目的 研究高钙离子浓度通过Janus激酶(Janus kinase，JAK)2/信号转导和转录激活因子(signal transducer and activator of transcription，STAT)3通路调控人腹膜间皮细胞上皮间质转化(epithelial-mesenchymal transition，EMT)的作用及机制。 方法 培养人腹膜间皮细胞HMrSV5，分为对照组(钙离子浓度1.25 mmol/L)、1.75 mmol/L钙离子组、2.25 mmol/L钙离子组、溶剂对照组(钙离子浓度1.25 mmol/L、二甲亚砜体积分数0.1%)、2.25 mmol/L钙离子+溶剂组(钙离子浓度2.25 mmol/L、二甲亚砜体积分数0.1%)、2.25 mmol/L钙离子+S3I-201组(钙离子浓度2.25 mmol/L、STAT3抑制剂S3I-201浓度100 μmol/L)、2.25 mmol/L钙离子+Stattic组(钙离子浓度2.25 mmol/L、STAT3抑制剂Stattic浓度1.5 μmol/L)。处理48h后检测侵袭数目，α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)、E-钙粘蛋白(E-cadherin)的mRNA和蛋白表达水平，JAK1、JAK2、STAT3、STAT6的磷酸化水平。 结果 1.75 mmol/L钙离子组、2.25 mmol/L钙离子组的细胞侵袭数目(t=6.901、10.870，均P＜0.001)、α-SMA的mRNA(t=5.375、9.564，均P＜0.001)和蛋白表达水平(t=7.018、10.859，均P＜0.001)、JAK2(t=7.031、11.001，均P＜0.001)和STAT3(t=5.425、9.846，P值分别为0.001、＜0.001)的磷酸化水平均高于对照组，E-cadherin的mRNA(t=4.457、10.749，P值分别为0.002、＜0.001)和蛋白表达水平(t=6.519、11.351，均P＜0.001)低于对照组；1.75 mmol/L钙离子组、2.25 mmol/L钙离子组和对照组的JAK1和STAT6的磷酸化水平比较无统计学差异(F=0.423、0.311，P=0.619、0.674)。2.25 mmol/L钙离子+S3I-201组和2.25 mmol/L钙离子+Stattic组的细胞侵袭数目(t=7.259、6.995，均P＜0.001)、α-SMA的mRNA(t=5.230、5.851，P值分别为0.001、＜0.001)和蛋白表达水平(t=7.684、7.833，均P＜0.001)、STAT3的磷酸化水平(t=6.753、8.574，均P＜0.001)均低于2.25 mmol/L钙离子+溶剂组，E-cadherin的mRNA(t=7.500、8.173，均P＜0.001)和蛋白表达水平(t=13.021、9.340，均P＜0.001)高于2.25 mmol/L钙离子+溶剂组；JAK2的磷酸化水平与2.25 mmol/L钙离子+溶剂组比较无差异(t=0.754、1.091，P=0.473、0.307)。 结论 高钙离子浓度通过促进STAT3通路磷酸化激活的方式促进人腹膜间皮细胞EMT。
Objective To investigate the role and mechanism of high calcium ion concentration in regulating epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Methods Human peritoneal mesothelial cells HMrSV5 were cultured and divided into the following groups: control group (Ca2+ 1.25 mmol/L), 1.75 mmol/L Ca2+ group, 2.25 mmol/L Ca2+ group, solvent control group (Ca2+ 1.25 mmol/L, 0.1% dimethyl sulfoxide), 2.25 mmol/L Ca2+ + solvent group (Ca2+ 2.25 mmol/L, 0.1% dimethyl sulfoxide), 2.25 mmol/L Ca2+ + S3I-201 (STAT3 inhibitor) group (Ca2+ 2.25 mmol/L, S3I-201 100 μmol/L), and 2.25 mmol/L Ca2+ + Stattic (STAT3 inhibitor) group (Ca2+ 2.25 mmol/L, Stattic 1.5 μmol/L). After 48 hours of treatment, number of invasive cells, mRNA and protein expression levels of α-smooth muscle actin (α-SMA) and E-cadherin, and phosphorylation levels of JAK1, JAK2, STAT3 and STAT6 were measured. Results In 1.75 mmol/L Ca2+ group and 2.25 mmol/L Ca2+ group, number of invasive cells (t=6.901 and 10.870, P＜0.001), α-SMA mRNA (t=5.375 and 9.564, P＜0.001), α-SMA protein (t=7.018 and 10.859, P＜0.001), phosphorylation of JAK2 (t=7.031 and 11.001, P＜0.001) and phosphorylation of STAT3 (t=5.425 and 9.846, P=0.001 and ＜0.001) were significantly higher than those in control group; E-cadherin mRNA (t=4.457 and 10.749, P=0.002 and ＜0.001) and E-cadherin protein (t=6.519 and 11.351, P＜0.001) were lower than those in control group; while phosphorylation levels of JAK1 and STAT6 (F=0.423 and 0.311, P=0.619 and 0.674) had no differences as compared with those of control group. In 2.25 mmol/L Ca2+ + S3I-201 group and 2.25 mmol/L Ca2++ Stattic group, number of invasive cells (t=7.259 and 6.995, P＜0.001), α-SMA mRNA (t=5.230 and 5.851, P=0.001 and ＜0.001), α-SMA protein (t=7.684 and 7.833, P＜0.001), and phosphorylation of STAT3 (t=6.753 and 8.574, P＜0.001) were lower than those of 2.25 mmol/L Ca2+ + solvent group; E-cadherin mRNA (t=7.500 and 8.173, P＜0.001) and E-cadherin protein (t=13.021 and 9.340, P＜0.001) were higher than those of 2.25 mmol/L Ca2+ + solvent group; while phosphorylation of JAK2 (t=0.754 and 1.091, P=0.473 and 0.307) had no difference as compared with that of 2.25 mmol/L Ca2+ + solvent group. Conclusion High Ca2+ concentration activates the signaling pathway via phosphorylation of STAT3 to promote EMT of human peritoneal mesothelial cells.
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