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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 construct the nucleic acid vaccine pIRSFV-HN based on the RNA replicon of Semliki forest virus (SFV) and the HN gene of Newcastle disease virus (NDV), and to explore its antitumor activity in vitro and in vivo. Methods: Human colon cancer cell line HCT-116 was transfected with SFV RNA replicon and Newcastle disease virus HN gene. The expression of HN in HCT-116 cells was detected by Western blotting. AO / EB double staining was used to detect the apoptosis of tumor cells. MTT assay was used to detect the inhibition of HCT-116 cell proliferation. The C57BL / 6 mice were injected subcutaneously with H22 ascites tumor cells in the right hind limbs. The mice were injected with pIRSFV-HN (3 times in total), and the levels of IL-2, IL-4, IL-10 and IFN- Specific CTL activity. Results: The nucleic acid vaccine pIRSFV-HN based on SFV RNA replicon and Newcastle disease virus HN gene was successfully constructed. The expression of HN protein in HCT-116 colon cancer cell line was significantly higher than that in HCT-116 colon cancer cell line, and the proliferation of HCT-116 cells was inhibited obviously with the maximum inhibition rate of 55.34% . After transplanted tumor in vivo, the levels of IL-2 and IFN-γ in tumor-bearing mice were significantly increased (P <0.05), and the CTL activity was also significantly increased (P <0.01). Conclusion: The nucleic acid vaccine pIRSFV-HN based on the SFV RNA replicon and the HN gene of Newcastle disease virus can effectively inhibit the tumor cells in vitro. It can promote the immune predominance of Th1 in vivo and improve the immunity level of the tumor suppressor gene.