摘要
目的探讨mTOR抑制剂雷帕霉素对体外培养的瘢痕疙瘩成纤维细胞生物学特性的影响和自噬的作用，以及其对mTOR信号通路和自噬相关非编码RNA表达的调控。方法以瘢痕疙瘩成纤维细胞为实验模型，经浓度为10、50、100nmol／L的雷帕霉素处理后，采用MTS法检测细胞增殖；应用流式细胞仪检测细胞凋亡；透射电镜观察自噬体形成情况，Western Blot检测自噬相关蛋白LC3的表达；实时荧光定量PCR检测细胞外基质相关基因、mTOR信号通路基因、自噬相关非编码RNA的表达。结果雷帕霉素各浓度处理组的成纤维细胞吸光度A的比值与对照组相比较，差异均有统计学意义（P〈0．05）。同时，下调其细胞外基质相关基因collagen-1、α—SMA和Fibronectin的mRNA表达（P〈0．05）。流式细胞仪检测结果显示，雷帕霉素对瘢痕疙瘩成纤维细胞凋亡无明显诱导作用（P〉0．05）。透射电镜观察发现，瘢痕疙瘩成纤维细胞中可见自噬小体的双层膜结构，并伴有自噬相关蛋白LC3的表达增加；mTOR通路下游基因4EBPl和p70S6K的表达均有下调；同时，自噬相关miRNA（miR-885—3p、miR-204、miR-101、miR-376b）和lncRNA FLJ11812表达增加，而miR-30a、lncRNA HULC表达减少（P〈0．05）。结论雷帕霉素对瘢痕疙瘩成纤维细胞的生长具有抑制作用，对其凋亡无显著影响，但可以通过抑制mTOR信号通路的活化以及调控自噬相关非编码RNA的表达，激活其自噬的发生。
Objective To investigate the effect of rapamycin on biological characteristics and autophagy of keloid fibroblasts, and the regulation of rapamycin in mTOR （mammalian target of rapamycin） signaling pathway and autophagy-related non-coding RNAs in keloid fibroblasts. Methods After Keloid fibroblasts were treated with rapamycin（10,50,100 nmol/L） , and MTS assay was used to test the cell proliferation. The apoptosis of cells was tested by the flow cytometry analysis. The formation of autophagy was observed by TEM, and the Western Blot was used to detect the expression of autophagy-related protein LC3. Real-time PCR was used to detect the expression of genes of involued in roTOR pathway and autophagy-related non-coding RNAs. Statistical significance was determined using Paired-Samples t Test, P value less than 0.05 was considered statistically significant. Results The ratio of 490nm was decreased significantly in rapamycin-treated keloid fibroblasts compared with that in untreated cells （ P 〈 0. 05 ）. Meanwhile the mRNA expressions of extracellular matrix （ECM） genes, including collagen-1 ,α-SMA and Fibronectin, were inhibited by rapamycin（ P 〈 0.05 ）. The flow cytometry analysis showed that the percent of apoptosis cells was not increased in rapamycin-induced cells （ P 〉 0. 05 ）. The double-layer membrane structure of autophagosomes could be observed under the TEM in rapamycin-treated fibroblasts, accompanied by the increased expression of autophagy-related protein LC3. The mRNA expressions of downstream genes of mTOR pathway, 4EBP1 and p70S6K, were down-regulated in rapamycin-treated fibroblasts, while the expressions of autophagy-related miRNAs, including miR-885-3p, miR-204, miR- 101, miR-376b and lncRNA FLJll812 were enhanced, and miR-30a, lncRNA HULC5 was decreased in rapamycin-treated fibroblasts （P 〈 0. 05 ）. Conclusions Rapamycin could inhibit the proliferation of keloid fibroblasts, and could not affect the apoptosis of cells. However, rapamycin induced the autophagy of
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2016年第3期208-214,共7页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金（30871433,81171817）
