肿瘤相关性巨噬细胞通过TNF-α/B7H3调节人膀胱癌细胞增殖的研究  

A study of tumor-associated macrophages regalating the proliferation of human bladder cancer cells through TNF-α/B7H3

作　　者：李仔祥 王苏贵 张先云 卢建文 嵇宏声 姜福金 Li Zaixiang;Wang Sugui;Zhang Xianyun;Lu Jianwen;Ji Hongsheng;Jiang Fujin(Department of urinary surgery,Huaian Hospital affiliated to Xuzhou Medical University,Huaian 223002,China)

机构地区：[1]徐州医科大学附属淮安医院泌尿外科,江苏淮安223002

出　　处：《中华临床医师杂志（电子版）》2024年第1期64-71,共8页Chinese Journal of Clinicians(Electronic Edition)

基　　金：江苏省第十六批“六大人才高峰”项目资助(2019-WSW-218);淮安市自然科学基金资助项目(HAB201730,HAB202117);徐州医科大学附属医院发展基金资助项目(XYFY202243,XYFM202245)。

摘　　要：目的探讨肿瘤相关性巨噬细胞对人膀胱癌细胞增殖的影响及机制。方法通过佛波酯(PMA)、细菌内毒素(LPS)、干扰素-γ(IFN-γ)刺激人单核白血病细胞系THP-1,建立肿瘤相关性巨噬细胞(TAM)模型。TAM模型与人膀胱癌细胞(T24、5637细胞系)在体外共培养,采用酶联免疫吸附实验(ELISA)检测TAM培养液上清肿瘤坏死因子-α(TNF-α)的变化采用CCK8法检测TAM上清液和TAM上清液联合TNF-α中和抗体阿达木单抗处理后人膀胱癌细胞的增殖情况;采用蛋白质印迹法检测人膀胱癌细胞,细胞与TAM和TAM联合阿达木单抗共培养后B7H3的表达。使用CCK8法检测B7H3基因过表达及干扰的人膀胱癌细胞系的增殖情况。结果80 ng/mlPMA联合20 ng/mlINF-γ可诱导THP-1细胞分化为TAM,其细胞表面标志物CD68表达率为(41.2±6.7)%。将TAM与T24及5637细胞共培养72 h后,细胞相对增殖率[(125.4±3.9)%和(191.4±13.4)%]较对照组(均为100%)明显升高(P<0.05)。ELISA检测TAM与T24及5637细胞共培养上清液中TNF-α明显升高[(279.5±12.1)pg/ml和(17.4±3.2)pg/ml](P<0.001),同时细胞内B7H3的表达量较对照组明显升高。使用阿达木单抗处理共培养上清液72 h后,ELISA检测上清液中TNF-α明显下降(40.5±2.2)pg/ml,B7H3的表达量较对照组也明显下降,T24及5637细胞相对增殖率([108.8±6.0)%和(98.4±9.0)%]较共培养组([136.6±7.3)%和(131.4±0.4)%]明显下降(P<0.01)。B7H3基因过表达组T24及5637细胞相对增殖率[(129.7±11.5)%和(125.7±6.7)%]较空载体转染组[(102.8±5.4)%和(91.9±13.7)%]显著增加(P<0.05),而B7H3基因干扰组细胞相对增殖率[(82.6±4.5)%和(73.5±9.1)%]较空载体转染组显著降低(P<0.05)。结论TA M通过分泌TNF-α诱导膀胱癌细胞内B7H3蛋白的表达上调,从而促进膀胱癌细胞的增殖。Objective To investigate the impact and underlying mechanisms of tumor-associated macrophages on the proliferation of human bladder cancer cells.Methods Human monocyte leukemia cell line THP-1 was employed as a platform for the induction of tumor-associated macrophages(TAMs)through stimulation with phorbol myristate acetate(PMA),lipopolysaccharide(LPS),and interferon-γ(IFN-γ).Subsequently,a co-culture model was established,uniting the TAM model with human bladder cancer cells(T24 and 5637 cell lines)in an extracellular environment.Alterations in the levels of tumor necrosis factor-alpha(TNF-α)in the supernatant of TAM cultures were assessed using enzyme-linked immunosorbent assays(ELISA).The proliferative tendencies of human bladder cancer cells following exposure to TAM-conditioned media and TAM-conditioned media,which had undergone TNF-αneutralization with the monoclonal antibody adalimumab,were examined utilizing the CCK-8 assay.Additionally,the expression of B7H3 in bladder cancer cells was scrutinized after co-cultivation with TAMs and TAMs in conjunction with adalimumab treatment,employing the Western blot technique.Furthermore,the proliferative behavior of bladder cancer cell lines with B7H3 gene overexpression and gene silencing was assessed through the CCK-8 assay.Results The differentiation of THP-1 cells into TAMs was successfully induced by the combination of 80 ng/ml PMA and 20 ng/ml INF-γ,with a notable surface expression of the CD68 marker observed at a rate of(41.2±6.7)%.Following a 72-hour co-cultivation of TAMs with T24 and 5637 cells,a significant increase in relative cell proliferation rates[(191.4±13.4)%and(173.1±11.5)%,respectively]was observed when compared to the control group(both set at 100%)(P<0.05).ELISA assays unveiled a substantial elevation of TNF-αlevels in the supernatant of TAMs co-cultured with T24 and 5637 cells[(279.5±12.1)pg/ml and(17.4±3.2)pg/ml,respectively](P<0.001).Concurrently,intracellular expression of B7H3 showed a marked increase compared to the control gr

关 键 词：肿瘤相关性巨噬细胞 膀胱癌 肿瘤坏死因子Α B7H3 增殖 

分 类 号：R737.14[医药卫生—肿瘤]