新型人肿瘤坏死因子基因在血管内皮细胞中的靶向表达
作者：肖畅 朱迅 赵显玲 石缨 王烨 刘丽 刘立忠 谢宝树 冷爱军 杨彦 曹颖&5.(]c;, 百拇医药
单位：肖畅 朱迅(白求恩医科大学免疫学教研室， 吉林 长春 130021)；赵显玲(东北师范大学遗传所， 吉林 长春 130024)；石缨 王烨 刘丽 刘立忠 谢宝树 冷爱军 杨彦 曹颖 (空军总医院临床分子生物学中心， 北京 100036)&5.(]c;, 百拇医药
关键词：启动子；TNF-α；逆转录病毒载体；靶向表达；基因治疗&5.(]c;, 百拇医药
免疫学杂志010108 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。&5.(]c;, 百拇医药
分类号：R392.11 文献标识码：A&5.(]c;, 百拇医药
文章编号：1000-8861(2001)01-0030-04&5.(]c;, 百拇医药
Endothelial cell-targeted expression of human tumor necrosis factor α mutant with higher activity from retroviral vectors&5.(]c;, 百拇医药
SHI Ying(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China)&5.(]c;, 百拇医药
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);ix, 百拇医药
WANG Ye(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
LIU Li(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
ZHU Xun(Department of Immunology， Norman Bethune University of Medical Sciences， Changchun 130021， China);ix, 百拇医药
LIU Li-zhong(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
XIE Bao-shu(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
LENG Ai-jun(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
YANG Yan(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
CAO Ying(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
Abstract：Objective To 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 therapyw!6;/&, http://www.100md.com
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.netw!6;/&, http://www.100md.com
参考文献：w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[2]Folkman J. What is the evidence that tumors are angiogenesis dependent?[J]. J Natl Cancer Inst，1990，82(1)：4-6.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[4]Klagsbrun M， D’amore PA. Vascular endothelial growth factor and its receptors[J].Cytokine Growth Factor Rev，1996，7(3)：259-270.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
收稿日期：2000年8月29日w!6;/&, http://www.100md.com
修稿日期：2000年10月8日w!6;/&, http://www.100md.com
出版日期：2001年1月15日(作者：肖畅 朱迅 赵显玲 石缨 王烨 刘丽 刘立忠 谢宝树 冷爱军 杨彦 曹颖)
单位：肖畅 朱迅(白求恩医科大学免疫学教研室， 吉林 长春 130021)；赵显玲(东北师范大学遗传所， 吉林 长春 130024)；石缨 王烨 刘丽 刘立忠 谢宝树 冷爱军 杨彦 曹颖 (空军总医院临床分子生物学中心， 北京 100036)&5.(]c;, 百拇医药
关键词：启动子；TNF-α；逆转录病毒载体；靶向表达；基因治疗&5.(]c;, 百拇医药
免疫学杂志010108 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。&5.(]c;, 百拇医药
分类号：R392.11 文献标识码：A&5.(]c;, 百拇医药
文章编号：1000-8861(2001)01-0030-04&5.(]c;, 百拇医药
Endothelial cell-targeted expression of human tumor necrosis factor α mutant with higher activity from retroviral vectors&5.(]c;, 百拇医药
SHI Ying(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China)&5.(]c;, 百拇医药
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);ix, 百拇医药
WANG Ye(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
LIU Li(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
ZHU Xun(Department of Immunology， Norman Bethune University of Medical Sciences， Changchun 130021， China);ix, 百拇医药
LIU Li-zhong(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
XIE Bao-shu(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
LENG Ai-jun(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
YANG Yan(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
CAO Ying(Research Center of Molecular Biology， Airforce General Hospital， Beijing 100036，China);ix, 百拇医药
Abstract：Objective To 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 therapyw!6;/&, http://www.100md.com
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.netw!6;/&, http://www.100md.com
参考文献：w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[2]Folkman J. What is the evidence that tumors are angiogenesis dependent?[J]. J Natl Cancer Inst，1990，82(1)：4-6.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[4]Klagsbrun M， D’amore PA. Vascular endothelial growth factor and its receptors[J].Cytokine Growth Factor Rev，1996，7(3)：259-270.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
[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.w!6;/&, http://www.100md.com
收稿日期：2000年8月29日w!6;/&, http://www.100md.com
修稿日期：2000年10月8日w!6;/&, http://www.100md.com
出版日期：2001年1月15日(作者：肖畅 朱迅 赵显玲 石缨 王烨 刘丽 刘立忠 谢宝树 冷爱军 杨彦 曹颖)