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目的 分离纯化雪旺细胞浆神经营养蛋白并研究其生物活性。方法 取 30 0只出生后 1～ 3天SD大鼠双侧坐骨神经 ,纯化培养 ,收集雪旺细胞 ,超声粉碎 ,超速离心 ,不同孔径分子筛滤膜 (PM10、30、5 0 )超滤浓缩 ,收集分子量 10～ 30 ku,30～ 5 0 ku和 >5 0 ku三种组份胞浆蛋白浓缩液 ,分别加入体外无血清培养的 E14SD胎鼠脊髓运动神经元培养液中 ,用 MTT酶标法检测证实 10～ 30 ku和 >5 0 ku两组份蛋白有神经营养活性。再通过Diol- 15 0高效液相分子层析进一步从雪旺细胞胞浆中纯化分离出分子量为 2 6 ku和 5 8ku两种胞浆蛋白 ,并对其神经生物活性进行研究。结果 2 6 ku和 5 8ku雪旺细胞胞浆蛋白能维持体外无血清培养的脊髓运动神经元存活 ,其最佳生物活性浓度为 2 0 ng/孔。结论 雪旺细胞胞浆内含有分子量为 2 6 ku和 5 8ku的运动神经营养蛋白
Objective To isolate and purify Schwann cell cytoplasmic neurotrophic protein and study its biological activity. Methods 30 sciatic nerves of SD rats were collected from 1 to 3 days after birth. Purified and cultured Schwann cells were sonicated, ultracentrifuged and ultrafiltered by different pore size molecular sieve filters (PM10, 30, 50) The cytosolic protein concentrates with molecular weights of 10-30 ku, 30-500 ku and> 50 ku were respectively added into the culture media of E14SD fetal rat spinal motor neurons cultured in serum without serum and detected by MTT enzyme-linked immunosorbent assay Confirmed that 10 ~ 30 ku and> 50 ku two components of protein neurotrophic activity. The two cytoplasmic proteins with molecular weights of 26 ku and 58 kU were further purified from the cytoplasm of Schwann cells by Diol-150 high performance liquid chromatography and their neurobiological activities were studied. Results Cytoplasmic proteins of 26 ku and 58 ku were stable in serum-free culture of spinal motor neurons. The best bioactivity concentration was 20 ng / well. Conclusion The Schwann cell cytoplasm contains motor neurotrophic proteins with molecular weights of 26 ku and 58 ku