骨髓源性细胞促进坐骨神经体外预变性实验研究  

BONE MARROW DERIVED CELLS PROMOTING PRE-DEGENERATION OF SCIATIC NERVE IN VITRO

作　　者：吴敏[1] 王晓盼[1] 肖玉周[1] 周建生[1] 

机构地区：[1]蚌埠医学院第一附属医院骨科、组织移植安徽省重点实验室安徽蚌埠,233003

出　　处：《中国修复重建外科杂志》2013年第5期554-558,共5页Chinese Journal of Reparative and Reconstructive Surgery

摘　　要：目的探讨周围神经体外预变性的新方法,以便在短期内获得大量高效雪旺细胞,以期为组织工程神经构建提供大量种子细胞。方法采用转绿色荧光蛋白基因C57BL/6小鼠的骨髓源性细胞(bone marrow derived cells,BMDCs)与C57BL/6小鼠坐骨神经体外共培养,建立体外预变性模型的实验组(A组),无BMDCs参与的单纯坐骨神经体外培养为对照组(B组)。培养7 d后行大体观察及免疫荧光染色观察BMDCs能否在体外进入坐骨神经内参与预变性;将变性后的神经酶消化行雪旺细胞培养,通过细胞免疫荧光染色及流式细胞仪检测各组细胞量及鉴定原代培养后的雪旺细胞纯度,评价各组雪旺细胞增殖情况。结果培养7 d后大体观察示两组坐骨神经断端开始形成神经瘤样结构,A组较B组明显;免疫荧光染色示A组大量BMDCs浸润至神经内部,其中部分细胞表达F4/80,为单核巨噬系统细胞。经细胞培养,A、B组获得的雪旺细胞产量分别为(5.59±0.19)×104个/mg和(3.20±0.21)×104个/mg,差异有统计学意义(t=2.14,P=0.03)。细胞接种后48 h经p75NTR荧光染色鉴定示,两组可见双极或三极样雪旺细胞,细胞核为蓝色且较小,胞体为红色;成纤维细胞呈扁平多角形,细胞核及核仁清晰,大而不透光,多位于雪旺细胞下且p75NTR染色为阴性。A组混杂的成纤维细胞较少,B组较多。A、B组雪旺细胞纯度分别为88.4%±5.8%和76.1%±3.7%,差异有统计学意义(t=2.38,P=0.04)。经流式细胞仪定量分析A组雪旺细胞纯度为89.6%,B组为74.9%。结论 BMDCs与周围神经体外共培养是一种有效获得大量雪旺细胞的方法,为组织工程神经构建中种子细胞的获取提供了新方法。Objective To explore a new method for the pre-degeneration of peripheral nerve in vitro for obtaining many effective Schwann cells so as to provide a large number of seed cells for the research and application of tissue engineered nerves. Methods The bone marrow derived cells （BMDCs） from transgenic green fluorescent protein C57BL/6 mouse and the sciatic nerve segments from the C57BL/6 mouse were co-cultured to prepare the pre-degeneration of sciatic nerve in vitro （experimental group, group A）, and only sciatic nerve was cultured （control group, group B）. At 7 days after culture, whether BMDCs can permeate into the sciatic nerve in vitro for pre-degeneration was observed by gross and immunohistofluorescence staining. And then Schwann cells were obtained from the sciatic nerves by enzymic digestion and cultured. The cell number was counted, and then the purity of primary Schwann cells was determined using immunohistofluorescence staining and flow cytometer analysis. Results At 7 days after pre-degeneration, gross observation showed that enlargement was observed at nerve stumps, and neuroma-like structure formed; the group A was more obvious than group B. Immunohistofluorescence staining showed many BMDCs permeated into the nerve segments, with positive F4/80 staining in group A. After culture, the yield of Schwann cells was （5.59 ± 0.19） × 104/mg in group A and （3.20 ± 0.21） × 104/mg in group B, showing significant difference （t=2.14, P=0.03）. At 48 hours after inoculation, the cells had blue bipolar or tripolar cell nuclei with small size and red soma by immunohistofluorescence staining; fibroblasts were fiat polygonal with clear nucleus and nucleolus, showing negative p75NTR staining; and there were few of fibroblasts in group A. The purity of Schwann cells was 88.4% ± 5.8% in group A and 76.1% ± 3.7% in group B, showing significant difference （t=2.38, P=O.04）. And the flow cytometer analysis showed that the purity was 89.6% in group A and 74.9% in group B. Conclusion BMDCs can

关 键 词：坐骨神经 雪旺细胞 骨髓源性细胞 体外预变性 小鼠 

分 类 号：R651.3[医药卫生—外科学]