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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 study the effect of clock gene Period2 (Per2) on the proliferation, differentiation and apoptosis of chronic myeloid leukemia K562 cell line and its possible molecular mechanism. Methods Per2 expression plasmid pcDNA3.1-Per2 and control plasmid pcDNA3.1 were transfected into K562 cells by cationic liposome respectively, and Per2 stably expressing cells were screened by G418. Wright’s staining was used to observe the cell morphology, trypan blue exclusion and MTT assay K562 cell proliferation, cell cycle and apoptosis analysis by flow cytometry, apoptosis was observed by electron microscopy, RT-PCR and Western blot detection of the cell line Proliferation, apoptosis-related genes p53, cyclin B1 and c-myc expression. Results The K562 / Per2 cells stably expressing Per gene were screened. Compared with the untransfected and untransfected cells, K562 / Per2 cells were reduced in size but not differentiated obviously. The trypan blue exclusion assay And Perkin-Elmer (MTT) assay showed that Per2 inhibited the cell growth and proliferation. Flow cytometry showed that G2 phase cells were increased in K562 / Per2 cells (36.1 ± 5.5%) and 12.5 ± 2.9% (9.7 ± 2.3)%, P <0.05]. Apoptosis assay showed that apoptosis in transfected cells was significantly increased (14.8%, P <0.05). The results of electron microscopy showed chromatin condensation, fragmentation, nuclear condensation and apoptosis Peroxisome proliferator-activated receptor 2 (PER2) and peroxisome proliferator-activated receptor 2 (PER2) were detected by RT-PCR and Western blot. Per2 significantly up-regulated the mRNA and protein expression of p53 gene and inhibited the expression of mR-NA and c-myc mRNA. Conclusion Peri gene can inhibit the growth and proliferation of K562 cells and induce the apoptosis of K562 cells. However, Per2 has no obvious effect on differentiation. The mechanism may be through the regulation of cell cycle related genes and prevent the cell cycle progression to achieve.