白细胞介素12和共刺激分子B_(7-1)联合修饰肿瘤细胞疫苗诱导抗肿瘤免疫治疗的实验研究

Study of the therapeutic effects of costimulatory molecules B_(7-1) and interleukin 12 modified tumor cells vaccines on ovarian epithelial carcinoma through induction of anti-cancer immunity of the body

目的 探讨经白细胞介素 1 2 (IL 1 2 )和共刺激分子 (costimulatorymolecule)B7 1 基因联合修饰的肿瘤细胞疫苗诱导机体抗肿瘤免疫反应 ,对大鼠卵巢上皮癌腹腔转移瘤的治疗效果。方法采用酶切、去磷酸化、连接、重组的方法 ,构建携带B7 1 基因的逆转录病毒表达载体pLmB7 1 SN ,以脂质体介导法 ,建立高表达细胞株NuTu 1 9/B7 1 、NuTu 1 9/IL 1 2及双基因联合表达细胞株NuTu 1 9/IL 1 2 B7 1 ,并以转染空载体pLXSN的细胞NuTu 1 9/Neo为对照组。观察各组细胞在大鼠体内的致瘤性 ;将经丝裂霉素C处理的各组肿瘤细胞 ,以皮下接种方式免疫动物 ,观察其对卵巢癌腹腔转移大鼠模型生存期的影响 ,及对大鼠脾淋巴细胞增殖及诱导细胞毒性T淋巴细胞 (CTL)杀伤活性的作用。结果 本研究构建pLmB7 1 SN成功。建立的NuTu 1 9/B7 1 、NuTu 1 9/IL 1 2和NuTu 1 9/IL 1 2 B7 1 细胞株中mB7 1 和mIL 1 2稳定表达。与对照组细胞相比 ,各组肿瘤细胞在动物体内的致瘤性有不同程度下降。B7 1 组[NuTu 1 9/BT 1 (B7 1 单独免疫 ) ]和IL 1 2组 [NuTu 1 9/IL 1 2 (IL 1 2单独免疫 ) ]大鼠的脾淋巴细胞增殖指数 (PI)分别为 2 4和 4 6 ,后者与对照组 (PI=1 0 5)相比 ,差异有显著性 (P <0 0 5) ,而联合组 [NuTu 1 9/IL 1 2 B7

Objective To study the biological characters of tumor cell vaccines modified by interleukin 12 (IL 12) and co stimulatory molecule B 7 1 and their combination therapeutic effect on rat ovarian cancer animal model Methods Retroviral vector pLmB 7 1 SN, which expressed murine B 7 1 was constructed An epithelial ovarian cancer cell line NuTu 19 was infected with pLmIL 12SN and/or pLmB 7 1 SN by lipofectin mediated gene transfer system Cell lines that could highly express either or both of these cytokines were named NuTu 19/IL 12, NuTu 19/B 7 1 or NuTu 19/IL 12 B 7 1 , according to the results of enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) NuTu 19/Neo, the cell line that was transfected by vector pLXSN was used as a control Various types of cytokine modified tumor cells were injected subcutaneously into syngeneic rats Fischer 344 and their tumorigenecities were recorded After being immunized twice by various types of mitomycin C treated gene modified tumor cells, the survival time of the intraperitoneal disseminating ovarian cancer animal model was observed, and the anti tumor mechanisms of different gene modified tumor cell lines were discussed Results The reconstruction of pLmB 7 1 SN was successful Stable IL 12 and B 7 1 expression in cell lines NuTu 19/B 7 1 , NuTu 19/IL 12 and NuTu 19/IL 12 B 7 1 were confirmed by ELISA and FCM Tumorigenecities of various gene modified tumor cells decreased in syngeneic rats The splenic lymphocytes proliferation indices (PI) in B 7 1 group (NuTu 19/B 7 1 immunized group) and IL 12 group (NuTu 19/IL 12 immunized group) were 2 4 and 4 6(the letter P <0 05 compared with that in control group), while PI in B 7 1 and IL 12 combination group (NuTu 19/IL 12 B 7 1 immunized group) increase to 10 2,being of significantly difference compared with those in control or B 7 1 or IL 12 group ( P <0 01) More significant cytotoxic effects of cytotoxic lymphocytes (CTLs) on syngeneic tumor cells could be observed in B 7 1 group (15 0%) or IL 12 group (31 5%) ( P <0 05 or P <0 01, compared with 2 6% in control group), while coexpression of IL 12 and B 7 1 was of great importance in enhancing CTL activity (52 4%), compared with that in control or!B 7 1 or IL 12 group ( P <0 05, respectively) The life spans of ovarian cancer bearing rats immunized by IL 12 (59 8 d) or B 7 1 (56 2 d) tumor cell caccines were a little longer than that in control group (53 4 d), but no significant differences existed ( P >0 05), whereas in the animal models who had received IL 12 and B 7 1 co immunization, prolonged survival time was showed which had statistical significance compared with that in control group (66 0 d, P <0 05) Conclusion B 7 1 and IL 12 modified tumor cell vaccines can induce anti tumor immunity through stimulating lymphocytes proliferation and inducing recognition and cytotoxic effects of CTLs on tumor cells An obvious synergistic effect exists when the two cytokines are combined Combined immunogene therapy with IL 12 and B 7 1 should prove to be a promising therapeutic strategy in ovarian cancer