【摘要】 目的:观察糖尿病早期大鼠，视网膜神经细胞凋亡与caspase3表达变化的关系。方法:健康雄性8周龄SD大鼠36只，建立糖尿病大鼠模型18只，正常对照18只。再各自分为4，8，12wk 3组，每组6只大鼠。TdT介导DNA缺口末端的dUTP标记(TUNEL)法检测视网膜神经细胞的凋亡程度;免疫组织化学方法检测半胱氨酸天冬氨酸蛋白酶3(caspase3)蛋白表达水平，实时荧光定量聚合酶链反应(PCR)法检测caspase3 mRNA表达情况。结果:正常对照组4，8，12wk大鼠均未见视网膜神经细胞凋亡。糖尿病组大鼠于建模后4wk即出现视网膜神经细胞凋亡，8wk和12wk大鼠视网膜神经凋亡程度较同期正常对照组大鼠均明显增强。神经细胞凋亡主要位于视网膜神经节细胞层及内核层。正常对照组4，8，12wk大鼠视网膜均未见caspase3蛋白阳性表达，糖尿病组大鼠4wk即可见caspase3蛋白阳性表达，8和12wk时表达增强。正常对照组4,8,12wk大鼠caspase3 mRNA表达RQ值分别为1.6±0.6，1.5±0.5，1.6±0.3;糖尿病组4，8，12wk大鼠caspase3 mRNA表达RQ值分别为5.7±1.2，12.6±2.3，14.3±2.1;较同期对照组均明显升高，差异有统计学意义(P<0.05)。结论:糖尿病早期视网膜神经细胞凋亡可能与capase3表达增强有关。
【关键词】 糖尿病;视网膜;神经细胞;凋亡;半胱氨酸天冬氨酸蛋白酶
Relationship between the apoptosis of retinal neurons and change of caspase3 expression in early diabetic rats
XiaoYan Li, MaoNian Zhang, YuLi Pi
1Department of Ophthalmology, the First Hospital Affiliated to Chinese PLA General Hospital, Beijing 100048, China;2Department of Ophthalmology, General Hospital of Chinese PLA, Beijing 100853, China
Correspondence to:MaoNian Zhang.Department of Ophthalmology, General Hospital of Chinese PLA, Beijing 100853, [email protected]
AbstractAIM: To probe the relationship between the apoptosis of diabetic rats retinal neurons and change of caspase3 expression in early diabetic rats. METHODS: Eighteen male 8weekaged SD rats were injected with a single dose of streptozotocin (60mg/kg) to induce the diabetic model, and then they were divided into 3 groups at 4, 8 and 12 weeks (each group, n=6). Another 18 male 8weekaged SD rats were the normal control group, and were raised at the same time without any intervertion, also divided into 3 groups at 4, 8 and 12 weeks (each group, n=6). Apoptosis of retinal neurons was detected by TdTmediated dUTP nick end label (TUNEL) assay. The protein expression of caspase3 was detected by immunohistochemistry. Caspase3 mRNA levels were determined by SYBR Green Realtime PCR Master Mix. RESULTS: There were no apoptosis neurons in control groups retina at 4, 8, 12 weeks. Apoptosis of the retinal neurons occurred 4 weeks after the onset of diabetes, and the apoptosis degrees were significantly higher than that of agematched control groups at 8 and 12 weeks. The apoptosis of the retinal neurons located in the rentinal ganglion cells layer (GCL) and the inner nuclear layer (INL). Caspase3 protein expression was not observed in the control rats retina at 4, 8, 12 weeks. Positive staining of caspase3 occurred 4 weeks after the onset of diabetes, and enhanced at 8 and 12 weeks. In control rats at 4, 8 and 12 weeks, caspase3 mRNA levels were 1.6±0.6, 1.5±0.5, 1.6±0.3 respectively. Caspase3 mRNA levels in diabetic rats that had diabetes for 4, 8 and 12 weeks were 5.7±1.2, 12.6±2.3, 14.3±2.1 respectively, which were greater than that in the control groups (P<0.05). CONCLUSION: The apoptosis of the retinal neurons in diabetic rats may be related with the enhance of the caspase3 expression.
KEYWORDS: diabetes; retina; neuron; apoptosis; capsase3
0引言
糖尿病视网膜病变(diabetic retinopathy, DR)是糖尿病最常见的慢性并发症之一，以往，众多学者认为DR是微血管病变，其视功能的下降是由于微血管的损伤。然而，近年来越来越多的研究表明，DR不仅存在视网膜微血管病变，同时也有视网膜神经元的损害[1，2]，主要表现为高血糖引起的视网膜神经细胞凋亡。因而，探讨DR神经病变的发病机制，从而寻找安全有效的神经保护措施，对于提高糖尿病患者的视功能状态具有重要意义。
1材料和方法
1.1材料
实验用封闭群雄性健康SpragueDawley(SD)大鼠36只， 8周龄，军事医学科学院实验动物中心提供。随机抽取18只大鼠诱导糖尿病大鼠模型，建模成功后再于建模后4，8，12wk 3个时间点分为3组，每组各6只大鼠。另外18只大鼠作为正常对照组不进行干预，与糖尿病组大鼠同时饲养，也按糖尿病组相同时间点分为4，8，12wk 3组，每组各6只大鼠。每组6只大鼠中，取双眼：左眼眼球制作石蜡组织病理学切片行TUNEL检测及caspase3免疫组化检测;右眼眼球提取视网膜组织RNA后行基因检测。链脲佐菌素(streptozotocin，STZ)(美国Sigma公司);原位细胞凋亡(TUNEL)检测试剂盒、兔抗鼠半胱氨酸天冬氨酸蛋白酶3(caspase3)多克隆抗体(武汉博士德生物技术有限公司);SYBR Green Realtime PCR Master Mix试剂:引物(北京奥科生物技术有限公司合成)：大鼠caspase3序列[3](U49930)：5AAT TCA AGG GAC GGG TCA TG3(上游)，5GCT TGT GCG CGT ACA GTT TC3(下游);大鼠βactin序列[4](V01027)：5CCT GCT TGC TGA TCC ACA3(上游)，5CTG ACC GAG CGT GGC TAC3(下游)。柱式动物RNA提取试剂盒(天泽基因工程有限公司)，逆转录试剂盒(Promega公司)，Real Master Mix(SYBR Green)(天根生化科技公司)。
1.2方法
STZ按60mg/kg一次性ip诱导糖尿病模型。模型建立标准：给药后48h剪尾法测血糖、尿糖试纸测尿糖，血糖>16.7mmol/L，尿糖+++以上者为糖尿病大鼠模型建立成功;给药后48h测血糖，均>16.7mmol/L，尿糖均在++以上。实验过程中，无血糖自行恢复者。每日饮水量、尿量明显多于同期对照组，随鼠龄增加体重初期改变不明显或略有增加，之后呈逐渐下降趋势，最终呈现消瘦状态。100g/L水合氯醛麻醉大鼠，快速取出左眼眼球固定于40g/L多聚甲醛固定液中15～20min后，从角膜缘剪开眼球、小心去除眼前节和部分玻璃体，余下“眼杯”再次固定2h左右，依次梯度乙醇脱水、二甲苯透明后浸蜡包埋。连续4μm切片，切片位置选择在距视盘2mm处，用于caspase3免疫组织化学染色和TUNEL法凋亡细胞检测。
1.2.1视网膜神经细胞凋亡的检测
石蜡切片常规脱蜡至水化;30mL/L H2O2室温处理10min;Proteinase K 37℃消化10min;取TdT和DIGdUTP各1μL,加入18μL标记缓冲液混匀后滴加至标本片，37℃湿盒内标记2h;加封闭液室温处理30min;用抗体稀释液1∶100稀释生物素化抗地高辛抗体，混匀后加至标本片，37℃湿盒内反应30min;用抗体稀释液1∶100稀释SABC，混匀后加至标本片，37℃湿盒内反应30min;DAB显色;Mayer苏木素轻度复染;水洗、脱水、透明、封片，显微镜观察。
1.2.2 caspase3表达的检测
石蜡切片常规脱蜡至水化;30mL/L H2O2抑制内源性过氧化物酶;将切片浸入0.01mol/L枸橼酸钠缓冲液(pH=6.0)95℃水浴10min，自然冷却后滴加复合消化液修复抗原;滴加正常山羊封闭血清;滴加1∶300稀释的兔抗鼠caspase3多克隆抗体4℃过夜;生物素化山羊抗兔IgG 37℃孵育30min;辣根酶标记链霉卵白素试剂37℃孵育15min;AEC显色剂室温下显色;Mayer苏木素衬染细胞核;水溶性封片剂封片，显微镜观察。另麻醉后的大鼠，迅速摘除右眼眼球，显微镜下剥离视网膜，标记分装至冻存管中置于80℃冰箱保存待测。视网膜组织总RNA的提取：采用柱式动物RNA提取方法。所有实验用品均预先进行去RNA酶处理，整个过程在冰浴中进行。逆转录：采用逆转录酶及随机引物进行逆转录，严格按Promega公司逆转录试剂盒说明书操作。Real Master Mix(SYBR Green) RealTime PCR的扩增步骤：采用高温启动法进行实时荧光定量PCR循环，扩增的每个标准均作内参对照，每个指标均有正常组作对照。7500型荧光定量PCR仪进行PCR循环扩增(40个循环)，循环条件(两步法，退火/延伸温度60℃)。取相对定量RQ值作结果进行统计分析。
统计学分析:应用SPSS 13.0统计软件进行数据分析。本研究中的计量资料以均数±标准差(±s)表示，正常对照组与糖尿病组大鼠各时间点视网膜caspase3 mRNA表达水平采用两因素方差分析，以P<0.05作为差异有统计学意义。
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