肿瘤相关性巨噬细胞诱导膀胱癌细胞对吉西他滨耐药的实验研究

Investigation on the drug resistance to Gemcitabine induced by tumor-associated macrophages in bladder cancer cells

目的探讨肿瘤相关巨噬细胞(TAM)在膀胱癌细胞对吉西他滨耐药中的作用和耐药机制。方法通过佛波酯、白细胞介素4、白细胞介素13刺激人单核细胞白血病细胞系THP-1, 建立TAM模型。运用免疫荧光技术检测TAM表面分子CD206的表达;使用MTT比色法检测吉西他滨对TAM/膀胱癌T24、5637细胞共培养组和空白对照组的杀伤情况。运用Western blot法检测TAM上清及TAM上清联合吉西他滨处理前后T24、5637细胞内p-STAT3、LDHA的表达。使用MTT比色法检测吉西他滨对野生型及LDHA过表达的T24、5637细胞的杀伤情况。结果 TAM模型建立成功后其表面因子CD206表达明显增加, TAM与膀胱癌细胞共培养可明显削弱吉西他滨对膀胱癌细胞的杀伤, 同时明显上调p-STAT3、LDHA的表达(P<0.05)。吉西他滨处理后, LDHA基因过表达组肿瘤细胞增殖较野生型对照组明显增加(P<0.05)。结论 TAM通过上调膀胱癌细胞表达p-STAT3蛋白来促进肿瘤细胞内LDHA蛋白的表达, 从而诱导膀胱癌细胞对吉西他滨耐药。

Objective To investigate the role and mechanism of tumor-associated macrophages(TAM)in Gemcitabine resistance in bladder cancer cells.Methods The model of TAM was established by stimulating human leukemia cell line THP-1 with phorbol myristate acetate,Interleukin-4 and Interleukin-13.Immunofluorescence was used to detect the expression of CD206 in TAM.MTT was used to detect the effective concentration and time of Gemcitabine on bladder cancer cell lines T24 and 5637,and the killing effect on bladder cancer cells cultured alone and co-cultured with TAM.Western blot was used to detect the expression of p-STAT3 and LDHA in bladder cancer cells before and after co-culture with TAM and TAM combined with Gemcitabine.The killing effect of gemcitabine on wild-type and LDHA-overexpressing T24 and 5637 cells was detected by MTT.Results Expression of CD206 on THP-1 cells differentiated from TAM was significantly increased.Co-culture of TAM with bladder cancer cells could significantly weaken the killing effect of Gemcitabine on bladder cancer cells and up-regulate p-STAT3 and LDHA.After treatment of Gemcitabine,the proliferation of tumor cells increased significantly in LDHA gene overexpression group(P<0.05).Conclusion TAM up-regulates the expression of p-STAT3 to promote the expression of LDHA in bladder cancer cells,thus inducing bladder cancer cells to be resistant to Gemcitabine.