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目的探讨以人β防御素-2为佐剂的HPV16E7c免疫功能及细胞免疫应答。方法将30只小鼠按随机数字表法分为3组:PBS组、pcDNA3.1(+)/E7c组、pEGFP-C1/HBD2-E7c组,每组10只。PBS组采用PBS100μg·只-1行股四头肌肌内注射,进行初次免疫;pcDNA3.1(+)/E7c组采用pcDNA3.1(+)/E7c质粒100μg·只-1行股四头肌肌内注射,进行初次免疫;pEGFP-C1/HBD2-E7c组采用pEGFP-C1/HBD2-E7c联合质粒100μg·只-1行股四头肌肌内注射,进行初次免疫。3组初次免疫后,再间隔2周,以上述方法和剂量加强免疫2次。共免疫3次。3组小鼠免疫1周后,断颈处死小鼠,取小鼠脾脏称重,计算脾脏脏器指数及计数脾淋巴细胞数量,流式细胞仪检测淋巴细胞亚群CD4+、CD8+细胞数量及比值;采用摘眼球采血法收集小鼠血清,采用ELISA法检测小鼠血清E7抗体水平。结果 pcDNA3.1(+)/E7c组、pEGFP-C1/HBD2-E7c组小鼠脾脏脏器指数均明显低于PBS组(均P<0.01)。pEGFP-C1/HBD2-E7c组、pcDNA3.1(+)/E7c组小鼠CD4+、CD8+阳性细胞比例、绝对数均明显高于PBS组(均P<0.05),pEGFP-C1/HBD2-E7c组、pcDNA3.1(+)/E7c组小鼠CD4+/CD8+比值均明显低于PBS组(均P<0.05),pEGFP-C1/HBD2-E7c组、pcDNA3.1(+)/E7c组小鼠血清E7抗体水平均明显高于PBS组(均P<0.01),pEGFP-C1/HBD2-E7c组小鼠血清E7抗体水平明显高于pcDNA3.1(+)/E7c组(P<0.05)。结论人β防御素-2可明显改善HPV16E7c基因的免疫原性,提高HPV16E7特异性抗体水平,升高免疫小鼠脾CD4+和CD8+T淋巴细胞数量。
Objective To investigate the immune function and cellular immune response of HPV16E7c adjuvanted with human β-defensin-2. Methods Thirty mice were randomly divided into three groups: PBS group, pcDNA3.1 (+) / E7c group and pEGFP-C1 / HBD2-E7c group, with 10 mice in each group. The PBS group was injected with 100μg · -1 quadriceps PBS for primary immunization. The pcDNA3.1 (+) / E7c group was treated with pcDNA3.1 (+) / E7c plasmid Intramuscular injection, the initial immunization; pEGFP-C1 / HBD2-E7c group using pEGFP-C1 / HBD2-E7c plasmid 100μg · only -1 quadriceps intramuscular injection, the initial immunization. Three groups after the first immunization, and then two weeks apart, with the above methods and doses to strengthen the immune 2 times. A total of 3 immunizations. Three weeks after immunization, mice were sacrificed and the spleens were sacrificed. The spleen of mice were weighed, the indexes of spleen organ were counted and the number of splenic lymphocytes was counted. The numbers of CD4 + and CD8 + cells in lymphocyte subsets and their ratio were determined by flow cytometry The serum of mice was collected by taking eyeball blood collection method and the level of serum E7 antibody was detected by ELISA. Results The indexes of spleen organs in pcDNA3.1 (+) / E7c group and pEGFP-C1 / HBD2-E7c group were significantly lower than those in PBS group (all P <0.01). The absolute numbers of CD4 + and CD8 + positive cells in pEGFP-C1 / HBD2-E7c group and pcDNA3.1 (+) / E7c group were significantly higher than those in PBS group (all P <0.05), while pEGFP-C1 / HBD2-E7c group , And the ratio of CD4 + / CD8 + in the pcDNA3.1 (+) / E7c group was significantly lower than that in the PBS group (all P <0.05). The serum levels of pEGFP-C1 / HBD2-E7c and pcDNA3.1 (+) / E7c (P <0.01). The level of serum E7 antibody of mice in pEGFP-C1 / HBD2-E7c group was significantly higher than that in pcDNA3.1 (+) / E7c group (P <0.05). Conclusion Human β-defensin-2 can significantly improve the immunogenicity of HPV16E7c gene, raise the level of HPV16E7-specific antibody and increase the number of splenic CD4 + and CD8 + T lymphocytes in immunized mice.