内质网应激前列腺癌细胞通过STAT3信号途径 促进巨噬细胞M2极化的研究

Endoplasmic Reticulum Stressed Prostate Cancer Cells Promotes Macrophage M2 Polarization Through STAT3 Signaling Pathway

目的探讨前列腺癌细胞内质网应激相关外泌体能否通过STAT3调控巨噬细胞M2极化。方法以有无毒胡萝卜素(TG)刺激,将前列腺癌细胞(PC3)细胞分为TG组和Con组。TG组分别以不同浓度TG同样时间和同样浓度TG不同时间刺激PC3细胞,通过观察ERS标志蛋白GRP78的表达,确定最佳刺激浓度与时间;Western blot检测Con组和TG组外泌体标志蛋白CD63和TSG101表达,透射电子显微镜鉴定外泌体;激光共聚焦显微镜观察巨噬细胞摄取外泌体;以Con组和TG组外泌体刺激巨噬细胞分为M-Con组和M-TG组,化学发光法检测M-TG组和M-Con组巨噬细胞炎症因子IL-1β、IL-6、IL-10与TNF-α表达情况;Western blot检测M-TG组和M-Con组巨噬细胞PD-L1表达及STAT3通路蛋白表达。结果以TG刺激PC3细胞,结果显示当以3μmol/L的TG刺激PC3细胞24 h,可明显升高GRP78蛋白的表达,可以构建较好的前列腺癌细胞ERS模型。Western blot及透视电镜结果证实成功分离PC3外泌体,且TG组外泌体表达CD63与TSG101多于Con组(P<0.05);激光共聚焦显微镜显示,外泌体能够被巨噬细胞所吞噬;化学发光结果显示,M-TG组分泌的炎症因子较M-Con组多(P<0.05),能够完成巨噬细胞的M2极化;与M-Con组比较,M-TG组能增加巨噬细胞PD-L1蛋白表达,并且激活巨噬细胞STAT3通路(P<0.05)。结论内质网应激的前列腺癌细胞可能通过释放外泌体,激活巨噬细胞STAT3通路,上调巨噬细胞PD-L1蛋白表达,促进巨噬细胞M2极化。

Objective To investigate whether endoplasmic reticulum stress-associated exosomes in prostate cancer cells can regulate macrophage M2 polarization via STAT3.Methods Prostate cancer cells(PC3)were stimulated with non-toxic carotene(TG)and divided into TG group and Con group.In the TG group,PC3 cells were stimulated at the same time with different concentrations of TG and at different times with the same concentration of TG,and the optimal stimulation concentration and time were determined by observing the expression of ERS marker protein GRP78;the expression of exosome marker proteins CD63 and TSG101 in Con group and TG group was detected by Western blot,and exosomes were identified by transmission electron microscope.Laser confocal microscope was used to observe the uptake of exosomes by macrophages;macrophages stimulated by exosomes in Con group and TG group were divided into M-Con group and M-TG group.The expressions of inflammatory factors IL-1β,IL-6,IL-10 and TNF-αin M-TG group and M-Con group were detected by chemiluminescence method.Western blot was used to detect PD-L1 expression and STAT3 pathway protein expression in macrophages of M-TG and M-Con groups.Results PC3 cells were stimulated by TG,the results showed that the expression of GRP78 protein was significantly increased when PC3 cells were stimulated by 3μmol/L TG for 24 h,and a better ERS model of prostate cancer cells could be constructed.Western blot and transmission electron microscopy confirmed the successful isolation of PC3 exosomes,and the expression of CD63 and TSG101 in the exosomes of the TG group was higher than that of the Con group(P<0.05).Laser confocal microscopy showed that exosomes could be swallowed by macrophages;the chemiluminescence results showed that the M-TG group secreted more inflammatory factors than the M-Con group(P<0.05),which could complete the M2 polarization of macrophages.Compared with the M-Con group,the M-TG group could increase the expression of PD-L1 protein in macrophages and activate the STAT3 pathway in macrophages(P<0.05).Conclusion Endoplasmic reticulum stress prostate cancer cells may activate STAT3 pathway of macrophages by releasing exosomes,up-regulate PD-L1 protein expression of macrophages,and promote M2 polarization of macrophages.