作者：刘丽娟，李茂欣，汤旭磊，高林，朱任之
Expression of PDGFB in retina of rats with earlystage diabetic retinopathy
【Abstract】 AIM: To observe the expression of plateletderived growth factorB chain (PDGFB) mRNA in the retina of diabetic rats at the early stage of diabetic retinopathy. METHODS: A total of 60 healthy adult male Wistar rats were randomly divided into control and diabetic groups. Streptozotocin （60 mg/kg） was injected into the tail vein to induce diabetes in 35 rats in diabetic group, while 25 rats in control group were treated with the same volume of citric acid buffer solution. On the 3rd day after injection, the rats whose urine glucose was more than ++ and fasting blood glucose ≥16.7 mmol/L were chosen as laboratory objects. At the end of 1st, 2nd and 3rd months, 6 rats in both groups were randomly sacrificed and the retinas were dissected and total RNA was isolated for the detection of mRNA for PDGFB by semiquantitative reversetranscription polymerase chain reaction (RTPCR). The retinas of the other rats in both groups were taken and prepared as the ultrathin and observed by transmission electron microscope. RESULTS: The levels of mRNA encoding PDGFB in the retinas of diabetic rats were significantly higher than those in controls at each observed time point （P<0.05 or P<0.01 at the end of 1st and 3rd month）. Obvious ultrastructural damages, which were in relation to the course of diabetes, could be seen in the retinas of diabetic rats. CONCLUSION: Diabetes upregulates PDGFB mRNA levels in the retinas of diabetic rats in a timedependent manner.
【Keywords】 diabetic retinopathy； pericyte； plateletderived growth factorB chain； RTPCR
【摘要】 目的：探讨血小板源生长因子B（PDGFB）mRNA表达水平在糖尿病(DM)大鼠视网膜中的变化规律及其作用. 方法：雄性Wistar大鼠60只，随机分为正常对照组（25只）与造模组（35只）. 造模组以链脲佐菌素(STZ)尾iv，3 d后测定血糖≥16.7 mmol/L，尿糖以上者定为DM大鼠. 分别于造模成功后1，2，3 mo末各组随机取6只大鼠，采用半定量逆转录聚合酶链反应（RTPCR）方法检测PDGFB mRNA在大鼠视网膜中的表达水平；电镜观察DM大鼠视网膜超微结构的改变. 结果：随着病程的延长，视网膜中PDGFB mRNA表达水平在DM组明显升高，与正常对照组相比于造模后1 mo末时即差异具有统计学意义（P＜0.05），3 mo末时差异显著（P<0.01）；DM大鼠视网膜超微结构的损害随病程进展逐渐加重. 结论：PDGFB mRNA在视网膜中的表达水平随DM病程进展逐步升高.
【关键词】 糖尿病视网膜病变；周细胞；血小板源生长因子B； 逆转录聚合酶链反应
0引言
视网膜周细胞的渐进性功能丧失及死亡为糖尿病(diabetes melitus, DM)视网膜病变（diabetic retinopathy, DR）的早期典型病理特征. 周细胞对于保持微血管形态与功能的完整都有重要作用，缺乏周细胞将导致内皮细胞增生及血管形态异常［1］，并且促使DR进展［2］. 血小板源生长因子B（plateletderived growth factorB，PDGFB）是周细胞最重要的生长因子，与周细胞表达的受体PDGFRβ结合维持着周细胞的密度和数量. PDGFB可以显著减少因缺血缺氧导致的周细胞死亡、减少缺氧对体外培养的视网膜微血管的损害［3］. 因此，探讨PDGFB在早期DR中表达的变化规律及其作用，可为DR的早期检测、防治提供理论依据.
1材料和方法
1.1材料
雄性Wistar大鼠60只，体质量(200±20) g，由兰州军区总医院实验动物中心提供；链脲佐菌素（streptozotocin,STZ）（美国Alexis公司）；Trizol（BBI产品）；罗氏血糖仪及试纸条；PDGFB的引物为：5′CGCTCCTTTGATGACCTTC3′, 5′GCACTCGGCGATTACGG3′（长177 bp），βactin引物为：5′GAGGGAAATCGTGCGTGAC3′, 5′GGAGCCAGGGCAGTAATC3′（长351 bp），由大连宝生物公司合成；Takara一步法RTPCR试剂盒；PE 2400型PCR仪（Applied Biosystems公司）；UVIpro凝胶成像与分析系统（华粤公司）.
1.2方法
雄性Wistar大鼠60只，体质量(200±20) g，随机分为正常对照组（25只）与造模组（35只）. 造模组禁食12 h后，将STZ以0.1 mol/L，pH 4.4柠檬酸缓冲液溶解配制成20 g/L的溶液后以60 mg/kg一次性尾iv，正常对照组仅注射等体积的柠檬酸缓冲液. 3 d后尾静脉采血测定血糖≥16.7 mmol/L，尿糖以上者定为DM大鼠，分笼喂养，以后每 2 wk称体质量、测血糖2次取均值. DM组和对照组大鼠均喂养普通颗粒饲料，实验期间自由摄食饮水，自然昼夜照明. 于造模后1，2，3 mo末DM大鼠组随机取2只大鼠用作电镜观察. 摘取眼球、剥离视网膜组织切成15 mm×15 mm的小块, 25 g/L戊二醛固定2 h, 10 g/L锇酸后固定1 h, 梯度乙醇脱水, 环氧树脂包埋, 聚合LKB超薄切片机切片, 醋酸双氧铀及枸椽酸铅双重染色, 于日本JEM100CX透射电镜下观察. 分别于造模后1，2，3 mo末各组取大鼠6只，称体质量、测空腹血糖后处死轻摘眼球，剥离视网膜，每2只眼为一组，将分离的视网膜置于去RNA酶的冻存管中，液氮中保存待用. Trizol提取视网膜总RNA，测定纯度及含量，10 mL/L甲醛变性凝胶电泳测定其完整性. 按Takara一步法RTPCR试剂盒说明操作，反应条件均为逆转录过程50℃ 30 min； 94℃预变性 3 min；扩增94℃ 30 s; 50℃ 30 s; 72℃ 1 min; 30个循环；72℃延伸7 min. PCR产物以15 g/L琼脂糖电泳. 以UVIpro凝胶成像与分析系统检测电泳结果，以PDGFB与βactin PCR产物DNA条带的像素亮度总和之比作为反映PDGFB mRNA水平的相对指标.
统计学处理： 数据用SPSS 10.0进行统计学分析，采用成组设计资料两样本均数比较t检验, 结果以x±s表示. P＜0.05表示差异有统计学意义.
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