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白细胞介素12基因的体外抗结肠癌效应
引用本文:邢春根,钱建新,周丽英,陈易人.白细胞介素12基因的体外抗结肠癌效应[J].苏州大学学报(自然科学版),2004,24(4):436-439.
作者姓名:邢春根  钱建新  周丽英  陈易人
作者单位:苏州大学附属第二医院 江苏苏州215004 (邢春根,钱建新,周丽英),苏州大学附属第一医院 江苏苏州215006(陈易人)
摘    要:目的 构建共表达P35、P4 0二个亚基的逆转录病毒载体 (Rv mIL12 )、建立PA317 mIL12包装细胞系并评估其IL12的生物学活性及经PA317 mIL12作用后淋巴细胞对结肠癌细胞 (C2 6 )的体外杀伤作用。方法 将经PCR扩增mIL12 p35、p4 0获取的 p35和 p4 0双亚基cDNA片段与经NotI及SalI酶切的逆转录病毒载体 (PL P35 P P4 0 SN)DNA在T4噬菌体DNA连接酶作用下进行粘端连接反应。然后将构建的 pGCXEXPN mIL12经电穿孔法转录单嗜性逆转录病毒包装细胞系 (PE5 0 1) ,再以其病毒上清加Polybrene感染PA317,筛选出病毒滴度最高的携有mIL12逆转录病毒载体的包装细胞系 :PA317 mIL12。采用脾淋巴细胞增殖法检测PA317 mIL12的生物学活性。另外 ,将PA317 mIL12、淋巴细胞以及人结肠癌细胞 (LOVO)按不同浓度比体外共同培养 ,采用MTT法检测经PA317 mIL12作用后的淋巴细胞对LOVO的杀伤率。结果 经筛选的携有mIL12逆转录病毒载体包装细胞系PA317 mIL12的上清mIL12浓度达 2 7ng/ ( 10 6cells·4 8h) ,其促脾淋巴细胞增殖的生物学活性也与mIL12标准品活性相近 ,经PA317 mIL12作用后的淋巴细胞体外杀伤试验结果则显示 :随着PA317 mIL12浓度的增高 ,其淋巴细胞对结肠癌细胞杀伤作用也逐渐增强 ,与对照组相比统计学有显著性差异 (P

关 键 词:白细胞介素12基因  结肠癌  抗肿瘤效应  生物学活性  治疗
文章编号:1000-5749(2004)04-0436-04
修稿时间:2004年4月10日

The Antitumor Effects of Interleukin 12 Gene on Colon Cancer in Vitro
XING Chun-gen,QIAN Jian-xing,ZHOU Li-ying,et al.The Antitumor Effects of Interleukin 12 Gene on Colon Cancer in Vitro[J].Suzhou University Journal of Medical Science,2004,24(4):436-439.
Authors:XING Chun-gen  QIAN Jian-xing  ZHOU Li-ying  
Abstract:Objective To construct a Retroviral vector (GCXEXPN-mIL12) which can coexpress p35 and p40 and establish a PA317-mIL12 packaging cell system which has a sufficient expression quantity of mIL12 and a good biological activity. To observe the effects of PA317-mIL12 on the cancer cell cidal action of human periperal lymphocyte in vitro. Methods The full-lenth cDNA encoding mouse IL12 subunits p35 and p40 were amplificated by polymerase chain reaction (PCR) seperately. The polycistronic retroviral vector (pGCXEXPN-mIL12) was constructed in which both p35 and p40 were linked with internal ribosome entry site (IRES) from encephalomyocarditis virus and poliovirus by cohesive end ligation action.Subsequently the monophilic retrovirus packaging cell strain (PE 501) was transfected by pGCXEXPN-mIL12 with the method of electroporation. Then the packaging cells (PA317) transfected with bi-ophilic retroviral in viral supernatant were monocloned after screening with G-418 for 4 weeks. The PA317-mIL12 was generated and its virus titre was determined by NIH373. The mIL12 concentration in supernatant of PA317-mIL12 was measured by enzyme-linked immunosorbent assay (ELISA) and its biological activity measured by the method off spleen lymphocyte proliferation. The human colon adenocarcinoma cells ( Lovo ) were co-incubated with human periperal blood mononuclear cells and PA317-mIL12. The killing rate of lymphocytes to Lovo was measured by MTT assay. Results The concentration of mIL12 in the supernatant of PA317-mIL12 reached 27 ng/106/48hrs and PA317-mIL12 could promote the spleen lymphocytic proliferation just like standard mIL12. The study also showed that the cell-mediated cytotoxicity of lymphocyte was enhanced by PA317-mIL12. The MTT result of experiment group was significantly differ from that of control group ( P <0.05 ). Conclusion The mIL12 gene transfer system (PA317-mIL12 ) which can express both p35 and p40 and has potential biological activity can obviously enhance the cell-mediated cytotoxicity of lymphocyte by PA317-mIL12.
Keywords:interleukin 12 gene  colon adenocarcinoma  antitumor effection
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