野生型p16基因对人瘢痕疙瘩成纤维细胞生长及代谢影响的实验研究

目的　探讨野生型 p16基因对人瘢痕疙瘩成纤维细胞 (keloidfibroblasts,KFb)生长增殖及DNA合成代谢的影响。　方法　构建含p16cDNA片段的真核表达载体 pcDNA3 p16 ,采用脂质体介导的基因转染法 ,将其导入体外培养的KFb中 ,并用G4 18筛选阳性克隆。随后对已转染及未转染的KFb进行免疫细胞化学染色 ,观察p16蛋白的表达 ,并通过绘制细胞生长曲线及采用氚标记胸腺嘧啶脱氧核苷 (3 H TdR)掺入法 ,比较转染前后细胞在生长增殖及DNA合成代谢方面的变化。结果　经酶切鉴定证实 ,pcDNA3 p16构建成功。已转染的KFb经G4 18筛选 ,出现阳性克隆 ,并有 p16蛋白表达 ;与正常KFb比较 ,其生长增殖速度明显减缓 ,DNA合成代谢能力明显减弱 (P <0 .0 5 )。　结论　p16基因对KFb的生长增殖及DNA合成代谢有明显的抑制作用。

An experimental study on the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts

Objective To investigate the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts. Methods Eukaryotic expression vector pcDNA3-p16 was constructed and imported into KFb by gene transfection mediated by liposome. And the positive clones were screened by G418. The transfected and untransfected KFbs were stained by Immunocytochemical method. The expression of p16 protein was observed. The changes of the proliferation and DNA synthesis of KFb before and after transfection were observed and compared by drafting cell growth curve and by 3H TdR incorporation method. Results The recombinant vector pcDNA3 p16 was successfully constructed and identified by enzyme digestion. The positive clones were identified by G418 selection for 10 days from transfected KFb and with p16 protein expression. The growth rate of transfected KFb slowed down obviously and its DNA synthesis decreased significantly (P< 0.05) when compared with those of normal KFb.Conclusion p16 gene might inhibit the growth and DNA synthesis of KFb. [