摘要
目的探讨前列地尔对体外培养类缺血再灌注损伤模型视网膜神经节细胞(retinal ganglion cells,RGC)生长的影响及其作用机制。方法以原代培养SD大鼠RGC为研究对象,将培养的细胞随机分为正常组、模型组、模型+前列地尔组。建立类缺血再灌注损伤模型,模型+前列地尔组在缺血期与再灌注期均加入浓度分别为15μg·L-1、45μg·L-1、135μg·L-1前列地尔。类缺血模型建立后24h、48h、72h应用MTT比色法分别测定各组细胞的吸光度值(A值),确定前列地尔的最佳有效作用浓度与时间;采用荧光定量PCR方法分别测定各组细胞中Bax、Bcl-2的相对表达量。结果 RGC培养48h后,可见RGC伸出突起,随着培养时间的延长,细胞突起变长,部分细胞突起相互连接。使用抗大鼠Thy-1单克隆抗体对RGC进行细胞免疫组织化学检测,可见RGC胞浆及轴突染成棕黄色。模型建立后24h、48h、72h时,与正常组、模型+前列地尔组比较,模型组A值均明显降低,差异均有统计学意义(均为P<0.05);模型建立后24h时,模型+45μg·L-1前列地尔组A值与正常组差异无统计学意义(P>0.05),而正常组、模型+45μg·L-1前列地尔组与模型+15μg·L-1前列地尔组、135μg·L-1前列地尔组比较,差异均有统计学意义(均为P<0.05),模型+15μg·L-1前列地尔组与模型+135μg·L-1前列地尔组组间差异无统计学意义(P>0.05);模型建立后48h、72h时,正常组、模型+前列地尔组之间A值差异均无统计学意义(均为P>0.05)。正常组Bcl-2的相对表达量为0.999±0.035,模型组为0.657±0.012,模型+45μg·L-1前列地尔组为1.715±0.016(P<0.05);正常组Bax相对表达量为1.001±0.048,模型组为1.712±0.089,模型+45μg·L-1前列地尔组为1.508±0.061(P<0.05)。结论前列地尔对体外培养类缺血再灌注损伤模型RGC具有保护作用;前列地尔可能通过上调细胞内Bcl-2的表达,同时下调Bax的表达,进而发挥细胞保护作用。
Objeetive To explore the effects of alprostadil on retinal ganglion cells（RGC） in vitro induced by ischemia-like-reperfusion and its mechanisms. Methotis Primary cultured Sprague-Dawley rats＇ RGC were randomly divided into three groups：normal group,ischemia-like-reperfusion group and alprostadil group（ RGC was cultured with 15 μg · L^-1 ,45μg·L^-1 and 135μg · L^-1 alprostadil during the ischemialike-reperfusion）. The A values of living RGC in each group were tested by MIT at 24 hours,48 hours and 72 hours after reperfusion. The most effective concentration of alprostadil and the best time were determined. The Bax and Bcl-2 of RGC in each group were assayed by real-time fluorescence quantitative PCR.Results RGC cultured for 48 hours presented short protuberance, the protuberance became longer with prolong of culture time, and part of the protuberance contacted with each other. The cytoplasm and protuberance of RGC appeared brown-yellow staining treated with anti-rat Thy-1 monoclonal antibody detected by cell immunohistochemistry. Compared with normal group and alprostadil group,the A value of living RGC in the ischemia-like-reperfusion group was evidently lower at 24 hours,48 hours and 72 hours after reperfusion,and there were significant differences between them （ all P 〈 0. 05 ）. At 24 hours after ischemla and reperfusion,the A values of living RGC in the normal group and alprostadil（45 μg·L^-1 ） group had no statistical difference（P 〉 0.05 ）, there were statistical differences among normal grouP,talprostadil（ 15 μg·L^-1） group and alprostadil （135 μg·L^-1）group（ all P 〈0.05 ） ,and there was no significant difference between talprostadfl（ 15 μg·L^-1 ） group and alprostadil （ 135 μg·L^-1 ） group （P 〉 0.05 ）. At 72 hours after ischemia and reperfusion, the A values of living RGC in normal group and alprostadil group had no statistical difference（P 〉 0. 05 ）. The relative quantity of Bcl-2 in normal group was 0. 999± 0. 035, ischemia-like-reperfusion group 0.657±0. 012,alprostadil（45 μg·L^-1） group 1. 715±0. 016,and there was significant difference among them （P 〈 0.05 ）. The relative quantity of Bax in normal group was 1. 001 ± 0. 048, ischemia-like-reperfusion group 1. 712 ±0.089,alprostadil（45 μg·L^-1） group 1. 508 ± 0. 061, and there was significant difference among them（P 〈 0. 05）. Conclusion Alprostadil can protect RGC in vitro induced by ischemia-like-reperfusion; Alprostadil can protect RGC orobably by increasing the exoression of Bcl-2 and decreasing the expression of Bax.
出处
《眼科新进展》
CAS
北大核心
2013年第5期423-426,共4页
Recent Advances in Ophthalmology
基金
广西自然科学基金资助(编号:2010GXNSFA013171)~~
