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  

Filtering glucose concentration and culture time of Wistar rat retinal neurons in high glucose model

作　　者：张凤久[1] 张丽敏[1] 林安岭[1] 彭向东[1] 刘兵[1] 杨建玲[1] 于玲燕[1] 王海明[1] 

机构地区：[1]赤峰学院附属医院眼科,内蒙古自治区赤峰市024000

出　　处：《眼科新进展》2016年第12期1113-1115,共3页Recent Advances in Ophthalmology

基　　金：国家自然科学基金地区项目(编号:61465001)~~

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1~3 d Wistar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0 mmol·L^(-1)(正常对照组),B组浓度为10 mmol·L^(-1),C组浓度为15 mmol·L-1,D组浓度为25 mmol·L^(-1),E组浓度为35 mmol·L^(-1)。分别培养24 h、48 h、72 h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24 h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。2~3 d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5~7 d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24 h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,48 h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)。72 h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24 h后视网膜神经元凋亡率差异无统计学意义(P>0.05)。48 h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)。72 h后各组凋亡率比较差异有统计学意义(P<0Objective To filter glucose concentration and culture time of Wistar rat retinal neurons in high glucose model. Methods The retinal neurons of Wistar rat with 1 -3 day old were isolated to passage culture, and the cells were identified using Nissl staining. The experiment was divided into 5 groups ： The glucose concentration in the culture solution of group A was 0 mmol ·L^-1 （control group）, group B was 10 mmol·L^-1 ,group C was 15 mmol ·L^-1 ,group D was 25 mmol ·L^-1 ,and group E was 35 mmol ·L^-1, respectively. The cell viability was detected by MTT assay, and the apop- totic rat was measured by flow cytometry at 24 hours ,48 hours and 72 hours, respective- ly. Results The inverted microscope was used to observe the isolation and culture of cells, most of the ceils 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 neuri- tis 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 nu- clei 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 val- ue of group D had the significant difference compared with group A at 48 hours （P 〈 0. 05 ）, and OD value of group E had the significant 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 inthe apoptotic rate of each ex

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 

分 类 号：R774[医药卫生—眼科]