【摘要】：目的:Tet-on系統(tǒng)的優(yōu)勢(shì)在于其調(diào)控的嚴(yán)謹(jǐn)、可控、背景表達(dá)低,因此使其成為最具潛力的基因調(diào)控系統(tǒng),鑒于Tet-on系統(tǒng)這些優(yōu)點(diǎn),我們構(gòu)建Dox誘導(dǎo)調(diào)控BMP-2表達(dá)的單質(zhì)粒Tet-on表達(dá)系統(tǒng),并且轉(zhuǎn)染大鼠脂肪來(lái)源的間充質(zhì)干細(xì)胞,研究不同濃度的誘導(dǎo)劑Dox誘導(dǎo)大鼠BMP2表達(dá)對(duì)ADSCs成骨分化的影響。方法:應(yīng)用PCR技術(shù)擴(kuò)增大鼠BMP2基因序列并插入到單質(zhì)粒Tet-on載體中,構(gòu)建單質(zhì)粒pTet-BMP2重組質(zhì)粒,與pMD2.G及psPAX2一起共轉(zhuǎn)染293T,在48h與72h兩個(gè)時(shí)間點(diǎn)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(xì)胞,并且對(duì)ADSCs進(jìn)行傳代培養(yǎng)、流式與干性的鑒定;觀察不同濃度的誘導(dǎo)劑Dox對(duì)ADSCs增值與凋亡的影響;將濃縮病毒轉(zhuǎn)染ADSCs,并使用不同濃度誘導(dǎo)劑Dox對(duì)ADSCs進(jìn)行表達(dá)調(diào)控;應(yīng)用Western Blot技術(shù)檢測(cè)BMP-2表達(dá)量與誘導(dǎo)劑Dox不同濃度之間的關(guān)系;茜素紅染色研究成骨分化鈣結(jié)節(jié)數(shù)量與誘導(dǎo)劑Dox的誘導(dǎo)劑量之間的關(guān)系;qPCR方法摸索成骨分化標(biāo)志基因(ALP、OCN、COL I)的表達(dá)水平情況與Dox的誘導(dǎo)劑量和誘導(dǎo)時(shí)間的關(guān)系。結(jié)果:成功構(gòu)建pTet-BMP2重組質(zhì)粒和嚴(yán)格調(diào)控BMP2表達(dá)的Tet-on慢病毒載體并且轉(zhuǎn)染脂肪干細(xì)胞;BMP2蛋白的表達(dá)量與誘導(dǎo)劑Dox之間存在嚴(yán)格的劑量依賴性關(guān)系,Dox終濃度在0-5ug/ml時(shí),ADSCs細(xì)胞內(nèi)BMP2表達(dá)量隨Dox濃度增加而增多,在誘導(dǎo)劑Dox劑量為5ug/ml時(shí)BMP2蛋白的表達(dá)量最高;茜素紅鈣鹽染色實(shí)驗(yàn)顯示,添加誘導(dǎo)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(jié)果顯示添加誘導(dǎo)劑Dox骨分化早期標(biāo)志基因和其他相關(guān)基因mRNA的表達(dá)水平均上調(diào),且與誘導(dǎo)劑Dox之間存在劑量依賴性關(guān)系。結(jié)論:本研究成功構(gòu)建了嚴(yán)格調(diào)控BMP2表達(dá)的單質(zhì)粒Tet-on調(diào)控系統(tǒng)的ADSCs細(xì)胞株,且ADSCs成骨分化的生物學(xué)行為與單質(zhì)粒Tet-on調(diào)控系統(tǒng)BMP2表達(dá)之間存在嚴(yán)格的劑量依賴性關(guān)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R68

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