The Effects And Mechanism Of Rapamycin On Keloid Fibroblastic Cells

In fibrotic diseases, the abnormal activation mTOR (mammalian target of rapamycin) signaling pathway not only involved in the regulation of the proliferation, survival, cell cycle progression of keloid fibroblastic cells and many other cellular functions, but also may be associated with the resistance of keloids on drugs. Previous studies have demonstrated that key molecules of mTOR signaling pathway can be taken as targets for the treatment of fibrotic diseases, therefore, as the targeted inhibitor of mTOR, rapamycin has broad prospects for the treatment of fibrotic diseases.Therefore, taking keloid fibroblastic cells as experimental subjects in this study, to explore the effects of the mTOR inhibitor rapamycin on keloid fibroblastic cells and its epigenetic mechanism, providing experimental basis for the clinical treatment application of rapamycin in keloids.Objective:1. To confirm the inhibitory role and mechanism of the mTOR inhibitor rapamycin on the proliferation of keloid fibroblastic cells. To confirm the effects of rapamycin on the expressions of collagen genes.2. To initially discuss rapamycin promoting the autophagy of keloid fibroblastic cells, and further to provide experimental evidence for clarifying the feasibility of the association treatment of rapamycin. To explore the effects of rapamycin on the mTOR signal transduction pathways of keloid fibroblastic cells, as well as its regulation mechanisms on cell cycle.3. To confirm the effects of rapamycin on autophagy-related non-coding RNAs in keloid fibroblastic cells, providing a theoretical basis for further deeply exploring the role of non-coding RNAs in the regulation of cell autophagy process.Methods:MTT assay was used to test the cell pro life ration.The apoptosis of cells were tested by DNA agarose gel electrophoresis and flow cytometry. Flow cytometry was used to test the effects of rapamycin on cell cycle.The formation of autophagy was observed by TEM, and 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 mTOR pathway and autophagy-related non-coding RNAs.Results:1. Rapamycin could directly inhibit the growth of keloid fibroblastic cells in dose-and time-dependent manners. Rapamycin did not induce the apoptosis of cells, but could block the cell cycle of keloid fibroblastic cells in G1 phase and down-regulated the expression of c-Myc mRNA, and up-regulated the expression of P27 mRNA. Rapamycin could inhibit the expression of collagen genes, and down-regulate the expression levels of mRNA.2. Rapamycin could induce autophagy, accompanied by the increased expression of autophagy-related protein LC3, and changes of cell ultra-structures such as the swelling mitochondria, the double-layer membrane structure of autophagosomes and chromatin aggregation could be observed under the TEM. Rapamycin had inhibitory effects on the expression of downstream genes 4EBP1 and p70S6K of mTOR pathway.3. Rapamycin could cause changes of autophagy-related non-coding RNAs expression, providing experimental evidence for clinical practice.Conclusion:The inhibitory effects of rapamycin on the growth of keloid fibroblastic cells, as well as its regulation mechanisms on cell cycle and the expressions of collagen genes; to explore the effects of rapamycin on the mTOR signal transduction pathways of keloid fibroblastic cells; The effects of rapamycin on autophagy-related non-coding RNAs in keloid fibroblastic cells.