Han Jun-t, Chen Bi, Liu Shu-juan, Tang Chao-wu
Department of Burns, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi Province, PR China.
Zhonghua Shao Shang Za Zhi. 2003 Aug;19(4):226-8.
To investigate the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts.
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 (3)H-TdR incorporation method.
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.
p16 gene might inhibit the growth and DNA synthesis of KFb.
探讨野生型p16基因对人瘢痕疙瘩成纤维细胞增殖和代谢的影响。
构建真核表达载体pcDNA3-p16，通过脂质体介导的基因转染将其导入瘢痕疙瘩成纤维细胞（KFb）。并用G418筛选阳性克隆。采用免疫细胞化学方法对转染及未转染的瘢痕疙瘩成纤维细胞进行染色，观察p16蛋白的表达情况。通过绘制细胞生长曲线及采用³H-TdR掺入法观察并比较转染前后瘢痕疙瘩成纤维细胞增殖及DNA合成的变化。
成功构建重组载体pcDNA3-p16，并经酶切鉴定。经G418筛选10天，从转染的瘢痕疙瘩成纤维细胞中鉴定出阳性克隆且有p16蛋白表达。与正常瘢痕疙瘩成纤维细胞相比，转染后的瘢痕疙瘩成纤维细胞生长速度明显减慢，其DNA合成显著减少（P < 0.05）。
p16基因可能抑制瘢痕疙瘩成纤维细胞的生长及DNA合成。
