高表达Gal-9的肝癌血浆外泌体诱导自然杀伤细胞功能失调的机制研究

Mechanism of dysfunction of natural killer cells induced by plasma exosomes with high expression of Gal-9 in hepatocellular carcinoma

目的:在肝细胞癌(HCC)血浆外泌体中探究半乳糖凝集素-9(Gal-9)表达及其对自然杀伤(NK)细胞功能的影响及机制。方法:收集10例HCC患者(HCC组)与10例健康志愿者(NC组)空腹静脉血,采用外泌体分离试剂盒分离血浆中外泌体,采用透射电镜(TEM)、纳米粒径分析(NTA)和Western blot对外泌体进行鉴定,并检测Gal-9在两组患者外泌体中的表达;流式细胞术检测两组NK细胞表面Gal-9受体、T细胞免疫球蛋白和黏附因子3(Tim-3)表达差异;分离并培养健康人群外周血NK细胞,并将其分为2组:Gal-9组(2μg/ml人工重组Gal-9处理)、PBS组(等体积PBS处理),流式细胞术及LDH释放法检测不同处理条件下NK细胞CD107a表达及NK细胞对HepG2细胞的杀伤活性;Western blot检测上述两组NK细胞中MAPK信号通路JNK蛋白磷酸化水平和Caspase-3表达变化。结果:成功分离血浆外泌体。与NC组相比,HCC组血浆外泌体中Gal-9表达显著增加(P<0.01),且NK细胞Tim-3表达明显升高(P<0.01);与PBS组相比,Gal-9组NK细胞Tim-3表达显著增加(P<0.05),细胞凋亡率明显升高(P<0.05),细胞表面IFN-γ表达显著降低(P<0.05),NK细胞对肝癌细胞HepG2的杀伤活性明显降低(P<0.05);Western blot结果显示,与PBS组相比,Gal-9组NK细胞中JNK蛋白磷酸化水平及Caspase-3表达均显著上升(P<0.05)。结论:HCC血浆外泌体中高表达的Gal-9可促进NK细胞表面Tim-3表达,并通过MAPK信号通路诱导NK细胞凋亡,并抑制其对肿瘤细胞的杀伤活性。

Objective:To investigate expression of galectin-9(Gal-9)in plasma exosomes of hepatocellular carcinoma(HCC)and its effect on function of natural killer(NK)cells and its mechanism.Methods:Fasting venous blood samples were collected from 10 patients with HCC(HCC group)and 10 normal controls(NC group).Exosomes were separated by exosome separation kit.Exosomes were identified by transmission electron microscopy(TEM),nanoparticle size analysis(NTA)and Western blot.Gal-9 expressions in exosomes of two groups were detected;Gal-9 receptor,T cell immunoglobulin and adhesion factor 3(Tim-3)expressions on NK cells were detected by flow cytometry;NK cells were isolated and cultured from peripheral blood of healthy people,which were divided into two groups:Gal-9 group(treated with 2μg/ml recombinant Gal-9)and PBS group(treated with equal volume of PBS).CD107a expression and cytotoxicity of NK cells to HepG2 were detected by flow cytometry and LDH release assay;phosphorylation level of JNK protein and expression of Caspase-3 were detected by Western blot.Results:Plasma exosomes were successfully separated.Compared with NC group,expression of Gal-9 of plasma exosomes in HCC group was significantly increased(P<0.01),and expression of Tim-3 in NK cells was significantly increased(P<0.01).Compared with PBS group,expression of Tim-3 in NK cells in Gal-9 group was significantly increased(P<0.05),apoptosis rate was significantly increased(P<0.05),IFN-γexpression on cell surface was significantly decreased(P<0.05),and cytotoxicity of NK cells to HepG2 cells was also significantly decreased(P<0.05).Western blot results showed that compared with PBS group,JNK protein phosphorylation level and Caspase-3 expression of NK cells in Gal-9 group were significantly increased(P<0.05).Conclusion:High expression of Gal-9 in plasma exosomes of HCC can promote expression of Tim-3 on surface of NK cells,induce apoptosis of NK cells through MAPK signaling pathway,and inhibit its cytotoxic activity to tumor cells.