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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 investigate the effects of caveolae on the proliferation and TGF-β / Smad signaling pathway of alveolar type Ⅱ epithelial cells (AECⅡ) induced by methyl β-cyclodextrin (MβCD). Methods AECⅡ was isolated and cultured in vitro. The expression and distribution of caveolin-1 and TGFβ receptor Ⅰ (TβR-Ⅰ) were detected by double immunofluorescence staining. Caveat was destroyed by MβCD (5 mmol / L), and blank control group was established. The distribution of TβR-Ⅰ and Caveolin-1 on the membrane was detected by lipofluorescence with non-ionic detergent method. Western blot was used to detect the expression of Caveolin-1 and phosphorylation Smad2 (pSmad2) expression; MTT assay of cell proliferation. Results The results of fluorescence double labeling and lipid raft extraction indicated that TβR-Ⅰ mainly distributed in Caveolae region. After MβCD destroyed Caveolae, TβR-Ⅰ redistributed in non-lipid rafts of cell membrane; Caveolin-1 protein expression in MβCD interference group (24.53 ± 3.24) (P <0.01). The expression of pSmad2 protein in the downstream of TGF-β / Smad signaling pathway (10.93 ± 1.11)% was significantly higher than that of the control group (P <0.05) <0.05). The proliferation rate of MβCD group (31.00 ± 4.18)% was significantly lower than that of control group (49.20 ± 4.44)% (P <0.01). Conclusion MβCD can down-regulate Caveolae expression and inhibit the proliferation of AECⅡ. The mechanism may be related to the accumulation of TβR-Ⅰ in non-lipid raft region and the increase of TGF-β / Smad signaling pathway induced by down-regulation of Caveolin-1.