摘要
目的探讨褪黑激素对人增生性瘢痕成纤维细胞增殖的影响及作用机制。方法收集人皮肤增生性瘢痕标本,原代培养人皮肤增生性瘢痕成纤维细胞(HSFBs)。将HSFBs细胞根据不同的处理方法分为空白对照组、低浓度褪黑激素组、中浓度褪黑激素组、高浓度褪黑激素组、NVP-BEZ235处理组、NVP-BEZ235+褪黑激素组、褪黑激素+si RNA-PTEN组、褪黑激素+si RNA-scramble组。MTT法检测各组细胞的增殖情况,q RT-PCR和Western blot检测各组细胞中磷酸酶与张力蛋白同源物(PTEN)、Cyclin D1和Caspase-3的表达以及PI3k/AKt/m TOR通路相关蛋白p-Akt和p-m TOR的蛋白水平。结果与空白对照组比较,从低到高浓度褪黑激素均能够减少HSFBs细胞同一时间点OD值,上调PTEN和Caspase-3表达量,同时抑制Cyclin D1、p-Akt和p-m TOR表达水平,组间差异具有统计学意义(P<0.05)。同时与高浓度褪黑激素组比较,NVP-BEZ235+褪黑激素组中细胞增殖显著被抑制,差异具有统计学意义(P<0.05);沉默PTEN后拮抗褪黑激素对HSFBs细胞增殖和PI3K/Akt/m TOR信号通路有抑制作用,差异具有统计学意义(P<0.05)。结论褪黑激素通过上调PTEN表达,抑制PI3K/Akt/m TOR信号通路活化从而抑制HSFBs细胞增殖。
Objective To study the effect and mechanism of melatonin on proliferation of fibroblasts in human hypertronhic scar. Methods The human skin hypertrophic scar(HSFBs) specimens were collected. Fibroblasts from hypertrophic scar were isolated by tissue culturing method. HSFBs were divided into different groups: blank control group, low concentration melatonin group, middle concentration melatonin group, high concentration melatonin group,NVP-BEZ235 treatment group, NVP-BEZ235 + melatonin group, melatonin + si RNA-PTEN group, melatonin + si RNA-scramble group. Cells proliferation was detected by MTT. The expression of phosphatase and tensin homolog(PTEN), Cyclin D1, Caspase-3 as well as the protein levels of PI3k/AKt/m TOR pathway related protein p-Akt and p-m TOR were detected by quantitative real-time polymerase chain reaction(q RT-PCR) and western blot respectively.Results Compared with the blank control group, from the low to high concentrations of melatonin can significantly decrease the OD value of HSFBs cells at the same time, increase the expression of PTEN and Caspase-3, meanwhile inhibited the expression levels of Cyclind1, p-Akt and p-m TOR, with statistically significant differences between the four groups(P<0.05). Compared with the high concentration melatonin group, the cell proliferation was significantly inhibited in the NVP-BEZ235+ melatonin group, and the difference was statistically significant(P<0.05). The inhibitory effect of melatonin +si RNA-PTEN group on HSFBs cell proliferation and PI3K/Akt/m TOR signaling pathway was observed,and the difference was statistically significant(P<0.05). Conclusion Melatonin could inhibit HSFB cells proliferation by upregulating the expression of PTEN and inhibiting the activation of PI3K/Akt/m TOR signaling pathways.
出处
《海南医学》
CAS
2016年第18期2927-2931,共5页
Hainan Medical Journal
