CAJ | 학술논문

目的利用shRNA干扰技术抑制恶性脑胶质瘤细胞TGFβ2/Smads通路中Smad2/3基因的表达,探讨其在恶性胶质瘤细胞增殖中的作用.方法将表达Smad2/3 shRNA干扰序列的特异性质粒及阴性对照质粒用脂质体转染法,分别转入到恶性脑胶质瘤细胞U251中.应用Real-TimePCR和Western blot法分别检测细胞中Smad2/3基因mRNA和蛋白的表达;转染后的细胞经TGFβ2(+/-)处理,采用CCK-8法评价两组的增殖率.结果 (1)两种特异性质粒能够分别下调细胞中Smad2/3基因的表达(P=0.01);(2)Smad2/3的表达量在Smad3/2表达下调组中明显高于对照组(P =0.018,P=0.008);(3)Smad2/3表达下调组的细胞增值率明显高于对照组(P=0.001);(4)Smad3表达下调的细胞,在TGFβ2(+/-)处理的两组中增殖率差异无统计学意义(P=0.258).结论在恶性胶质瘤细胞U251中,Smad2/3基因表达的下调能促进细胞的增殖,而TGFβ2介导的细胞增殖依赖于TGFβ2/Smad3通路;同时Smad2/3基因的表达下调增强了Smad3/2基因的表达.
목적 이용shRNA간우기술억제악성뇌효질류세포TGFβ2/Smads통로중Smad2/3기인적표체,탐토기재악성효질류세포증식중적작용.방법 장표체Smad2/3 shRNA간우서렬적특이성질립급음성대조질립용지질체전염법,분별전입도악성뇌효질류세포U251중.응용Real-TimePCR화Western blot법분별검측세포중Smad2/3기인mRNA화단백적표체;전염후적세포경TGFβ2(+/-)처리,채용CCK-8법평개량조적증식솔.결과 (1)량충특이성질립능구분별하조세포중Smad2/3기인적표체(P=0.01);(2)Smad2/3적표체량재Smad3/2표체하조조중명현고우대조조(P =0.018,P=0.008);(3)Smad2/3표체하조조적세포증치솔명현고우대조조(P=0.001);(4)Smad3표체하조적세포,재TGFβ2(+/-)처리적량조중증식솔차이무통계학의의(P=0.258).결론 재악성효질류세포U251중,Smad2/3기인표체적하조능촉진세포적증식,이TGFβ2개도적세포증식의뢰우TGFβ2/Smad3통로;동시Smad2/3기인적표체하조증강료Smad3/2기인적표체.
Objective Using shot hairpin RNA (shRNA) interference to specifically deplete cells expression of Smad2/3 gene of TGF-β2/Smads signal pathway and to investigate their effects on the proliferation of hunman glioblastoma mutiforme (GBM) cells.Methods GBM cell line U251 was transfected with plasmids taking psh-Smad2 or psh-Smad3 that expressed shRNA targeting Smad2 or Smad3 gene,and the negative control plasmid.The expression levels of Smad2/3 mRNA and protein were measured by Real-time fluorescence Quantitative PCR (Real-time PCR) and Western blot.Cells were treated by TGF-β2 (+ /-) after being transfected with plasmids.The proliferation rate was evaluated by CCK-8 assay.Results (1) Real-time PCR and Western blot illustrated that the Smad2/3 expression levels were dramatically down-regulated in U251 cells by shRNA interference (P =0.01).(2)The proliferation of cells transfected with psh-Smad2 and psh-Smad3 group was obviously higher than that of negative control group(P =0.018,P =0.008).(3)The rate of cell proliferation was similar between the cells treated with and without TGF-β2 when Smad3 was knocked down (P =0.001).(4)Additionally,the expression level of Smad3 in psh-Smad2 group was significantly higher than that of the psh-NC group and the mRNA expression of Smad2 in psh-Smad3 group also was significantly higher than that of the psh-NC group(P =0.258).Conclusions Knockdown of Smad2/3 both enhanced the cellular proliferation.U251 cell proliferation inducted by TGFβ2 was dependent on Smad3 but not Smad2.And depletion of Smad2 enhanced Smad3 expression,meanwhile depletion of Smad3 also enhanced Smad2 expression.