單質粒Tet-BMP2載體的構建及其對ADSCs調控作用的研究
發(fā)布時間：2018-12-20 07:01
【摘要】：目的:Tet-on系統(tǒng)的優(yōu)勢在于其調控的嚴謹、可控、背景表達低,因此使其成為最具潛力的基因調控系統(tǒng),鑒于Tet-on系統(tǒng)這些優(yōu)點,我們構建Dox誘導調控BMP-2表達的單質粒Tet-on表達系統(tǒng),并且轉染大鼠脂肪來源的間充質干細胞,研究不同濃度的誘導劑Dox誘導大鼠BMP2表達對ADSCs成骨分化的影響。方法:應用PCR技術擴增大鼠BMP2基因序列并插入到單質粒Tet-on載體中,構建單質粒pTet-BMP2重組質粒,與pMD2.G及psPAX2一起共轉染293T,在48h與72h兩個時間點收集病毒液上清與濃縮病毒;從大鼠腹股溝與大網(wǎng)膜提取大鼠脂肪肝細胞,并且對ADSCs進行傳代培養(yǎng)、流式與干性的鑒定;觀察不同濃度的誘導劑Dox對ADSCs增值與凋亡的影響;將濃縮病毒轉染ADSCs,并使用不同濃度誘導劑Dox對ADSCs進行表達調控;應用Western Blot技術檢測BMP-2表達量與誘導劑Dox不同濃度之間的關系;茜素紅染色研究成骨分化鈣結節(jié)數(shù)量與誘導劑Dox的誘導劑量之間的關系;qPCR方法摸索成骨分化標志基因(ALP、OCN、COL I)的表達水平情況與Dox的誘導劑量和誘導時間的關系。結果:成功構建pTet-BMP2重組質粒和嚴格調控BMP2表達的Tet-on慢病毒載體并且轉染脂肪干細胞;BMP2蛋白的表達量與誘導劑Dox之間存在嚴格的劑量依賴性關系,Dox終濃度在0-5ug/ml時,ADSCs細胞內BMP2表達量隨Dox濃度增加而增多,在誘導劑Dox劑量為5ug/ml時BMP2蛋白的表達量最高;茜素紅鈣鹽染色實驗顯示,添加誘導劑Dox組較未添加Dox組染色具有明顯差異,且隨著Dox濃度的增加,染色區(qū)域越明顯;qPCR結果顯示添加誘導劑Dox骨分化早期標志基因和其他相關基因mRNA的表達水平均上調,且與誘導劑Dox之間存在劑量依賴性關系。結論:本研究成功構建了嚴格調控BMP2表達的單質粒Tet-on調控系統(tǒng)的ADSCs細胞株,且ADSCs成骨分化的生物學行為與單質粒Tet-on調控系統(tǒng)BMP2表達之間存在嚴格的劑量依賴性關系。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R68
本文編號：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R68
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