雷帕霉素对人角质形成细胞系HaCaT细胞自噬诱导效应及其分子机制的初步研究

Research on rapamycin-induced autophagy and its mechanisms in HaCaT cells

目的:初步探讨雷帕霉素在不同浓度和不同孵育时间下,对人角质形成细胞系HaCaT细胞自噬的诱导效应及其分子机制。方法:不同浓度雷帕霉素处理HaCaT细胞4 h或12 h后,利用四甲基偶氮唑盐(MTT)方法检测雷帕霉素对细胞活力的影响,并提取蛋白进行蛋白印迹实验,检测LC3-Ⅱ表达水平,作为自噬的检测方法;通过透射电镜分析自噬体形成,确认雷帕霉素对自噬的诱导效果。检测自噬调控上游关键元件哺乳动物雷帕霉素靶蛋白(mTOR)蛋白表达及其se^(2481)和ser^(2448)磷酸化产物水平,分析mTOR活性;并分析自噬调控下游的另一关键基因Atg7的表达,初步分析雷帕霉素诱导HaCaT细胞的分子机制。结果:不同浓度雷帕霉素处理4 h,对HaCaT细胞的活力没有显著的影响。高剂量雷帕霉素(80 nmol/L和100 nmol/L)在处理12 h后对HaCaT细胞的活力产生毒性效应。20 nmol/L和50 nmol/L雷帕霉素孵育4 h诱导HaCaT细胞LC3-Ⅱ表达上调,而20 nmol/L和50 nmol/L雷帕霉素孵育12 h仍具有诱导效应。相较于其他浓度,在50 nmol/L雷帕霉素孵育诱导的LC3-Ⅱ表达上调效应最为显著,并且在药物孵育4h时超微结构水平诱导产生胞质内大量自噬体形成。1~50nmol/L浓度的雷帕霉素孵育4 h或12 h均可诱导HaCaT细胞mTOR ser^(2481)和mTOR ser^(2448)磷酸化产物水平降低:然而Atg7表达不能观测到显著的差异。结论:50 nmol/L雷帕霉素孵育4 h具有显著的诱导HaCaT细胞自噬效应。HaCaT细胞mTOR分子对雷帕霉素处理敏感,可被其诱导产生蛋白活化抑制效应。这种mTOR信号抑制可能参与了雷帕霉素对HaCaT细胞的自噬诱导效应,但Atg7没有参与这种调控效应。

Objectives： The aims of this study were to investigate the effects of rapamycin on autophagy in human ker- atinocyte cell line, HaCaT cells, and its molecular mechanisms. Methods： HaCaT cells were treated with different concentrations of rapamycin for 4or 12hours. MTr assay was used to detect the cell viability. Total protein from cell lysis was subjected to western- blotting to determine the expression levels of LC3- Ⅱ, a marker of autophagy process. Transmission electron microscopy was used to evaluate the formation of autophagosomes and to validate the induction of autphagy. The expression levels of phosphorylated mTOR at ser2448 and ser2481 sites, the up-stream of autophagy process, were analyzed to determine the activation of mTOK In addition, Atg7, a key regulatory gene of down-steam for autophagy process, was examined. These preliminary results were used to determine the potential mechanisms whereby rapamycin induces autophagy in HaCaT cells. Results： Incubation with different doses of ra- pamycin for 4hours did not affect the viability of HaCaT cells. However, incubation with 80 and 100nmol/L rapamycin for 12 hours showed toxic effects Expression levels of LC3-Ⅱ in HaCaT cells were up-regulated by 20 and 50 nmol/L rapamycirr Moreover, up- regulation of LC3-Ⅱ expression was observed at 12 hours after incubation with either 20 or 50 nmoFL rapamycim Compared with other doses, 50 nmol/L rapamycin was most effective in up-regulation of LC3-Ⅱ expression in HaCaT cells. Additionally, numerous autophagosomes were observed within HaCaT cells after incubation with 50nmol/L rapamycin for 4hours. Furthermore, the levels of phosphorylated roTOR at ser2448 and ser2481 sites were significantly down-regulated in HaCaT cells treated with 1 to 50nmol/L ra- pamycin for either 4 or 12hours, whereas changes in Atg7 expression were not significant. Conclusion： Treatment with 50nmol/L ra- pamycin for 4houm is most effective in the induction of autophagy in HaCaT cells. The mTOR was sensitive to rapamycin treatment. Inactivation of mTOR signaling, but not Atg7, is involved in rapamycin-induced autophagy in HaCaT cells.