CAJ | 학술논문

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],G1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.
목적 탐토Bcl-2기인대인이선암SW1990세포증식급조망적영향.방법 설계병합성파향Bcl-2기인적sgRNA(Bcl-2-sgRNA),통과CRISPR-Cas9계통장기결합도CRISPR재체Cas9,경측서험증후전염인이선암세포주SW1990,사선Bcl-2기인고제은전세포,이야생형SW1990세포작위대조.채용CCK-8법측정세포생장곡선,통과극륭형성실험계수세포극륭수,운용류식세포의검측세포주기급조망.결과 성공획득Bcl-2기인고제적인이선암SW1990세포주,기Bcl-2단백표체결실.여야생SW1990세포비교,고제Bcl-2기인적SW1990세포적생장피억제,세포극륭형성수량현저감소[(160.7±10.0)개비(285.3±14.2)개],G1기세포비례현저증가[(84.51±0.97)％비(57.49±1.08)％],S기세포비례현저감소[(12.82±0.99)％비(27.56±1.65)％],세포조망솔현저증가[(12.67±0.59)％비(0.37±0.35)％],차이균유통계학의의(P치균＜0.01).결론 고제Bcl-2기인가억제이선암SW1990세포적생장,강저세포극륭형성능력,사세포조체재G1기,병현저증가세포조망솔.
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 significandy 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.