Bcl-2基因敲除对人胰腺癌细胞增殖及凋亡的影响

Effects of knocking out Bcl-2 gene on proliferation and apoptosis of human pancreatic cancer cells SW1990

目的探讨Bcl-2基因对人胰腺癌SW1990细胞增殖及凋亡的影响。方法设计并合成靶向Bcl-2基因的sgRNA（Bcl-2-sgRNA），通过CRISPR—Cas9系统将其结合到CRISPR载体Cas9，经测序验证后转染人胰腺癌细胞株SW1990，筛选Bcl-2基因敲除稳转细胞，以野生型SW1990细胞作为对照。采用CCK-8法测定细胞生长曲线，通过克隆形成实验计数细胞克隆数，运用流式细胞仪检测细胞周期及凋亡。结果成功获得Bcl-2基因敲除的人胰腺癌SW1990细胞株，其Bcl-2蛋白表达缺失。与野生SW1990细胞比较，敲除Bcl-2基因的SW1990细胞的生长被抑制，细胞克隆形成数量显著减少[（160．7±10．0）个比（285．3±14．2）个]，G．期细胞比例显著增加[（84．51±0．97）％比（57．49±1．08）％]，S期细胞比例显著减少[（12．82±0．99）％比（27．56±1．65）％]，细胞凋亡率显著增加[（12．67±0．59）％比（0．37±0．35）％]，差异均有统计学意义（P值均〈0．01）。结论敲除Bcl-2基因可抑制胰腺癌SW1990细胞的生长，降低细胞克隆形成能力，使细胞阻滞在G1期，并显著增加细胞凋亡率。

Objective To investigate the effect of Bcl-2 gene expression on the proliferation and apoptosis of human pancreatic cancer SW1990 cells. Methods Bcl-2 short guide RNA （Bcl-2-sgRNA） was designed and synthesized, and it was combined with CRISPR-Cas 9. After confirmation by gene sequencing, it was transfected into human pancreatic cancer cell line SW1990, then the cells with stable Bcl-2 gene knock-out were selected, and wild type SW1990 cells were used as control. The cell growth curve was determined by CCK-8 method. The number of clone formation was measured. Flow cytometry was used to measure cell cycle and apoptosis. Results Human pancreatic cancer cell line SW1990 with Bcl-2 gene knock-out was successful constructed. Compared with wild type SW1990 cells, the growth of SW1990 cells with Bcl-2 gene knock-out was inhibited, the number of clone formation was significantly decreased [ （160.7 ± 10.0） vs （285.3 ± 14.2） ], the proportion of G1 cells was significantly increased [ （ 84.51 ± 0.97 ） % vs （ 57.49 ± 1.08 ） % ], the proportion of S phase cells significantly decreased [ （ 12.82 ± 0.99 ） % vs （ 27.56 ± 1.65 ） % ], and apoptosis rate was remarkably increased [ （ 12.67 ± 0.59 ） % vs （ 0. 37 ± 0.35 ） % ], and the difference between the two groups was statistically significant （P 〈 0.01 ）. Conclusions Knock-out of Bcl-2 gene can inhibit the growth of human pancreatic cancer cell line SW1990, decrease the ability of clone formation, block the cell in G1 phase and greatly increase cell apoptosis rate.