不同方法诱导大鼠脂肪基质细胞定向分化为神经细胞的比较研究

Direct induction versus neurosphere induction of neural cells from rat adipose stromal cells： a comparative study

目的探索提高大鼠脂肪基质细胞（ADSCs）向神经细胞定向诱导分化比例的实验方法。方法根据所采用的不同细胞诱导分化方法分为A组f直接诱导组：以含血清及神经营养因子的Neurobasal培养基直接诱导ADSCs向神经细胞分化）和B组[神经球诱导组：先以无血清Neurobasal培养基将ADSCs诱导为神经干细胞CNSCs）球，再以含血清及营养因子的Neurobasal培养基诱导神经球干细胞向神经细胞分化]。通过细胞形态学观察以及免疫细胞化学检测巢蛋白（nestin）、β-微管蛋白Ⅲ（β-tubulin Ⅲ）、微管相关蛋白2（MAP2ab）和胶质纤维酸性蛋白（GFAP）表达，鉴定细胞分化结果。结果免疫细胞化学结果证实，两组细胞均有效表达nestin和β-tubulin Ⅲ。A组细胞在诱导6h时nestin表达可达高峰（73．8％±6．5％），并很快下降（24h为50.3％±3．8％，72h为10．5％±3．0％），72h后即检测不到；β-tubulinⅢ在诱导后24h开始表达（33．5％±6．6％），1周达高峰（84.3％±33％）。B组nestin的高表达持续整个神经球阶段，β-tubulinⅢ在神经球阶段有少量表达（成球后7d为14．1％±3.3％），神经球细胞分化后其表达明显增加（分化后3d为46．4％±6．1％）；B组在由NSCs向神经细胞诱导一定时间后可检测到一定比例的MAP2ab阳性细胞（24．5％）。结论ADSCs在体外稳定扩增传代并在适宜诱导条件下，可以向神经细胞定向分化。应用神经球诱导法和直接诱导法均可得到较高比例的nestin及β-mbulinⅢ阳性细胞，神经球诱导组nestin表达稳定，并有一定比例MAP2ab阳性细胞出现。

Objective To enhance the differentiation efficiency of adipose-derived stromal ceils （ADSCs） into neural cells. Methods ADSCs were isolated from the adipose tissue of SD rats and induced to differentiate into neural cells using two different protocols, namely direct induction （group A） and neurosphere induction （group B） protocols. For direct induction, the ADSCs were cultured in Neurobasal medium containing fetal bovine serum （FBS） and neurotrophic factors （NTs）. Neurosphere induction protocol was carried out by culturing the ADSCs in serum-free Neurobasal medium to induce the formation of neurospheres, which were further induced in serum- and NT-eontaining Neurobasal medium into neural cells. Morphological observation and immunohistochemistry for nestin, β-tubulin m, MAP2ab and GFAP were used to identify the differentiated cells. Results Primary cultured ADSCs appeared polymorphous, from which the mixed cells were removed by adherent culture. The fifth passage of ADSCs showed homogenous morphology in regular alignment, and the phenotype could be maintained after further passaging. Immunohistochemistry showed that the two induction protocols both resulted in high expression of nestin and β-tubulin HI in the induced ADSCs, and nestin expression in group A reached the peak level of （73.8±6.5）% at 6 h of the induction, followed by rapid reduction to （50.3±3.8）% at 24 h and （10.5±30）% at 72 h, becoming almost undetectable afterwards; β-tubulin Ⅲ expression occurred at 24 h following the induction [（33.5±6.6）%] and reached the peak level of （84.3±33）% at 1 week. In group B, high nestin expression persisted throughout the neurosphere stage, while low levels of β-tubulin Ⅲ expression was detected during the neurosphere stage [（14.1 ±3.3）% at 7 days after neurosphere formation], but its expression increased obviously with time after neurosphere differentiation [（46.4±6.1）% at 3 days after the differentiation. In group B, 24.5% of the cells were found positive for MAP2ab after the differentiation of the neurospheres into neural cells. Conclusion ADSCs can be induced into neural cells under appropriate conditions. Both of the induction protocols can obtain high rate of nestin- and β-tubulin Ill-positive cells, and the neurosphere induction protocol produces more stable nestin expression in the differentiated cells, which contain a proportion of MAP2ab-positive cells.