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目的探讨不同部位取材分离纯化雪旺细胞(Schwann cells,SCs)的纯度、活性以及数量差异,为神经缺损修复提供优质、足量种子细胞奠定理论基础。方法 6只5日龄SD大鼠,雌雄不限,体重8～10 g;切取背根神经节(实验组)和坐骨神经(对照组)。采用联合酶消化加机械吹打法分离培养SCs,并进行纯化及传代培养。取第1代SCs,用计数法绘制8 d内SCs生长曲线,MTT法检测8 d内SCs增殖情况,抗S-100免疫细胞化学检测SCs纯度,ELISA法检测脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)浓度。结果每只大鼠可切取背根神经节36～43个。消化成单个细胞后计数,实验组可获取(7.5±0.6)×106个SCs,对照组可获取(3.5±0.4)×106个SCs,差异有统计学意义(t=13.175,P=0.000)。两组SCs第3天开始进入对数增长期,随培养时间延长,细胞数及细胞增殖吸光度(A)值均呈上升趋势;且培养3、4、5、6 d时,实验组细胞数及A值明显高于对照组,差异均有统计学意义(P<0.05)。经S-100免疫细胞化学检测,实验组SCs纯度为92.08%±3.45%,对照组为77.50%±3.57%,差异有统计学意义(t=6.869,P=0.001)。ELISA法检测示实验组培养3 d和5 d时BDNF浓度均高于对照组,差异有统计学意义(P<0.05)。结论与坐骨神经相比,取材于背根神经节能获得数量更多、纯度和活性更高的SCs,为神经损伤修复创造了必要条件。
Objective To investigate the purity, activity and quantity differences of Schwann cells (SCs) extracted from different parts of the Schwann cells and lay the theoretical foundation for providing high quality and sufficient seed cells for nerve defect repair. Methods Six 5-day-old SD rats were randomly divided into male and female. Body weight was 8-10 g. The dorsal root ganglia (experimental group) and sciatic nerve (control group) were excised. SCs were isolated and cultured by combined enzyme digestion and mechanical blowing method, and purified and subcultured. The first generation of SCs was taken and the growth curve of SCs within 8 days was counted by counting method. The proliferation of SCs within 8 days was detected by MTT assay, the purity of SCs was detected by anti-S-100 immunocytochemistry, and the brain-derived neurotrophic factor (brain- derived neurotrophic factor (BDNF) concentration. Results Each rat can cut 36 ~ 43 dorsal root ganglia. After digestion into single cells, the number of SCs in the experimental group was (7.5 ± 0.6) × 106 SCs, and that in the control group was (3.5 ± 0.4) × 106 SCs. The difference was statistically significant (t = 13.175, P = 0.000). The SCs of both groups began to enter the phase of logarithmic growth on the 3rd day, and the numbers of cells and the proliferative absorbance (A) increased with the prolongation of culture time. At 3, 4, 5 and 6 days, A value was significantly higher than the control group, the difference was statistically significant (P <0.05). The results of S-100 immunocytochemistry showed that the purity of SCs in the experimental group was 92.08% ± 3.45% and that in the control group was 77.50% ± 3.57%, the difference was statistically significant (t = 6.869, P = 0.001). The results of ELISA showed that BDNF concentrations in experimental group were higher than those in control group on the 3rd and 5th day of culture, the difference was statistically significant (P <0.05). Conclusion Compared with the sciatic nerve, SCs derived from dorsal root ganglion can acquire more, more pure and more active SCs, which has created the necessary conditions for nerve injury repair.