IL-2基因转染的G422胶质母细胞瘤体外增殖及体内致瘤性的改变

In vitro proliferation and in vivo tumorigenicity of IL 2 gene modified G422 mouse glioblastoma

目的：探讨ＩＬ－２基因治疗胶质瘤的可能性，并对其免疫机制进行初步分析。方法：重组的复制缺陷腺病毒作为基因转移的载体，将鼠ＩＬ－２基因转染Ｇ４２２小鼠胶质母细胞瘤细胞（Ｇ４２２－ｍＩＬ２），ＲＴ－ＰＣＲ检测Ｇ４２２－ｍＩＬ２细胞中目的基因的ｍＲＮＡ表达，采用ＩＬ－２依赖细胞株检测Ｇ４２２－ｍＩＬ２培养上清中ｍＩＬ－２的活性，ＭＴＴ法检测Ｇ４２２－ｍＩＬ２体外增殖能力的改变。将野生型Ｇ４２２细胞、ＬａｃＺ基因转染的Ｇ４２２细胞（Ｇ４２２－ＬａｃＺ）、Ｇ４２２－ｍＩＬ２接种到小鼠皮下，观察皮下肿瘤的生长和荷瘤小鼠的存活期，采用４小时５１Ｃｒ释放法检测荷瘤小鼠脾细胞ＬＡＫ、ＣＴＬ的杀伤活性。结果：Ｇ４２２－ｍＩＬ２细胞有目的基因ｍＲＮＡ的表达，体外培养上清中分泌高水平的ｍＩＬ－２，和野生型Ｇ４２２、Ｇ４２２－ＬａｃＺ相比，其体外的细胞增殖能力无显著差异（Ｐ＞０．０５），而接种皮下后肿瘤生长延缓，小鼠的存活期延长（Ｐ＜０．０１），有４／１０的小鼠长期无瘤生存（＞９０天）。Ｇ４２２－ｍＩＬ２接种组小鼠脾细胞的ＬＡＫ、ＣＴＬ活性显著增强（Ｐ＜０．０１）。结论：ＩＬ－２基因转染的Ｇ４２２细胞的体内致瘤性显著降低，其机理可能?

Objective: To evaluate the possibility of Interleukin 2 gene therapy to gliomas, and analyzed the preliminary immunological mechanisms. Methods: Mouse IL 2 gene was introduced into mouse glioblastoma cell line G422 by recombinant adenovirus vector. RT PCR was used to detect mRNA expression of target gene in gene modified tumor cells(G422 mIL2). IL 2 secretion by G422 mIL2 was assayed with the use of IL 2 dependent cell line. In vitro proliferation potential of G422 mIL2 by MTT assay. We investigated the tumor size and survival of tumor bearing mice were observed after wild type G422, LacZ gene transfected G422(G422 LacZ), G422 mIL2 were inoculated subcutaneously. The splenic LAK, CTL cytotoxicity were analyzed by standard 51 Cr release assay. Results: We detected mRNA transcription of target gene in G422 mIL2. High level of mouse IL 2 could be detected in the culture supernatant of G422 mIL2. There was no significant change of the in vitro proliferation potential (P>0 05). When inoculated subcutaneously, the tumor growth of G422 mIL2 was significantly inhibited as compared to wild type G422 and G422 LacZ. The survival of mice inoculated with G422 mIL2 was significantly prolongated (P<0 01) with 4/10 long term survival mice (>90 days), and the splenic LAK and CTL cytotoxicity were also significantly enhanced (P<0 01). Conclusion: The reduction of in vivo tumorigenicity of IL 2 gene modified G422 may due to enhanced specific and non specific antitumor immunity of tumorbearing mice.