模拟热放疗制备的高效伴侣分子抗原肽的抗癌免疫作用

The chaperone-peptide-rich vaccine derived from colo-carcinoma cells subjected to heat stress followed by radiation elicits potent anticancer efficacy

目的研究高效伴侣分子-抗原肽肠癌疫苗制备方法并为优化肠癌的热、放疗和免疫治疗提供基础资料。方法将CT26肿瘤细胞置于含榄香烯的培养基中以不同热应激温度培养，继以高能X射线（200cGy）照射；超声裂解细胞后低温离心；盐析法分离上清蛋白；透析去除低于50kD和高于300kD的分子。凝胶过滤结合SDS-PAGE法收取70、90、110和170kD峰段蛋白并合并。Western blotting法对所获蛋白定性；BCA法测量浓度。检测不同条件制备的伴侣分子肽混合物的细胞增殖指数、NK和CTL细胞活性。结果成功提取制备了富伴侣分子-抗原肽瘤苗；不同条件制备的瘤苗的T细胞增殖指数、NK和CTL细胞活性均明显提高。结论凝胶过滤结合SDS-PAGE可以较好提取制备主要伴侣分子肽；热放疗可使伴侣分子肽的细胞表达量增加，从而诱导瘤苗产生更强的抗癌免疫作用。其中42℃热应激序贯照射组的瘤苗抗癌免疫作用最强。

Objective To prepare a high-performance and chaperone-peptide-rich anticancer vaccine, and further explore the elementary data for the grading-up synthetic therapy including thermo-radiation therapy and immunotherapy. Methods CT26 colo-carcinoma cells were cultured in the 0.1% elemene 1640 medium at high temperatures,followed by 200cGy X-ray radiation, to induce the expression of the molecular chaperones and promote the synthesis of antigen peptide. Groups of these cells treated under the different conditions were lysed by the sonic disintegration, and the lysates were centrifuged. The raw-purified proteins were obtained from the supernatants by precipitating with ammonium sulfate and removing the molecules below 50kD and above 300kD in molecular weight via dialysis. Furthermore, the proteins with the weights in 70, 90, t10 and 170kD were collected through gel filtration and SDS-PAGE. The purified proteins were examined by Western blotting and BCA, and inspected on the lymphocyte proliferation and the activities of NK and CTL. Results Major of the chaperone-peptides of anticancer immunity, were satisfactorily extracted that the multi-chaperone-peptide-rich composites were successfully obtained. The composites prepared under various conditions could all enhance lymphocyte proliferation and the activities to some extent. Conclusion The multi-chaperone-peptide-rich composites were successfully prepared via dialysis, gel filtration and SDS-PAGE; The heat stress, elemene and radiation could all increase the composites synthesis, of which that treated by 42℃ heat stress followed by radiation was found to be of the strongest anticancer immunological efficacy.