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Tgf-b1 induces a nucleus pulposus-like phenotype in notch 1 knockdown rabbit bone marrow mesenchymal stem cells

机译:Tgf-b1在Notch 1敲低的兔骨髓间充质干细胞中诱导髓核样表型

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We have investigated the effects of Notch1 knockdown and treatment with TGF-b1 on the regulation of the directional differentiation of mesenchymal stem cells (MSCs). MSCs were isolated from the femur bone of New Zealand rabbits and purified by using discontinuous gradient density centrifugation. Notch1 siRNAs were designed, synthesised and transiently transfected into these MSCs, and treated with TGF-b1. The MSCs were examined for morphology, stained with toluidine blue for proteoglycan analysis and gene and protein expression were measured with qRT-PCR and Western blotting respectively. They had an ellipse or fusiform shape and gathered in nests or swirls after being cultured for 7 days. Notch1 expression was knocked down with Notch1 siRNA (the silence rate was 47%; P < 0.001). After knockdown and TGF-b1 treatment, MSCs expressed more proteoglycan (P < 0.01), and higher levels of collagen II mRNA and protein than control cells (P < 0.001). Thus knockdown of Notch1 expression in MSCs may be useful in the treatment of intervertebral disc degeneration.
机译:我们已经研究了Notch1基因敲低和TGF-b1处理对间充质干细胞(MSCs)定向分化的调节作用。从新西兰兔的股骨中分离MSC,并通过不连续梯度密度离心法纯化。将Notch1 siRNA设计,合成并瞬时转染到这些MSC中,并用TGF-b1处理。检查MSC的形态,用甲苯胺蓝染色以进行蛋白聚糖分析,并分别通过qRT-PCR和蛋白质印迹法测量基因和蛋白质表达。它们培养了7天后呈椭圆形或梭形,聚集在巢或漩涡中。 Notch1 siRNA敲低Notch1表达(沉默率为47%; P <0.001)。敲除和TGF-b1处理后,与对照细胞相比,MSCs表达更多的蛋白聚糖(P <0.01),并表达更高的II型胶原蛋白mRNA和蛋白(P <0.001)。因此,MSC中Notch1表达的敲低可能在治疗椎间盘退变中有用。

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