论文部分内容阅读
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 a shRNA eukaryotic expression vector targeting human γ-synuclein gene and observe the effect of γ-synuclein gene on the biological behavior of human colon cancer cell line in vitro. Methods: shRNA eukaryotic expression vector of γ-synuclein gene was designed and constructed based on the human γ-synuclein sequence. The γ-synuclein interference plasmid was transfected into human colon cancer cell line HCT116 by liposome transfection, and then screened by G418 Stably transfected cell lines. The effect of γ-synuclein interference on the proliferation of HCT116 cells was detected by CCK8 assay. The ability of colony formation in vitro was detected by soft agar colony formation assay. The in vitro migration ability of cells was detected by scratch assay. Transwell chamber assay was used to detect the invasion ability of HCT116 cells. Results: The shRNA sequence of γ-synuclein was successfully inserted into the pGCsi-U6 / neo / GFP plasmid by sequencing. The constructed interference plasmids could significantly inhibit the expression of γ-synuclein mRNA and protein. After γ-synuclein was inhibited, the number of HCT116 cell proliferation decreased from 48h, and continued for 72h. The difference was statistically significant (P <0.05) compared with empty plasmid group and untransfected group. The rate of colony formation in siRNA group was (9.6 ± 2.9)%, which was significantly lower than that in non-transfected group (29.4 ± 4.5%) and empty plasmid group (32.1 ± 5.8)%, P <0.05 . In the cell scratch assay, the arrest of γ-synuclein cell scratch injury significantly slowed the rate of healing. In the invasion assay, the number of invasive cells in the siRNA group that passed through the Matrigel and Transwell chamber membranes was 20.1 ± 5.26, which was significantly lower than that in the untransfected group 100 and the blank plasmid group 105.4 ± 12.5, P < <0.05. Conclusion: Interfering with the expression of γ-synuclein can significantly inhibit the proliferation, colonization and migration of colon cancer cells in vitro.