因此使其成为最具潜力的基因调控系统,鉴于Tet-on系统这些优点,我们构建Dox诱导调控BMP-2表达的单质粒Tet-on表达系统,并且转染大鼠脂肪来源的间充质干细胞,研究不同浓度的诱导剂Dox诱导大鼠BMP2表达对ADSCs成骨分化的影响。方法:应用PCR技术扩增大鼠BMP2基因序列并插入到单质粒Tet-on载体中,构建单质粒pTet-BMP2重组质粒,与pMD2.G及psPAX2一起共转染293T,在48h与72h两个时间点收集病毒液上清与浓缩病毒;从大鼠腹股沟与大网膜提取大鼠脂肪肝细胞,并且对ADSCs进行传代培养、流式与干性的鉴定;观察不同浓度的诱导剂Dox对ADSCs增值与凋亡的影响;将浓缩病毒转染ADSCs,并使用不同浓度诱导剂Dox对ADSCs进行表达调控;应用Western Blot技术检测BMP-2表达量与诱导剂Dox不同浓度之间的关系;茜素红染色研究成骨分化钙结节数量与诱导剂Dox的诱导剂量之间的关系;qPCR方法摸索成骨分化标志基因(ALP、OCN、COL I)的表达水平情况与Dox的诱导剂量和诱导时间的关系。结果:成功构建pTet-BMP2重组质粒和严格调控BMP2表达的Tet-on慢病毒载体并且转染脂肪干细胞;BMP2蛋白的表达量与诱导剂Dox之间存在严格的剂量依赖性关系,Dox终浓度在0-5ug/ml时,ADSCs细胞内BMP2表达量随Dox浓度增加而增多,在诱导剂Dox剂量为5ug/ml时BMP2蛋白的表达量最高;茜素红钙盐染色实验显示,添加诱导剂Dox组较未添加Dox组染色具有明显差异,且随着Dox浓度的增加,染色区域越明显;qPCR结果显示添加诱导剂Dox骨分化早期标志基因和其他相关基因mRNA的表达水平均上调,且与诱导剂Dox之间存在剂量依赖性关系。结论:本研究成功构建了严格调控BMP2表达的单质粒Tet-on调控系统的ADSCs细胞株,且ADSCs成骨分化的生物学行为与单质粒Tet-on调控系统BMP2表达之间存在严格的剂量依赖性关系。
[Abstract]:Objective: the advantage of Tet-on system lies in its precise, controllable and low background expression, which makes it the most potential gene regulation system. In view of these advantages of Tet-on system, We constructed a single plasmid Tet-on expression system induced by Dox to regulate BMP-2 expression and transfected rat adipose derived mesenchymal stem cells (MSCs) to investigate the effect of different concentrations of Dox on ADSCs osteogenic differentiation in rats. Methods: the sequence of rat BMP2 gene was amplified by PCR and inserted into the single plasmid Tet-on vector. The recombinant plasmid of single plasmid pTet-BMP2 was constructed and cotransfected with pMD2.G and psPAX2 into 293T. Virus supernatant and concentrated virus were collected at 48h and 72h. Rat fatty liver cells were extracted from rat groin and omentum, and ADSCs was subcultured, and the effects of different concentrations of Dox on the proliferation and apoptosis of ADSCs were observed. The concentrated virus was transfected into ADSCs, and the expression of ADSCs was regulated by different concentration of Dox, and the relationship between the expression of BMP-2 and the concentration of Dox was detected by Western Blot technique. Alizarin red staining was used to study the relationship between the number of osteogenic differentiation calcium nodules and the induced dose of inducer Dox, and the relationship between the expression of ALP,OCN,COL I gene and the induction dose and time of Dox by qPCR method. Results: the recombinant plasmid of pTet-BMP2 and the Tet-on lentivirus vector which strictly regulated the expression of BMP2 were successfully constructed and transfected into adipose stem cells. There was a strict dose-dependent relationship between the expression of BMP2 protein and the inducer Dox. When the final concentration of Dox was 0-5ug/ml, the expression of BMP2 in ADSCs cells increased with the increase of Dox concentration. The expression of BMP2 protein was the highest when the dose of Dox was 5ug/ml. The staining results of alizarin red calcium salt showed that there was significant difference in staining between Dox group and Dox group, and the more obvious the staining area was with the increase of Dox concentration. The results of qPCR showed that the expression of mRNA in the early stage of bone differentiation of Dox and other related genes were up-regulated, and there was a dose-dependent relationship between the expression of mRNA and the inducer Dox. Conclusion: in this study, we successfully constructed the ADSCs cell line of single plasmid Tet-on regulatory system which strictly regulated the expression of BMP2, and there was a strict dose-dependent relationship between the biological behavior of ADSCs osteogenic differentiation and the BMP2 expression of single plasmid Tet-on regulatory system.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R68
本文编号：2387615
[Abstract]:Objective: the advantage of Tet-on system lies in its precise, controllable and low background expression, which makes it the most potential gene regulation system. In view of these advantages of Tet-on system, We constructed a single plasmid Tet-on expression system induced by Dox to regulate BMP-2 expression and transfected rat adipose derived mesenchymal stem cells (MSCs) to investigate the effect of different concentrations of Dox on ADSCs osteogenic differentiation in rats. Methods: the sequence of rat BMP2 gene was amplified by PCR and inserted into the single plasmid Tet-on vector. The recombinant plasmid of single plasmid pTet-BMP2 was constructed and cotransfected with pMD2.G and psPAX2 into 293T. Virus supernatant and concentrated virus were collected at 48h and 72h. Rat fatty liver cells were extracted from rat groin and omentum, and ADSCs was subcultured, and the effects of different concentrations of Dox on the proliferation and apoptosis of ADSCs were observed. The concentrated virus was transfected into ADSCs, and the expression of ADSCs was regulated by different concentration of Dox, and the relationship between the expression of BMP-2 and the concentration of Dox was detected by Western Blot technique. Alizarin red staining was used to study the relationship between the number of osteogenic differentiation calcium nodules and the induced dose of inducer Dox, and the relationship between the expression of ALP,OCN,COL I gene and the induction dose and time of Dox by qPCR method. Results: the recombinant plasmid of pTet-BMP2 and the Tet-on lentivirus vector which strictly regulated the expression of BMP2 were successfully constructed and transfected into adipose stem cells. There was a strict dose-dependent relationship between the expression of BMP2 protein and the inducer Dox. When the final concentration of Dox was 0-5ug/ml, the expression of BMP2 in ADSCs cells increased with the increase of Dox concentration. The expression of BMP2 protein was the highest when the dose of Dox was 5ug/ml. The staining results of alizarin red calcium salt showed that there was significant difference in staining between Dox group and Dox group, and the more obvious the staining area was with the increase of Dox concentration. The results of qPCR showed that the expression of mRNA in the early stage of bone differentiation of Dox and other related genes were up-regulated, and there was a dose-dependent relationship between the expression of mRNA and the inducer Dox. Conclusion: in this study, we successfully constructed the ADSCs cell line of single plasmid Tet-on regulatory system which strictly regulated the expression of BMP2, and there was a strict dose-dependent relationship between the biological behavior of ADSCs osteogenic differentiation and the BMP2 expression of single plasmid Tet-on regulatory system.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R68
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