大鼠脂肪源性干细胞来源的施万样细胞的增殖能力

Proliferation ability of Schwann-like cells derived from rat adipose-derived stem cells and its significance

目的:通过检测大鼠脂肪源性干细胞(ADSCs)来源的施万样细胞的增殖能力,探讨其在神经修复再生领域的应用及其意义。方法:取大鼠附睾旁脂肪组织进行ADSCs的分离与培养;将第4代ADSCs在β-巯基乙醇(β-ME)、反式维甲酸(ATRA)、血小板衍生因子AA(PDGF-AA)、碱性成纤维细胞生长因子(bFGF)、腺苷酸环化酶激活剂(Forskolin)和Heregulin-β的联合作用下诱导分化为施万样细胞。采用免疫荧光法、Western blotting法和Real-time PCR法检测施万细胞(SCs)标记物S-100β、P75和胶质纤维酸性蛋白(GFAP)的表达水平;采用MTT法检测施万样细胞的增殖能力,观察其有丝分裂增殖现象。结果:诱导分化后的施万样细胞呈梭形、栅栏样排列。S-100β、P75和GFAP蛋白免疫荧光为Cy3/FITC标记,阳性产物位于细胞浆及其突起。Western blotting和Real-time PCR法检测,施万样细胞中S-100β、P75和GFAP蛋白和mRNA表达水平明显高于ADSCs(P<0.01)。MTT法检测,施万样细胞有较强的有丝分裂增殖能力。结论:ADSCs能够成功被诱导分化为施万样细胞,并表达SCs标记物;施万样细胞有较强的有丝分裂增殖能力,可作为神经组织工程理想的种子细胞。

Objective： To investigate the application and significance of Schwann-like cells in nerve regeneration field by detecting the proliferation ability of Schwann-like cells derived from rat adipose-derived stem cells（ADSCs）.Methods： The ADSCs obtained from rat adipose tissue were isolated and cultured.The ADSCs at passage 4 were induced to differentiate into Schwann cells under the treatment of inducing factors,such as β-ME,ATRA,PDGF-AA,bFGF,Forskolin, and Heregulin-β. Immunofluorescence,Western blotting and Real-time PCR were performed to detect the expresson levels of the markers of Schwann cells（SCs） S100β,P75, and glial fibrillary acidic protein （GFAP）.The proliferation ability of the Schwann like cells were analyzed using an MTT assay and the mitotic feature of Schwann like cells was also assessed.Results： After induction and differentiation,the Schwann-like cells were spindle and arranged as fence. S-100β,P75, and GFAP protein immunofluorescence were marked by Cy3/FITC,and the positive products were located in the cytoplasm and its processes.The Western blotting and Real-time PCR results showed that S-100β,P75, and GFAP protein and mRNA expression levels in the Schwann-like cells were obviously higher than those in ADSCs（P〈0.01）.Moreover,the Schwann-like cells possessed a strong proliferation ability of mitosis.Conclusion： ADSCs can be successfully induced to differentiate into Schwann-like cells and express the markers of SCs.In addition,the Schwann-like cells possess a strong proliferation ability of mitosis,revealing that they would be used as the seed cells for nerve tissue engineering.