【作者】 植玉婷；

【导师】 张庆平；

【作者基本信息】 广西医科大学 ， 眼科学， 2007， 硕士

【摘要】 目的:观察并比较不同浓度酸性成纤维细胞生长因子(acidic fibroblastgrowth factor,aFGF)及其改构体(Modified acidic fibroblast growthfactor,MaFGF)对培养大鼠视网膜神经节细胞(retinal ganglion cells,RGCs)生长、存活的影响,并比较研究aFGF的神经保护活性对促有丝分裂活性的依赖程度方法:采用胰酶消化法将30只生后3天的Sprague-Dawley大鼠视网膜制成细胞悬液后接种于96孔板进行细胞培养。通过Thy-1单克隆抗体免疫组织化学检查法对培养的RGCs进行鉴定,进行免疫阳性细胞计数,计算有轴突生长细胞的百分率。实验分为对照组(DMEM培养液)、25ng/ml、100ng/ml、400ng/ml aFGF组(A1组、A2组、A3组)和25ng/ml、100ng/ml、400ng/ml MaFGF组(Bl组、B2组、B3组),记录RGCs存活时间,并将培养3天、5天、7天的RGCs行四甲基偶氮唑盐(methylthio-terazole.MTT)法测量吸光度(A)值。结果:①Thy-1单克隆抗体免疫组织化学检查显示培养第3天,大部分存活细胞为免疫阳性,培养至第5天、第7天后的存活细胞中免疫阳性细胞数渐渐减少。培养第3天时,实验各组和对照组比较,免疫阳性细胞数及有轴突生长的细胞比率均无明显差异(均P＞0.05);培养第5天、第7天时实验各组免疫阳性细胞数及有轴突生长的细胞比率均比对照组较高,差异均有统计学意义(均P＜0.05)。②细胞存活期间各实验组和对照组细胞大小及形态均无明显差别,实验组比对照组细胞存活时间长3—4天。③培养第3天、第5天和第7天MTT法测量吸光度(A)值:培养第3天时,A、B各浓度组A值与对照组比较均无显著性差异(均P＞0.05);培养第5天和第7天时,A、B各浓度组A值均比对照组较高,差异均有统计学意义(均P＜0.01);相同培养天数的A1组与A2组比较,及A2组与A3组比较,A2比A1、A3较高,差异均有统计学意义(均P＜0.05);A1组与A3组比较,无显著性差异(P＞0.05);相同培养天数的Bl组与B2组比较,及B2组与B3组比较,B2比B1、B3较高,差异均有统计学意义(均P＜0.05);B1组与B3组比较,无显著性差异(P＞0.05);相同浓度的aFGF各组与MaFGF各组比较,在相同培养天数时差异均无统计学意义(均P＞0.05)结论:①aFGF及MaFGF均能促进培养大鼠RGCs的存活,并能延长RGCs的存活时间,且对RGCs的大小、形态无影响。②aFGF保护及促进RGCs存活的作用不依赖其有丝分裂活性。更多还原

【Abstract】 Obstract: To observe the effect of acidic fibroblast growth factor (aFGF) and modified acidic fibroblast growth factor (MaFGF) with different concentrations on the growth and surival of rat’s retinal ganglion cells (RGCs) in vitro.Methods: The retinaes of 30 Sprague-Dawley rats which were 3 days after birth were dissociated into suspension with 0.25%trypin digestion. The cultured RGCs were identified with immunohistochermistry method using anti-r at Thy-1 monoclonal antibody after cultured 3,5 and 7 days. Then we counted the number of the cell of immuntional positive and the rate of cells with axon group ing .Cultured RGCs were divided into control group , the 25,100,400ng/ml aFGF group (A1,A2 and A3 group) and MaFGF group (B1,B2 and B3 group) respectively. The duration of living RGCs were recored. The A value of living cells was tested by methylthio-tetrazole colorimetric microassay After the RGCs were cultured for 3,5,7 days.Result: (1) The immunohistochermistry examination showed that most of living cells cultured for 3 days were RGCs. After 5,7 days,the number of RGCs was decreased. In culture for 3 days ,no different of the number of RGCs and the rate of the cells had axon was observed in control group , aFGF groups and MaFGF groups (P>0.05);but has differern in culture 5 and 7 days(P<0.05). (2) No volume increase and shape change of RGCs were observed in all groups. The duration of the living RGCs was prolonged 3 to 4 days in aFGF groups and MaFGF groups compared with the control group. (3)We measured the A valure of living RGCs cultured for 3,5,and 7 days:at 3rd days the different among aFGF groups , MaFGF groups and control group were insignificant(all P>0.05);but at 5th days and 7th days ,there had significant different among aFGF groups, MaFGF groups and control group (all P<0.01),the A values of aFGF groups , MaFGF groups were all higher than control group;in aFGF groups, at the same culture days,the A values of A2 group was higher than Al and A2 group,the differern were were significant(all P<0.05);the different between Al group and A3 group were insignificant(P>0.05);in MaFGF groups ,at the same culture days,the A values of B2 group was higher than B1 and B2 group,the differern were significant(all P<0.05);the different betweenB1 group and B3 group were insignificant(P>0.05); the different among the same concentrations of aFGF group and MaFGF group at the same culture days were insignificant(P>0.05).Conclusion: (1)aFGF and MaFGF both could facilitate survival of RGCs in vitro and prolong the duration of living RGCs. aFGF and MaFGF have no effect on the conformation and volume of RGCs. (2)The function of protect and facilitate survival of RGCs of aFGF is not depend on it’s mitosis activity.更多还原