透明质酸水凝胶体外促进骨髓间充质干细胞神经分化的研究

目的观察以透明质酸(hyaluronic acid,HA)水凝胶作细胞支架,骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)在HA水凝胶中的生长、增殖和分化情况,探讨HA水凝胶支持、促进BMSCs分化为神经元细胞的作用。方法采用Percoll法从SD雄鼠的骨髓中分离纯化BMSCs,传代培养,倒置显微镜观察BMSCs的生长情况和形态变化。各组以不同的方法诱导BMSCs分化。采用免疫细胞化学法检测各组BMSCs在相应时间点神经元特异性烯醇化酶(NSE)和神经丝蛋白(NF)及巢蛋白(nestin)的阳性细胞数。RT-PCR检测各组BMSCs相应时间点的NSE mRNA、NF mRNA和nestin mRNA表达。结果传代后BMSCs形似纤维细胞。诱导后,HA水凝胶组(A组)和脑组织提取液组(B组)BMSCs分化为神经元样细胞:A组细胞黏附在HA水凝胶表面,长出较多突起和分支;B组细胞出现突起及少量分支。诱导后A组和B组NSE、NF阳性细胞增多(P<0.05)。诱导2d后及7d后A组和B组nestin阳性细胞均增多:2d后,A组高于B组(P<0.05);7d后,A组低于B组(P<0.05)。诱导7d后A组和B组的NSE mRNA、NF mRNA表达均增加,A组增加明显(P<0.05)。诱导2d后A组和B组的nestin mRNA表达增加,A组更显著(P<0.05);7d后A组和B组的nestin mRNA表达则降低,A组更明显(P<0.05)。结论 HA水凝胶有利于BMSCs的黏附,为BMSCs生长、增殖和分化提供了结构支持和适宜的微环境,促使BMSCs向神经元细胞分化、增殖并表达神经元特异性蛋白。

Effect of Hyaluronic Acid on Promoting Bone Marrow-Derived Mesenchymal Stem Cells to Differentiate into Neural Cells in vitro

Objective To determine the effect of hyaluronic acid(HA) on promoting bone marrow-derived mesenchymal stem cells(BMSCs) to differentiate into neural cells.Methods BMSCs were purified and monitored.NSE+/NF+ cells and nestin+ cells were detected by immunocytochemistry.The mRNA levels of NSE,NF and nestin were determined by RT-PCR.Results The BMSCs adhered to HA hydrogel,with many surface projections,branches or crossing(Group A).Those cultured in brain tissue culture medium grew projections with fewer branches(Group B).Group A had more NSE+ cells(92.58±15.84) than Group B(80.26±16.47),and more NF+ cells(71.25±17.44) than Group B(52.37±14.75)(P<0.05).At day 2 post-stimulation,more nestin+ cells were found in Group A(48.3±7.7) compared with Group B(34.6±5.2)(P<0.05).At day 7,the mRNA levels of NSE and NF increased in both groups,but more in Group A.Nestin mRNA increased at day 2 post-stimulation and dropped at day 7 in both groups,where more significant in Group A.Conclusion HA hydrogel provides structural support and proper microenvironment for the growth,proliferation and differentiation of BMSCs,and promotes BMSCs to differentiate into neuron cells.