摘要
目的观察甲基-β-环糊精(methyl-β-cyclodextrin,MβCD)破坏小窝(caveolae)对肺泡Ⅱ型上皮细胞(AECⅡ)增殖和TGF-β/Smad信号通路的影响。方法分离培养大鼠AECⅡ,免疫荧光双标检测小窝蛋白-1(caveolin-1)和TGF-β受体Ⅰ(TβR-Ⅰ)表达和分布关系。以MβCD(5 mmol/L)破坏AECⅡ细胞膜Caveolae,设空白对照组,非离子去污剂法提取脂筏检测TβR-Ⅰ与Caveolin-1在细胞膜上的分布;Western blot检测Caveolin-1和磷酸化Smad2(pSmad2)表达;四甲基偶氮唑盐(MTT)法检测细胞增殖能力。结果荧光双标和脂筏提取结果提示TβR-Ⅰ主要分布于Caveolae区域,MβCD破坏Caveolae后,TβR-Ⅰ重新分布于细胞膜上非脂筏区域;MβCD干扰组Caveolin-1蛋白表达(24.53±3.24)%较正常对照组(54.83±5.67)%显著下调(P<0.01),而TGF-β/Smad信号通路下游分子pSmad2蛋白表达(10.93±1.11)%较对照组(8.36±0.64)%上调(P<0.05);MβCD干扰组细胞增殖率(31.00±4.18)%较对照组(49.20±4.44)%显著降低(P<0.01)。结论 MβCD破坏Caveolae结构可抑制AECⅡ增殖,其机制可能与TβR-Ⅰ在非脂筏区域的聚集和Caveolin-1下调导致的TGF-β/Smad信号通路增强有关。
Objective To study the influences of methyl-β-cyclodextrin（MβCD）-caused caveolae destruction on proliferation of type Ⅱ alveolar epithelial cells（AECs Ⅱ） and on TGF-β/Smad signaling pathway in AECs Ⅱ.Methods Rat AECs Ⅱ were isolated through enzyme digestion,and then identified through immunofluorescence assay.The distribution of caveolin-1（a caveolae-specific protein） and type I TGF-β receptor（TβR-Ⅰ） in AECs Ⅱ cell membranes was analyzed with double-labeling immunofluorescence assay and confocal laser scanning microscopy.AECs Ⅱ were divided into a treatment group and a control group.MβCD（5 mmol/L in DMEM） was added into the treatment group to destroy caveolae of AECs Ⅱ,while DMEM was added into the control group.Lipid rafts were extracted from AECs Ⅱ by nonionic detergent method,and the distribution of caveolin-1 and TβR-Ⅰ in cell membranes of treated AECs Ⅱ was analyzed through SDS-PAGE.The expression of caveolin-1 and phosphorylated Smad2（pSmad2,a downstream molecule of TGF-β/Smad signaling pathway） in AECs Ⅱ was analyzed through Western blotting.The proliferation rate of AECs Ⅱ was analyzed through methyl thiazolyl tetrazolium method.Results The double-labeling immunofluorescence assay and lipid raft extraction showed that TβR-Ⅰ was mainly distributed in caveolae of cell membrane and,after MβCD treatment,was re-distributed in non-raft domains.The expression of caveolin-1 in AECs Ⅱ of the treatment group was significantly lower than that of the control group [（24.53±3.24）% vs（54.83±5.67）%,P0.01].The expression of pSmad2 in AECs Ⅱ of the treatment group was significantly higher than that of the control group [（10.93±1.11）% vs（8.36±0.64）%,P0.05].The proliferation rate of AECs Ⅱ of the treatment group is significantly lower than that of the control group（31.00±4.18）% vs（49.20±4.44）%,P0.01）.Conclusion MβCD-caused caveolae destruction can inhibit the proliferation of AECs Ⅱ,probably through a mechanism of enhancing TGF-β/Smad signaling pathway with TβR-Ⅰ accumulation in non-raft domains and caveolin-1 downregulation.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第8期780-784,共5页
Journal of Third Military Medical University
基金
国家自然科学基金(30700346)~~
