Si-PTN抑制瘢痕成纤维细胞生长

摘要

目的 分析多效生长因子(si-PTN)对瘢痕成纤维细胞生长的影响.方法 选取2015年1月至2017年10月于北部战区总医院就诊的18例瘢痕增生患者,从18例患者术中切取的新鲜增生性瘢痕组织中提取原代瘢痕成纤维细胞,构建si-PTN细胞系.构建成功后,将对数生长期瘢痕成纤维细胞分为si-PTN组和对照组,分别转染2μg si-PTN及对照无意义小链.采用MTT、PI染色及Annexin V-FITC/PI实验分别检测si-PTN组和对照组细胞的增殖和凋亡情况.通过Western blot和RT-PCR检测si-PTN和对照组细胞中Cyclin D1、Bcl-2和Bax的含量.结果 si-PTN抑制瘢痕成纤维细胞增殖,1、2、3、4 d时si-PTN对细胞生长的抑制率分别为14.49％、13.24％、20.78％和23.12％,与对照组比较差异均具有统计学意义(P<0.05).细胞周期实验结果显示si-PTN组G0-G1期所占比例为(60.79±3.34)％,高于对照组的(50.54±1.80)％,差异具有统计学意义(P=0.02).si-PTN组S期细胞比例为(29.02±2.07)％,低于对照组的(40.08±2.89)％,差异具有统计学意义(P=0.03).细胞凋亡检测结果显示,si-PTN组早期及晚期凋亡细胞比例分别为(8.17±0.57)％和(6.80±0.74)％,均高于对照组的(4.35±0.46)％和(4.70±0.48)％,差异均具有统计学意义(P<0.05).Western Blot和RT-PCR结果显示,与对照组相比si-PTN组Cyclin D1下调43.76％,Bcl-2下调43.56％,Bax上调31.97％,差异均具有统计学意义(P<0.05).结论 si-PTN可抑制瘢痕成纤维细胞的生长.%Objective To analyze the effects of PTN in the growth of hypertrophic scar fibroblasts. Methods The primary scar fibroblasts were extracted from fresh hypertrophic scar tissues of 18 patients, to restrict the si-PTN cell line. After successful construction, the scar fibroblasts in logarithmic growth were divided into si-PTN group and control group, transfected with 2 μg si-PTN and control nonsense chains, respectively. MTT, PI staining and Annexin V-FITC/PI assay were used to detect the proliferation and apoptosis of si-PTN and control groups. Western blot and RT-PCR were used to detect the expression level of Cyclin D1, Bcl-2 and Bax in si-PTN and control groups. Results The inhibition rates of si-PTN on the proliferation of scar fibroblasts were 14. 49％, 13. 24％, 20. 78％ and 23. 12％ at 1, 2, 3 and 4 days, respectively. The difference was statistically significant compared with the control group (P<0. 05). The results of cell cycle experiments showed that the proportion of G0-G1 phase in the si-PTN group was (60. 79±3. 34)％, which was higher than that of the control group (50. 54±1. 80)％ (P=0. 02). The proportion of S phase cells in the si-PTN group was (29. 02±2. 07)％, which was lower than that in the control group (40. 08±2. 89)％ (P=0. 03). The results of apoptosis assay showed that the proportion of early and late apoptotic cells in si-PTN group were (8. 17±0. 57)％ and (6. 80±0. 74)％, respectively, which were higher than that of the control group ( 4. 35 ± 0. 46 )％, and ( 4. 70 ± 0. 48 )％ ( P<0. 05 ) . Western Blot and RT-PCR results showed that compared with the control group, the si-PTN group was down-regulated by Cyclin D1 ( 43. 76％) and Bcl-2 ( 43. 56％) , Bax was up-regulated ( 31. 97％) ( P<0. 05) . Conclusions si-PTN inhibited the growth of scar fibroblasts.