Li Lang, Dong Liang, Chen Yizhe, Hui Jiaojie
Department of Critical Care Medicine, Taizhou Central Hospital (Taizhou University Hospital), Taizhou 318000, Zhejiang, China.
Department of Critical Care Medicine, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, Jiangsu, China. Corresponding author: Dong Liang, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2019 Jun;31(6):750-755. doi: 10.3760/cma.j.issn.2095-4352.2019.06.018.
To explore the effects of Hippo pathway on differentiation, proliferation, and migration of bone marrow mesenchymal stem cells (BMSCs) in vitro.
BMSCs of C57BL/6 mice were identified using fluorescence-activated cellsorting analysis and the capabilities of osteogenic, chondrogenic and adipogenic differentiation were evaluated. The differentiation of BMSCs to type II alveolar epithelial cells (AEC II) was induced by indirect co-culture with mouse lung epithelial cells (MLE-12) and small airway epithelial cell growth medium (SAGM). The Hippo pathway was regulated by 2-deoxy-D-glucose (2-DG) and 9E1, the effects of 2-DG and 9E1 on the expression of BMSCs surface proteins (SPB, SPC and SPD) mRNA and pro-SPC protein were detected by real time quantitative polymerase chain reaction (qRT-PCR) and Western Blot. The effect of Hippo pathway on differentiation of BMSCs to AEC II cells was evaluated. The effect of Hippo pathway on the proliferation of BMSCs was evaluated by methyl thiazolyl tetrazolium (MTT) assay (intervention of 0.1, 0.5, 1.0, 5.0 mmol/L 2-DG). The scratch test and Transwell chamber test were used to analyze the effect of Hippo pathway on migration ability of BMSCs to conditioned medium of acute respiratory distress syndrome (ARDS) lung tissue.
2-DG could activate Hippo pathway in a dose-dependent manner and promote the differentiation to AEC II and proliferation of BMSCs, the maximum effects were observed after 5 mmol/L of 2-DG treatment [SPB mRNA (2): 2.42±0.28 vs. 1.89±0.11, SPC mRNA (2): 8.06±0.68 vs. 6.59±0.79, SPD mRNA (2): 6.45±0.37 vs. 5.27±0.28, pro-SPC/β-actin: 5.80±1.86 vs. 4.93±1.18, proliferation rate: (145.46±18.18)% vs. (98.91±4.36)%, all P < 0.05], but 9E1 could reverse those effects through inhibition of Hippo pathway [SPB mRNA (2): 1.32±0.17 vs. 1.89±0.11, SPC mRNA (2): 3.91±0.34 vs. 6.59±0.79, SPD mRNA (2): 3.38±0.25 vs. 5.27±0.28, pro-SPC/β-actin: 2.48±0.17 vs. 4.93±1.18, proliferation rate: (80.00±7.27)% vs. (98.91±4.36)%, all P < 0.05]. The ability of horizontal migration [wound healing: (27.17±3.53)% vs. (52.45±6.52)%, P < 0.05] and homing BMSCs to conditioned medium of ARDS lung tissue [cell count (fold, relative to control): 2.77±0.21 vs. 1.90±0.19, P < 0.05] were increased after activation of Hippo pathway by 2-DG treatment, but those effects were reversed after inhibition of Hippo pathway by 9E1 treatment [wound healing: (79.89±8.42)% vs. (52.45±6.52)%, cell count (fold, relative to control): 1.69±0.13 vs. 1.90±0.19, both P < 0.05].
Activation of Hippo pathway could enhance differentiation of BMSCs to AEC II, promote proliferation and ability of horizontal migration and homing BMSCs to conditioned medium of ARDS lung tissue in vitro.
探讨Hippo信号通路对体外培养的骨髓间充质干细胞（BMSC）分化、增殖及迁移能力的影响。
采用荧光激活细胞分选分析鉴定C57BL/6小鼠的BMSC，并评估其成骨、成软骨及成脂分化能力。通过与小鼠肺上皮细胞（MLE-12）及小气道上皮细胞生长培养基（SAGM）间接共培养，诱导BMSC向II型肺泡上皮细胞（AEC II）分化。用2-脱氧-D-葡萄糖（2-DG）和9E1调控Hippo信号通路，采用实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测2-DG和9E1对BMSC表面蛋白（SPB、SPC和SPD）mRNA及前体SPC蛋白表达的影响，评估Hippo信号通路对BMSC向AEC II细胞分化的作用。采用甲基噻唑基四氮唑（MTT）法（分别用0.1、0.5、1.0、5.0 mmol/L 2-DG干预）评估Hippo信号通路对BMSC增殖的影响。采用划痕试验和Transwell小室试验分析Hippo信号通路对BMSC向急性呼吸窘迫综合征（ARDS）肺组织条件培养基迁移能力的影响。
2-DG可呈剂量依赖性激活Hippo信号通路，促进BMSC向AEC II分化及增殖，2-DG 5 mmol/L处理后作用最明显[SPB mRNA（2）：2.42±0.28比1.89±0.11，SPC mRNA（2）：8.06±0.68比6.59±0.79，SPD mRNA（2）：6.45±0.37比5.27±0.28，前体SPC/β-肌动蛋白：5.80±1.86比4.93±1.18，增殖率：（145.46±18.18）%比（98.91±4.36）%，均P<0.05]，而9E1可通过抑制Hippo信号通路逆转上述作用[SPB mRNA（2）：1.32±0.17比1.89±0.11，SPC mRNA（2）：3.91±0.34比6.59±0.79，SPD mRNA（2）：3.38±0.25比5.27±0.28，前体SPC/β-肌动蛋白：2.48±0.17比4.93±1.18，增殖率：（80.00±7.27）%比（98.91±4.36）%，均P<0.05]。2-DG处理激活Hippo信号通路后，BMSC的水平迁移能力[伤口愈合：（27.17±3.53）%比（52.45±6.52）%，P<0.05]及向ARDS肺组织条件培养基归巢能力[细胞计数（倍数，相对于对照）：2.77±0.21比1.90±0.19，P<0.05]增强，但9E1处理抑制Hippo信号通路后上述作用逆转[伤口愈合：（79.89±8.42）%比（52.45±6.52）%，细胞计数（倍数，相对于对照）：1.69±0.13比1.90±0.19，均P<0.05]。
激活Hippo信号通路可增强体外培养的BMSC向AEC II的分化能力，促进其增殖及水平迁移能力，并增强其向ARDS肺组织条件培养基的归巢能力。
