腺病毒介导hPDGF-BB转染表皮干细胞对其体外细胞增殖的实验研究

发布时间：2018-03-09 01:35

本文选题：表皮干细胞　切入点：增殖　出处：《暨南大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的：(1)构建人血小板源性生长因子hPDGF-BB基因腺病毒表达载体； (2)观察hPDGF-BB腺病毒表达载体修饰对hESCs增殖的影响。方法：(1)通过PCR方法合成PDGF-BB,与pAD-track-CMV质粒连接后构建穿梭质粒pAD-PDGF-BB, pAD-PDGF-BB进行测序,检测PDGF-BB序列；取骨架质粒pAD-easy I和穿梭质粒pAD-PDGF-BB在BJ5183细菌中进行同源重组获得阳性重组子rad-PDGF, Pac I限制性内切酶切鉴定rad-PDGF,0.8%琼脂糖凝胶电泳检测；用lipofectaminTM 2000脂质体将rad-PDGF导入HEK293细胞,细胞出毒后将细胞反复冻融3次,高速离心收集上清液作为第一代病毒,检测其病毒滴度,PCR鉴定rad-PDGF携带目的片段PDGF-BB后进行病毒扩增与纯化。 (2)取健康青年人包皮来源的表皮干细胞(hESCs)作为靶细胞,体外分离和纯化hESCs,用免疫组织化学法检测表皮干细胞表达K19情况；用hPDGF-BB腺病毒表达载体(rad-PDGF)修饰表皮干细胞后,荧光显微镜观察修饰后的hESCs荧光蛋白表达情况,Western blot分析PDGF-BB的表达,用MTT法检测细胞增殖情况,并进行统计学分析。结果：(1)PDGF-BB cDNA成功连接到穿梭载体pAD-track-CMV上,DNA测序结果与Genebank上PDGF-BB基因CDS区完全一致；rad-PDGF经Pac I限制性内切酶切可分离出一条为4.5kbp特异性大小的基因片段；包装成功的腺病毒颗粒,其病毒滴度可达到1×109pfu/ml,通过PCR方法可以获得大小为744bp大小的基因片段。 (2)分离后的表皮干细胞表达K19；在荧光显微镜下观察约85%的rad-PDGF修饰后的hESCs有绿色荧光蛋白表达；rad-PDGF修饰的hESCs可表达hPDGF-BB蛋白；与对照组相比较,rad-PDGF修饰促进了hESCs增殖(P0.05)。结论：(1)通过细菌BJ5183同源重组的方法成功构建PDGF-BB重组腺病毒表达载体rad-PDGF; (2) rad-PDGF能高效的导入hESCs中,并能在hESCs表达hPDGF-BB蛋白,rad-PDGF修饰促进了hESCs的增殖。
[Abstract]:Objective to construct adenovirus expression vector of human platelet-derived growth factor (hPDGF-BB) gene. (2) to observe the effect of hPDGF-BB adenovirus expression vector modification on the proliferation of hESCs. Methods PCR method was used to synthesize PDGF-BB. the shuttle plasmid pAD-PDGF-BBB was constructed after being ligated with pAD-track-CMV plasmid, and pAD-PDGF-BB was sequenced to detect the PDGF-BB sequence. The recombinant plasmid rad-PDGF was obtained by homologous recombination of the skeleton plasmid pAD-easy I and the shuttle plasmid pAD-PDGF-BB in BJ5183 bacteria, and the Pac I restriction endonuclease digestion was performed to identify rad-PDGFI 0.8% agarose gel electrophoresis. Rad-PDGF was introduced into HEK293 cells by lipofectaminTM 2000 liposome. The supernatant was collected as the first generation virus by high speed centrifugation. The virus titer was detected by PCR to identify the PDGF-BB fragment carried by rad-PDGF and then the virus was amplified and purified. (2) Epidermal stem cells derived from prepuce of healthy young people were used as target cells, hESCs were isolated and purified in vitro, the expression of K19 in epidermal stem cells was detected by immunohistochemical method, and epidermal stem cells were modified with hPDGF-BB adenovirus expression vector. The expression of hESCs fluorescence protein was observed by fluorescence microscope. The expression of PDGF-BB was detected by Western blot, and the proliferation of cells was detected by MTT assay. Results the DNA sequence of PDGF-BB cDNA ligated to the shuttle vector pAD-track-CMV was identical to that of the CDS region of PDGF-BB gene on Genebank. A 4.5kbp specific gene fragment was isolated by Pac I restriction endonuclease digestion, and the adenovirus particles were successfully packaged. The titer of the virus can reach 1 脳 10 9 pFu / ml, and a 744 BP gene fragment can be obtained by PCR method. (2) the isolated epidermal stem cells expressed K19, the hESCs modified with about 85% rad-PDGF was observed to express hPDGF-BB protein by green fluorescent protein (GFP), and compared with the control group, the proliferation of hESCs was promoted by P0.05. Conclusion the recombinant adenovirus expression vector rad-PDGF of PDGF-BB was successfully constructed by bacterial BJ5183 homologous recombination. 2) rad-PDGF can be efficiently introduced into hESCs, and the expression of hPDGF-BB protein rad-PDGF in hESCs can promote the proliferation of hESCs.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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