摘要
目的 探讨JAK2小分子抑制剂AG490对肿瘤相关巨噬细胞(TAM)衍生外泌体(Exo)治疗后宫颈癌细胞自噬、凋亡和干性特性的影响.方法 使用佛波酯(PMA)和重组人IL-4将THP-1细胞转化为M2型TAM.采用差速离心法提取Exo,通过透射电镜和纳米颗粒跟踪技术观察Exo形态和大小分布.采用Western blot检测Exo标记物蛋白表达.采用Dil联合PKH67染色检测宫颈癌细胞系HeLa细胞对TAM衍生Exo的摄取.将HeLa细胞分为3组:对照组、Exo组和Exo+AG490组,分别用TAM衍生Exo和AG490处理各组细胞;采用流式细胞术和mRFP-GFP-LC3双荧光检测细胞凋亡、自噬情况;采用Western blot检测自噬指数(LC3-Ⅱ/LC3-Ⅰ、Beclin-1、p62)、凋亡指标(Bax、Bcl-2、Cleaved caspase-3)及肿瘤干细胞指数(CD133、OCT4、SOX2)的表达水平;采用肿瘤细胞颗粒形成实验检测细胞干性.结果 给予PMA和IL-4,THP-1细胞的形态发生改变,CD86的表达减少、CD206的表达增加(P＜0.05).分离的颗粒被鉴定为直径为140 nm的球形囊泡,Alix、CD63和TSG101在颗粒中有明显表达.HeLa细胞能够成功地分离和内化来自M2型TAM的Exo.与对照组比较,Exo组HeLa细胞凋亡率、GFP和mRFP荧光点、LC3-Ⅱ/LC3-Ⅰ比值、Beclin-1、Bax和caspase-3表达水平显著降低(P＜0.05),p62和Bcl-2表达水平显著增加(P＜0.05),细胞球状性增加;此外,CD133、OCT4、SOX2表达显著上调(P＜0.05).AG490孵育可逆转Exo组上述指标表达趋势(P＜0.05).结论 AG490可能增强M2型TAM Exo处理的宫颈癌细胞自噬诱导的凋亡,抑制宫颈癌的致瘤特性.
Abstract
Objective To examine the impact of the Janus kinase 2(JAK2)small molecule inhibitor AG490 on autophagy,apoptosis,and desiccation properties in cervical cancer cells following treatment with TAM-derived exosomes.Methods PMA and human IL-4 were applied to differentiate human peripheral blood mononuclear cells THP-1 into M2 type TAMs;exosomes were isolated by differential centrifugation,and TEM and nanoparticle tracking technologies were used to analyze exosome morphology and size distribution;Western blot was used to determine whether exosomal marker proteins were expressed;Dil in conjunction with PKH67 staining was utilized to ascertain the internalization of TAM-derived exosomes by the HeLa cervical cancer cell line.The HeLa cells were segregated into 3 experimental groups:control,Exo,and Exo+AG490.Subsequently,the HeLa cells were exposed to TAM-derived exosomes and AG490 in accordance with their respective groups.An apoptosis rate was demonstrated via flow cytometry,and autophagy was quantified via double-fluorescence mRFP-GFP-LC3 assay.Western blot analysis was utilized to evaluate the stemness of cancer cells by measuring the protein levels of autophagy-and apoptosis-associated indicators.Further,the levels of tumor stem cell-associated proteins CD 133,OCT4,and SOX2 were also assessed.Results The induction of PMA and IL-4 resulted in significant alterations in the morphology of THP-1 cells,characterized by a decrease in the expression ratio of CD86 and an increase in the expression ratio of CD206(P＜0.05).Approximately 140 nm-sized spherical vesicles were found in the individual particles,with significant expression of exosomal marker proteins Alix,CD63,and TSG101.These findings suggested successful isolation of M2-type TAM-derived exosomes,which were found to be internalized by HeLa cells.In comparison to the control group,apoptosis rate,fluorescence intensity of GFP and mRFP,LC3-Ⅱ/LC3-1 ratio,and the expression levels of Beclin-1,Bax,and caspase-3 were significantly decreased in the Exo group(P＜0.05);the expression levels of p62 and Bcl-2 were significantly increased(P＜0.05),and the cellular globularity of cells was increased in the Exo group;the up-regulation of CD 133,OCT4,and SOX2 was found to be statistically significant(P＜0.05).The incubation of AG490 reversed the expression trend of the above indicators in the Exo group(P＜0.05).Conclusion Our findings suggest that AG490 may have the potential to induce autophagy-mediated apoptosis in cervical cancer cells exposed to M2-type TAM-derived exosomes,while also demonstrating the capacity to suppress the tumorigenic properties of cervical cancer.
维普
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