人乳头瘤病毒16E7DNA疫苗微针给药效果研究

The effect of HPV16E7 DNA vaccine transdermal delivery with microneedle array

目的探讨DNA疫苗微针给药方法的有效性。方法构建pcDNA3．1-HPV16E7重组质粒，在体外透皮条件下，观察pcDNA—HPV16E7皮肤透过量，再利用微针给药方式免疫BALB／c小鼠，分三组（实验组、空载体对照组、阴性对照组），每组10只，每2周免疫1次，共3次，每次免疫剂量为200μg／只，对照组参照实验组进行，最后1次免疫后2周采血，分别分离血清和淋巴细胞，用间接免疫荧光试验检测小鼠的体液免疫功能，用淋巴细胞转化试验检测小鼠的细胞免疫功能。结果体外透皮试验显示DNA疫苗微针给药可以透过皮肤，而且透过量随着时间的延长而逐步增加，在第30小时时可以达到0．73819mg／cm^2；通过微针给药方法DNA疫苗可以诱导小鼠产生特异性抗体，淋巴细胞转化试验显示：实验组（平均淋巴细胞转化率为47．25％）和阴性对照组（平均淋巴细胞转化率为30．00％）之间比较，Χ^2=12．903，P〈0．001，差异有显著统计学意义；空载体组（平均淋巴细胞转化率为43．00％）和阴性对照组之间比较，Χ^2=7．292，P=0．007，差异有显著统计学意义；实验组和空载体组之间比较，Χ^2=0．817，P=0．366，差异无统计学意义。结论HPV16E7DNA疫苗经微针给药可以透过皮肤吸收并诱导小鼠产生体液免疫和细胞免疫反应。

Objective To study the effects of DNA vaccine transdermal delivery with microneedle array. Methods The pcDNA3. 1-HPV16E7 recombinant vector acting as gene vaccine was established. The infiltration quantity of pcDNA3. 1-HPV16E7 getting across the microchannels generated by microneedle arrays in vitro was observed. 30 BALB/c mice were divided into 3 groups （experimental group, in vain plasmid group, negative control）. Each group had 10 mice. Then immunized BALB/c mice with a dose of 200 μg with microneedle array every two weeks. The control groups did the same as that as the study groups. Two weeks after the third immunization, the serum and lymphocytes were separated to detect the functions of humoral immunity with indirect immunofluorescence test, while, the functions of cellular immunity with lymphocyte transformation test was also detected. Results The DNA vaccine could easily get across the microchannels generated by microneedle arrays in vitro. Moreover, the course was permanent and the whole infiltration quantity was comparatively high, reaching 0. 738 19 mg/cm2 at the 30th hour. And among immunized BALB/c mouse, DNA vaccine transdermal delivery with microneedle array could induce specific antibodies. Lymphocyte transformation test showed that there was significant difference for the lymphocyte transformation rate between experiment （the average of lymphocyte transformation rate was 47.25% ） and control group（the average of lymphocyte transformation rate was 30. 00% ）（Χ^2 = 12. 903, P〈0. 001 ）. Also, the difference was found between in vain plasmid group（ the average of lymphocyte transformation rate was 43.00% ） and negative control（ Χ^2 =7. 292, P =0. 007） . While, no difference was observed in the experimental group and in vain plasmid group （Χ^2 = 0. 817, P = 0. 366）. Conclusion The DNA vaccine combined administering with microneedle array might get across the microchannels generated by microneedle arrays in vitro and induce humoral and cellular immune response in vivo.