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目的:研究高迁移率族蛋白B1(HMGB1)对放疗后残存胰腺癌细胞的干性转化作用。方法:酶联免疫吸附实验(ELISA)检测在不同放疗剂量下(0、4、8、10、12 Gy)胰腺癌SW1990细胞上清液中HMGB1的含量;通过不同剂量(0、4、6、8、10 Gy)放疗后,流式细胞术检测残存SW1990细胞获得CD133~+细胞的比例;获取10 Gy放疗剂量下SW1990细胞的上清液,然后将8 Gy放疗后残存SW1990细胞分成4组:PBS组、上清液+EP(ethyl pyruvate,丙酮酸乙酯,为胞外HMGB1特异性抑制剂)组、上清液组、HMGB1(100 ng/mL)组,流式细胞术检测各组细胞CD133~+细胞的比例,同时蛋白质印迹实验检测干性标志分子SOX2和OCT4蛋白表达。结果:ELISA结果显示,胰腺癌SW1990细胞上清液中HMGB1的浓度在10 Gy放疗剂量时最大(217.3±34.97)ng/mL。流式细胞术检测结果显示,在8 Gy放疗后SW1990细胞中CD133~+细胞比例最大(22.63±0.74)%。同时流式细胞术检测结果显示,上清液+EP组、上清液组、HMGB1(100 ng/mL)组中CD133~+细胞相对于PBS组的比值分别是2.2±0.11、3.2±0.19和6.4±0.32(F=143.4,P<0.01)。蛋白质印迹结果显示,上清液组与HMGB1(100 ng/mL)组中干性相关蛋白SOX2和OCT4的表达明显上调(P<0.01)。结论:放疗后胰腺癌SW1990上清液中存在HMGB1,并且HMGB1会调控残存SW1990细胞干性的获得。
OBJECTIVE: To investigate the effect of high mobility group box 1 protein (HMGB1) on the survival of pancreatic cancer cells after radiotherapy. Methods: The levels of HMGB1 in the supernatant of pancreatic cancer SW1990 cells were detected by enzyme-linked immunosorbent assay (ELISA) at different doses of radiation (0, 4, 8, 10, 12 Gy) 8,10 Gy), the percentage of CD133 ~ + cells in SW1990 cells was detected by flow cytometry; the supernatant of SW1990 cells was obtained at the dose of 10 Gy, and then the remaining SW1990 cells after radiotherapy were divided into 4 groups: PBS group, supernatant + EP (ethyl pyruvate), supernatant group, HMGB1 (100 ng / mL) group, and the cells in each group were detected by flow cytometry CD133 ~ + cells, Western blotting assay was used to detect the expression of SOX2 and OCT4. Results: The results of ELISA showed that the concentration of HMGB1 in the supernatant of SW1990 cells was the highest (217.3 ± 34.97) ng / mL at the dose of 10 Gy. Flow cytometry results showed that the proportion of CD133 ~ + cells in SW1990 cells was the highest (22.63 ± 0.74)% after 8 Gy radiotherapy. Meanwhile, the results of flow cytometry showed that the ratio of CD133 ~ + cells in the supernatant + EP group, supernatant group and HMGB1 (100 ng / mL) group to PBS group were 2.2 ± 0.11, 3.2 ± 0.19 and 6.4 ± 0.32 (F = 143.4, P <0.01). Western blotting showed that the expressions of SOX2 and OCT4 in the supernatant group and HMGB1 (100 ng / mL) group were significantly increased (P <0.01). Conclusion: HMGB1 is present in the supernatant of pancreatic cancer SW1990 after radiotherapy, and HMGB1 regulates the survival of surviving SW1990 cells.