Hippo通路对小鼠骨髓间充质干细胞分化增殖及迁移的调控作用

Effects of Hippo pathway on differentiation, proliferation, migration of bone marrow mesenchymal stem cells in vitro

目的探讨Hippo通路对小鼠骨髓间充质干细胞(BMSCs)向Ⅱ型肺泡上皮细胞(AECⅡ)分化、增殖、迁移能力的调控效应.方法采用直接贴壁法分离培养C57BL/6小鼠原代BMSCs,取第6~7代细胞,通过流式细胞仪检测和诱导其成骨、成软骨、成脂分化并进行鉴定,通过非接触共培养诱导BMSCs向AECⅡ细胞分化.通过2-脱氧-D-葡萄糖(2-DG)及哺乳动物不育系20样激酶1(MST1)抑制剂9E1调节Hippo通路,用实时荧光定量聚合酶链反应(qRT-PCR)和蛋白质免疫印迹试验(Western Blot)检测2-DG及9E1对BMSCs表面活性蛋白(SPB、SPC和SPD)mRNA和表面活性蛋白C前体(pro-SPC)蛋白表达的影响,以评估Hippo通路对BMSCs向AECⅡ细胞分化的作用;用四甲基偶氮唑盐比色法(MTT)检测BMSCs的数量变化(用0.1、0.5、1.0、5.0 mmol/L的2-DG干预),以评估Hippo通路对BMSCs增殖能力的影响;用划痕实验及Transwell小室迁移实验评估Hippo通路对BMSCs水平迁移及向急性呼吸窘迫综合征(ARDS)肺组织归巢能力的影响.结果2-DG呈浓度依赖性激活Hippo通路并促进BMSCs向AECⅡ细胞分化及增殖,其效应在2-DG浓度5 mmol/L时最为明显〔SPB mRNA(2^-ΔΔCT):2.42±0.28比1.89±0.11,SPC mRNA(2^-ΔΔCT):8.06±0.68比6.59±0.79,SPD mRNA(2^-ΔΔCT):6.45±0.37比5.27±0.28,pro-SPC/β-actin:5.80±1.86比4.93±1.18,细胞增殖:(145.46±18.18)%比(98.91±4.36)%,均P<0.05〕;9E1可抑制BMSCs向AECⅡ细胞分化及增殖〔SPB mRNA(2^-ΔΔCT):1.32±0.17比1.89±0.11,SPC mRNA(2^-ΔΔCT):3.91±0.34比6.59±0.79,SPD mRNA(2^-ΔΔCT):3.38±0.25比5.27±0.28,pro-SPC/β-actin:2.48±0.17比4.93±1.18,细胞增殖:(80.00±7.27)%比(98.91±4.36)%,均P<0.05〕.2-DG活化Hippo通路能促进BMSCs的水平迁移〔划痕愈合程度:(27.17±3.53)%比(52.45±6.52)%,P<0.05〕及向ARDS肺组织归巢〔迁移细胞数(倍,相对于对照):2.77±0.21比1.90±0.19,P<0.05〕,而9E1抑制Hippo通路能逆转上述效应〔划痕愈合程度:(79.89±8.42)%比(52.45±6.52)%,迁移细胞数(倍,相对于对照):1.69±0.13比1.90±0.19,均P<0.05〕.结论活化Hippo通路能促进BMSCs向AECⅡ细胞分化、增殖、水平迁移,并增强BMSCs向ARDS肺组织归巢的能力.

Objective To explore the effects of Hippo pathway on differentiation, proliferation, and migration of bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods 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 typeⅡalveolar epithelial cells (AECⅡ) 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 AECⅡ 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. Results 2-DG could activate Hippo pathway in a dose-dependent manner and promote the differentiation to AECⅡ and proliferation of BMSCs, the maximum effects were observed after 5 mmol/L of 2-DG treatment [SPB mRNA (2^-ΔΔCT): 2.42±0.28 vs. 1.89±0.11, SPC mRNA (2^-ΔΔCT): 8.06±0.68 vs. 6.59±0.79, SPD mRNA (2^-ΔΔCT): 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^-ΔΔCT): 1.32±0.17 vs. 1.89±0.11, SPC mRNA (2^-ΔΔCT): 3.91±0.34 vs. 6.59±0.79, SPD mRNA (2^-ΔΔCT): 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]. Conclusion Activation of Hippo pathway could enhance differentiation of BMSCs to AECⅡ, promote proliferation and ability of horizontal migration and homing BMSCs to conditioned medium of ARDS lung tissue in vitro.