p27KIP1过表达对人胃癌细胞的细胞周期和细胞凋亡的影响

目的研究p27KIP1基因对胃癌细胞的细胞周期和细胞凋亡的潜在作用. 方法采用脂质体转染法将p27KIP1全长cDNA转入胃癌细胞系SCG7901中,通过Western Blot以及RNA斑点杂交检测p27KIP1基因在蛋白质和mRNA水平的表达;细胞活力实验和软琼脂集落形成实验显示转染p27KIP1基因对细胞增殖的作用;DNA电泳、流式细胞仪、TUNEL染色及透射电镜等方法观察目的基因对该细胞的细胞周期和细胞凋亡的影响. 结果转染p27KIP1的SCG7901细胞在mRNA和蛋白质水平均有高水平p27KIP1的表达.细胞活力检测显示在外加Zn离子48h后细胞生长被抑制42%;软琼脂集落形成率明显少于亲代细胞(P<0.01);p27KIP1的过表达能够显著地增加G1期的细胞数,由33.68%增加到69.29%,P<0.01;在G1期前出现1个亚二倍体的凋亡峰,占14.68%,并且凋亡指数也显著增加(P<0.01).结论 p27KIP1基因能够诱导SCG7901细胞凋亡和细胞周期在G1期延长.因此,p27KIP1基因可能成为肿瘤基因治疗的靶基因.

Effect of p27KIP1 on cell cycle and apoptosis in SCG7901 cells

Objective To elucidate the effect of p27 KIP1 on cell cycle and apoptosis regulation in gastric carcinoma cells. Methods The whole length of p27 KIP1 cDNA was transfected into human gastric cancer cells SCG7901 by the method of lipofectin transfection. Expression of p27 KIP1 in protein or mRNA level were analyzed by Western blot and RNA dot blotting. Effect of p27 KIP1 on cell growth was observed by trypan blue exclusion assay and anchorage-independent growth in soft agar. DNA laddering was performed on electrophoresis. Flow cytometry, TUNEL and electron microscope were applied to assess the effect of p27 KIP1 on cell cycle and apoptosis.Results Expression of p27 KIP1 in protein or mRNA increased evidently in SCG7901 cells transfected with p27 KIP1. The cell growth was reduced 42% 48 h after induction with zinc as determined by cell viability assay. The alteration of cell malignant phenotype was evidently indicated by the loss of anchorage-independent growth ability in soft agar. p27 KIP1 over-expression caused cell arrest with 36%(from 33.68% to 69.29%, P<0.01) increase in G 1 population. And prolonged p27 KIP1 expression induced apoptotic cell death reflected by pre-G 1 peak in the histogram of FACS, which was also confirmed by TUNEL assay and electron microscope . Conclusion p27 KIP1 can prolong cell cycle in G 1 phase and lead to apoptosis. Therefore, p27 KIP1 may be a good candidate for cancer gene therapy.