野生型p^(53)基因导入对培养的兔血管平滑肌细胞生长的抑制作用

Inhibition of wild type p53 gene on the growth of cultured rabbit vascular smooth muscle cells

为探索腺病毒载体重组野生型ｐ５３基因转染平滑肌细胞的效率及其应用于动脉粥样硬化和动脉再狭窄的基因治疗的可能性，构建了野生型ｐ５３基因的生长缺陷型重组腺病毒载体。在体外培养条件下转染兔血管平滑肌细胞。应用生化染色法、免疫组化法及聚合酶链技术等检测了外源基因的转染效率及表达效果。应用细胞计数及同位素参入技术测定了ｐ５３基因对平滑肌细胞的抑制效果。流式细胞仪检测了平滑肌细胞。结果显示：腺病毒载体可快速有效地将外源基因转导进入平滑肌细胞，在转染细胞内可检测到外源性ｐ５３ｃＤＮＡ，并可高效表达ｐ５３蛋白。ｐ５３重组腺病毒载体转染细胞后，可显著抑制细胞生长，细胞计数减少，ＤＮＡ合成减少，并可导致细胞死亡。流式细胞仪检测有部分细胞体积缩小，ＤＮＡ减少。结果提示，野生型ｐ５３基因重组腺病毒载体转染平滑肌细胞可有效抑制细胞生长，有可能成为动脉粥样硬化和再狭窄的基因治疗手段。

In order to study the transfection efficiency of wild type p53 gene mediated by recombinant adenovirus vector and the possibility of using p53 gene in gene therapy of atherosclerosis and artery restenosis, wild type p53 gene recombinant replication defective adenovirus vector was constructed and transfected into cultured rabbit vascular smooth muscle cells (SMC). The efficiency of transfection and expression was detected by biochemical stainig immunohistochemical and polymerase chain reaction techniques. Condition of the genomic p53 gene was tested, too. Proliferation and cell cycle of SMC were investigated by cell counting, isotope incorporation and cytometric tests. The results showed that wild type p53 gene could be quickly and effectively transferred into the cells by adenovirus vector. The p53 protein and p53 cDNA could be detected within the transfected cells. Normal SMC contain genomic p53 gene but do not express enough p53 protein. The transferred p53 gene could inhibit the proliferation and DNA synthesis of SMC. It could also cause cell death. This result suggested that the p53 recombinant adenovirus vector might play a role in gene therapy of atheroscerosis and artery restenosis.