体内外检测乙型肝炎DNA疫苗诱生小鼠特异性CTL活性的比较研究  

Study on detection efficiency in vivo/in vitro of the special CTL responses of mice after inoculated with HBV DNA vaccine

作　　者：王松[1] 孙梅芹[2] 许诚[1] 何清[1] 周陶友[3] 陈守春 赵连三[3] 

机构地区：[1]深圳市东湖医院,广东深圳518020 [2]成都军区总医院 [3]四川大学华西医院 [4]成都地奥集团

出　　处：《中西医结合肝病杂志》2006年第4期224-225,共2页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：深圳市卫生科技计划资助(No.199906007)

摘　　要：目的:了解体内外两种方法检测乙型肝炎DNA疫苗诱生BALB/c小鼠特异性细胞毒性T淋巴细胞(CTL)活性的特点,比较其异同,建立与体外法互为补充,且更加直观、简便的活体CTL活性检测模型。方法:用HBV-S真核表达质粒pVAX1/S对SP2/0细胞进行转染,经鉴定后作为目的靶细胞(稳定转染并表达HBV主蛋白,命名为SP2/0-S细胞);对照靶细胞为稳定转染pVAX1空载质粒的细胞(命名为SP2/0-P细胞)备用。活体诱生实验:用目的或对照靶细胞分别使小鼠荷瘤,观察免疫及非免疫小鼠在各靶细胞负荷下的生存时间与肿瘤的生长情况,设置不同组合做同期对照观察,小鼠生存率的差异可以提示体内特异性CTL是否存在。体外诱生实验:采用经典CTL活性检测方法,LDH释放法成品药盒。SP2/0-S细胞与SP2/0-P细胞作为靶细胞,免疫鼠与非免疫鼠的脾细胞为效应细胞,按照不同效/靶比共孵育后以酶标仪检测LDH的释放活性。结果:活体诱生实验:免疫小鼠存活时间明显长于对照小鼠(P<0·05)。体外诱生实验:免疫鼠CTL活性在效/靶比为50/1时出现最大杀伤活性,为36·9%,明显优于对照组(P<0·05)。结论:体内外两种方法均能够检测到乙型肝炎DNA疫苗诱生BALB/c小鼠特异性CTL活性,实验中发现体外LDH释放法CTL活性并不高,但是总体上仍然能够反映CTL活性。而活体诱生实验操作相对简便,表现更为直观,可望成为与经典方法相互补充的新型实验模型。Objective： To study the detection efficiency in vivo of special CTL response, of which the magnitudy and strengh after DNA vaccine of eukaryotic expression plasmids encoded HBV S protein was inoculated in BALB/c mice. Methods： BALB/c mice were inoculated with HBV DNA vaccine （pVAXI/S）, the detection efficiency of CTL response was compared between the two methods. The target cell line of stable transfected with eukaryotic expression plasmid encoded HBV S protein and expressing the protein （named SP2/0-S） was developed, and that of stable transfection with eukaryotic expression plasmids vector pVAX1 （named SP2/0-P） was for control. The animal model of CTL test in vivo was studied as a more convenient test compared with that in vivo . The different survival rate of immuned mice which were inoculated with target or control cells could be considered the reflection of CTL response in existence or not （ in vivo）. The LDH released assay was used in classical CTL test （in vivo）. Slpeen cells of immuned mice （uninmmuned mice for control） used as effect cells. SP2/0-S and SP2/0-P cells served as target cells or control. Results： In vivo test, the immuned mice showed a significant prolonged survival length relative to that of the unimmuned mice （P 〈 0. 05 ） . In vitro test, the optimal CTL activities （36. 9% ） of the immuned mice were showed the E/T rate in 50/1 with a significant superior in that of the uninmmuned mice （ P 〈 0. 05 ）. Conclusion ： The special CTL response induced by eukaryotic expression plasmids encoded HBV S protein （ S domain） in BALB/c mice could be detected both in vivo and in vivo. The CTL activities observed with LDH released assay in vivo was a briefness and comprehensible method which may be served as a estimate animal model and a supplementary to classical CTL test.

关 键 词：DNA疫苗 细胞毒性T淋巴细胞 动物模型 

分 类 号：R392[医药卫生—免疫学] R446.1[医药卫生—基础医学]