端粒酶在整倍体与非整倍体细胞中的表达差异

Differences of telomerase expression in euploidy and aneuploidy cells

目的探讨端粒酶在HCT116整倍体细胞与非整倍体细胞中的表达差异及其端粒酶抑制剂,叠氮脱氧胸苷(AZT)对整倍体和非整倍体细胞h TERT基因表达及端粒酶活性变化的影响。方法取HCT116细胞加入盐酸强力霉素16 h后撤药,设为非整倍体组;另取HCT116细胞不作任何处理,称为整倍体组;在撤药后第11天,分别采用0、100、250μmol/L的AZT处理两组细胞72 h,分别设立空白对照组和AZT处理组。采用染色体滴定法对两组细胞的染色体进行计数。采用Western blot检测撤药后第3、6、11天MAD2L1蛋白的表达。运用RT-PCR法检测整倍体组、非整倍体组及AZT处理组h TERT基因的表达,端粒酶活性试剂盒检测端粒酶活性。结果盐酸强力霉素可以通过破坏MAD2L1诱导非整倍体,非整倍体率可达到78%;整倍体组和非整倍体组细胞在第6、8、11天h TERT mRNA基因的表达量分别为(1︰1.19±0.05、1︰1.21±0.02、1︰1.46±0.04),端粒酶活性分别为(2.87±0.01︰3.14±0.10、2.89±0.01︰3.25±0.03、2.79±0.03︰4.26±0.15),非整倍体组细胞的h TERT mRNA基因的表达量、端粒酶活性明显高于整倍体,两组细胞在第6、8、11天比较具有统计学差异(P<0.05);整倍体组细胞在100、250μmol/L处的h TERT mRNA基因下降率分别为5%、32%,端粒酶活性下降率为14.69%、25.40%;非整倍体组细胞在100、250μmol/L处的h TERT mRNA基因下降率分别为38.46%、51.28%,端粒酶活性下降率为17.00%、74.59%。两组细胞h TERT mRNA基因下降率在100、250μmol/L处具有统计学差异(P<0.05)。两组细胞端粒酶活性下降率在250μmol/L处具有统计学差异(P<0.05)。结论盐酸强力霉素可以诱导非整倍体形成,非整倍体细胞的端粒酶活性及h TERT基因表达高于整倍体细胞,AZT可以抑制整倍体细胞和非整倍体细胞h TERT基因的表达及端粒酶活性,非整倍体细胞对AZT更敏感。

Objective To study the differences of telomerase expression in HCT 116 euploidy and aneuploidy cells and the influence of telomerase inhibitor,azidothymidine(AZT)on euploidy and aneuploidy cells of hTERT gene expression and telomerase.Method HCT 116 cells were added into doxycycline hydrochloride(doxycycline)for 16 hours,known as aneuploidy group;The control group(known as euploidy group)was added nothing;After the withdrawal of doxycycline of 11 days,the two groups were added AZT of 0,100,250μmol/L for 72 hours,established as control group and AZT-dealt group respectivily.The chromosomes numbers of two groups were counted by karyotype analysis,The expression of MAD2L1 protein was detected by western blot at day 3,6,11.RT-PCR was used to detect the hTERT gene expression of Euploidy,Aneuploidy and AZT-dealt group.the telomerase activity was detected by the Telomeric Repeat Amplification Protocol(TRAP).Results Doxycycline can induce aneuploidy by destroying MAD2L1,the aneuploidy rate of cells can reach to 78%.The hTERT gene expression were 1︰ 1.19±0.05,1︰ 1.21±0.02,1︰ 1.46±0.04 of euploidy and aneuploidy group at day 6,8,11.The telomerase activity of euploidy and aneuploidy group were 2.87±0.01︰ 3.14±0.10,2.89±0.01︰ 3.25±0.03,2.79±0.03︰ 4.26±0.15 at day 6,8,11.The hTERT gene expression and telomerase activity of aneuploidy group were significantly higher than the euploidy group,there were statistically different of two groups(P<0.05).The hTERT gene expression was concentration-dependent with about 5,32%decrease for euploidy group and approximately 38.46,51.28%decline for aneuploidy group after exposure to 100,250μM of AZT for 72 h,respectively.The telomerase activity was concentration-dependent with about 14.69,25.40%decrease for euploidy group and approximately 17.00,74.59%decline for aneuploidy group after exposure to 100,250μM of AZT for 72 h,respectively.There were statistically different of two groups of hTERT gene decline rate in 100,250μM(P<0.05).There were statistically different of two groups of telomerase activity decline rate in 250μmol/L(P<0.05).Conclusion Doxycycline can induce aneuploidy,the hTERT gene expression and telomerase activity of aneuploidy group were significantly higher than the euploidy group,AZT can inhibit hTERT gene expression and telomerase activity of euploidy and aneuploidy cells.Aneuploidy cells were more sensitive to telomerase inhibitors.