摘要：目的探索一条肿瘤特异性基因治疗的新途径。方法将新型人TNF-αD11a基因和KDR特异性启动子插入到3’LTR缺失299bp的逆转录病毒载体pLXSN中，构建成pLXSN-D299-KDRp-TNF-αD11a重组逆转录病毒载体，使目的基因转录受KDR启动子驱动。通过脂质体介导将该重组载体转染PA317包装细胞，并用病毒上清感染血管内皮细胞和NIH3T3细胞。分别经ELISA和MTT法测定TNF-αD11a在血管内皮细胞中的表达及其生物学活性。结果成功构建了携带TNF-αD11a和KDR启动子的逆转录病毒载体，TNF-αD11a在血管内皮细胞中的表达水平及细胞增殖抑制作用均高于NIH3T3细胞。结论KDR启动子可使外源TNF-αD11a在血管内皮细胞中特异性表达。

分类号：R392.11文献标识码：A

文章编号：1000-8861（2001）01-0030-04

Endothelial cell-targeted expression of human tumor necrosis factor α mutant with higher activity from retroviral vectors

SHI Ying（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

XIAO Chang（Department of Immunology， Norman Bethune University of Medical Sciences， Changchun 130021， China）

ZHAO Xian-ling（Institute of Genetics， Northeast Normal University，Changchun 130024， China）

WANG Ye（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

LIU Li（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

ZHU Xun（Department of Immunology， Norman Bethune University of Medical Sciences， Changchun 130021， China）

LIU Li-zhong（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

XIE Bao-shu（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

LENG Ai-jun（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

YANG Yan（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

CAO Ying（Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China）

Abstract：ObjectiveTo develop a new approach in specific gene therapy of tumor.Methods TNF-α D11a was inser-ted downstream of KDR promoter in retroviral vector pLXSN in which 299 bp of 3’ LTR had been deleted. The constructed retroviral vectors pLXSN-D299-KDRp-TNF-α D11a were transfected into PA317 packaging cells by lipofectamine reagent. Then the recombinant retroviruses were used to infect endothelial cells and NIH3T3 cells. Expression and bioactivity of TNF-α D11a in culture medium of endothelial cells were identified by ELISA and MTT， respectively. Results Retroviral vectors， carrying TNF-α D11a and KDR promoter， were constructed successfully. Expression level of TNF-α D11a in endothelial cells was higher than that in NIH 3T3， and inhibitory effects of TNF-α D11a released from endothelial cells on tumor cells prolife-ration were also stronger than those from NIH3T3 cells.Conclusion Endothelial cell-targeted expression of TNF-α D11a was regulated by KDR promoter.

Keywords：promoter； TNF-α； retroviral vector； targeted expression； gene therapy

作者简介：石缨（1970－），女，山东招远市人，主治医师，博士，主要从事肿瘤基因治疗研究。Tel：（010）66928758； E-mail：shiying@263.net参考文献：

［1］Lin P，Sankar S， Shan S， et al. Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor［J］． Cell Growth Differ，1998，9（1）：49－58.

［2］Folkman J. What is the evidence that tumors are angiogenesis dependent?［J］． JNatl Cancer Inst，1990，82（1）：4－6.

［3］Miller DG，Adam MA， Miller AD. Gene transfer by retrovirus vectors occurs only in cell that are actively replicating at the time of infection［J］． Mol Cell Biol，1990，10（8）：4 239－4 242.

［4］Klagsbrun M， D’amore PA. Vascular endothelial growth factor and its receptors［J］．Cytokine Growth Factor Rev，1996，7（3）：259－270.

［5］刘丽，田生礼，冯彪，等.一种高活性人新型TNF的研制［J］． 中华微生物学和免疫学杂志，1996，16（4）：254－258.

［6］刘丽，谢宝树，冷爱军，等.一种高活性人TNF-α对人肝癌裸鼠移植瘤的抗肿瘤作用［J］． 解放军医学杂志，1997，22（2）：91－92.

［7］刘丽，赵建增，谢宝树，等. rhTNF-α D11a对S-180肉瘤的体内抗肿瘤作用［J］． 免疫学杂志，1997，13（2）：88－89.

［8］李景泰，刘丽，赵建增，等. rhTNF-α D11a在体外对肿瘤细胞的细胞毒作用研究［J］． 解放军医学杂志，1997，22（4）：284－285.

［9］Olson P，Nelson S， Dornburg R. Improved self-inactivating retroviral vectors derived from spleen necrosis virus［J］． J Virol，1994，68（11）：7 060－7 066.

收稿日期：2000年8月29日

修稿日期：2000年10月8日

出版日期：2001年1月15日