用ribozyme抑制增殖细胞核抗原的表达对HaLa细胞增殖的影响

增殖细胞核抗原(PCNA)是DNA聚合酶δ的辅助蛋白,它是细胞染色体DNA复制所必需的。人工设计的ribozyme具有可特异地切割PCNA mRNA的性质,将此ribozyme的自修剪体内表达质粒导入HeLa细胞,从细胞总RNA中分离相应部分能在体外切割靶RNA片段,证明此表达质粒在细胞内能表达出有活性的ribozyme分子。与对照相比,导入ribo-zyme表达质粒的HeLa细胞进入S期的时间从12 h推迟到20 h,而突变ribozyme的对照表明反义抑制对细胞进入S期的影响较小(推迟到15 h)。证明该ribozyme能有效抑制He-La细胞DNA复制,同时亦证明PCNA对于细胞DNA复制及细胞周期进程的重要性。

EFFECT OF INHIBITION OF EXPRESSION PCNA WITH RIBOZYME ON THE PROLIFERATION OF HELA CELLS

The Proliferating Cell Nuclear Antigen (PCNA), which is an auxiliary protein for DNA polymerase 6, is found to be essential for cellular DNA replication. A designed hammerhead ribozyme, with high efficiency to cleave the PCNA mRNA site-specificly in vitro, was constructed into a self-trimming expression plas-mid, and then was introduced into HeLa cells by lipofectin reagent. Small molecular RNAs, isolated from total cellular RNA with the same length as active ribozyme, can cleave the target RNA in vitro, which suggested that this expression plasmid can yield active ribozyme molecules in cells. In comparison with the vector control, the entrance of S phase of the HeLa cells transfected by the ribozyme expression plasmid was delayed 8 hours after serum stimulation. Meanwhile, those cells transfected by mutant inactive ribozyme as antisense RNA control was delayed only 3 hours. These results demonstated that this ribozyme can inhibit the DNA replication in HeLa cells effectively and could be used as a potential tool to study the function of PCNA in cellular DNA replication and cell cycle progression.