[1]张凤久,张丽敏,林安岭,等.Wistar大鼠视网膜神经元高糖模型葡萄糖浓度和培养时间的筛选[J].眼科新进展,2016,36(12):1113-1115.[doi:10.13389/j.cnki.rao.2016.0297]
　ZHANG Feng-Jiu,ZHANG Li-Min,LIN An-Ling,et al.Filtering glucose concentration and culture time of Wistar rat retinal neurons in high glucose model[J].Recent Advances in Ophthalmology,2016,36(12):1113-1115.[doi:10.13389/j.cnki.rao.2016.0297]
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Wistar大鼠视网膜神经元高糖模型葡萄糖浓度和培养时间的筛选
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
36卷
期数:
2016年12期
页码:
1113-1115
栏目:
实验研究
出版日期:
2016-12-05

文章信息/Info

Title:
Filtering glucose concentration and culture time of Wistar rat retinal neurons in high glucose model
作者:
张凤久; 张丽敏; 林安岭; 彭向东; 刘兵; 杨建玲; 于玲燕; 王海明
024000　内蒙古自治区赤峰市，赤峰学院附属医院眼科
Author(s):
ZHANG Feng-Jiu ; ZHANG Li-Min ;  LIN An-Ling; PENG Xiang-Dong ;  LIU Bing; YANG Jian-Ling; YU Ling-Yan; WANG Hai-Ming
Department of Ophthalmology,the Affiliated Hospital of Chifeng College,Chifeng 024000,Nei Monggol Autonomous Region,China
关键词:
大鼠视网膜神经元细胞; 高糖模型; 葡萄糖浓度; 培养时间
Keywords:
rat retinal neuronal cells; high glucose model;  glucose concentration;  incubation time
分类号:
Ｒ７７４
DOI:
10.13389/j.cnki.rao.2016.0297
文献标志码:
A
摘要:
目的　筛选Ｗｉｓｔａｒ大鼠视网膜神经元高糖模型的葡萄糖浓度及培养时间。方法　取出生１～３ｄＷｉｓｔａｒ大鼠分离视网膜神经元进行传代培养，尼氏染色法鉴定细胞；实验分５组：Ａ组培养液葡萄糖浓度为０ｍｍｏｌ·Ｌ－１（正常对照组），Ｂ组浓度为１０ｍｍｏｌ·Ｌ－１，Ｃ组浓度为１５ｍｍｏｌ·Ｌ－１，Ｄ组浓度为２５ｍｍｏｌ·Ｌ－１，Ｅ组浓度为３５ｍｍｏｌ·Ｌ－１。分别培养２４ｈ、４８ｈ、７２ｈ后用ＭＴＴ法检测各组细胞ＯＤ值，流式细胞仪检测各组细胞凋亡率。结果　倒置显微镜观察分离培养细胞，大部分细胞于接种２４ｈ后贴壁，少数细胞长出较短的突起，并有向中央聚集生长的现象。２～３ｄ后长出突起的神经元数目增多，突起长度增加，约为自身胞体长度的１倍。培养５～７ｄ后神经元突起进一步增多、变长。经尼氏染色后细胞质呈蓝紫色，尼氏小体颗粒状结构清晰。非神经元细胞胞质基本不着色，细胞核呈淡紫色、圆形，核仁清晰可辨，神经元率达９１％。各实验组在培养２４ｈ后ＯＤ值均低于正常对照组，但差异无统计学意义（Ｐ＞０．０５）。随培养时间延长ＯＤ值继续下降，４８ｈ后各组ＯＤ值比较差异有统计学意义（Ｐ＜０．０１）；其中Ｂ组及Ｃ组与Ａ组比较差异无统计学意义（均为Ｐ＞０．０５）；Ｄ组ＯＤ值明显下降，与Ａ组比较差异有统计学意义（Ｐ＜０．０５），Ｅ组下降更明显，与Ａ组比较差异有统计学意义（Ｐ＜０．０１）。７２ｈ后各组ＯＤ值比较差异有统计学意义（Ｐ＜０．０１）；其中Ｂ组及Ｃ组与Ａ组比较差异无统计学意义（均为Ｐ＞０．０５）；Ｄ组及Ｅ组与Ａ组比较差异有统计学意义（均为Ｐ＜０．０１）。各组在培养２４ｈ后视网膜神经元凋亡率差异无统计学意义（Ｐ＞０．０５）。４８ｈ后各组凋亡率比较差异有统计学意义（Ｐ＜０．０１）；其中Ｂ组及Ｃ组与Ａ组比较差异无统计学意义（均为Ｐ＞０．０５）；Ｄ组凋亡率明显升高，与Ａ组比较差异有统计学意义（Ｐ＜０．０５）；Ｅ组凋亡率升高更明显，与Ａ组比较差异有统计学意义（Ｐ＜０．０１），且与Ｄ组比较亦有统计学意义（Ｐ＜０．０５）。７２ｈ后各组凋亡率比较差异有统计学意义（Ｐ＜０．０１）；其中Ｂ组及Ｃ组与Ａ组比较差异无统计学意义（均为Ｐ＞０．０５），Ｄ组及Ｅ组与Ａ组比较差异有统计学意义（均为Ｐ＜０．０１）。结论　葡萄糖浓度为２５ｍｍｏｌ·Ｌ－１培养４８ｈ是视网膜神经元高糖模型最佳葡萄糖浓度及干预时间。
Abstract:
Objective To filter glucose concentration and culture time of Wistar rat retinal neurons in high glucose model. Methods The retinal neurons of Wistar rat with l - 3 day old were isolated to passage culture,and the cells were identified using Nissl stairung. The experiment was divided int0 5 groups:The glucose concentration in the culture solution of group A was o mmol . L’l ( control group) , group B was 10 mmol . L’I ,group C was 15 mmol . L" ,group D was 25 mmol . L-’,and group E was 35 mmol . L’l ,respectively. The cell viability was detected by MTT assay,and the apoptotic rat was measured by flow cytometry at 24 hours,48 hours and 72 hours,respectively. Results The inverted microscope was used to observe the isolation and culture of cells,most of the cells were attached to the wall after inoculation with 24 hours,and a few cells grew out of the short neurite with the phenomenon of growing to the central. After 2 - 3 days,the number of neurons with neuritis increased,and the length of neuritis increased,which was about twice of the length of cell. After 5 - 7 days,the neuritis were more increased and long. After the staining,the cytoplasm was blue and purple, and the granular structure was clear. Non neuronal cells were without coloring,the nuclei were pale purple round,nucleolus were clear,the proportion of neurons was 91% . OD values of the experimental group showed no significant difference compared with group A when cultured 24 hours( P > 0. 05 ) ; OD value of group B and group C had no significant difference compared with group A at 48 hours,72 hours ( all P > 0. 05) ;OD value of group D had the siõÂificant difference compared with goup A at 48 hours ( P < 0. 05) ,and OD value of goup E had the sigruficant difference compared with group A at 48 hours( P < 0.01) ;There was a significant difference in OD value of group D and group E compared with group A at 72 hours ( P < 0. 01). There was no significant difference in the apoptotic rate of each experimental group compared with group A at 24 hours ( P > 0. 05 ) ,and there was no significant difference in the apoptotic rate of group B and group C compared with group A at 48 hours,72 hours ( all P > 0. 05) ;There was significant difference of group D compared with group A at 48 hours ( P < 0. 05 ) ,there was significant difference of group E compared with group A at 48 hours ( P < 0. 01) ,and there was sigruficant difference compared with group D( P < 0.05) ;There was siguficant difference of group D and group E compared with group A at 72 hours( P < 0. 01). Conclusion For high sugar model of retinal neurons,the best glucose concentration is 25 mmol . L’l ,and the best incubation time is 48 hours.

备注/Memo

备注/Memo:
国家自然科学基金地区项目（编号：６１４６５００１）
更新日期/Last Update: 2016-12-19