缺氧诱导因子-2α经转化生长因子-β2促进胶质母细胞瘤细胞化疗抵抗OA
Hypoxia-inducible factor-2α promotes chemoresistance in glioblastoma cells via transforming growth factor-β2
目的 肿瘤微环境中的缺氧是导致胶质母细胞瘤化疗抵抗的关键因素.本研究通过体外研究初步探讨缺氧条件下缺氧诱导因子-2α(hypoxia-inducible factor-2α,HIF-2α)与转化生长因子-β2(transforming growth factor-β2,TGF-β2)促进胶质母细胞瘤细胞化疗抵抗.方法 常氧-缺氧培养U87、U118细胞(n=3)并予替莫唑胺处理24、48、72 h后经CCK-8检测细胞增殖,培养48 h后流式细胞仪检测细胞凋亡.常氧培养为对照,缺氧培养U87、U118细胞(n=3)4、8、12及24 h后采用RT-qPCR检测HIF-2α、TGF-β2表达,进一步缺氧培养12、24 h及48 h后,Western blot检测HIF-2α、TGF-β2表达.敲除HIF-2α、TGF-β2后,缺氧培养细胞(n=3)并给予替莫唑胺(temozolomide,TMZ)处理24、48、72 h后经CCK-8检测细胞增殖,培养24 h后在部分细胞的培养基中添加TGF-β2(50 ng/mL)继续缺氧培养细胞(n=3)24 h后流式细胞仪检测细胞凋亡.Western blot检测敲除HIF-2 α细胞缺氧培养 48 h后TGF-β 2表达.结果 CCK-8和流式细胞检测提示,低氧+TMZ组较常氧+TMZ组细胞的存活率增高(P<0.05)、凋亡率降低(P<0.05).RT-qPCR检测显示,与常氧组相比,缺氧培养细胞的HIF-2α、TGF-β2表达随着缺氧时间的延长,表达水平逐渐升高(P<0.05),Western blot也证实HIF-2α、TGF-β2表达缺氧培养后呈时间依赖性升高.敲除HIF-2α、敲除TGF-β2表达后的细胞存活率较对照未敲除组降低(P<0.05),细胞凋亡率较对照未敲除组升高(P<0.05).敲除HIF-2α细胞继续缺氧培养后提示TGF-β2表达较对照未敲除HIF-2α细胞组水平下降.结论 HIF-2α经TGF-β2促进胶质母细胞瘤细胞化疗抵抗.
Objective Hypoxia in the tumor microenvironment is a critical factor contributing to chemoresistance in glioblastoma.This study preliminarily investigates the role of hypoxia-inducible factor-2α(HIF-2α)and transforming growth factor-β2(TGF-β2)in promoting chemotherapy resistance of glioblastoma cells under hypoxic conditions.Methods U87 and U118 cells(n=3)were cultured under normoxic and hypoxic conditions and treated with temozolomide(TMZ)for 24,48,and 72 h.Cell proliferation was assessed by CCK-8 assay,and apoptosis was detected by flow cytometry after 48 h of culture.Using normoxic culture as control,U87 and U118 cells(n=3)were subjected to hypoxic culture for 4,8,12,and 24 h,followed by RT-qPCR to measure HIF-2α and TGF-β2 mRNA expression.After further hypoxic culture for 12,24,and 48 h,the protein levels of HIF-2α and TGF-β2 were detected by Western blotting.After knockdown of HIF-2α and TGF-β2,the cells(n=3)were cultured under hypoxia and treated with TMZ for 24,48,and 72 h.Then,the cell proliferation was detected by CCK-8 assay.After 24 h of culture,TGF-β2(50 ng/mL)was added to the culture medium of some cells,and the cells were further cultured under hypoxia for another 24 h,followed by flow cytometry for cell apoptosis.Western blotting was applied to detect TGF-β2 expression in HIF-2α knockdown cells after 48 h of hypoxic culture.Results CCK-8 and flow cytometry assays indicated that the hypoxia+TMZ group showed increased cell viability(P<0.05)and decreased apoptotic rate compared to the normoxia+TMZ group(P<0.05).RT-qPCR revealed that compared with the normoxia group,the expression levels of HIF-2α and TGF-β2 in hypoxia-cultured cells gradually increased with the prolongation of hypoxic time(P<0.05),and Western blotting also confirmed that the protein expression of HIF-2α and TGF-β2 increased in a time-dependent manner after hypoxic culture.Lower cell viability(P<0.05)and higher apoptotic rate(P<0.05)were observed in the HIF-2α-knockdown and TGF-β2-knockdown cells compared with the control non-knockdown group.Continued hypoxic culture of HIF-2α-knockdown cells indicated decreased TGF-β2 expression compared with the control non-knockdown HIF-2α group.Conclusion HIF-2α chemoresistance in glioblastoma cells via TGF-β2.
赵璐;汪攀;龚胜;廖彬;吴南
重庆医科大学,重庆重庆市人民医院神经外科,重庆市脑胶质瘤精准医学研究中心,重庆重庆市人民医院神经外科,重庆市脑胶质瘤精准医学研究中心,重庆重庆医科大学,重庆||重庆市人民医院神经外科,重庆市脑胶质瘤精准医学研究中心,重庆重庆医科大学,重庆||重庆市人民医院神经外科,重庆市脑胶质瘤精准医学研究中心,重庆
医药卫生
胶质母细胞瘤缺氧缺氧诱导因子-2α转化生长因子-β2
glioblastomahypoxiahypoxia-inducible factor-2αtransforming growth factor-β2
《陆军军医大学学报》 2026 (6)
701-708,8
国家自然科学基金面上项目(82473430)重庆市科卫联合医学科研项目重大项目(2023DBXM004)重庆市科卫联合医学科研青年项目(2023QNXM043) Supported by the General Program of National Natural Science Foundation of China(82473430),the Major Joint Medical Research Project of Chongqing Science and Technology Commission and Chongqing Health Commission(2023DBXM004),and the Joint Youth Medical Research Project of Chongqing Science and Technology Commission and Chongqing Health Commission Chongqing(2023QNXM043).
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