本文選題：大鼠骨髓間充質(zhì)干細(xì)胞 + 成堿性纖維細(xì)胞因子��； 參考：《福建醫(yī)科大學(xué)》2010年碩士論文

【摘要】：目的:探討構(gòu)建攜帶人堿性成纖維生長因子(bFGF)的重組慢病毒載體方法并觀察堿性成纖維細(xì)胞生長因子基因轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞后bFGF目的基因的表達(dá)情況。 方法:采用RT—PCR擴(kuò)增的方法獲取人源性的bFGF與FGF4基因,將目的基因與載體pGC-FU連接[含綠色熒光蛋白(GFP)]。產(chǎn)生pGC FU- FGF4-bFGF慢病毒載體。PCR篩選陽性克隆,測序鑒定。通過lipofectamine2000的介導(dǎo)把pGC FU- FGF4-bFGF載體,pHelper 1.0載體和pHelper 2.0載體三質(zhì)粒共轉(zhuǎn)染至包裝細(xì)胞293T.應(yīng)用Real time-PCR法鑒定和測定滴度。而后轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞并檢測其表達(dá)。MTT法檢測到間充質(zhì)干細(xì)胞經(jīng)過基因修飾以后和未經(jīng)過基因修飾的干細(xì)胞生長情況 結(jié)果: 1.流式細(xì)胞儀顯示體外分離培養(yǎng)的第3代大鼠骨髓間充質(zhì)干細(xì)胞表面抗原表達(dá): CD11b/c陽性細(xì)胞表達(dá)率為14.2%±0.7%,CD34陽性細(xì)胞表達(dá)率為1.3%±0.5%,CD44陽性細(xì)胞表達(dá)率97.8%±0.9%,CD90陽性細(xì)胞表達(dá)率96.9%±1.5%。 2. PCR鑒定證實(shí)重組慢病毒有人FGF4信號肽-人BFGF ,病毒滴度為2.00E+9 TU/ml。 3.轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞后,行RT-PCR檢測到BFGF基因轉(zhuǎn)入干細(xì)胞內(nèi),其大小約500bp。 4.在熒光顯微鏡下,觀察到轉(zhuǎn)染的干細(xì)胞內(nèi)GFP的表達(dá);通過Western Blot檢測轉(zhuǎn)染的細(xì)胞可以觀察到36～55KDr之間有明顯表達(dá),與FGF4-BFGF -GFP融合蛋白(~21+28KDr=~49KDr)基本吻合。(備注:構(gòu)建的pGC-FU-FGF4-BFGF基因插入片斷大小549bp)綜上:基本判斷FGF4-BFGF表達(dá)正常。ELISA檢測結(jié)果表明轉(zhuǎn)染bFGF的細(xì)胞培養(yǎng)上清液中堿性成纖維細(xì)胞生長因子濃度顯著高于空白對照。 5.MTT法檢測到間充質(zhì)干細(xì)胞經(jīng)過基因修飾以后和未經(jīng)過基因修飾的干細(xì)胞生長情況對比,基因修飾后的目的細(xì)胞明顯優(yōu)于未經(jīng)修飾的干細(xì)胞。 結(jié)論:成功構(gòu)建的攜帶人成堿纖維生長因子的慢病毒載體能在293T細(xì)胞擴(kuò)增獲得足夠高的病毒滴度,并轉(zhuǎn)染入大鼠骨髓間充質(zhì)干細(xì)胞,經(jīng)檢測可以有效的表達(dá)。可以為下一步基因治療大鼠下肢缺血提供很好的基礎(chǔ)。
[Abstract]:Aim: to investigate the construction of recombinant lentivirus vector carrying human basic fibroblast growth factor (bFGF) and observe the expression of bFGF target gene in rat bone marrow mesenchymal stem cells transfected with basic fibroblast growth factor gene. Methods: human bFGF and FGF4 genes were obtained by RT-PCR. The target gene was ligated with vector pGC-FU [GFP containing green fluorescent protein]. The positive clones of lentivirus vector pGC FU- FGF4-bFGF were screened and sequenced. PGC FU-FGF4-bFGF vector pHelper 1.0 and pHelper 2.0 were co-transfected into packaging cell line 293T by lipofectamine2000. Real time PCR method was used to identify and determine the titer. Then transfected rat bone marrow mesenchymal stem cells and detected its expression. MTT assay detected the growth of mesenchymal stem cells after gene modification and without gene modification: 1. Flow cytometry showed that the expression rate of CD11b / c positive cells in the third passage of rat bone marrow mesenchymal stem cells in vitro was 14.2% 鹵0.710%. The positive rate of CD34 positive cells was 1.3% 鹵0.5%. The expression rate of CD44 positive cells was 97.8% 鹵0.9m + CD90 positive cells. The expression rate of CD11b / c positive cells was 96.9% 鹵1.5.2%. PCR confirmed the recombinant lentivirus human FGF4 signal peptide human bFGF, the titer of the virus was 2.00E 9 TU / ml. 3. After transfection of rat bone marrow mesenchymal stem cells, the expression of bFGF gene was detected by RT-PCR and its size was about 500bp.4. The expression of GFP in the transfected stem cells was observed under fluorescence microscope, and the expression of GFP in transfected cells was detected by Western Blot, which was consistent with the FGF4-BFGF -GFP fusion protein (21 28 K Dr). (note: the size of pGC-FU-FGF4-BFGF gene insertion fragment was 549bp). The results of Elisa showed that the concentration of basic fibroblast growth factor in the supernatant of bFGF transfected cell culture was significantly higher than that of blank control. 5. MTT assay showed that the expression of FGF4-BFGF was normal. The growth of mesenchymal stem cells after gene modification was compared with that of unmodified mesenchymal stem cells. Conclusion: the successfully constructed lentivirus vector carrying human alkali-derived fibroblast growth factor can obtain high enough virus titer in 293T cells. It was transfected into rat bone marrow mesenchymal stem cells and expressed effectively. It can provide a good basis for gene therapy of lower extremity ischemia in rats.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

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相關(guān)期刊論文 前2條

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本文編號：1993014