Background Both p16^INK4 and p21waf1 are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16^INK4 could be activated by p21waf1 through transc...Background Both p16^INK4 and p21waf1 are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16^INK4 could be activated by p21waf1 through transcriptional factor Spl in HeLa cells. This study was undertaken to determine the effects of p16^INK4 on the expression and functions of p21^waf1. Methods Human diploid fibroblast 2BS cells were stably transfected with sense （2BS/p16^INK4）, antisense p16^INK4 （2BS/asp16^INK4） or empty vector （2BS/neo） .Then they were assayed by reverse-transcription polymerase chain reaction （RT-PCR）, fluorescence activated cell sorting （FACS） and Western blot. Results 2BS/p16^INK4 cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21^waf1 protein levels increased twofold in the 2BS/p16^INK4 cells, but not decreased in the 2BS/asp16^INK4 cells. P21^waf1 mRNA levels were not affected in neither 2BS/p16^INK4 nor 2BS/asp16^INK4 cells. Conclusion p16^INK4 may play an important role in the regulation of cellular senescence by modulating the p21^waf1 protein level via the posttranscriptional mechanism.展开更多
目的研究亨廷顿蛋白相关蛋白1(Huntingtin-associated protein 1,Hap1)基因对成纤维细胞增殖的影响。方法体外分离培养获得Hap1基因敲除(Hap1^(-/-))的原代成纤维细胞,鉴定后采用EdU增殖实验、细胞周期(流式细胞术)检测验证Hap1敲除后...目的研究亨廷顿蛋白相关蛋白1(Huntingtin-associated protein 1,Hap1)基因对成纤维细胞增殖的影响。方法体外分离培养获得Hap1基因敲除(Hap1^(-/-))的原代成纤维细胞,鉴定后采用EdU增殖实验、细胞周期(流式细胞术)检测验证Hap1敲除后成纤维细胞增殖的改变;将野生型和Hap1^(-/-)成纤维细胞送转录组测序筛选增殖相关基因,实时荧光定量PCR(qPCR)验证相关基因表达水平的改变。对小鼠进行皮肤损伤造模检测Hap1敲减(Hap1^(+/-))小鼠皮肤损伤修复情况;PCNA免疫组化染色检测损伤修复过程中成纤维细胞的增殖水平。结果成功培养原代Hap1^(-/-)成纤维细胞;与野生型成纤维细胞相比,Hap1^(-/-)成纤维细胞EdU阳性比例减少,进入S期的细胞比例减少;原代成纤维细胞转录组测序筛选出Cdc25C、E2f7、E2f8和Ccl5四个差异表达的增殖相关基因,qPCR验证发现E2f7在Hap1敲除后表达增多。小鼠皮肤损伤结果显示,Hap1^(+/-)小鼠伤口面积较相同时间点野生型小鼠伤口面积大且愈合速度减慢,成纤维细胞增殖阳性密度低于野生型小鼠。结论 Hap1可能通过抑制细胞周期负性调节因子E2f7的表达对成纤维细胞增殖起正向调节作用,其缺失将抑制成纤维细胞进入S期,从而减少细胞增殖,影响伤口修复。展开更多
基金grants from the National Basic Research Program of China(No.2007CB507400)National Nature Science Foundation of China(No.30671064).
文摘Background Both p16^INK4 and p21waf1 are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16^INK4 could be activated by p21waf1 through transcriptional factor Spl in HeLa cells. This study was undertaken to determine the effects of p16^INK4 on the expression and functions of p21^waf1. Methods Human diploid fibroblast 2BS cells were stably transfected with sense （2BS/p16^INK4）, antisense p16^INK4 （2BS/asp16^INK4） or empty vector （2BS/neo） .Then they were assayed by reverse-transcription polymerase chain reaction （RT-PCR）, fluorescence activated cell sorting （FACS） and Western blot. Results 2BS/p16^INK4 cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21^waf1 protein levels increased twofold in the 2BS/p16^INK4 cells, but not decreased in the 2BS/asp16^INK4 cells. P21^waf1 mRNA levels were not affected in neither 2BS/p16^INK4 nor 2BS/asp16^INK4 cells. Conclusion p16^INK4 may play an important role in the regulation of cellular senescence by modulating the p21^waf1 protein level via the posttranscriptional mechanism.
文摘目的研究亨廷顿蛋白相关蛋白1(Huntingtin-associated protein 1,Hap1)基因对成纤维细胞增殖的影响。方法体外分离培养获得Hap1基因敲除(Hap1^(-/-))的原代成纤维细胞,鉴定后采用EdU增殖实验、细胞周期(流式细胞术)检测验证Hap1敲除后成纤维细胞增殖的改变;将野生型和Hap1^(-/-)成纤维细胞送转录组测序筛选增殖相关基因,实时荧光定量PCR(qPCR)验证相关基因表达水平的改变。对小鼠进行皮肤损伤造模检测Hap1敲减(Hap1^(+/-))小鼠皮肤损伤修复情况;PCNA免疫组化染色检测损伤修复过程中成纤维细胞的增殖水平。结果成功培养原代Hap1^(-/-)成纤维细胞;与野生型成纤维细胞相比,Hap1^(-/-)成纤维细胞EdU阳性比例减少,进入S期的细胞比例减少;原代成纤维细胞转录组测序筛选出Cdc25C、E2f7、E2f8和Ccl5四个差异表达的增殖相关基因,qPCR验证发现E2f7在Hap1敲除后表达增多。小鼠皮肤损伤结果显示,Hap1^(+/-)小鼠伤口面积较相同时间点野生型小鼠伤口面积大且愈合速度减慢,成纤维细胞增殖阳性密度低于野生型小鼠。结论 Hap1可能通过抑制细胞周期负性调节因子E2f7的表达对成纤维细胞增殖起正向调节作用,其缺失将抑制成纤维细胞进入S期,从而减少细胞增殖,影响伤口修复。
