化疗诱导损伤相关分子模式强化CIK对肺癌A549细胞的抑制作用

Inhibitory effects of CIK on lung cancer A549 cells intensified by chemotherapyinduced damaged associated molecular pattern

目的:探讨化疗诱导损伤相关分子模式(damaged associated molecular patterns,DAMP)对CIK细胞抑制RAS突变A549肺腺癌细胞的影响及其机制。方法:体外分离人外周血单个核细胞且培养CIK细胞,以顺铂(cisplatin,DDP)和多柔比星(doxorubicin,ADM)单独或联合作用于A549细胞,镜下观察A549细胞的形态,将药物后的A549细胞上清加入CIK细胞共培养,流式细胞术检测共培养后CIK细胞免疫表型,MTT实验检测A549细胞上清诱导CIK细胞对A549肺腺癌细胞增殖的抑制,ELISA实验检测多种浓度化疗药物杀伤A549细胞上清中CRT、ATP、HMGB1含量。结果:低质量浓度化疗药杀伤A549细胞后呈现较多的免疫原性死亡特征。杀伤后A549细胞上清加入CIK细胞共培养使CIK细胞CD8+、CD56+的比例较对照CIK细胞明显升高(均P<0.05)。A549细胞损伤后上清诱导CIK细胞对A549肺腺癌细胞增殖抑制率明显高于同质量浓度化疗药组[DDP组(31.34±1.51)%vs(5.97±1.74)%,ADM组(45.46±1.78)%vs(6.22±1.34)%,DDP+ADM组(45.78±1.14)%vs(11.94±3.11)%,均P<0.05],并且低质量浓度化疗药物杀伤A549细胞上清诱导的CIK对A549细胞的抑制率高于较高浓度化疗药物杀伤后细胞上清诱导的CIK(均P<0.05)。低质量浓度化疗药物杀伤A549细胞上清中免疫原性死亡相关分子CRT、ATP、HMGB1含量增加得最多(均P<0.05)。低质量浓度组该上清诱导的CI对A549肺腺癌细胞的增殖抑制率随着CRT、ATP、HMGB1水平的增高而增加。结论:较低质量浓度的DDP和ADM单独或联合用药更易引起A549细胞免疫原性死亡并释放较高水平的DAMP分子,能更强的促进CIK对肺癌A549细胞的抑制作用。

Objective： To investigate the effect of DAMP （damaged associated molecular pattern） on the inhibition of RAS-mutant A549 lung adenocarcinoma cells by CIK cells and its mechanism. Methods： Human peripheral blood mononuclear cells were isolated in vitro and CIK cells were cultured. A549 cells were treated with cisplatin （DDP） and doxorubicin （ADM） alone or in combination,and the morphology of A549 cells was observed under a microscope. The supernatant of A549 cells was co-cultured with CIK cells.Flow cytometry was used to detect the CIK cell immunophenotype after co-culture. MTT assay was used to detect the inhibition of A549 lung cancer cell proliferation induced by A549 cell supernatant. The concentration of chemotherapeutic drugs kills A549 cell supernatant CRT, ATP, HMGB1 content. Results： Low-level chemotherapeutic drugs showed more immunogenic death characteristics after killing A549 cells. The ratio of CD8＋ and CD56＋ in CIK cells was significantly higher than that in control CIK cells （P〈0.05）. The inhibition rate of CIK cells induced by A549 cells after injury on A549 lung adenocarcinoma cells was significantly higher than that of the same dose chemotherapy group [DDP group （31.34±1.51）% vs （5.97±1.74）%, ADM group （45.46±1.78）% vs （6.22±1.34）%, DDP＋ADM group （45.78±1.14）% vs （11.94±3.11）%, all P〈0.05], and low-mass chemotherapeutic agents killed C549 induced by A549 cell supernatant on A549 The inhibition rate of the cells was higher than that of the supernatant induced by the higher concentration of che-motherapeutic drugs （all P〈0.05）. The level of CRT, ATP, and HMGB1 in immunogenicity-related molecules in the supernatant of A549 cells was significantly increased by low-concentration chemotherapy drugs （all P〈0.05）. In the low-concentration group, the supernatant-induced inhibition of the proliferation of A549 lung adenocarcinoma cells increased with the increase of CRT, ATP, and HMGB1 levels. Conclusion： The combination of lower concentration of DDP and ADM alone or in combination could more easily induce the immunogenic death of A549 cells and release higher levels of DAMP molecules, which could promote the inhibitory effect of CIK on lung cancer A549 cells.