摘要
目的观察低氧微环境对胶质瘤细胞增殖和凋亡的影响;探索不同时间梯度和细胞密度在低氧微环境下对HIF-1α表达的影响。方法(1)构建脑胶质瘤细胞(T98G)生长的缺氧微环境,将细胞设为低氧组(1%氧气含量)和常氧组(21%氧气含量),在不同氧浓度下培养4~72 h后观察细胞生长情况;利用western blot印迹法检测低氧诱导因子-1α(HIF-1α)表达情况;(2)将低氧组T98G细胞设计为四组不同细胞密度,在1%O2的低氧浓度下培养24 h,通过Western blot检测四组细胞HIF-1α表达情况。结果(1)倒置显微镜和Hoechest染色均未见低氧组和常氧组细胞发生明显的细胞凋亡,MTT法检测二组细胞均呈增殖状态。与常氧组相比,低氧组细胞HIF-1α蛋白明显高表达(P<0.001)。低氧组细胞在4、6、12、24 h(<24 h)HIF-1α蛋白表达量快速升高(P<0.05),24、48、72 h(>24 h)HIF-1α蛋白表达量逐渐下降;(2)低氧微环境下四组不同细胞密度中,24 h后高密度组(8.5万个细胞/cm^2)细胞的HIF-1α表达最高,细胞的过高密度或过低密度生长都会导致HIF-1α表达降低(P<0.05)。结论低氧微环境(1%O2)不能诱导T98G胶质瘤细胞发生凋亡;在低氧微环境(1%O2)中,T98G细胞HIF-1α的蛋白表达不是恒定不变的,其表达量会随时间和细胞密度的变化而变化。低氧处理的T98G细胞24 h内HIF-1α蛋白表达量快速升高,24 h后逐渐降低。在缺氧环境下T98G细胞汇合度过高或过低均会降低HIF-1α的表达,在95%左右时其表达最高。
Objectives To explore the effects of hypoxic microenvironment on the proliferation and apoptosis of glioma cells.To explore the effects of different time gradients and cell densities on hypoxia-inducible factor-1α(HIF-1α)expression in hypoxic microenvironment.Methods(1)A hypoxic microenvironment was constructed and the glioma cells(T98 G)were set to a hypoxic group(1%oxygen content)and a normoxic group(21%oxygen content)cultured at different oxygen concentrations for 4-72 h.Cell growth was observed after 72 h.The expression of HIF-1αwas detected by western blott.(2)The T98 G cells in the hypoxia group were designed into four groups with different cell densities.The cells were cultured for 24 h at low oxygen concentration of 1%O2.The expression of HIF-1αin the four groups was detected by Western blot.Results(1)Inverted microscope and Hoechest staining showed no obvious apoptosis in hypoxic and normoxic cells.MTT assay showed that 2 groups showed proliferative status.Compared with the normoxic group,the HIF-1αprotein was significantly higher in the hypoxic group(P<0.001).The expression of HIF-1αprotein increased rapidly in hypoxic cells at 4,6,12,24 h(<24 h)(P<0.05),and the expression of HIF-1αprotein gradually decreased at 24,48,72 h(>24 h)(P<0.05).(2)Among the 4 different cell densities in the hypoxic microenvironment,the expression of HIF-1αwas highest in the high-density group(8.5 million cells/cm^2)after 24 h,and the the expression of HIF-1αwas low in the cell density too high or too low(P<0.05).Conclusions Hypoxia microenvironment(1%O2)cannot induce apoptosis of T98 G glioma cells,the protein expression of HIF-1αin T98 G cells is not constant,and its expression level changes with change of time and cell density.The expression of HIF-1αprotein in hypoxic-treated T98 G cells increases rapidly within 24 hours and gradually decreases after 24 hours.Higher or lower confluence of T98 G cells in hypoxic environment can reduce HIF-1αexpression,and the expression can reach around 95%.
作者
余朝军
赵迁浩
赵宁辉
YU Chao-jun;ZHAO Qian-hao;ZHAO Ning-gui(Dept.of Neurosurgery,Jiangyou 903 Hospital,Jiangyou Sichuan 621700;Dept.of Neurosurgery,The 2nd Affiliated Hospital of Kunming Medical University,Kunming Yunnan 650101;Dept.of Neurosurgery,Kunming Children’s Hospital,Kunming Yunnan 650034,China)
出处
《昆明医科大学学报》
CAS
2020年第7期11-16,共6页
Journal of Kunming Medical University
基金
国家自然科学基金资助项目(81960459)。
关键词
低氧
低氧诱导因子1Α
T98G细胞株
增殖
凋亡
Hypoxia
Hypoxia-inducible factor 1α
T98G cell lines
Proliferation
Apoptosis
