首页|低氧微环境下脂滴包被蛋白2对胶质瘤干细胞干性的影响

低氧微环境下脂滴包被蛋白2对胶质瘤干细胞干性的影响

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目的 探究胶质瘤干细胞(GSC)中脂滴包被蛋白2(PLIN2)在低氧微环境中的表达,及对胶质瘤干细胞干性及生长的影响.方法 将在华中科技大学附属同济医院收取的胶质母细胞瘤样本构建胶质瘤干细胞系WF4.将WF4分为两组,分别置于常氧(21%O2)和低氧(1%O2)培养24 h,采用蛋白质印迹法(Western blot)检测两组细胞中缺氧诱导因子-1α(HIF-1α)和PLIN2的相对表达量.分别设计合成靶向抑制HIF-1α和PLIN2的短发夹RNA(shRNA),转染WF4细胞,分为对照组(shNT组)和敲低组(shHIF-1α、shPLIN2组),细胞均低氧处理24 h.Western blot实验检测敲低HIF-1α后,对照组和敲低组的PLIN2蛋白的相对表达.敲低PLIN2后,Western blot实验检测shNT组和shPLIN2组胶质瘤干细胞干性指标性别决定区Y-box2(SOX2)和少突胶质细胞转录因子2(OLIG2)的相对表达量,Cell Titer-Glo法检测细胞活力,成球实验检测GSCs成球能力,使用BODIPY 493/503荧光探针2 µmol/L浓度孵育细胞30 min,免疫荧光(IF)和流式细胞术检测细胞荧光强度.两组间差异比较使用独立样本t检验,多组间差异比较使用One-way ANOVA分析.结果 低氧处理 WF4 后,低氧组中 HIF-1α(0.38±0.02 比 0.07±0.01,t=17.313,P<0.01)和 PLIN2(0.68±0.03比0.46±0.04,t=6.566,P<0.01)表达高于常氧组,差异有统计学意义.shHIF-1α组中PLIN2 表达量低于 shNT 组(0.87±0.01,0.82±0.01 比 1.18±0.06,F=55.191,P<0.01).shPLIN2 组中 SOX2(1.30±0.13、1.17±0.08 比 2.74±0.10,F=138.535,P<0.01)和 OLIG2(0.76±0.09、0.92±0.11 比 1.65±0.10.F=44.497,P<0.01)相对表达量低于 shNT 组.成球实验显示shPLIN2组细胞成球能力下降低于shNT组(0.34±0.01、0.40±0.01比1.00±0.08,F=115.823,P<0.01).免疫荧光(IF)和流式细胞术结果显示,shPLIN2组BODIPY 493/503荧光探针荧光强度低于shNT组(2.67±0.47、2.33±0.47 比 6.00±0.82,F=22.200,P<0.01;27 440.20±117.80、25 908.73±375.21 比 40 681.50±413.55,F=667.206,P<0.01).结论 在胶质瘤干细胞中,PLIN2低氧上调,并受HIF-1α调控,同时敲低PLIN2影响胶质瘤干细胞干性、增殖和脂滴数量.
Impact of perilipin 2 on the stemness of glioblastoma stem cells under hypoxic microenvironment
Objective To explore the expression of perilipin 2(PLIN2)in glioblastoma stem cells(GSC)under hypoxic microenvironment and its impact on the stemness and growth of GSC.Methods Construct a glioma stem cell line WF4 from glioblastoma samples collected at Tongji Hospital affiliated with Huazhong University of Science and Technology.Divide WF4 into two groups,culture under normoxic(21%O2)and hypoxic(1%O2)conditions for 24 hours,and use Western blotting to detect the relative expression of hypoxia-inducible factor-1α(HIF-1α)and PLIN2 proteins in the two groups of cells.Design and synthesize short hairpin RNA(shRNA)targeting HIF-1α and PLIN2,transfect WF4 cells,divided into control group(shNT group)and knockdown group(shHIF-1α,shPLIN2 group),and treat all cells under hypoxic conditions for 24 hours.Use Western blotting to detect the relative expression of PLIN2 protein in the control group and knockdown group after knocking down HIF-1α,and to detect the relative expression of glioma stem cell stemness indicators sex-determining region Y-box 2(SOX2)and oligodendrocyte tran-scription factor 2(OLIG2)in the shNT group and shPLIN2 group after knocking down PLIN2.Use the BODIPY 493/503 fluorescence probe at a concentration of 2 μmol/L to incubate cells for 30 mins,and de-tect cell fluorescence intensity by immunofluorescence(IF)and flow cytometry.compare differences be-tween two groups using independent sample t-tests,and compare differences among multiple groups using One-way ANOVA analysis.Results After hypoxia treatment of WF4 cells,the expression of HIF-1α(0.38±0.02 vs.0.07±0.01,t=17.313,P<0.01)and PLIN2(0.68±0.03 vs.0.46±0.04,t=6.566,P<0.01)in the hypoxia group was higher than that in the normoxia group,with statistically signif-icant differences.In the shHIF-1α group,the expression of PLIN2 was lower than that in the shNT group(0.87±0.01,0.82±0.01 vs.1.18±0.06,F=55.191,P<0.01).In the shPLIN2 group,the relative expression of SOX2(1.30±0.13,1.17±0.08 vs.2.74±0.10,F=138.535,P<0.01)and OLIG2(0.76±0.09,0.92±0.11 vs.1.65±0.10,F=44.497,P<0.01)was lower than that in the shNT group.Sphere formation assays showed that the sphere formation ability of cells in the shPLIN2 group was lower than that in the shNT group(0.34±0.01,0.40±0.01 vs.1.00±0.08,F=115.823,P<0.01).Immunofluorescence(IF)and flow cytometry results indicated that the fluorescence intensity of BODIPY 493/503 probe in the shPLIN2 group was lower than that in the shNT group(2.67±0.47,2.33±0.47 vs.6.00±0.82,F=22.200,P<0.01;27 440.20±117.80,25 908.73±375.21 vs.40 681.50±413.55,F=667.206,P<0.01).Conclusion In GSC,PLIN2 is upregulated under hypoxia and regula-ted by HIF-1α.Knockdown of PLIN2 affects the stemness,proliferation,and lipid droplet number of GSC.

Glioblastoma stem cellsHypoxic microenvironmentPerilipin 2StemnessLipid droplets

鲍俊霖、王宝峰

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华中科技大学同济医学院附属同济医院神经外科,武汉 430030

胶质瘤干细胞 低氧微环境 脂滴包被蛋白2 干性 脂滴

2024

中华实验外科杂志
中华医学会

中华实验外科杂志

CSTPCD
影响因子:0.759
ISSN:1001-9030
年,卷(期):2024.41(11)