中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (11): 1927-1932.doi: 10.3969/j.issn.1673-8225.2012.11.007

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

携带人血管内皮生长因子165慢病毒表达载体的构建与表达*★

易  超,王喜艳,李海军,王  海   

  1. 新疆医科大学附属肿瘤医院肝胆胰腺肿瘤外科,新疆维吾尔自治区乌鲁木齐市    830011
  • 收稿日期:2011-12-15 修回日期:2012-01-12 出版日期:2012-03-11
  • 通讯作者: 李海军,博士,主任医师,副教授,新疆医科大学附属肿瘤医院肝胆胰腺肿瘤外科,新疆维吾尔自治区乌鲁木齐市 830011 lhjun3408@163.com
  • 作者简介:易超★,男,1985年生,新疆维吾尔自治区乌鲁木齐市人,汉族,新疆医科大学在读硕士,主要从事肝胆胰腺肿瘤诊断与治疗方面的基础与临床研究。yinlanglff000@163.com
  • 基金资助:

    新疆维吾尔自治区自然基金面上项目(2010211A28),资质单位:新疆维吾尔自治区科学技术厅。

Construction and expression of human vascular endothelial growth factor 165 gene lentiviral expression vector

Yi Chao, Wang Xi-yan, Li Hai-jun, Wang Hai   

  1. Department of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of Xinjing Medical University, Urumqi  830011, Xinjiang Uygur Autonomous Region, China
  • Received:2011-12-15 Revised:2012-01-12 Online:2012-03-11
  • Contact: author: Li Hai-jun, Doctor, Chief physician, Associate professor, Department of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of Xinjing Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China lhjun3408@163.com
  • About author:Yi Chao★, Studying for master’s degree, Department of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of Xinjing Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China yinlanglff000@163.com
  • Supported by:

    the General Program of the Natural Science Foundation of Xinjiang Uygur Autonomous Region, No. 2010211A28*

摘要:

背景:已有研究证实血管内皮生长因子在正常肝脏肝部分切除后余肝的再生过程中发挥着重要的作用,但关于其对肝硬化肝脏是否也有相同作用国内外鲜有报道。
目的:构建携带人血管内皮生长因子165基因的慢病毒载体,体外转染BLR 3A大鼠肝细胞并观察该细胞中人血管内皮生长因子165基因的表达。
方法:采用DNA重组技术将人血管内皮生长因子165基因克隆入pLenti6/V5-D-TOPO慢病毒表达载体中,筛选出阳性克隆与慢病毒包装系统ViraPowerTM Packaging Mix共转染293T细胞产生病毒颗粒,通过实时定量-聚合酶链反应法测定病毒滴度;携带人血管内皮生长因子165基因的慢病毒载体体外转染BLR 3A大鼠肝细胞72 h后,利用反转录-聚合酶链反应及Western blot法检测细胞中人VEGF165 mRNA及蛋白的表达。
结果与结论:成功构建了表达人VEGF165基因的慢病毒载体pLenti6/V5-D-TOPO-VEGF165,测得病毒滴度为1.18×     107 VP/mL。重组慢病毒载体转染BLR 3A大鼠肝细胞72 h后,荧光蛋白表达率超过80%,反转录-聚合酶链反应及Western blot法测得转染组人血管内皮生长因子165 mRNA及血管内皮生长因子165蛋白表达阳性。提示构建的携带人血管内皮生长因子165的慢病毒载体可有效转染BLR 3A大鼠肝细胞,并促使该细胞表达人血管内皮生长因子165 mRNA及蛋白。
关键词:人血管内皮生长因子165;慢病毒载体;BLR 3A大鼠肝细胞;转染;基因治疗
doi:10.3969/j.issn.1673-8225.2012.11.007

关键词: 人血管内皮生长因子165, 慢病毒载体, BLR 3A大鼠肝细胞, 转染, 基因治疗

Abstract:

BACKGROUND: Studies have confirmed that vascular endothelial growth factors (VEGF) play an important role in the course of normal liver regeneration, while, there are very few reports on the same role in the cirrhotic liver nationally and internationally.
OBJECTIVE: To construct the lentivirus vector containing human VEGF165 gene, and to examine the expression of VEGF165 in normal rat liver cells after transfecting BLR 3A rat liver cells, in order to lay a foundation for the study about the effects of VEGF165 on the regeneration power of remnant liver tissue after hepatectomy in the rats with cirrhosis.
METHODS: The VEGF165 gene was cloned to a lentiviral expression vector by recombinant DNA technology. The positive clones were screened, and lentiviral packaged systems (ViraPowerTM Packaging Mix) were co-transfected to package virus in 293T cells by lipofection with target gene plasmid. Real-time PCR technique was used o test the titer of pLenti6/V5-D-TOPO-VEGF165. The BLR 3A rat liver cells were transfected by pLenti6/V5-D-TOPO-VEGF165 and the expression of VEGF165 mRNA and protein was detected by reverse transcriptase-PCR and Western blot technique.
RESULTS AND CONCLUSION: The lentivirus vector containing VEGF165 was successfully constructed. Virus titer could reach as high as 1.18×107 VP/mL. The transfer efficiency of green fluorescent protein was more than 80% in the BLR 3A rat liver cells after transfection with pLenti6/V5-D-TOPO-VEGF165 for 72 hours. Reverse transcriptase-PCR and Western blot results showed that the VEGF165 gene expression of the transfected group was positive. Lentivirus vector pLenti6/V5-D-TOPO-VEGF165 could transfect BLR 3A rat liver cells effectively, and VEGF165 gene was expressed after transfection.
 

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