摘要目的 探讨通过大肿瘤抑制因子2(LATS2)介导Hippo信号途径对小鼠骨髓间充质干细胞(BMSCs)生物学行为的调控效应.方法 体外培养C57BL/6小鼠BMSCs,传至第6～7代用于实验.采用慢病毒载体转染分别构建高表达或低表达LATS2的BMSCs模型;根据不同转染试剂设置空白对照组(MSC组)、空载体对照组(MSC-eGFP组)、高表达LATS2组(MSC-LATS2组)、空干扰病毒转染组(MSC-shcontrol组)和干扰LATS2病毒转染组(MSC-shLATS2组).采用流式细胞仪检测慢病毒转染效率;采用反转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹试验(Western Blot)检测LATS2及其下游共转录因子磷酸化Yes相关蛋白(p-YAP)、14-3-3的mRNA与蛋白表达;采用茜素红、油红O染色观察BMSCs成骨、成脂分化情况;采用CCK-8细胞增殖检测试剂盒检测BMSCs数量;采用划痕实验及Transwell迁移实验评估Hippo信号途径对BMSCs水平和垂直迁移能力的影响.结果 慢病毒载体转染效率高达93.1%～97.1%.MSC-LATS2组LATS2、p-YAP、14-3-3表达均较MSC-eGFP组显著增高〔LATS2 mRNA(2-ΔΔCT):2.55±0.13比1.08±0.05,LATS2/GAPDH :2.63±0.11比1.06±0.08,p-YAP/总YAP :1.67±0.11比1.00±0.04,14-3-3/β-actin :2.22±0.20比0.98±0.06,均P＜0.05〕,MSC-shLATS2组均较MSC-shcontrol组显著降低〔LATS2 mRNA(2-ΔΔCT):0.10±0.01比1.01±0.05, LATS2/GAPDH :0.09±0.01比1.05±0.06,p-YAP/总YAP :0.10±0.02比1.10±0.09,14-3-3/β-actin :0.05±0.01比0.90±0.08,均P＜0.05〕,说明高表达及低表达LATS2可以活化或抑制Hippo信号途径.光镜下观察显示, MSC-LATS2组诱导成骨、成脂分化后,钙质沉积及脂质颗粒沉积较MSC-eGFP组显著减少,MSC-shLATS2组均较MSC-shcontrol组显著增加,说明高表达及低表达LATS2可抑制或促进BMSCs成骨、成脂分化.MSC-LATS2组BMSCs增殖率较MSC-eGFP组显著下降,MSC-shLATS2组BMSCs增殖率较MSC-shcontrol组显著增高,说明高表达及低表达LATS2可抑制或促进BMSCs增殖.划痕实验和Transwell迁移实验显示,MSC-LATS2组划痕弥合程度较MSC-eGFP组显著减小〔(22.11±3.02)%比(45.99±6.58)%〕,迁移至Transwell小室膜下层细胞数显著减少(个/中倍视野:20.82±3.05比111.33±13.28,均P＜0.05);MSC-shLATS2组划痕弥合程度较MSC-shcontrol组显著增加〔(70.32±7.17)%比(39.28±2.98)%〕,而迁移至Transwell小室膜下层细胞数显著增加(个/中倍视野:206.19±30.58比120.10±25.10,均P＜0.05),说明高表达及低表达LATS2可抑制或促进BMSCs的水平和垂直迁移能力.结论 通过LATS2可介导Hippo信号途径调控BMSCs的分化、增殖和迁移能力.

abstractsObjective To investigate the regulatory effect of Hippo signaling pathway mediated by large tumor suppressor gene 2 (LATS2) on biological behavior of mice bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods BMSCs of C57BL/6 mice were cultured in vitro and passed to the 6th to 7th generations for experiment. BMSCs with activated and inactivated LATS2 were constructed with lentiviral vectors transfections. The BMSCs were allocated to blank control group (MSC group), empty vector control group (MSC-eGFP group), LATS2-overexpressing group (MSC-LATS2 group), empty vector without LATS2 shRNA control group (MSC-shcontrol group) and LATS2-underexpressing group (MSC-shLATS2 group). The transduction efficiencies mediated by the lentiviral vectors were evaluated by flow cytometry. The mRNA and protein expressions of LATS2, phosphorylation of Yes associated protein (p-YAP) and 14-3-3 were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot respectively. Osteogenic and adipogenic differentiation of BMSCs were evaluated through alizarin red and oil red O staining. Proliferation of BMSCs was evaluated using the CCK-8 assay. The effect of Hippo pathway on horizontal and vertical migration ability of BMSCs was measured by the scratch test and Transwell chamber test. Results The transduction efficiencies mediated by the lentiviral vectors were 93.1%-97.1%. Compared with MSC-eGFP group, the expressions of LATS2, p-YAP and 14-3-3 in MSC-LATS2 group were significantly elevated [LATS2 mRNA (2-ΔΔCT): 2.55±0.13 vs. 1.08±0.05, LATS2/GAPDH: 2.63±0.11 vs. 1.06±0.08, p-YAP/total YAP: 1.67±0.11 vs. 1.00±0.04, 14-3-3/β-actin: 2.22±0.20 vs. 0.98±0.06, all P < 0.05], however, compared with MSC-shcontrol group, the expressions in MSC-shLATS2 group were significantly reduced [LATS2 mRNA (2-ΔΔCT): 0.10±0.01 vs. 1.01±0.05, LATS2/GAPDH: 0.09±0.01 vs. 1.05±0.06, p-YAP/total YAP: 0.10±0.02 vs. 1.10±0.09, 14-3-3/β-actin: 0.05±0.01 vs. 0.90±0.08, all P < 0.05]. It suggested that high and low expression of LATS2 could activate or inhibit Hippo pathway. The osteogenic and adipogenic differentiation and proliferation rate of BMSCs in MSC-LATS2 group were significantly lower than those in MSC-eGFP group, however, those in MSC-shLATS2 group were significantly higher than MSC-shontrol group (all P < 0.05). It suggested that high and low expression of LATS2 could inhibit or promote osteogenesis, adipogenesis and cell proliferation of BMSCs. Scratch test and Transwell chamber test showed that the degree of scratch healing in MSC-LATS2 group was significantly lower than that in MSC-eGFP group [(22.11±3.02)% vs. (45.99±6.58)%], while the number of cells migrating to the subventricular layer of Transwell was significantly reduced (cells/MP:20.82±3.05 vs. 111.33±13.28, both P < 0.05); the degree of scratch healing in MSC-shLATS2 group was significantly higher than that in MSC-shcontrol group [(70.32±7.17)% vs. (39.28±2.98)%], the number of cells migrating to the subventricular layer of Transwell was increased significantly (cells/MP: 206.19±30.58 vs. 120.10±25.10, both P < 0.05). It suggested that high and low expression of LATS2 could inhibit or promote the horizontal and vertical migration of BMSCs. Conclusion LATS2-mediated alteration of Hippo pathway could modulate differentiation, proliferation, and migration of mesenchymal stem cells in vitro.