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目的探讨兔坐骨神经匀浆上清液对脂肪来源干细胞(adipose-derived stem cells,ADSCs)的诱导分化作用。方法取4月龄健康新西兰大白兔(体重2.0~2.5 kg)体外分离、培养ADSCs并传至第3代;用b FGF预诱导后,加入正常坐骨神经匀浆上清液(B组)及损伤3、7、14 d的坐骨神经匀浆上清液(分别为C、D、E组)对其进行诱导,空白对照组(A组)仅加入D-Hank液。观察细胞形态变化,并采用实时荧光定量PCR检测各组细胞神经组织蛋白(S-100)、神经元特异性烯醇化酶(neuron-specific enolase,NSE)及巢蛋白(Nestin)基因表达,免疫细胞化学染色检测S-100蛋白表达。结果 C、D、E组细胞经诱导后逐渐出现雪旺细胞或神经细胞样形态改变,以D组变化最为明显;A、B组细胞形态未见明显改变,仍以梭形为主。S-100免疫细胞化学染色示,C、D、E组染色均为阳性,以D组阳性细胞最多;A、B组细胞无阳性表达。实时荧光定量PCR检测示,S-100基因表达量随损伤时间延长呈上升趋势,C、D组达高峰,之后缓慢下降;C、D组表达量显著高于A、B、E组(P<0.05),C、D组间差异无统计学意义(P>0.05)。NSE基因表达量呈相同趋势,D组达高峰,之后缓慢下降;D、E组表达量显著高于A、B、C组(P<0.05),D、E组间差异亦有统计学意义(P<0.05)。Nestin基因表达量各组间差异无统计学意义(P>0.05)。结论兔损伤坐骨神经匀浆上清液在体外能够有效诱导ADSCs分化为雪旺细胞和神经细胞,伤后早期(3~7 d)可作为细胞移植的最佳时间。
Objective To investigate the differentiation and differentiation of adipose-derived stem cells (ADSCs) from rabbit sciatic nerve homogenate supernatant. Methods ADSCs were isolated and cultured in healthy New Zealand white rabbits (weighing 2.0-2.5 kg) at 4 months of age. The ADSCs were cultured and passaged to passage 3. After pre-induction with bFGF, normal sciatic nerve homogenate supernatant (group B) , Sciatic nerve homogenate supernatant (group C, D and E respectively) on day 7 and day 14 were induced. The blank control group (group A) only received D-Hank solution. The morphological changes of the cells were observed. The expression of S-100, NSE and Nestin was detected by real-time fluorescence quantitative PCR. The immune cells Chemical staining was used to detect S-100 protein expression. Results The morphology of Schwann cells or neurons gradually appeared in C, D and E groups, the most obvious change was in group D. The morphology of cells in groups A and B did not change obviously. S-100 immunocytochemical staining, C, D, E group were stained positive to D group of the most positive cells; A, B group of cells without positive expression. Real-time quantitative PCR showed that the expression of S-100 gene increased with the extension of injury time and peaked in groups C and D, then decreased slowly. The expression of S-100 in group C and D was significantly higher than that in groups A, B and E (P < 0.05). There was no significant difference between C and D groups (P> 0.05). NSE gene expression showed the same trend, the peak in D group, then decreased slowly; the expression of D, E group was significantly higher than that of A, B, C group (P <0.05), and the difference between D and E group was also statistically significant P <0.05). There was no significant difference in the expression of Nestin between groups (P> 0.05). CONCLUSIONS: Rabbit sciatic nerve homogenate supernatant can effectively induce ADSCs to differentiate into Schwann cells and nerve cells in vitro, and the optimal time for cell transplantation can be obtained in early stage of injury (3-7 d).