胚胎大鼠背根神经节酶消化法的评价

Study of sensory neuron dissociation methods from the embryonic rat dorsal root ganglion

目的为胚胎大鼠背根神经节(dorsal root ganglion,DRG)神经元耳蜗植入建立优化的体外分离培养方法。方法①比较3种组合酶消化法对胚胎大鼠DRG神经细胞的分散度以及神经细胞存活时间的差异。实验组:木瓜蛋白酶、胶原酶II/胰蛋白酶、DNA酶I;对照组I:木瓜蛋白酶、胶原酶II/中性蛋白酶II、DNA酶I;对照组II:胶原酶II、胰蛋白酶。②通过激光共聚焦显微镜观察体外培养神经元神经烯醇化酶(neuronal speci?c enolase,NSE)、高分子量神经微丝(neuro?lament H,NF-H)的表达,比较不同酶消化法对神经元轴突长度的影响。结果 DRG神经细胞分散度分别为:实验组80%～90%,对照组I 60%～70%,对照组II 30%～40%;神经细胞存活时间:实验组(12.0±3.0)d,对照组I(7.5±1.5)d,对照组II(4.5±0.5)d。3种组合酶处理的神经元均有NSE和NF-H阳性表达,实验组轴突长度在培养第3、5、7天均明显长于另外两组,差异有统计学意义(F分别为32.612、6.093、9.334,P均<0.05),神经元轴突长度测量:实验组>对照组I>对照组II。结论木瓜蛋白酶、胶原酶II/胰蛋白酶、DNA酶I组合消化法对胚胎大鼠DRG神经细胞体外分离最为理想,可为基因修饰神经元耳蜗植入的实验提供大量的、存活时间长且活性好的细胞来源。

OBJECTIVE To establish an optimized method for dissociating sensory neuron from the embryonic rat dorsal root ganglion（DRG）in vitro for cell transplantation therapy in cochlear.METHODS Comparing the effect of three kinds of enzyme combinations in DRG neuron＇s growth： the dispersing degree and cell survival time.The experimental group contains papain,collagenase type II/trypsin and DNase I.Control I contains papain,collagenase type II/dispase type II and DNase I.Control II contains collagenase type II and trypsin.Neuron–specific enolase（NSE）and Neurofilament H（NF-H）were detected by immunohistochemistry and the lengths of axon outgrowth were determined by laser scanning confocal microscope（LSCM）in these groups.RESULTS The range of dispersing degree of DRG neuron was： Experimental group 80%-90%,Control I 60%～70%,Control II 30%～40%.The cell survival time were： Experimental group（12.0±3.0）days,Control I（7.5±1.5）days and Control II（4.5±0.5）days.NSE and NF-H were positive expressed in all DRG neuron of three groups.The lengths of axon outgrowth of experimental group were obviously longer than the others in 3d,5d,7d respectively,statistics shows a significant difference between them（F=32.612、6.093、9.334,P0.05）.The ranges of axon length in three groups were： Experimental group Control IControl II.CONCLUSION The embryonic rat DRG neuron may be dissociated fully and migrated plentifully by the combination of administration of papain,collagenase type II/trypsin and DNase I,and the neuron survived well and the axon developed very healthy also.This method provided an abundant viable sensory neuron for xenografts transplantation.