基于SFVRNA复制子核酸疫苗pIRSFV-HN的构建及其体内外抑瘤作用  

Construction of SFV RNA replicon-based nucleic acid vaccine pIRSFV-HN and its anti-tumor effect in vivo and in vitro

作　　者：张钰[1] 金宁一[1] 李霄[1] 朱继红[2] 陈漉[2] 刘立明[3] 李昌[1] 佟敬山[1] 李群[1] 陈勇志[4] 

机构地区：[1]军事医学科学院全军基因工程重点实验室 [2]吉林大学第一临床医院,长春130021 [3]吉林农业大学动物科学技术学院,长春130118 [4]吉林大学畜牧兽医学院,长春130062

出　　处：《中国肿瘤生物治疗杂志》2008年第1期20-24,共5页Chinese Journal of Cancer Biotherapy

基　　金：吉林省科技发展计划项目(No20060566)~~

摘　　要：目的:构建基于塞姆利基森林病毒(Semliki forest virus,SFV)RNA复制子和新城疫病毒HN基因的核酸疫苗pIRSFV-HN,并探讨其体内外的抑瘤作用。方法:基于SFV RNA复制子和新城疫病毒HN基因构建核酸疫苗pIRSFV-HN,pIRSFV-HN转染人结肠癌细胞HCT-116,以Western blotting检测转染后HCT-116细胞HN的表达水平,以AO/EB双荧光染色检测肿瘤细胞的凋亡,以MTT法检测疫苗对HCT-116细胞增殖的抑制。构建C57BL/6小鼠右后肢皮下荷H22腹水瘤细胞模型,瘤体内注射pIRSFV-HN(共3次),检测该小鼠血清IL-2、IL-4、IL-10和IFN-γ水平和特异性CTL活性。结果:成功构建基于SFV RNA复制子和新城疫病毒HN基因的核酸疫苗pIRSFV-HN。结肠癌细胞HCT-116被疫苗转染后可有效表达HN蛋白,对照细胞则无表达;转染后HCT-116细胞出现凋亡的征象;该细胞的增殖受到明显抑制,最大抑制率达55.34%。移植瘤体内注射疫苗后,荷瘤小鼠血清IL-2、IFN-γ水平显著升高(P<0.05),其CTL活性也显著升高(P<0.01)。结论:基于SFVRNA复制子和新城疫病毒HN基因的核酸疫苗pIRSFV-HN在体外可有效地抑制肿瘤细胞;在体内可促使免疫趋向Th1优势,提高其抑瘤免疫水平。Objective： To prepare semliki forest virus （SFV） RNA replicon-based nucleic acid vaccine pIRSFV-HN and investigate its role on tumor suppression in vivo and in vitro. Methods： SFV RNA replicon and newcastle disease virus HN gene-based nucleic acid vaccine pIRSFV-HN was prepared and was used to transfect human colonic cancer cell line HCT-116. The expression of HN gene was detected by Western blotting after transfecttion and the apoptosis of tumor cells was detected by AO/EB staining. MTT assay was used to examine the inhibition of HCT-116 cells after transfection. C57BL/6 mice model beating H22 hepatoma was prepared by transplanting H22 cells into the tight hind limb of the mice and pIRSFV-HN was injected into the tumor （ for 3 times） ; IL-2, IL-4, IL-10, and IFN-γ levels in the sera and the CTL activity of splenocytes were detected. Results： SFV RNA replicon - based nucleic acid vaccine pIRSFV-HN was success- fully prepared. HCT-116 tumor cells transfected with pIRSFV-HN effectively expressed HN protein. HCT-116 tumor cell transfected with pIRSFV-HN showed a typical apoptosis morphology and was obviously suppressed, with the maximal inhibitory rate being 55.43%. The levels of IL-2 and IFN-γ was increased significantly after injection of pIRSFV-HN into the tumors （P 〈0.05）, and the activity of CTL was also increased significantly （P 〈0.01）. Conclusion： SFV RNA replicon-based nucleic acid vaccine pIRSFV-HN can effectively suppress tumor cells proliferation in vitro and promote Th1 immune response, increase the tumor suppression in vivo.

关 键 词：塞姆利基森林病毒 RNA复制子 新城疫病毒 核酸疫苗 结肠癌 抑瘤作用 细胞毒性T淋巴细胞 

分 类 号：R735.3[医药卫生—肿瘤] R730.5[医药卫生—临床医学]