| Objective To observe the effects of Notchl gene on the directional differentiation of rabbit marrow mesenchymal stem cells (MSCs) into a nucleus pulposus-like phenotype through silenced Northl gene expression by small interfering RNA (siRNA) and induced by TGF-β1.Methods MSCs were obtained from New Zealand rabbits (4-6week-old) femur bone marrow and purified by discontinuous gradient density centrifugation. The short hairpin RNA (shRNA) aimed at Notchl gene was designed according to the siRNA principle, and transient transfect into MSCs. Cells were divided into three groups:induced by TGF-β1only (group1); induced by TGF-β1and transfect by nonsense siRNA (group2); induced by TGF-β1and transfect by shRNA-Notchl (group3). The expressions of proteoglycan were detected by toluidine blue staining. The expressions of collagen Ⅱ and proteoglycan were examined in gene and protein level with RT-PCR and Western blot.Results The MSCs were ellipse or fusiform shape and gathered in nest or swirl. Notchl expression was down-regulated by shRNA-Notchl, and the silencing rate was47%(P<0.001). The positive expressions in shRNA-Notchl group were higher than the others by toluidine blue staining. RT-PCR showed that the expression of collagen Ⅱ in group1was2.55times than group1(P<0.001),2.53times than group2(P<0.001), and the expression of proteoglycan in group3was1.51times than group1(P<0.01),1.53times than group2(P<0.01). Western-blot showed that the expression of collagen Ⅱ in group1was1.25times than group1(P<0.05),1.37times than group2(P<0.05), and the expression of proteoglycan in group3was1.99times than group1(P<0.05),1.46times than group2(P<0.05).Conclusions This study demonstrates that MSCs could be purified by discontinuous gradient density centrifugation. TGF-β1can induce the MSCs to excrete collagen Ⅱand proteoglycan, and the down-regulation of Notchl gene could increase these excretion. Objective To investigated the effects of Notchl knockdown and treatment with TGF-β1on the regulation of the directional differentiation of mesenchymal stem cells (MSCs) in suppressing the degeneration of punctured intervertebral discs in rabbits.Methods MSCs were isolated from the femur bone of New Zealand rabbits and purified with discontinuous gradient density centrifugation. Notchl siRNAs were designed, synthesized, and transiently transfected into these MSCs, and then treated with TGF-β1.Ten New Zealand rabbits were invited in this study.The rabbits’discs in L3/4, L4/5and L5/6were stabbed by a needle. Two weeks after the operation, the discs were examined with Magnetic Resonance Images(MRI). And the L3/4discs were treated by pYr1.1transfected MSCs treated with TGF-β1, L4/5discs treated by shRNA-Notch-1transfected MSCs treated with TGF-β1, L5/6discs were treated by the MSCs treated with TGF-β1only. Four weeks after the treatment, the discs were examined with MRI. The expression of proteoglycan in nucleus pulposus was evaluated by using toluidine blue, and the expressions of collagen II and proteoglycan were examined with RT-PCR and Western-blot. Results Four weeks after the treatment, the signal intensity of T2weightedimages of the L4/5discs was higher than the others, with a significant difference found between them. The toluidine blue staining showed that the expression of proteoglycan in the L4/5discs was higher than that in the others. RT-PCR and Western-blot showed that the expression of collagen Ⅱ and proteoglycan mRNA and protein in the L4/5discs were significant higher than that in the L3/4and L5/6disc.Conclusion Transplantation of shRNA-Notch-1transfected MSCs treated with TGF-β1restrain the degeneration of punctured discs. |
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