论文部分内容阅读
该文研究端粒酶在人结肠癌HCT116高非整倍体变异组及低非整倍体变异组细胞中的表达差异及其端粒酶抑制剂3′-叠氮-3′-脱氧胸苷(3′-Azido-3′-deoxythymidine,AZT)对2组细胞增殖及凋亡的影响。取HCT116细胞加入盐酸强力霉素16 h后撤药,称为高非整倍体变异组;另取HCT116细胞不作任何处理,设为对照组,称为低非整倍体变异组;在撤药后第11 d,采用100、250μmol/L的AZT处理2组细胞72 h,并分别设立空白对照组(未加AZT的高非整倍体变异组及低非整倍体变异组)。采用染色体滴定法进行染色体计数。采用Western blot检测MAD2L1、PUMA、BAX、P21、γ-H2AX蛋白质水平。采用荧光定量PCR检测h TERT、PUMA、BAX、NOXA、P21基因表达,端粒酶活性试剂盒检测端粒酶活性,CCK-8法检测细胞生存率。实验结果表明,采用盐酸强力霉素诱导HCT116细胞的非整倍体率可达到77.33%;高非整倍体变异组细胞h TERT基因的表达及端粒酶活性明显高于低非整倍体变异组;加入AZT后,高非整倍体变异组P21、γ-H2AX蛋白质水平上升程度较整倍体明显,低非整倍体变异组PUMA、BAX蛋白质水平上升程度较高非整倍体变异组明显。该研究表明,盐酸强力霉素可以诱导非整倍体形成,高非整倍体变异组的端粒酶活性及h TERT基因表达高于低非整倍体变异组,AZT可以对高非整倍体变异组和低非整倍体变异组细胞产生增殖抑制作用、DNA损伤作用、细胞周期阻滞作用、诱导凋亡作用。
In this paper, telomerase human colon cancer HCT116 high aneuploidy mutation group and low aneuploidy mutation group cells expression and telomerase inhibitor 3’-azido-3’-deoxythymidine (3’-Azido-3’-deoxythymidine, AZT) on cell proliferation and apoptosis in two groups. HCT116 cells were treated with doxycycline hydrochloride 16 h after withdrawal, called high aneuploidy mutation group; the other to take HCT116 cells without any treatment, as the control group, known as low aneuploidy mutation group; withdrawal of drugs On the eleventh day, the two groups of cells were treated with 100,250μmol / L AZT for 72 hours, and blank control group (high aneuploidy group without AZT and low aneuploidy group) were established. Chromosome titration was used for chromosome counting. Western blot was used to detect the protein levels of MAD2L1, PUMA, BAX, P21 and γ-H2AX. The mRNA expression of hTERT, PUMA, BAX, NOXA and P21 were detected by real-time PCR. Telomerase activity was detected by telomerase activity kit. Cell viability was detected by CCK-8 assay. The experimental results showed that the rate of aneuploidy of HCT116 cells induced by doxycycline hydrochloride reached 77.33%. The expression of h TERT gene and telomerase activity in high aneuploidy group were significantly higher than those in low aneuploidy Group. After adding AZT, the protein levels of P21 and γ-H2AX in high aneuploidy group were higher than those in euploidy group, and the levels of PUMA and BAX protein in low aneuploidy group were higher than those in aneuploidy group obvious. The study showed that doxycycline hydrochloride can induce aneuploidy formation, high aneuploidy mutation group telomerase activity and h TERT gene expression higher than low aneuploidy mutation group, AZT can be high aneuploidy The gene mutation group and the low aneuploidy mutation group had cell proliferation inhibition, DNA damage, cell cycle arrest and apoptosis induction.