γ-synuclein对结肠癌细胞系体外生物学行为影响的观察  

Effects of γ-synuclein on biological features of colon cancer cell line in vitro

作　　者：叶青[1] 黄峰[1] 龚福生[1] 许杨梅[1] 王晓盈[1] 黄丽洁[1] 应敏刚[1] 

机构地区：[1]福建医科大学教学医院.福建省肿瘤生物治疗重点实验室.福建省肿瘤医院腹部外科,福建福州350014

出　　处：《中华肿瘤防治杂志》2013年第14期1047-1052,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金青年科学基金(81101897);福建省自然科学基金青年创新项(2011J05061)

摘　　要：目的:构建针对人γ-synuclein基因的shRNA真核表达载体,观察γ-synuclein基因对人结肠癌细胞系体外生物学行为的影响。方法:根据人γ-synuclein序列设计并构建γ-synuclein基因的shRNA真核表达载体,以脂质体转染的方法将γ-synuclein干扰质粒转染至人结肠癌细胞株HCT116,经G418筛选出稳定转染的细胞株。应用CCK8法检测γ-synuclein干扰对HCT116细胞增殖的影响,软琼脂克隆形成实验检测细胞体外克隆形成能力,细胞划痕实验检测细胞体外迁移能力,Transwell小室法检测细胞体外侵袭能力。结果:经测序证明γ-synuclein的shRNA序列已成功插入pGCsi-U6/neo/GFP质粒中,构建的干扰质粒能够显著抑制γ-synuclein mRNA及蛋白的表达。γ-synuclein受抑制后,HCT116细胞增殖数目从48h后开始减少,持续72h,与空质粒组及未转染组相比,差异均有统计学意义,P<0.05。siRNA组细胞克隆形成率为(9.6±2.9)%,显著低于未转染组的(29.4±4.5)%和空质粒组的(32.1±5.8)%,差异均具有统计学意义,P<0.05。在细胞划痕实验中,γ-synuclein被抑制后细胞划痕损伤愈合的速度明显减慢。在侵袭实验中,穿过Matrigel胶及Transwell小室膜的siRNA组侵袭细胞数为20.1±5.26,显著低于未转染组的100和空质粒组的105.4±12.5,差异均有统计学意义,P<0.05。结论:干扰γ-synuclein的表达可显著抑制结肠癌细胞体外增殖、克隆形成和迁移侵袭能力。OBJECTIVE：To construct the shRNA against human 7-synuclein gene expression vector,and study the effects of y-synuclein on tlhe biological features of colon cancer cell line HCT116 in vitro. METHODS：The shRNA targe- ting y-synuclein mRNA plasmid was constructed and transfected into the colon cancer cell line HCT116 using Lipo- fectamine. The stable cell lines were selected with G-418 for 28 d,and the biological features of these cells were examined by CCK8 assay, soft agar assay, wound healing assay,and cell invasion assay in vitro. RESULTS：The sequence proved the y-synuclein gene was successfully inserted pGCsi-U6/neo/GFP shRNA plasmid. The interference plasmid could signifi- cantly inhibit the expression of 7-synuclein mRNA and protein. After y-synuclein knockdown,the number of proliferation cells was significantly reduced by 48,72,96 and 120 h （P〈 0.05）, respectively, and colony formation rate was （9.6 士 2.9） % in siRNA cells, compared with （29.4 士 4.5）% in control cells and （32.1 士 5.8）% in pGCsi-vector cells, respectively （P%0.05）. The wound healing assay showed that y-synuclein down-expression led to decreased healing speed of cell injury. In invasion assa y, the amount of invasion cells that passed through matrigel and chamber membrane was 20.1士 5.26 in siRNA cells,much less than 100 in control cells and 105.4士 12.5 in pGCsi-vector cells, respectively, P〈0.05. CONCLUSION：The expre,sion of Y-synuclein interference could inhibit colon cancer cell proliferation,colony formation, and migration and invasion ability in vitro.

关 键 词：结肠癌细胞 Γ-SYNUCLEIN SHRNA 生物学行为 

分 类 号：R735.35[医药卫生—肿瘤]