兔坐骨神经匀浆上清液对脂肪干细胞的诱导分化作用

INDUCTING DIFFERENTIATION EFFECT OF SCIATIC NERVE EXTRACTS ON RABBIT ADIPOSE-DERIVED STEM CELLS IN VITRO

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 study the inducting differentiation effect of the sciatic nerve extracts on rabbit adiposederived stem cells（ADSCs） in vitro. Methods The ADSCs were isolated from 2 healthy 4-month-old New Zealand rabbits（weighing, 2.0-2.5 kg） and cultured to passage 3, which were pretreated with 10 ng/m L basic fibroblast growth factor（b FGF） for 24 hours before induction. Then the induction media containing the extracts of normal sciatic nerve（group B） and injured sciatic nerve at 3, 7, and 14 days（group C, group D, and group E） were used, and D-Hank was used in group A as blank control group. The morphological changes of the cells were observed. At 7 days of induction, the gene expressions of neuron-specific enolase（NSE）, nestin（NES）, and S-100 were detected by real-time fluorescent quantitative PCR. The S-100 protein expression was tested by immunocytochemical staining. Results At 4 days after induction, some ADSCs of groups C, D, and E showed the morphology of Schwann-like cells or neuron-like cells, the change of group D was more obvious; and the ADSCs of group A and B had no obvious change, which were still spindle. The S-100 immunocytochemical staining showed positive expression in groups C, D, and E（more obvious in group D） and negative expression in groups A and B. The gene expression of S-100 displayed time-dependent increases in groups C and D, which was significantly higher than that of groups A, B, and E（P〈0.05）, but no significant difference was found between groups C and D（P〉0.05）. The gene expression of NSE showed the same tendency to S-100, which reached the peak in group D; the gene expression of NSE in groups D and E was significantly higher than that of groups A, B, and C（P〈0.05）, and groups D and E showed significant difference（P〈0.05）. However, the gene expression of Nestin showed no significant difference among different groups（P〉0.05）. Conclusion The ADSCs can be induced to differentiate into Schwann-like cells or neuron-like cells with sciatic nerve extracts; and the early stage（3-7 days） after injury is the best time for stem cell transplantation.