CAJ | 학술논문

目的探讨叉头状螺旋转录因子3基因(Forkhead box P3,FOXP3)的短发夹RNA(shRNA)对人胶质瘤细胞系U87和LN229增殖能力及凋亡的影响.方法用脂质体法将4个含有靶向FOXP3的shRNA序列(shRNA1 ～4)和1个含有shRNA无意义随机阴性对照序列(neg-shRNA)的质粒转染入人胶质瘤U87细胞和LN229细胞;应用Western blot检测转染后FOXP3蛋白的表达情况筛选出最有效的一个shRNA干扰质粒;应用流式细胞术、CCK-8法检测转染后U87细胞和LN229细胞的凋亡和增殖活性的变化.结果转染72 h后,Western blot结果显示空白对照组、shRNA-1、shRNA-2、shRNA-3、shRNA-4组和neg-shRNA组中FOXP3蛋白的表达受抑制最明显的是shRNA-1组.CCK-8结果显示转染72 h后,shRNA-1转染组U87细胞和LN229细胞增殖活性分别为(122.00±4.32)％、(118.36±2.49)％,与neg-shRNA组(98.55±4.34)％、(99.87±2.17)％和空白对照组100％相比均显著升高(P＜0.05);流式细胞术结果显示转染72 h后,shRNA-1转染的U87细胞和LN229细胞凋亡率分别为(7.03±3.36)％和(9.40±2.51)％,neg-shRNA转染U87细胞和LN229细胞的凋亡率分别为(17.70±4.39)％和(22.63±1.86)％,在U87和LN229细胞中空白对照组凋亡率分别为(16.57±2.30)％和(21.67±1.93)％,两种细胞的凋亡率在shRNA-1组与neg-shRNA组和空白对照组比较差异均有统计学意义(P＜0.05).结论靶向FOXP3基因的shRNA能够有效抑制该基因表达,抑制胶质瘤细胞的凋亡,促进细胞增殖.
목적 탐토차두상라선전록인자3기인(Forkhead box P3,FOXP3)적단발협RNA(shRNA)대인효질류세포계U87화LN229증식능력급조망적영향.방법 용지질체법장4개함유파향FOXP3적shRNA서렬(shRNA1 ～4)화1개함유shRNA무의의수궤음성대조서렬(neg-shRNA)적질립전염입인효질류U87세포화LN229세포;응용Western blot검측전염후FOXP3단백적표체정황사선출최유효적일개shRNA간우질립;응용류식세포술、CCK-8법검측전염후U87세포화LN229세포적조망화증식활성적변화.결과 전염72 h후,Western blot결과현시공백대조조、shRNA-1、shRNA-2、shRNA-3、shRNA-4조화neg-shRNA조중FOXP3단백적표체수억제최명현적시shRNA-1조.CCK-8결과현시전염72 h후,shRNA-1전염조U87세포화LN229세포증식활성분별위(122.00±4.32)％、(118.36±2.49)％,여neg-shRNA조(98.55±4.34)％、(99.87±2.17)％화공백대조조100％상비균현저승고(P＜0.05);류식세포술결과현시전염72 h후,shRNA-1전염적U87세포화LN229세포조망솔분별위(7.03±3.36)％화(9.40±2.51)％,neg-shRNA전염U87세포화LN229세포적조망솔분별위(17.70±4.39)％화(22.63±1.86)％,재U87화LN229세포중공백대조조조망솔분별위(16.57±2.30)％화(21.67±1.93)％,량충세포적조망솔재shRNA-1조여neg-shRNA조화공백대조조비교차이균유통계학의의(P＜0.05).결론 파향FOXP3기인적shRNA능구유효억제해기인표체,억제효질류세포적조망,촉진세포증식.
Objective To investigate the effects of short hairpin RNA (shRNA) targeting Forkhead box P3 (FOXP3) gene on the proliferation and apoptosis of human glioma cells U87 and LN229.Methods 4 plasmids containing different sequences of shRNA targeting FOXP3 gene and 1 plasmids containing random sequences were transfected into human glioma U87 cells and LN229 cells mediated by Lipofectamine 2000.The expression of FOXP3 protein was detected by Western blot to screen the most effective shRNA plasmid.The cell proliferation and apoptosis were detected by CCK-8 assay and flow cytometry,respectively.Results At 72 h after transfection,the expression of FOXP3 protein was most obviously suppressed by shRNA-1 sequence compared with other shRNA sequences.The CCK-8 assay results showed that the proliferation rates of U87 cell and LN229 cell tranfected with shRNA-1 plasmid were (122.00 ±4.32)％ and (118.36 ±2.49)％ at 72 h,respectively,which were significantly higher than that transfected with neg-shRNA plasmid (neg-shRNA group) and the non-transfection group (control group) (P ＜0.05).At 72 h after transfection,the apoptosis rates of U87 cell and LN229 cell in shRNA-1 group were (7.03 ± 3.36)％ and (9.40 ± 2.51)％,respectively,which were decreased significantly compared with those of the neg-shRNA group (17.70 ± 4.39)％ and (22.63 ± 1.86)％,and the control group (16.57 ±2.30)％ and(21.67 ± 1.93)％ (P ＜ 0.05).Conclusions The shRNA targeting FOXP3 gene could efficiently inhibit the gene expression and hence it could significantly inhibit the apoptosis of glioma cells and promote cell proliferation.