pSecTag2B-mIL-18真核分泌表达载体的构建及其抗肿瘤作用研究

Study on construction of secretion mouse interleukin-18 plasmid and its antitumor effects

目的:探索小鼠IL-18基因治疗肿瘤的方法和疗效。方法:构建pSecTag2B-mIL-18真核分泌表达载体,并用其转染HT-29细胞,RT-PCR和ELISA检测IL-18表达情况;将转染pSecTag2B-mIL-18质粒的HT-29培养上清,加入到小鼠T淋巴细胞培养液,ELISA、RT-PCR检测T淋巴细胞分泌IFN-γ;脂质体包被的pSecTag2B-mIL-18裸质粒,采用肌肉注射法转入Balb/c小鼠,检测IL-18蛋白表达情况;在Balb/c小鼠接种小鼠co-lon-26肿瘤细胞的同时,将表达载体pSecTag2B-mIL-18转入,观察抑制肿瘤生长的情况。结果:1)经酶切和测序鉴定,pSecTag2B-mIL-18真核分泌表达载体所表达的基因序列经比对完全正确。2)pSecTag2B-mIL-18转染HT-29细胞后,IL-18在mRNA和蛋白水平高表达。3)转染后的HT-29培养上清可刺激小鼠T淋巴细胞高水平分泌IFN-γ。4)用裸质粒肌肉注射法将pSecTag2B-mIL-18转入Balb/c小鼠,证明质粒均能够在小鼠肌肉中表达,且表达的蛋白均能分泌到外周血液循环中。5)接种co-lon26细胞的Balb/c小鼠,将pSecTag2B-mIL-18转入小鼠,结果表明IL-18单独应用能明显抑制肿瘤的生长。结论:pSecTag2B-mIL-18裸质粒肌肉注射具有显著的抗肿瘤作用。

OBJECTIVE： To explore the method and antitumor effects of gene therapy of interleukin-18, METttODS： The secretion pSecTag2B-mlL-18 was constructed by our laboratory, and expression and biological activity of the plasmids were observed in vitro. Then the naked plasmid incapsuled by liposome was injected into the skeletal muscle of Balb/c mouse, and expression status of the gene was tested by RT-PCR and ELISA. pSecTag2B-mlL 18 plasmids were transfered to Balb/c mice which were vaccined by colon26 tumor cells. The antitumor effects were observed. RESULTS： The full length cDNA sequence of mouse ILl8 assayed correctly was inserted into the mammalian secretion expression vector pSecTag2B. The ex- pression vectors （pSecTag2B-m-IL18） were transfected into HT-29 cells, and the concentrations of IL-18 and the supernatant of incubated HT29 cells were detected by ELISA significantly. Its expression in HT29 cells in mRNA level was also detected by RT-PCR. The secreted IL-18 stimulated T lymphocytes of mice to secrete interferon （IFN-gamma）. When the plasmids were transferred into the muscle of mice simultaneously by means of injection of naked plasmid DNA, the gene also expresses simultaneously in both mRNA and protein level. IFN-gamma was detected in the peripheric serum of the mice. In antitumor animal test, when 10^5 of colon26 tumor cells were im- planted subcutaneous into every Balb/c mouse of IL-18 group and control group, the IL-18 gene transferred into the mice alone suppressed significantly the growth of implanted tumor. CONCLUSION： The transfer of mouse IL-18 gene can be expressed successfully by in tramuscular injection of naked plasmids in vivo, leading to a significant antitumor effect.