Mesenchymal stem cell （MSC）-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical st...Mesenchymal stem cell （MSC）-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical stimuli, including into tenocytes. Cell elongation and cytoskeletal tension have been shown to be instrumental to the process of MSC differentiation. Previous studies have shown that inhibition of stress fiber formation leads MSCs to default toward an adipogenic lineage, which suggests that stress fibers are required for MSCs to sense the environmental factors that can induce differentiation into tenocytes. As the Rho/ROCK signal transduction pathway plays a critical role in both stress fiber formation and in cell sensation, we examined whether the activation of this pathway was required when inducing MSC tendon differentiation using rope-like silk scaffolds. To accomplish this, we employed a loss-of-function approach by knocking out ROCK, actin and myosin （two other components of the pathway） using the specific inhibitors Y-27632, Latrunculin A and blebbistatin, respectively. We demonstrated that independently disrupting the cytoskeleton and the Rho/ ROCK pathway abolished the expression of tendon differentiation markers and led to a loss of spindle morphology. Together, these studies suggest that the tension that is generated by MSC elongation is essential for MSC teno-differentiation and that the Rho/ROCK pathway is a critical mediator of tendon differentiation on rope-like silk scaffolds.展开更多
目的:探讨大黄素对脂多糖(LPS)诱导损伤的人脐静脉血管内皮细胞(HUVECs)骨架及通透性影响的作用机制。方法:HUECs分为五组:正常对照组、内皮炎症损伤模型组(0.2μg/m L LPS)、大黄素干预组(10μmol/L大黄素+0.2μg/m L LPS)、抑制剂对照...目的:探讨大黄素对脂多糖(LPS)诱导损伤的人脐静脉血管内皮细胞(HUVECs)骨架及通透性影响的作用机制。方法:HUECs分为五组:正常对照组、内皮炎症损伤模型组(0.2μg/m L LPS)、大黄素干预组(10μmol/L大黄素+0.2μg/m L LPS)、抑制剂对照组(0.2μg/m L LPS+50μmol/L Y27632)、西药对照组(10-5mol/L缬沙坦+0.2μg/m L LPS)。首先,以内皮细胞生物学功能PCR基因芯片技术筛选可能的基因改变。结合基因芯片结果和文献中各基因功能,拟定观察Rho/ROCK通路相关的和可能涉及调节细胞骨架的基因,如纤连蛋白(FN)、整合素A5(ITGA5)、Ras同源基因家族成员(Rho A)、肌球蛋白轻链磷酸酶(MLCP)、磷脂酰肌醇-4,5-二磷酸肌醇3-激酶(PI3K)、黏着斑激酶(FAK)、血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR2)的m RNA表达水平,进行实时荧光定量PCR验证,同时以Western blotting对ITGA5、Rho A、4,5-二磷酸磷脂酰肌醇(PIP2)和磷酸化的肌球蛋白轻链(PMLC)进行蛋白水平的研究。结果:1LPS组Rho/ROCK通路相关基因(FN、ITGA5、Rho A、MLCP、PI3K、FAK、VEGF、VEGFR2)表达显著升高,ITGA5、Rho A、PIP2、PMLC的蛋白表达亦升高,与正常对照组比较,差异均有统计学意义(P<0.05)。2与LPS组比较,Y27632组、缬沙坦组的FN、ITGA5、Rho A、PI3K、FAK、VEGF、VEGFR2基因表达下降,MLCP表达上升,差异均有统计学意义(P<0.05);ITGA5、PIP2的蛋白表达下降;缬沙坦组PMLC表达降低,差异有统计学意义(P<0.05)。3与LPS组比较,大黄素组FN、PI3K、FAK表达降低,MLCP表达上升,差异均有统计学意义(P<0.05);PIP2蛋白表达下降,差异有统计学意义(P<0.05)。结论:LPS可激活Rho/ROCK信号通路,成功构建HUVECs内皮损伤模型;Y27632和缬沙坦可阻断Rho/ROCK通路,对细胞骨架有较强的保护作用;大黄素可轻度抑制Rho/ROCK通路,同时可能通过调节PI3K、MLCP表达而影响其他通路(如AKT和/或FAK-PI3K信号通路)从而发挥对内皮细胞损伤的干预作用。展开更多
文摘Mesenchymal stem cell （MSC）-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical stimuli, including into tenocytes. Cell elongation and cytoskeletal tension have been shown to be instrumental to the process of MSC differentiation. Previous studies have shown that inhibition of stress fiber formation leads MSCs to default toward an adipogenic lineage, which suggests that stress fibers are required for MSCs to sense the environmental factors that can induce differentiation into tenocytes. As the Rho/ROCK signal transduction pathway plays a critical role in both stress fiber formation and in cell sensation, we examined whether the activation of this pathway was required when inducing MSC tendon differentiation using rope-like silk scaffolds. To accomplish this, we employed a loss-of-function approach by knocking out ROCK, actin and myosin （two other components of the pathway） using the specific inhibitors Y-27632, Latrunculin A and blebbistatin, respectively. We demonstrated that independently disrupting the cytoskeleton and the Rho/ ROCK pathway abolished the expression of tendon differentiation markers and led to a loss of spindle morphology. Together, these studies suggest that the tension that is generated by MSC elongation is essential for MSC teno-differentiation and that the Rho/ROCK pathway is a critical mediator of tendon differentiation on rope-like silk scaffolds.
文摘目的:探讨大黄素对脂多糖(LPS)诱导损伤的人脐静脉血管内皮细胞(HUVECs)骨架及通透性影响的作用机制。方法:HUECs分为五组:正常对照组、内皮炎症损伤模型组(0.2μg/m L LPS)、大黄素干预组(10μmol/L大黄素+0.2μg/m L LPS)、抑制剂对照组(0.2μg/m L LPS+50μmol/L Y27632)、西药对照组(10-5mol/L缬沙坦+0.2μg/m L LPS)。首先,以内皮细胞生物学功能PCR基因芯片技术筛选可能的基因改变。结合基因芯片结果和文献中各基因功能,拟定观察Rho/ROCK通路相关的和可能涉及调节细胞骨架的基因,如纤连蛋白(FN)、整合素A5(ITGA5)、Ras同源基因家族成员(Rho A)、肌球蛋白轻链磷酸酶(MLCP)、磷脂酰肌醇-4,5-二磷酸肌醇3-激酶(PI3K)、黏着斑激酶(FAK)、血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR2)的m RNA表达水平,进行实时荧光定量PCR验证,同时以Western blotting对ITGA5、Rho A、4,5-二磷酸磷脂酰肌醇(PIP2)和磷酸化的肌球蛋白轻链(PMLC)进行蛋白水平的研究。结果:1LPS组Rho/ROCK通路相关基因(FN、ITGA5、Rho A、MLCP、PI3K、FAK、VEGF、VEGFR2)表达显著升高,ITGA5、Rho A、PIP2、PMLC的蛋白表达亦升高,与正常对照组比较,差异均有统计学意义(P<0.05)。2与LPS组比较,Y27632组、缬沙坦组的FN、ITGA5、Rho A、PI3K、FAK、VEGF、VEGFR2基因表达下降,MLCP表达上升,差异均有统计学意义(P<0.05);ITGA5、PIP2的蛋白表达下降;缬沙坦组PMLC表达降低,差异有统计学意义(P<0.05)。3与LPS组比较,大黄素组FN、PI3K、FAK表达降低,MLCP表达上升,差异均有统计学意义(P<0.05);PIP2蛋白表达下降,差异有统计学意义(P<0.05)。结论:LPS可激活Rho/ROCK信号通路,成功构建HUVECs内皮损伤模型;Y27632和缬沙坦可阻断Rho/ROCK通路,对细胞骨架有较强的保护作用;大黄素可轻度抑制Rho/ROCK通路,同时可能通过调节PI3K、MLCP表达而影响其他通路(如AKT和/或FAK-PI3K信号通路)从而发挥对内皮细胞损伤的干预作用。
