中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2013年
11期
815-819
,共5页
陈旭%郭新云%徐松%张孟丽%金慧%邢美春%黄丹%任发亮%杜开和
陳旭%郭新雲%徐鬆%張孟麗%金慧%邢美春%黃丹%任髮亮%杜開和
진욱%곽신운%서송%장맹려%금혜%형미춘%황단%임발량%두개화
角蛋白细胞%自噬%细胞,培养的
角蛋白細胞%自噬%細胞,培養的
각단백세포%자서%세포,배양적
Keratinocytes%Autophagy%Cells,cultured
目的 研究血清饥饿处理对人角质形成细胞系HaCaT细胞自噬的调控效应,初步分析其分子机制.方法 采用血清饥饿处理体外培养的HaCaT细胞诱导自噬,分析血清饥饿对细胞形态和细胞活力(MTT法)的影响.透射电镜观察白噬体囊泡形成,使用Western印迹法进行白噬标志性分子LC3-Ⅰ→LC3Ⅱ转化分析和自噬关键基因Atg7的表达以及MDC染色标记自噬体囊泡.对mTOR的蛋白表达及其ser2448和ser2481位点磷酸化产物水平进行分析.结果 血清饥饿HaCaT细胞活力上升,电镜观察和MDC染色发现,血清饥饿处理的HaCaT细胞中有自噬体囊泡形成.Western印迹显示,血清饥饿细胞LC3-Ⅰ→LC3Ⅱ转化(LC3-Ⅱ/LC3-Ⅰ比率为3.508±0.415)较正常培养的细胞(0.538±0.038)显著上调(两组比较,t=13.32,P＜ 0.01)；白噬关键基因Atg7表达上调(对照细胞和血清饥饿细胞Atg7/GAPDH分别为0.021±0.006和0.048±0.011,f=7.27,P＜ 0.05).血清饥饿处理的细胞中,mTOR的ser2448位点、ser2481位点磷酸化产物水平显著降低(对照细胞和血清饥饿细胞间的磷酸化mTORser2448/mTOR比率分别为0.762±0.108和0.394±0.048,t=7.58,P＜ 0.05；磷酸化mTORser248 1/mTOR的比率分别为0.263±0.039和0.111±0.020,t=13.77,P＜ 0.01).结论 血清饥饿处理可以诱导HaCaT细胞发生自噬,并且导致细胞活力上升.这种白噬的诱导可能与自噬调控关键元件mTOR蛋白活化抑制相关.
目的 研究血清饑餓處理對人角質形成細胞繫HaCaT細胞自噬的調控效應,初步分析其分子機製.方法 採用血清饑餓處理體外培養的HaCaT細胞誘導自噬,分析血清饑餓對細胞形態和細胞活力(MTT法)的影響.透射電鏡觀察白噬體囊泡形成,使用Western印跡法進行白噬標誌性分子LC3-Ⅰ→LC3Ⅱ轉化分析和自噬關鍵基因Atg7的錶達以及MDC染色標記自噬體囊泡.對mTOR的蛋白錶達及其ser2448和ser2481位點燐痠化產物水平進行分析.結果 血清饑餓HaCaT細胞活力上升,電鏡觀察和MDC染色髮現,血清饑餓處理的HaCaT細胞中有自噬體囊泡形成.Western印跡顯示,血清饑餓細胞LC3-Ⅰ→LC3Ⅱ轉化(LC3-Ⅱ/LC3-Ⅰ比率為3.508±0.415)較正常培養的細胞(0.538±0.038)顯著上調(兩組比較,t=13.32,P＜ 0.01)；白噬關鍵基因Atg7錶達上調(對照細胞和血清饑餓細胞Atg7/GAPDH分彆為0.021±0.006和0.048±0.011,f=7.27,P＜ 0.05).血清饑餓處理的細胞中,mTOR的ser2448位點、ser2481位點燐痠化產物水平顯著降低(對照細胞和血清饑餓細胞間的燐痠化mTORser2448/mTOR比率分彆為0.762±0.108和0.394±0.048,t=7.58,P＜ 0.05；燐痠化mTORser248 1/mTOR的比率分彆為0.263±0.039和0.111±0.020,t=13.77,P＜ 0.01).結論 血清饑餓處理可以誘導HaCaT細胞髮生自噬,併且導緻細胞活力上升.這種白噬的誘導可能與自噬調控關鍵元件mTOR蛋白活化抑製相關.
목적 연구혈청기아처리대인각질형성세포계HaCaT세포자서적조공효응,초보분석기분자궤제.방법 채용혈청기아처리체외배양적HaCaT세포유도자서,분석혈청기아대세포형태화세포활력(MTT법)적영향.투사전경관찰백서체낭포형성,사용Western인적법진행백서표지성분자LC3-Ⅰ→LC3Ⅱ전화분석화자서관건기인Atg7적표체이급MDC염색표기자서체낭포.대mTOR적단백표체급기ser2448화ser2481위점린산화산물수평진행분석.결과 혈청기아HaCaT세포활력상승,전경관찰화MDC염색발현,혈청기아처리적HaCaT세포중유자서체낭포형성.Western인적현시,혈청기아세포LC3-Ⅰ→LC3Ⅱ전화(LC3-Ⅱ/LC3-Ⅰ비솔위3.508±0.415)교정상배양적세포(0.538±0.038)현저상조(량조비교,t=13.32,P＜ 0.01)；백서관건기인Atg7표체상조(대조세포화혈청기아세포Atg7/GAPDH분별위0.021±0.006화0.048±0.011,f=7.27,P＜ 0.05).혈청기아처리적세포중,mTOR적ser2448위점、ser2481위점린산화산물수평현저강저(대조세포화혈청기아세포간적린산화mTORser2448/mTOR비솔분별위0.762±0.108화0.394±0.048,t=7.58,P＜ 0.05；린산화mTORser248 1/mTOR적비솔분별위0.263±0.039화0.111±0.020,t=13.77,P＜ 0.01).결론 혈청기아처리가이유도HaCaT세포발생자서,병차도치세포활력상승.저충백서적유도가능여자서조공관건원건mTOR단백활화억제상관.
Objective To evaluate the regulatory effect of serum starvation on autophagy in human HaCaT keratinocytes,and to investigate its molecular mechanism.Methods HaCaT cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10％ fetal calf serum (control group) or in DMEM without fetal calf serum (starvation group),for four hours.Then,cell appearance was observed by light microscopy,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell viability,transmission electron microscopy to observe ultrastructural changes,monodansylcadaverine (MDC) staining to identify the formation of autophagosomes,and Western blot to measure the expression of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ,LC3-Ⅰ and autophagy related protein 7 (ATG7).Additionally,the phosphorylation level of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 was semiquantitatively analyzed by Western blot.Results Serum starvation increased the viability of HaCaT cells.Electron microscopy and MDC staining confirmed the formation of autophagosomes in serum-starved HaCaT cells.Compared with the control cells,the serum-starved HaCaT cells showed a significant increase in the transformation of LC3-Ⅰ to LC3-Ⅱ (LC3-Ⅱ/LC3-Ⅰ ratio:3.508 ± 0.415 vs.0.538 ± 0.038,t =13.32,P ＜ 0.01),and expression of ATG7 (ATG7/GAPDH ratio:0.048 ± 0.011 vs.0.021 ± 0.006,t =7.27,P ＜ 0.05).The phosphorylation level of mTOR at ser2448 and ser2481 was significantly lower in the serum-starved HaCaT cells than in the control cells (phosphorylated mTOR (ser2448)/mTOR ratio:0.394 ± 0.048 vs.0.762 ± 0.108,t =7.58,P＜ 0.05; phosphorylated mTOR (ser2481)/mTOR ratio:0.111 ± 0.020 vs.0.263 ± 0.039,t =13.77,P ＜ 0.05).Conclusion Serum starvation can induce autophagy in and increase the viability of HaCaT cells,likely by inhibiting the activation of mTOR.
