大鼠视网膜神经节细胞存活和突起生长与可溶性混合晶状体蛋白的促进作用:体外培养实验

Survival of rat retinal ganglion cell and growth of its processes in relation to the promoting effect of soluble mixed crystallin:an in vitro culture experiment

目的:观察不同培养基中血清和混合晶状体蛋白对视网膜神经节细胞的影响,探讨晶状体损伤后其促进视网膜神经节细胞存活和再生的作用。方法:实验于2004-04/07在解放军第三军医大学西南眼科医院眼科实验室完成。选择成年LongEvens大鼠8只用于可溶性混合晶状体蛋白的提取;选择新生0～2dLongEvens大鼠30只用于视网膜神经节细胞的培养。分别用下列3组培养基对离体视网膜神经节细胞进行培养:①100mL/L新生牛血清+900mL/LDMEM/F12培养基(新生牛血清+DMEM/F12组)。②100mL/L新生牛血清+100mL/L胎牛血清+800mL/LDMEM/F12培养基(新生牛血清+胎牛血清+DMEM/F12组)。③100mL/L胎牛血清+900mL/LDMEM/F12培养基(胎牛血清+DMEM/F12组)。每组又分为6个亚组,其中5个亚组分别加入5种不同终浓度的可溶性混合晶状体蛋白(1,0.5,0.02,0.01,0.005mg/L)溶液,一个亚组不加可溶性混合晶状体蛋白液。每天在倒置相差显微镜下观察细胞生长情况,记录存活细胞数和存活时间。培养2,4,6d后,计数每视野分散的、有突起生长的视网膜神经节细胞数和最长突起长度(每一视野随机取5个细胞进行测量,共30个视野,150个突起)应用LeicaQWin图像分析系统。结果:①视网膜神经节细胞存活时间:新生牛血清+DMEM/F12组培养细胞能存活六七天,新生牛血清+胎牛血清+DMEM/F12组和胎牛血清+DMEM/F12组培养细胞能存活七八天。②有突起生长的视网膜神经节细胞数目和最长突起长度:在相同培养天数(2,4,6d)和条件下(加入同一终浓度晶状体蛋白)新生牛血清+胎牛血清+DMEM/F12组细胞数目均多于新生牛血清+DMEM/F12组犤加入0.5mg/L晶状体蛋白,2d时(3.83±4.53比0.14±0.38)个/视野,(4d时14.7±9.62比2.5±3.89)个/视野,P<0.01;6d时(20.13±12.32比5.43±5.62)个/视野,P<0.05犦;在4d内胎牛血清+DMEM/F12组细胞突起长度明显优于新生牛血清+胎牛血清+DMEM/F12组(P<0.01);加入混合晶状体蛋白后细胞存活时间及突起长度明显长于未加晶状体蛋白组,晶状体蛋白液含量为0.01mg/L时,作用较明显。结论:加入晶状体蛋白液和不同血清的培养基两因素间无交互作用,分别独立地发挥作用。可溶性混合晶状体蛋白可以促进体外培养的视网膜神经节细胞存活和突起生长。

AIM: To observe the effects of blood serum and crystalline on retinal ganglion cell (RGCs )in different culture medium in order to probe into its promoting effect on survival and regeneration of RGCs following the crystalline lens injuring. METHODS:The experiment was conducted in the Laboratory of Ophthalmology, Southwest Ophthalmology Hospital, Third Military Medical University of Chinese PLA from April to July 2004. Eight Long Evens rats were used for the extraction of dissoluble mixed crystalline; and 30 newly born 0-2 day Long Evens rats were used for culturing retinal ganglion cells. The RGCS were cultured with the following 3-group culture medium: ①100 mL/L newly born calf serum +900 mL/L DMEM/F12(newly born calf serum+DMEM/F12 group).②100 mL/L newly born calf serum+100 mL/L fetal calf serum +800 mL/L DMEM/F12(newly born serum+fetal calf serum+DMEM/F12 group). ③100 mL/L fetal calf serum+900 mL/L DMEM/F12(fetal calf serum+DMEM/F12 group).Each group was divided into 6 subgroups, among which, 5 different concentrations of dissoluble mixed crystalline liquid(1,0.5,0.02,0.01,0.005 mg/L) were divided into 5 subgroups,1 subgroup without dissoluble mixed crystalline liquid. The number of the survival cells and survival time were recorded following the cells were observed under the reverse phase microscope every day. The number of RGCs with process and length of the longest process were measured with Leica Qwin image analysis system after cultured for 2, 4, 6 days (5 cells chosen randomly per vision were measured, totally 30 visions,150 processes). RESULTS: ①The survival time of the RGCs : the cultured cells in newly bone serum+DMEM/F12 group could survive for 6 to 7 days and those in the newly born calf serum+fetal calf serum+DMEM/F12 group and fetal calf serun+DMEM/F12 group could survive 7 to 8 days .②The number of RGCs with process and the length of the longest process: At the same cultured days(2, 4 and 6 days) and condition (the same concentration of crystallin at last), the number of RGCs in the newly calf serum+fetal calf serum+DMEM/F12 group was all larger than that in the newly born serum+DMEM/F12group[adding 0.5 mg/L crystallin,(3.83±4.53) vs (0.14±0.38) per vision at day 2,(14.7±9.62)vs(2.5±3.89)per vision at day 4 , P< 0.01;(20.13±12.32) vs (5.43±5.62) per vision at day 6,P< 0.05]; The length of process in the fetal calf serum+DMEM/F12 group was longer significantly than that in the newly calf born serum+fetal calf serum+DMEM/F12 group in 4 days(P< 0.01);The survival time and the length of the process in the group with mixed crystallin were superior to those in the group without crystallin. The effect of crystallin was obvious when its concentration was 0.01 mg/L. CONCLUSION:Interaction does not exist in the two groups after the crystallin and different culture mediums were addeds respectively. Crystallin and different culture mediums exert their effects independently. Dissoluble mixed crystallin can promote the survival and the growth of process of GRCs cultured in vivro.