摘要
目的神经元纯化是各种神经细胞实验研究必不可少的实验步骤,但目前少见神经元纯化过程中各种因素对神经轴突影响的实验报道。探讨简便有效的背根神经节神经元(dorsal root ganglion neurons,DRGn)细胞纯化方法和神经元培养体系内相关因素对β3-tubulin阳性神经轴突生长状态的影响。方法取新生3d的SD大鼠背根神经节(dorsal root ganglion,DRG)制成细胞悬液,根据玻片包被底物不同分为D-多聚赖氨酸(poly-D-lysine,PDL)组、PDL/层粘连蛋白(Laminin,LN)组和Ⅰ型胶原(collagenⅠ,ColⅠ)组,分别差速贴壁10、20、30、40、50、60、70、80、90、100min,倒置相差显微镜观察细胞贴壁情况,并采用免疫荧光染色观察各时间点DRGn细胞和非DRGn细胞贴壁数量。将DRG制备组织块培养72h,采用完全随机设计的方法观察底物(PDL、PDL/LN、ColⅠ)、FBS(0、5%、10%)、五氟尿嘧啶(5-fluorouracil,5-Fu,0、20、40μmol/L)和阿糖胞苷(cytrarabine,Ara-C,0、10、20μmol/L)不同水平对DRG整节培养中β3-tubulin阳性轴突长度和单位阳性轴突分支末梢数量的影响。结果倒置相差显微镜和倒置荧光显微镜观察显示,细胞接种后即开始贴壁,贴壁10min后移除细胞悬液仍可见DRGn细胞及非DRGn细胞贴壁生长。PDL组:贴壁10、30min,神经元特异性烯醇化酶(neuron-specific enolase,NSE)阴性(NSE-)细胞数高于NSE+细胞数(P<0.05);贴壁80、90、100min,NSE+细胞数大于NSE-细胞数(P<0.05)。PDL/LN组:贴壁10、20、30、40、50min,NSE+细胞数及NSE-细胞数差异无统计学意义(P>0.05);贴壁60、70、80、90、100min,NSE+细胞数大于NSE-细胞数(P<0.05)。ColⅠ组:贴壁10~40min,NSE-细胞数大于NSE+细胞数,但差异无统计学意义(P>0.05);贴壁70、80、90、100min,NSE+细胞数大于NSE-细胞数(P<0.05)。DRG整节培养72h后,底物水平为PDL/LN时轴突生长分化最好,与PDL水平和ColⅠ水平比较差异有统计学意义(P<0.05);FBS为5%水平时β3-tubulin单位阳性轴突分支末梢数最大(P<0.05),但阳性轴突长度在0、5%和10%3个浓度水平比较差异无统计学意义(P>0.05);5-Fu在0浓度水平时β3-tubulin单位阳性轴突分支末梢数及阳性轴突长度均最大,与20、40μmol/L浓度水平比较差异有统计学意义(P<0.05);Ara-C在0和10μmol/L浓度水平的阳性轴突分支末梢数相同,均高于20μmol/L水平(P<0.05);而阳性轴突长度在10μmol/L水平时最长,与0和20μmol/L浓度比较差异有统计学意义(P<0.05)。结论将DRGn细胞悬液在PDL包被的玻片上差速贴壁30min,再将悬液吸出进行接种培养,可在一定程度上起到分离纯化神经元细胞的作用。行DRG整节培养时,选择神经元专用培养基联合PDL/LN细胞培养底物和10μmol/LAra-C,可获得最理想的β3-tubulin阳性轴突生长分化效果。
Objective Neuron purification is essential to procedure of various nerve cell experimental research,however,at present there is few reports on the effect of various factors on neural axons during purification.To find out a simple method of neuron purification,and to investigate the influence factors of corresponding purification culture in dorsal root ganglion(DRG)tissue culture onβ3-tubulin positive axon.Methods The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension.Then,the amount of attached DRG neurons and nonneuronal cells in poly-D-lysine(PDL)group,PDL/Laminin(PDL/LN)group and collagen-I(Col I)group was observed from 10 to 100 minutes.Then,the extension and arborization ofβ3-tubulin positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates(PDL,PDL/LN,and Col I),FBS(0,5%,and 10%),5 fluorouracil(5-Fu,0,20,and 40μmol/L),and cytrarabine(Ara-C,0,10,and 20μmol/L).Results Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluoresence microscope;after cell suspension was removed,adherent growth of DRGn cells and non-DRGn cells were still seen.In PDL group,the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes(P0.05);the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80,90 and 100 minutes(P0.05).In PDL/LN gruop,there was no significant difference(P0.05)in the amount of NSE negative cells and NSE positive cells at 10,20,30,40,and 50 minutes;the amount of NSE positive cells was significantly higher(P0.05)than that of NSE negative cells at 60,70,80,90,100 minutes.In Col I group,the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes,but showing no significant difference(P0.05);the amount of NSE positive cells was significantly higher(P0.05)than that of NSE negative cells at 70-100 minutes.At 72 hours after DRG tissue culture,the best result of β3-tubulin positive axon extension and arborization was obtained when the substrate level was PDL/LN,and the average length of PDL/LN level was significantly larger than that of other two substrates(P0.05).The highest number ofβ3-tubulin positive axon distal end was obtained at 5%concentration level of FBS(P0.05),but showing no significant differences in β3-tubulin positive axon length among three levels(P0.05).Both the most ofβ3-tubulin positive axon distal ends and the longestβ3-tubulin positive axon average length were obtained at 0μmol/L concentration level of 5-Fu,showing significant differences between 0μmol/L level and 20,40μmol/L levels(P0.05).A similar result ofβ3-tubulin positive axon distal end was got at the 0μmol/L level and 10μmol/L level of Ara-C,which was significantly higher than that of 20μmol/L level(P0.05).Conclusion A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes.When DRG neuron culture,neuron special medium,PDL/LN substrate and 10μmol/L Ara-C are recommended inβ3-tubulin positive axon research.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2010年第2期172-179,共8页
Chinese Journal of Reparative and Reconstructive Surgery
关键词
背根神经节神经元
差速贴壁纯化
FBS
五氟尿嘧啶
阿糖胞苷
大鼠
Dorsal root ganglion neurons Differential attachment purification Fetal bovine serum 5-fluorouracil Cytrarabine Rat