hPDGF-BB腺病毒表达载体的构建及其在表皮干细胞中的表达

中国临床解剖学杂志 ›› 2010, Vol. 28 ›› Issue (4): 434-.

hPDGF-BB腺病毒表达载体的构建及其在表皮干细胞中的表达

李新平¹，　张鸣生^1,2，　白利明²，　邱家琦²，　白文芳²，　吴志东¹，　吴　博²

1.暨南大学第一临床学院, 广州 510632；　2.广东省医学科学院广东省老年医学研究所，广州 510080

收稿日期:2010-02-20 出版日期:2010-07-25 发布日期:2010-07-27
通讯作者: 张鸣生，教授，主任医师，硕士生导师，，E-mail:mszrch　@163.com E-mail:easepains@126.com
作者简介:李新平（1983-），男，在读硕士，医师，研究方向：创伤愈合
基金资助:
广东省自然科学基金（9151008901000199）

The construction of the recombinant PDGF-BB adenovirus vector and its expression in epidermal stem cells

LI Xin-ping, ZHANG Ming-sheng, BAI Li-ming, et al.

The First Clinical College, Jinan University, Guangzhou 510632, China

Received:2010-02-20 Online:2010-07-25 Published:2010-07-27

摘要/Abstract

摘要：

目的　构建hPDGE-BB(human platelet-derived growth factor-BB)腺病毒表达载体并观察其在人表皮干细胞(human epidermal stem cells, hESCs)中PDGF-BB蛋白的表达情况。方法　从质粒pCMV-SPORT6上通过PCR扩增hPDGF-BB基因，将其酶切后连接到穿梭质粒pAd-track-CMV上，线性化后在BJ5183细菌中和骨架质粒pAdeasy-1进行同源重组，筛选阳性克隆并酶切鉴定，线性化后转染入HEK293细胞中进行包装、扩增，得到重组腺病毒rAD-PDGF。原代培养hESCs，流式细胞术分析hESCs的纯度，用收集的腺病毒感染靶细胞hESCs后，Westorn blot检测其表达PDGF-BB情况。　结果　成功使重组腺病毒载体rAD-PDGF携带PDGF-BB基因，流式细胞术检测hESCs的纯度达88%以上，　rAD-PDGF成功导入hESCs后，Westorn blot检测hESCs能表达PDGF-BB蛋白。　结论　成功构建的重组腺病毒rAD-PDGF在hESCs中表达PDGF-BB蛋白。

关键词: PDGE-BB, 腺病毒, 表皮干细胞

Abstract:

Objective　To construct the recombinant adenovirus vector containing PDGF-BB gene and detect its expression in human epidermal stem cells (hESCs).　Methods　The core sequence of human PDGF-BB was amplified by PCR from pCMV-SPORT6, and then was cloned to pAd track-CMV. The linearized shuttle plasmid was homogenously recombined with pAdeasy-l in BJ5183, and the potential clone was analyzed by restriction endonuclease digestion. The correct clone was linearized and transfected into HEK293 cells for packing and amplifying so as to obtain adenovirus rAD-PDGF. hESCs were separated from the human prepuce, cultured in vitro, and identified by flow cytometry. hESCs were infected by the harvested virus, and the protein expression of PDGF-BB in hESCs was respectively detected by Western blot.　Results　PDGF-BB was correctly cloned into the adenovirus vector, and the percentage of putative epidermal stem cells showed expression of the molecular marker was about 88%. The recombinant adenovirus vector containing human PDGF-BB gene was constructed successfully, which could infect hESCs efficiently, with the expression of the PDGF-BB in the infected hESCs detected by Western blotting.　Conclusions　 The human ESCs infected by constructed adenovirus vector rAD-PDGF could express PDGF-BB successfully.

Key words: Platelet-derived growth factor (PDGE-BB), Adenovirus, Human epidermal stem cells(hESCs)

中图分类号:

李新平, 张鸣生, 白利明, 邱家琦, 白文芳, 吴志东, 吴　博. hPDGF-BB腺病毒表达载体的构建及其在表皮干细胞中的表达[J]. 中国临床解剖学杂志, 2010, 28(4): 434-.

LI Xin-Beng, ZHANG Ming-Sheng, BAI Li-Meng, QIU Jia-Qi, BAI Wen-Fang, TUN Zhi-Dong, TUN 　Bo. The construction of the recombinant PDGF-BB adenovirus vector and its expression in epidermal stem cells[J]. Chinese Journal Of Clinical Anatomy, 2010, 28(4): 434-.