摘要
目的研究钟基因Period2(Per2)对慢粒急变K562细胞株增殖、分化、凋亡的影响及可能的分子机制。方法将Per2表达质粒pcDNA3.1-Per2及空载对照质粒pcDNA3.1分别经阳离子脂质体介导转染K562细胞,用G418筛选出Per2稳定表达细胞株。瑞氏染色观察细胞形态,台盼蓝拒染法和MTT法检测K562细胞增殖,流式细胞仪分析细胞周期和凋亡,电镜观察细胞凋亡,RT-PCR及Western blot方法检测该细胞株中增殖、凋亡相关基因p53、cyclin B1和c-myc等的表达。结果筛选到稳定表达Per基因的稳定表达细胞株K562/Per2细胞,与空载体转染组及未处理对照组细胞相比,K562/Per2细胞体积缩小,但是分化不明显;台盼蓝拒染法和MTT实验发现Per2抑制细胞生长与增殖能力;流式细胞仪检测周期结果K562/Per2细胞中G2期细胞增多[转染组(36.1±5.5)%,空载组(12.5±2.9)%,未处理对照组(9.7±2.3)%,P<0.05],凋亡检测显示转染组细胞凋亡明显增加[14.8%P<0.05];电镜结果显示染色质浓集、断裂,核固缩和凋亡小体;RT-PCR及Western blot显示Per2明显上调p53基因的mRNA和蛋白表达,而抑制cyclin B1、c-myc基因mR-NA和蛋白的表达。结论钟基因Per2能抑制K562细胞的生长和增殖,并诱导其发生凋亡,但对分化无明显作用。其机制可能是通过对细胞周期相关基因的调控并阻止细胞周期进程来实现的。
Objective To investigate the effects of circadian clock gene Period2 （Per2） on the proliferation, differentiation and apoptosis of K562 cells and its probable molecular mechanism. Methods The Per2 expression plasmid pcDNA3. 1-Per2 and empty control plasmid were respectively transfected into K562 cells with cationic liposome, and the resistant cells stably expressing Per2 gene were obtained by G418 selection. Their morphological changes were observed under light microscope following Wright-Giemsa staining. Trypan blue excluding staining and MTF assay were employed to evaluate cell proliferation. Flow cytometry was performed to analyze cell cycle distribution and cell apoptosis, and electron microscopy was used to detect cell apoptosis. Meanwhile, the expressions of proliferation and apoptosis associated proteins, such as P53, Cyclin B1 and C-Mye, were respectively detected by RT-PCR and Western blot analysis at mRNA and protein level. Results The K562/Per2 cell line stably expressing Per2 gene was screened out. As compared with either the empty plasmid transfected group （K562/empty） or the untreated group （K562/untreated）, K562/Per2 cells was smaller in volume and showed no obvious cellular differentiation. Circadian clock gene Per2 could significantly inhibit both growth and proliferation of K562 cells. The percentage of K562 cells in Gz/M phase increased [ K562/Per2 group （36.1 ± 5.5 ） %, K562/empty group （ 12.5 ± 2.9 ） %, untreated group （ 9.7 ± 2.3 ） %, P 〈 0. 05 ] and more apoptotie eells were found in the transfeeted group （ 14.8%, P 〈 0. 05）. Nuelear condensation, fragmentation, karyopycnosis and apoptotic bodies were found under transmission electron mieroseope in the K562/Per2 group. P53 was significantly elevated at the mRNA and protein level, while Cyclin B1 and C-Myc were downregulated. Conclusion Expression of circadian clock gene Per2 can not only inhibit the growth and proliferation of K562 cells, but also induce massive apoptosis possibly through its regulation on cell cycle associated genes and subsequent inhibition on cell cycle progression.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第4期301-306,共6页
Journal of Third Military Medical University
基金
国家自然科学基金(30600748)~~
关键词
钟基因
增殖
凋亡
细胞周期
节律调节
circadian clock gene
proliferation
apoptosis
cell cycle
rhythm regulation
