融合基因pEGFP-C3-B7．2-hTERT诱导小鼠免疫应答及抑制肝癌移植瘤  

Fusion gene pEGFP-C3-B7.2-hTERT induces immune response in mice and restrains transplanted hepatoma

作　　者：艾敏[1] 杨胜利[2] 姜爱华[1] 

机构地区：[1]濮阳市人民医院检验科,河南濮阳457000 [2]郑州大学基础医学院食管癌研究室,河南郑州450052

出　　处：《中国肿瘤生物治疗杂志》2008年第1期51-55,共5页Chinese Journal of Cancer Biotherapy

基　　金：河南省自然科学基金资助项目(No0411042400)~~

摘　　要：目的:构建融合DNA疫苗pEGFP-C3-B7.2-hTERT,观察其诱导小鼠免疫应答的能力和抗小鼠肝癌H22细胞移植成瘤的作用。方法:通过RT-PCR扩增B7.2和hTERTcDNA,以pEGFP-C3为载体构建pEGFP-C3-B7.2-hTERT融合基因质粒,肌肉注射接种C57BL/6小鼠,间隔7d,共3次,以注射接种pEGFP-C3-B7.2、pEGFP-C3-hTERT、pEGFP-C3和PBS为对照。检测免疫小鼠脾淋巴细胞CTL杀伤活性、脾细胞培养上清IL-2和IFN-γ水平、外周血淋巴细胞亚群变化及血清中抗hTERT抗体水平。将融合基因pEGFP-C3-B7.2-hTERT免疫已负荷肝癌细胞H22/hTERT或H22/B7.2的小鼠,观察小鼠成瘤时间、生存时间。结果:琼脂糖凝胶电泳、酶切及序列测定证实,成功构建了融合基因质粒pEGFP-C3-B7.2-hTERT。融合基因免疫小鼠的脾淋巴细胞对B7.2-hTERT阳性靶细胞的杀伤活性明显高于各对照组(P<0.01),每只pEGFP-C3-B7.2-hTERT免疫小鼠都产生了抗体,免疫鼠外周血CD4+、CD8+T细胞和脾细胞培养上清中Th1类细胞因子IFN-γ、IL-2水平较各对照组明显升高(均P<0.01)。pEGFP-C3-B7.2-hTERT融合基因质粒免疫已负荷肝癌细胞H22/hTERT或H22/B7.2的小鼠后,小鼠60d未成瘤,其他各组22d时所有小鼠均已成瘤,60d时均死亡。结论:DNA疫苗pEGFP-C3-B7.2-hTERT在体内能诱导出明显的B7.2-hTERT特异性的抗肿瘤免疫应答,且能抑制体内已经存在的少量肿瘤细胞的成瘤。Objective： To construct a fusion DNA vaccine pEGFP-C3-B7.2-hTERT and to observe the capability of pEGFP-C3-B7.2-hTERT DNA vaccine to induce specific anti-tumor immune responses and to inhibit growth of the hepatoma transplanted from H22 cells. Methods： The B7.2 and hTERT eDNA （ amplified by RT-PCR）, together with pEGFP-C3 as the vector were used to construct fusion gene plasmid pEGFP-C3-B7.2-hTERT, which was then used to vaccinated C57BL/6 mice for 3 times at a 7 d interval. Animals vaccinated with pEGFP-C3-B7.2, pEGFP-C3-hTERT, pEGFP-C3 and PBS were taken as controls. Splenocytes CTLs of immunized mice, the levels of IL-2, IFN-γ in the culture supernatant, the levels of antibodies against hTERT, and the changes of the T lymphocyte subsets in the peripheral blood were all examined. The mice harboring hepatocarcinoma H22 cells were challenged with pEGFP-C3-B7.2-hTERT and the tumor forming time and the survival periods of mice were observed. Results： Agarose gel electrophoresis, nuclease digestion and sequencing confirmed the successful construction of pEGFP-C3-B7.2-hTERT. The CTLs activity of splenocytes from the mice immunized with pEGFP-C3-B7.2-hTERT fusion gene was significantly higher than those of the other groups （P 〈 0. 01 ）. All mice immunized with pEGFP-C3-B7.2-hTERT produced antibody. The counts of CD4^＋, CD8^＋ T cells and the levels of IFN-γ, IL-2 were significantly higher in pEGFP-C3-B7.2-hTERT immunized mice compared with those in other groups （ all P 〈 0.01 ）. No tumor formed in mice harboring hepatocarcinoma H22 cells after pEGFP-C3-hTERT immunization ; tumors were noticed in other groups 22 d after inoculation and all mice died 60 d after inoculation. Conclusion： The pEGFP-C3-B7.2-hTERT DNA vaccine can induce potent B7.2-hTERT specific cellular immune responses a- gainst tumors; it can also inhibit tumor forming by tumor cells in vivo.

关 键 词：DNA疫苗 B7.2 HTERT 免疫应答 移植瘤 免疫治疗 

分 类 号：R730.54[医药卫生—肿瘤] R735.7[医药卫生—临床医学]