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目的探讨绞股蓝皂苷(XLIX)调节可溶性T细胞免疫球蛋白和粘蛋白域3(sTim-3)和Tim-3比率在非小细胞肺癌(NSCLC)细胞免疫逃逸中的作用机制。方法收集临床NSCLC组织,培养A549 NSCLC细胞。使用Tim-3的小干扰RNA重组质粒(siTim-3)和阴性对照(siNC)转染A549细胞;使用5、20、80μg/ml的XLIX处理A549细胞48 h,A549分别对应的组别为对照组、XLIX-5μg、XLIX-20μg、XLIX-80μg组;MTT法检测细胞增殖活性;Transwell侵袭实验检测细胞侵袭率变化。另外,加入XLIX-80μg或给予XLIX-80μg+Tim-3抗体(anti-Tim-3)或XLIX-80μg+金属蛋白酶(ADAM)10,Western blot检测Tim-3、sTim-3、金属蛋白酶ADAM-10/-17、Tim-3配体Galectin-9、干扰素γ(INF-γ)、肿瘤坏死因子(TNF)-α的蛋白表达,ELISA法检测组织中sTim-3的表达水平。结果与癌旁组比,NSCLC组织中Tim-3水平升高,而sTim-3、ADAM-10及ADAM-17表达降低(均P<0.05)。A549细胞中,与siNC比,siTim-3的INF-γ、TNF-α的表达、细胞活性及侵袭率均下调(均P<0.05)。与对照组相比,XLIX-20μg和XLIX-80μg组抑制A549细胞增殖活性和侵袭率,上调A549细胞sTim-3/Tim-3的比率,但下调Galectin-9表达,差异均有统计学意义(均P<0.05)。另外,XLIX-80μg处理A549后,在此基础上使用anti-Tim-3处理,可以明显上调TNF-α和INF-γ表达,但是下调sTim-3/Tim-3的比率(均P<0.05)。而在XLIX-80μg处理基础上使用ADAM10处理,sTim-3上调,并抑制TNF-α和INF-γ表达(均P<0.05)。结论 XLIX可减少NSCLC细胞的免疫逃逸,XLIX促进sTim-3/Tim-3比率升高可能是其对NSCLC免疫逃逸抑制作用关键机制之一。
Abstract：This study was performed to explore the immune escape mechanism of non-small cell lung cancer(NSCLC) regulated by Gypenosides(XLIX)-mediated modulation of soluble Tim-3(sTim-3). Clinical NSCLC tis-sues was collected and A549 NSCLC cells were cultured. Then A549 cells were transfect with Tim-3 small interfer-ing RNA recombinant plasmid(siTim-3) or negative control plasmid(siNC); XLIX of 0, 5, 20, 80 μg/ml were used to treat A549 cells in control group, XLIX-5 μg, XLIX-20 μg, XLIX-80 μg groups. MTT method was used to detect cell proliferation activity; Transwell invasion test was performed to detect cell invasion. Furthermore, A549 were treated with XLIX-80 μg or XLIX-80 μg + Tim-3 antibody(anti-Tim-3) or XLIX-80 μg+ metalloproteinase(AD-AM) 10. Western blot was used to detect the protein expression of Tim-3, sTim-3, metalloproteinase ADAM-10/-17, Tim-3 ligand Galectin-9, interferon gamma(INF-γ), tumor necrosis factor alpha(TNF-α); while ELISA method was used to detect the expression level of sTim-3 in tissues. Compared with the paracancerous group, the level of Tim-3 in NSCLC increased, while the expression of sTim-3, ADAM-10 and ADAM-17 decreased(all P <0.05).Compared with siNC, the expression of INF-γ and TNF-α, cell activity and invasion rate of siTim-3 in A549 cellswere down-regulated(all P <0.05). Compared with thecontrol group, the XLIX-20 μg and XLIX-80 μg groupsdemonstrated lower the proliferation activity and invasion rate of A549 cells, higher ratio of A549 cells sTim-3/Tim-3, and lower expression of Galectin-9(both P <0.05). In addition, XLIX-80 μg + anti-Tim-3 treatment could signif-icantly increase TNF-α and INF-γ, but reduce the expression of sTim-3/Tim-3 ratio(both P<0.05) of A549 cells.While ADAM10 + XLIX-80 μg treatment could upregulate sTim-3, but inhibit TNF-α and INF-γ(all P<0.05).Taken together, XLIX can reduce the immune escape of NSCLC cells by promoting the increase of sTim-3/Tim-3 ra-tio.
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基本信息:
DOI：10.13431/j.cnki.immunol.j.20210071
中图分类号:R734.2
引用信息:
[1]谭先胜,李锴男,苗亚军.绞股蓝皂苷通过上调sTim-3/Tim-3比率抑制非小细胞肺癌细胞免疫逃逸因子的表达[J].免疫学杂志,2021,37(06):520-527.DOI:10.13431/j.cnki.immunol.j.20210071.
2021-06-01
2021-06-01