rhBMP-2诱导颅神经嵴细胞向神经元分化的体外研究

目的；观察重组人骨形成蛋白2（rhBMP-2）对颅神经嵴细胞的诱导发化作用。方法：分别用不含rhBMP-2的无血清培养液和含有不同浓度rhBMP-2的培养液对颅神经嵴细胞进行培养。10d后，观察两组细胞的形态学差别，用胶质纤维酸性蛋白（GFAP），神经纤维细丝蛋白（NF），颅神经嵴细胞的特异性抗体P75等抗体进行免疫组化染色，鉴定细胞的分化状态，并计算NF阳性细胞数的百分比及进行统计分析。结果：在含有rhBMP-2的培养液中，颅神经嵴细胞的形态由成纤维细胞样向神经元样的表型进行转变，可见较长的细胞突起出现。NF染色结果阳性，而GFAP，P75染色结果阴性。50ng/mL组中，NF的阳性细胞数比其它浓度组明显增多，并有显著性差异（P＜0．05）。结论：rhBMP-2可以诱导颅神经嵴细胞向神经元进行分化，50ng/mL是其中最有效的诱导浓度。

rhBMP-2 induced differentiation of cranial neural crest cells into neuron in vitro

AIM:To observe the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) on cranial neural crest cells(CNCC) and determine the appropriate dose for their differentiation. METHODS:CNCC were cultured in different doses of serum free medium with and without rhBMP-2. 10 days later, the morphology of cells in two groups was observed under light microscope. Antibodies of glial fibrillary acidic protein (GFAP), neurofilament (NF) and p75 were used to determine the differentiation state of CNCC in above groups. Percentage of NF-positive cells were counted and analysized. RESULTS: The shape of CNCC transformed from fibroblast-like to neuronal phenotype after treated with rhBMP-2. Long neurites were observed in rhBMP-2 treated cells. NF was stained positively in rhBMP-2 treated group. No positive signals were found in non-rhBMP-2 treated group. There were more NF immunoreactive cells in 50ng/mL rhBMP-2 groups than in other groups. There was a significant difference of NF-positive cell numbers between 50 ng/mL rhBMP-2 group and other doses of rhBMP-2 groups ( P <0.05). CONCLUSION: rhBMP-2 can induce the differentiation of CNCC into neurons?50 ng/mL rhBMP-2 was the most effective dose for differentiation of CNCC.