【作者】 李新平；

【导师】 张鸣生；

【作者基本信息】 暨南大学 ， 康复医学与理疗学， 2010， 硕士

【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(P<0.05)。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。更多还原

【Abstract】 Objectives:(1) To construct a recombinant adenovirus vector (rad-PDGF)containing PDGF-BB gene.(2) To observe the effects of proliferation of hESCs after Transfection by rad-PDGF.Methods:(1) hPDGF-BB was amplified by PCR and cloned into the transfer vector pAd-Track-CMV to construct a transfer plasmid pAd-Track-CMV-PDGF-BB. The plasmid cotransformed into E.coli BJ5183 cells with adenovirus genomic plasmid pAdEasy-1 to achieve homologous recombination (rad-PDGF). After being digested with Pac I, the DNA of identified recombinant plasmid was transfected into HEK293 cells with lipofectaminTM 2000 to package and amplify adenovirus. After the viruses were harvested, the infectious titer of the recombinant adenovirus was tested, and the PCR products of the viruses ware analyzed.(2) hESCs were separated from the human prepuce, cultured in vitro, and identified, and infected with the recombinant adenoviruses. The expression of green fluorescent protein in hESCs modificated by rad-PDGF was visualized in fluorescence microscope. The expression of the target gene was detected by Western blotting. Its effect on the proliferation of the infected hESCs was examined by MTT.Results:(1) PDGF-BB cDNA had been linked with pAd-Track-CMV vector with correct order. The result of DNA sequencing showed that the sequence of gene combined with pAd-Track-CMV-PDGF-BB was identical to the coding region of PDGF-BB gene in Genebank. Homologous recombination had completed successfully between pAd-Track-CMV-PDGF-BB and adenoviral backbone plasmid pAdEasy-1. The recombinant adenoviral plasmid could be digested with Pac I to produce a fragment whose size was 4.5kb. The infectious titer of adenovirus was 1×109pfu/ml. the size of PCR products is consistent with the size of the target gene.(2) hESCs strongerly expressed the K19. The percentage of putative hESCs infected by rad-PDGF which showed expression of GFP was about 85%. rad-PDGF can expressed PDGF-BB in infected hESCs. The proliferation of hESCs transfected with the recombinant virus was significantly increased in comparison with those of the control hESCs(P<0.05).Conclusion:(1) The rad-PDGF was successfully constructed;(2) rad-PDGF was transfected into hESCs and the PDGF-BB protein was expressed in infected hESCs successfully. hPDGF-BB can increase the growth of hESCs.更多还原