糖尿病大鼠视网膜早期病变中PDGF

1、糖尿病大鼠视网膜早期病变中PDGF作者：时间：2007-11-22 11:27:00 作者：刘丽娟，李茂欣，汤旭磊，高林，朱任之【关键词】 糖尿病视网膜病变 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

2、 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 vo

3、lume 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 dissec

4、ted 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 o

5、f mRNA encoding PDGFB in the retinas of diabetic rats were significantly higher than those in controls at each observed time point （P0.05 or P0.01 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 diab

6、etic 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

7、（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（P0.05），3 mo末时差异显著（P16.7 mmol/L 26只, 食量、饮水量及尿量显著增加，尿糖均在以上；同时体质量增长速度显著减缓，并出现皮毛枯黄、烂

8、尾，行动缓慢、反应迟钝.2.1视网膜超微结构观察正常对照组大鼠视网膜毛细血管内皮细胞、周细胞的核膜完整，染色质分布均匀，细胞器及核形态正常，基底膜连续（Fig 1A）. DM 1 mo组可见周细胞水肿,线粒体肿胀、嵴消失及结构紊乱，但毛细血管内皮细胞及基底膜未见明显改变（Fig 1B）. DM 2 mo组可见周细胞病变进一步加重，毛细血管内皮细胞线粒体肿胀，微绒毛形成（Fig 1C）. DM 3 mo组可见周细胞线粒体肿胀明显，嵴消失，甚至出现空泡性变，核染色质浓缩、边集，其他细胞器减少；内皮细胞水肿、变形,向血管腔内有指状突起, 微绒毛增多；毛细血管基底膜增厚，电子密度增大（Fig 1D）.

9、2.2PDGFB mRNA的表达量DM组大鼠视网膜中PDGFB mRNA的表达量(Fig 2)与同龄正常大鼠相比在1 mo病程时即增高，有统计学意义(P0.05); 在2 mo病程组仍继续增高(P0.05); 于3 mo病程末时PDGFB mRNA的表达量显著高于同龄正常对照组大鼠(P0.01, Tab 1).表1糖尿病大鼠视网膜PDGFB mRNA 的相对表达量(略)3讨论PDGFB与同一家族的血管内皮生长因子（vascular endothelial grouth factor,VEGF)相似，在视网膜缺氧及血管功能障碍时表达显著增加，二者共同参与了缺氧性视网膜病变.BB的合成便相应增高，

10、通过与特异性受体PDGFR结合发挥生物学功能. 本实验中PDGFB mRNA表达在1 mo时已增高，与Yokota等4研究结果一致；并且PDGFB mRNA表达随病程延长而持续升高，因此我们认为DR早期周细胞的破坏、丧失并不是由于PDGFB表达的降低所造成的. Hammes等5发现,与野生型啮齿动物相比，敲除PDGFB单等位基因的啮齿动物的视网膜中周细胞的数量下降30%；而以STZ诱发DM后其视网膜中周细胞减少50%，由此可见，缺少PDGFB足以使视网膜周细胞减少. 促使PDGFB在早期DR中表达持续增高的原因可能有如下几方面：高血糖及糖基化产物使内皮细胞产生PDGF增加；DR时眼内局部增高的

11、血管紧张素通过诱导周细胞自分泌释放及激活PKC、升高胞质钙浓度促进血管内皮细胞产生PDGFB增多；缺氧、凝血酶及各种细胞因子也可刺激视网膜细胞合成PDGF增加等. 另外还有一方面原因可能是视网膜的反馈性调节，DM状态促使周细胞丧失，视网膜必须代偿性高表达PDGFB以维持周细胞的数量、血管的正常形态与功能. 故PDGFB可作为DR早期诊断及病程进展的检测指标.PDGFB表达水平升高在早期对维持周细胞的存活可能是有利的，但PDGFB在DR中上调其他生长因子的表达水平，如VEGF，ET1等,促进视网膜微循环紊乱和新生血管的形成，这可能与作用部位及DR发展阶段有关. PDGFB对视网膜周细胞有独特的保

12、护作用，并且研究发现在缺氧性视网膜疾病时抑制PDGF会促使周细胞的丧失6. 因此，对于PDGFB这个可能对DR有双向调节作用的生长因子，我们认为不宜在PDGFB代偿性升高的阶段（即早期）使用PDGFB的抑制剂，那么，如何找到一个方法，既保护视网膜周细胞又能够有效抑制PDGFB高表达所引起的视网膜病变加剧，是值得进一步研究的.【参考文献】1 Hellstrom M, Gerhardt H, Kalen M, et al. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis J

13、. J Cell Biol,2001; 153(3):543-553.2 Enge M, Bjarnegard M, Gerhardt H, et al. Endotheliumspecific plateletderived growth factorB ablation mimics diabetic retinopathy J. EMBO J, 2002; 21(16): 4307-4316.3 Kodama T, Oku H, Kawamura H, et al. Plateletderived growth factorBB: A survival factor for the re

14、tinal microvasculature during periods of metabolic compromise J. Curr Eye Res, 2001; 23(2): 93-97.4 Yokota T, Ma RC, Park JY, et al. Role of protein kinase C on the expression of plateletderived growth factor and endothelin1 in the retina of diabetic rats and cultured retinal capillary pericytes J. Diabetes, 2003; 52: 838-845.5 Hammes HP, Lin J, Renner O, et al. Pericytes and the pathogenesis of diabetic retinopathy