重症监测治疗与研究“中青年优秀论文评奖” 机械牵张通过丝裂原活化蛋白激酶激酶6 诱导肺泡上皮细胞表达高迁移率族蛋白B1.docVIP

重症监测治疗与研究 “中青年优秀论文评奖” 机械牵张通过丝裂原活化蛋白激酶激酶6诱导肺泡上皮细胞表达高迁移率族蛋白B1 丁宁1，肖慧2，许立新1，佘守章1广州市第一人民医院麻醉科6 (mitogen-activated protein kinase kinase 6, MKK6)在机械牵张诱导肺泡上皮细胞(A549)表达高迁移率族蛋白B1 (high mobility group box 1 protein, HMGB1) 中的作用。方法 A549细胞分为空载体MKK6(E)细胞组和转染MKK6的显性无活性突变体MKK6(A)细胞组，各组细胞又分为施加牵张应变(20%)组和未施加牵张应变组。应用荧光显微镜和Western blot方法观察转染基因的表达情况；用激酶活性测定法检测细胞内MKK6的激酶活性；用Western blot和RT-PCR检测各组细胞HMGB1蛋白和mRNA的表达。结果　机械牵张可明显诱导A549细胞MKK6激活及HMGB1表达增加，MKK6活性突变体也可明显诱导A549细胞HMGB1表达增加，而MKK6无活性突变体则明显抑制机械牵张诱导HMGB1的表达。结论 机械牵张通过MKK6诱导了A549细胞表达HMGB1。 [关键词] 机械应力；腺病毒；丝裂原活化蛋白激酶激酶6；高迁移率族蛋白B1 The mechanical stretch induced high mobility group box 1 expression in A549 cells is mediated by MAP kinase kinase 6 DING Ning1, XIAO Hui2, XU Li-Xin1*, SHE Shou-Zhang1 Department of 1Anesthesiology and 2Out-patient, Guangzhou First Municipal Peoples Hospital, Guangzhou 510180, China [Abstract] Objective To investigate the role of mitogen-activated protein kinase kinase 6 (MKK6) in mechanical stretch induced high mobility group box 1 protein (HMGB1) expression in alveolar epithelial cells (A549). Methods A549 cells were cultured and divided into five groups: control group, adenovirus vector ad.RSV transfection group, recombinant adenovirus of MKK6 transfection group, recombinant adenovirus of constitutively active form of MKK6 [MKK6(E)] transfection group and recombinant adenovirus of dominant negative form of MKK6 [MKK6(A)] transfection group. The cells were exposed to cyclic mechanical stretch at 20% elongation using Flexercell 4000T cell stretching unit. Unstretched cells served as control. The infection and expression of gene were tested under fluorescent microscope examination and Western blot analysis. The activity of MKK6 in A549 cells was detected by protein kinase assay. The expression of HMGB1 protein and mRNA was detected by Western blot analysis and RT-PCR. Results Mechanical stretch significantly increased the activity of MKK6 and the expression of HMGB1 protein and mRNA in A549 cell