不同结合状态硫酸软骨素多糖链对体外二维三维培养背根节神经突起生长影响研究

目的构建鸡胚背根节细胞3D生长模型,观察CSPG对不同浓度的琼脂糖水溶胶培养基对鸡胚背根节神经突起生长的影响。方法①利用多肽缩合剂1’1羰基二咪唑介导CS-B长链与琼脂糖凝胶共价结合。②分别配制0.5%、0.75%、1%、1.25%、1.5%的SeaPrep琼脂糖凝胶溶液和共价结合了CS-B多糖长链的琼脂糖凝胶溶液。取孵育9~10天后鸡胚背根节加入上述凝胶溶液加培养液培养。③培养24小时之后在24 h、48 h7、2 h、96 h进行观察拍照。观察各时间点神经突起生长情况。结果①鸡胚背根节神经突起在1.25%的琼脂糖凝胶溶液中开始呈3D生长,在小于1%的琼脂糖溶液中只能2D生长。②在同一时间点,呈2D生长的背根节突起和呈3D生长的背根节突起在突起长度方面没有统计学差异(P>0.05)。③3D生长的DRG神经突起在共价结合了CS-B的琼脂糖凝胶中生长长度明显短于普通琼脂糖水凝胶(P<0.05),并且可观察到部分突起长出短而直的成束突起。但是对于呈2D生长的DRG突起长度在两种培养基中却未见差异(P>0.05)。结论鸡胚背根节神经元周围突起生长长度在3D培养中与在2D培养中有没有明显的差异。硫酸软骨素能够有效抑制呈3D生长的神经节突起生长,并且影响到神经突起的生长方式。但是在2D培养基中,未能看到上述的抑制作用。

Effect of CS-B on DRG neurite extension in three and two dimensional agarose gel culture

Objective To build a 3D culture model of neuron in vivo,Investigate the changes of chicken DRG neuritis growth under the 3D and 2D growth.Explore how the CSPGs covolent with agarose hydrogels affect the length of the DRG neurite extension.Method 9days of chicken DRG embedded in the SeaPrep agarose gel,a range of concentrations of agarose(0.5%,0.75%,1%,1.25%,1.5%) was used.the gel was combined with CS-B by covalence binding,snap the DRG at the 24 h,48 h,72 h,96 h.Results The chicken dorsal root ganglions can grow in three-dimensional way under the 1.25% concentrations agarose conditioned substrate.But under the concentrations of 1% agarose conditioned substrate,the DRG only can grow by two-dimensional manner.Under the same time point the average value and standard error of the mean of neurite length for each gel have no difference in statistic.(P>0.05),Results from ANOVA demonstrated statistically lower average neurites lengths between normal and CS-B covolent binding gels at each time point in three-dimensional growth in media culture(p<0.05).But have no difference in two-dimensional culture(P>0.05).Conclusion The CS-B can inhibit the neurite extend in three-dimensional culture but have no effect in two-dimensional culture.All our data was support the hypothesis that the scaffold surrounding the neuron plays an important role in the regenerating and extending of neurite.