高迁移率族蛋白B1调控放疗残存胰腺癌细胞的干性获得

Role of HMGB1 in the acquisition of stemness of post-radiation residual pancreatic carcinoma cell line SW1990 in vitro

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 stem cell transformation of the HMGB1 in the post-radiation residual pancreatic carcinoma cell line SW1990 in vitro.Methods：With various radiation doses（0,4,8,10,12 Gy）, ELISA assay was used to access the content of HMGB1 in the supernatant of pancreatic carcinoma cell line SW1990.With another various radiation doses（0,4,6,8,10 Gy）, the proportion of the CD133^＋ cells in the post-radiation residual SW1990 cells were accessed by flow-cytometry （FCM）.The 8 Gy post-radiation residual SW1990 cells were divided into four groups, including PBS group, supernatant＋EP（ethyl pyruvate, inhibitor of HMGB1）group, supernatant group, HMGB1（100 ng/mL） group, then the proportion of the CD133^＋ cells in each group were examined by flow-cytometry.At the same time, the expression levels of SOX2 and OCT4 proteins were detected by Western blotting.Results：ELISA assay showed that the concentration of the HMGB1 in the supernatant of the post-radiation residual pancreatic carcinoma cell line SW1990 was the highest at 10 Gy radiation dose[（217.3±34.97）ng/mL].FCM assay showed that the CD133^＋ cells ratio was the highest at the 8 Gy radiation dose in the varies doses[（22.63±0.74）%].Compared with PBS group, the CD133^＋ relative ratio of the supernatant＋EP group,supernatant group and HMGB1（100 ng/mL） group were （2.2±0.11）,（3.2±0.19） and （6.4±0.32）, respectively（F=143.4,P〈0.01）.Meanwhile supernatant group and HMGB1 （100 ng/mL） group up-regulated notably the expression levels of SOX2 and OCT4 （P〈0.01）.Conclusion：HMGB1 is detected in the supernatant of the post-radiation residual pancreatic carcinoma cell line SW1990 in vitro, and HMGB1 can positively induce the acquisition of stemness of post-radiation residual SW1990 cells in vitro.