敲除Brg1基因对小鼠肺泡Ⅱ型上皮细胞数量和功能的影响及其机制研究

Effect of Brahma-related gene 1 knockout on number and function of typeⅡalveolar epithelial cells in mice

目的研究染色质重构复合物核心催化亚基(Brahma-related gene 1,Brg1)对C57BL/6小鼠Ⅱ型肺泡上皮细胞数量和功能的影响及其可能的机制。方法取6~8周龄雌性野生型C57BL/6小鼠(Wild type,WT)及Ⅱ型肺泡上皮细胞(alveolar epithelial cell Ⅱ,AECⅡs)上特异性敲除Brg1的C57BL/6小鼠(Brg1fl/fl)作为实验对象,每组选取8只小鼠,收取标本,利用免疫组化及免疫荧光分析肺组织中表面活性物质相关蛋白C(surfactant-associated protein C,SFTPC)阳性的细胞率,流式细胞术分析小鼠肺组织中表面活性物质蛋白C前体蛋白(prosurfactant protein C,pro SP-C)阳性的细胞(即AECⅡs)数量。Western blot检测各组小鼠肺组织中pro SP-C、SFTPC、表面活性物质相关蛋白A(surfactant-associated protein A,SFTPA)、表面活性物质相关蛋白D(surfactant-associated protein D,SFTPD)的表达水平。Western blot检测细胞周期蛋白D1(CyclinD1)表达及增殖相关信号转导途径中关键蛋白EGFR、p-EGFR、PI3K、p-PI3K、AKT、p-AKT及NF-κB p65的活化水平。结果抗SFTPC免疫组化、免疫荧光显示其在肺泡上皮表达较WT小鼠增多(P<0.05);流式细胞术显示Brg1fl/fl组小鼠肺组织中pro SP-C+的细胞百分比较WT小鼠明显增高(P<0.05),Western blot检测结果显示AECⅡs的功能性蛋白pro SP-C、SFTPC、SFTPA及SFTPD在Brg1^fl/fl小鼠中明显增多;Brg1^fl/fl组小鼠肺组织中NF-κB p65及周期蛋白CyclinD1的表达增加,且p-EGFR/EGFR、p-PI3K/PI3K、p-AKT/AKT百分比显著提高,且差异均有统计学意义(P<0.05)。结论特异性敲除C57BL/6小鼠Ⅱ型肺泡上皮细胞中的Brg1可促使AECⅡs数量和分泌功能的增加,其可能机制与敲除Brg1促进EGFR/PI3K/AKT/NF-κB信号通路的激活,且与增殖周期蛋白CyclinD1表达的增加有关。

Objective To investigate the effect of brahma-related gene 1(Brg1)knockout on the number and function of type Ⅱ alveolar epithelial cells(AECⅡs)in C57BL/6 mice and explore the possible mechanisms.Methods Eight female wild-type(WT)C57BL/6 mice(6 to 8 weeks old)and 8 C57BL/6 mice with Brg1 gene knockout in type Ⅱ alveolar epithelial cells(Brg1fl/fl mice)were examined with immunohistochemistry and immunofluorescence assay for the number of surfactant-associated protein C(SFTPC)-positive cells in the pulmonary alveoli.The percentage of prosurfactant protein C(pro SP-C)positive cells in the lung tissues of the mice was analyzed with flow cytometry.Western blotting was used to detect the expression levels of pro SP-C,SFTPC,SFTPA and SFTPD in the lung tissues.The expression levels of cyclin D1 and the key proteins EGFR,p-EGFR,PI3K,p-PI3K,AKT,p-AKT,and NF-κB p65 in the proliferation-related signaling pathway were also detected with Western blotting.Results The results of immunohistochemistry and immunofluorescence assay revealed a significantly greater number of SFTPC-positive cells in Brg1^fl/fl mice than in the WT mice(P<0.05).Flow cytometry showed that the percentage of pro SP-C positive cells increased significantly in the lung tissues of Brg1fl/fl mice(P<0.05).The expression levels of the functional proteins in AECⅡs,including proSP-C,SFTPC,SFTPA and SFTPD,were all significantly increased in Brg1fl/fl mice(P<0.05);Brg1 knockout significantly promoted the expression of NF-κB p65 and CyclinD1(P<0.05)and increased the ratios of p-EGFR/EGFR,p-PI3K/PI3K and p-AKT/AKT in the lung tissue of the mice(P<0.05).Conclusion Specific knockout of Brg1 in AECⅡs of C57BL/6 mice increases the number and enhances the functionality of AECⅡs possibly due to the activation of EGFR/PI3K/AKT/NF-κB signaling pathways and an increased protein expression of CyclinD1.