microRNA在糖尿病大鼠视网膜中表达差异谱的研究

发布时间：2018-06-24 05:48

本文选题：microRNA + 糖尿病视网膜病变　；参考：《第二军医大学》2010年博士论文

【摘要】： 目的：观察链脲佐菌素(STZ)诱导的糖尿病(DM)大鼠早期视网膜的形态学改变,评价其作为早期糖尿病视网膜病变(DR)动物模型的应用价值；测定DM大鼠视网膜VEGF mRNA和PEDF mRNA于不同时间点的表达情况,探讨二者间的相互关系及其对于DR发生发展的相关意义,并从细胞因子角度进一步评价STZ-DM大鼠作为早期DR动物模型的应用价值。以lOw STZ-DM大鼠作为早期DR动物模型,检测视网膜microRNAs (miRNAs)的表达差异谱,并初步探讨其在DR发生发展过程中的作用。方法： 64只体重180±20g雄性SD大鼠随机分为CON组和DM组各32只,DM组按60mg／kg体质量腹腔注射STZ 1次建立DM大鼠模型；CON组腹腔注射等量柠檬酸缓冲液作为对照。DM成膜后每周测量各组体质量、血糖等一般生理指标,并于2w、4w、6w、8w和10w各时间点随机抽样行视网膜实时定量PCR(qRT-PCR)检测视网膜VEGF mRNA、PEDF mRNA的表达丰度。10w时各组随机抽样对视网膜组织进行HE染色、FITC-Dextran灌注视网膜血管铺片及透射电镜超微结构观察；免疫组化染色检测视网膜VEGF及PEDF的表达强度及部位；miRNA基因芯片筛选DM组与CON组视网膜表达显著差异的miRNAs。qRT-PCR检测目的miRNAs于每个时间点在视网膜中的表达丰度。并通过生物信息学分析预测表达差异显著miRNAs的可能靶基因。结果： 1.STZ腹腔注射DM成模率100%。注射前DM组平均体质量(187.5±5.4g)与CON组(185.0±6.9g)差异不显著(P0.05)；STZ腹腔注射后,DM组体质量增加不明显,后期甚至有所减轻,CON组体质量逐步增长,10w时DM组平均体质量(169.9±26.9g)与CON组(439.2±23.5g)差异显著(P0.001)。注射前DM组平均血糖(6.49±0.68mmol/l)与CON组(6.60±0.59mmol/L)差异不显著(P0.05)；STZ腹腔注射72h后,DM组平均血糖(26.63±4.54mmol／l)与CON组(6.37±0.49mmol／l)差异显著(P0.001),～10wDM组血糖均16.7mmol／l,CON组保持在5.6～7.4mmol／l。视网膜HE染色显示10w DM组大鼠视网膜毛细血管扩张,间质水肿；CON组视网膜未见明显异常。FITC-Dextran灌注视网膜显示10w DM组血管迂曲,管径不规则,未见毛细血管荧光渗漏、微血管瘤、视网膜无灌注区等；CON组血管管径均匀一致、分支自然流畅。透射电镜下DM组视网膜超微结构的改变主要有：毛细血管基底膜增厚,内皮细胞指状突起,线粒体肿胀、嵴脱落、空泡样变；周细胞线粒体肿胀、嵴脱落、空泡样变；内核层双极细胞线粒体肿胀、嵴脱落、空泡样变；感光细胞膜盘间隙增宽、数量减少；神经节细胞线粒体肿胀、嵴脱落、空泡样变。 2.经qRT-PCR检测,CON组视网膜有VEGF mRNA表达,表达量随时间无明显变化；DM组VEGF mRNA的表达,2w时较CON组有所增高,至6w表达量约为CON组的3倍(P0.05),10w表达量增高到CON组的6倍(P0.001)。PEDF mRNA也表达于CON组视网膜,DM组2w时的表达量较CON组有所减少,至4w其表达量约为CON组的1／2(P0.001),10w表达量降至CON组的1／3(P0.001)。视网膜免疫组化染色观察,CON组：VEGF主要分布于内核层和节细胞层,PEDF主要分布于内核层和节细胞层,其余各层也可见阳性表达；DM组VEGF表达较CON组明显增强,视网膜各层神经细胞均见有强阳性表达；PEDF表达较CON组明显减弱,其分布主要位于节细胞层及内丛状层。 3.miRNA芯片检测结果显示：和CON组比较,DM组共有168个miRNAs的表达出现明显变化。其中,主要有miR-182、miR-96、miR-183、miR-211、miR-204、miR-124、miR-592、miR-190b、miR-502-3p、miR-363、miR-29c*、miR-210等表达显著上调；miR-10b、miR-10a、miR-219-2-3p、miR-144、miR-338、miR-199a、miR-451、miR-34a、miR-542-5p、miR-142-3p、miR-223、miR-18a等表达显著下调。经qRT-PCR检测,大部分表达上调的miRNAs在视网膜中的表达量随病程发展逐渐上升；表达下调的12个miRNAs的表达量随病程发展均逐渐下降。生物信息学分析结果显示,这些表达显著差异的miRNAs可以调控许多与DR密切相关基因的表达,如miR-29c*、miR-199a可能靶向VEGFa,miR-363可能靶向PEDF,等等。结论： 1.STZ诱导的DM大鼠在病程10w时即已出现类似早期DR的相关改变,可作为人类早期背景型糖尿病视网膜病变(background diabetic retinopathy, BDR)的动物模型以用于DR的相关实验研究,并且该造模方法简单经济、用时较短、重复性好、成功率高。 2.作为主要血管生成刺激因子的VEGF和潜在血管生成抑制因子的PEDF,二者间的动态平衡对调节血管渗漏与新生血管形成起着至关重要的作用。生理状态下,VEGF在视网膜中呈低表达,而PEDF相对占有优势,这对维持视网膜血管完整、抑制新生血管形成是非常必要的。10w DM大鼠视网膜VEGF表达增多、PEDF减少,破环了二者间的平衡关系,继而可能导致视网膜出现血管渗漏和新生血管等。这就说明VEGF与PEDF的失衡可能是造成DR发生发展的重要因素之一,同时也从细胞因子角度进一步证明了10w STZ-DM大鼠可作为早期BDR的动物模型。 3.早期BDR大鼠模型视网膜中,miRNAs的表达谱较正常对照有明显差异；在这些表达明显差异的miRNAs中,有些随病程发展表达量逐渐上升,还有些随病程发展逐渐下降；这些表达差异的miRNAs可能靶向许多与DR发生发展密切相关的基因。提示：miRNAs在DR发生发展过程中可能起着重要的调控作用。
[Abstract]:Objective:
To observe the morphological changes of the early retina of streptozotocin (STZ) induced diabetic (DM) rats and evaluate its application as an animal model of early diabetic retinopathy (DR); to determine the expression of VEGF mRNA and PEDF mRNA in the retina of DM rats at different time points and to explore the relationship between the two and the occurrence of DR in the rats. The significance of the development was further evaluated from the angle of cytokine, and the value of STZ-DM rats as an early DR animal model was further evaluated. The lOw STZ-DM rat was used as an early DR animal model to detect the differential expression of microRNAs (miRNAs) in the retina, and to explore its role in the development of DR.
Method:
64 male SD rats weighing 180 20g were randomly divided into CON group and 32 DM group. DM group was injected with STZ 1 times to establish DM rat model according to 60mg / kg mass intraperitoneally, and the CON group was injected with equal amount of citric acid buffer as a control.DM membrane. Retina real-time quantitative PCR (qRT-PCR) was used to detect retinal VEGF mRNA and PEDF mRNA expression in.10w. The retinal tissue was stained with HE, the retinal vascular spread and transmission electron microscopy were observed by random sampling, and the expression intensity and location of retina VEGF and PEDF were detected by immunohistochemical staining. IRNA gene chip screened the expression abundance of miRNAs at each time point in the retina of the retina, which was significantly different between the DM group and the CON group, and predicted the possible target genes for the significant miRNAs expression by bioinformatics analysis.
Result:
The average body mass (187.5 + 5.4G) and CON group (185 + 6.9g) before 100%. injection of 100%. was not significant (P0.05). After STZ intraperitoneal injection, the mass increase of the DM group was not obvious, the latter even decreased, the mass of CON group increased gradually, and the average body mass of the group (169.9 +) was significantly different from that of the group (439.2 + 439.2). (P0.001). The average blood glucose (6.49 + 0.68mmol/l) and CON group (6.60 + 0.59mmol/L) before the injection were not significant (P0.05). After STZ intraperitoneal injection of 72h, the average blood glucose (26.63 + 4.54mmol / L) in the DM group was significantly different from that of the CON group (6.37 + CON). The retinal capillary dilatation and interstitial edema in the 10W DM group showed that there was no obvious abnormal.FITC-Dextran perfusion retina in group CON showed that the vascular circuitous in group 10W DM, irregular tube diameter, no capillary fluorescent leakage, microangioma, and retinal instillation area, and the vascular diameter of the CON group was uniform, and the branch was natural and fluent. Transmission electricity. Transmission electricity The ultrastructural changes in the retina of the DM group were mainly: the thickening of the capillary basement membrane, the finger like protruding of the endothelial cells, the swelling of the mitochondria, the exfoliation of the crista, the vacuoles like changes, the swelling of the mitochondria in the pericytes, the exfoliation of the crista, the vacuoles, the widening of the membrane disks of the photoreceptor cells, the widening of the membrane disc in the photoreceptor cells, and the decrease in the number of the photoreceptor cells. The mitochondria of the ganglion cells were swollen, the ridges were shedding and vacuoles changed.
2. by qRT-PCR, the expression of VEGF mRNA in the retina of group CON was not changed with time. The expression of VEGF mRNA in group DM was higher than that in CON group, and the expression amount of 6W was about 3 times of CON group (P0.05), and the expression amount increased to 6 times of the retina. The expression of 4W was about 1 / 2 (P0.001) in group CON and 1 / 3 (P0.001) in CON group. CON group: VEGF mainly distributed in the kernel layer and ganglion layer, PEDF mainly in the kernel layer and the ganglion layer, and the other layers were also positive, and VEGF expression in DM group was obviously enhanced than that of CON group. There were strong positive expression of neurons in all layers of the retina, and the expression of PEDF was significantly lower than that in CON group. The distribution was mainly in the ganglion cell layer and the inner plexiform layer.
The results of 3.miRNA chip detection show that compared with the CON group, there are 168 miRNAs expressions in the group DM, including miR-182, miR-96, miR-183, miR-211, miR-204, miR-124, miR-592. 51, miR-34a, miR-542-5p, miR-142-3p, miR-223, miR-18a and other expressions were significantly downregulated. By qRT-PCR, the expression of most of the up regulated miRNAs in the retina increased with the course of disease, and the expression of 12 miRNAs expressed down gradually decreased with the development of the disease. Bioinformatics analysis showed that these expressions were significantly poor. Different miRNAs can regulate many DR related genes expression, such as miR-29c*, miR-199a may target VEGFa, miR-363 may target PEDF, and so on.
Conclusion:
1.STZ induced DM rats had related changes similar to early DR during the course of disease 10W, which could be used as the animal model of background diabetic retinopathy (BDR) for early human background diabetic retinopathy (BDR) for the experimental study of DR, and the modeling method was simple, short, reproducible, and high success rate.
2. as the VEGF of the major vascular stimulation factors and the PEDF of the potential angiogenesis inhibitor, the dynamic balance between the two plays a vital role in regulating vascular leakage and neovascularization. In physiological state, VEGF is low in the retina, while PEDF is relatively dominant, which is new to the maintenance of retinal blood vessels. Angiogenesis is essential for the increase of VEGF expression in the retina of.10w DM rats, the decrease of PEDF, the equilibrium relationship between the two and the retinal vascular leakage and the neovascularization in the retina. This indicates that the imbalance of VEGF and PEDF may be one of the important factors that cause the development of DR, and also from the angle of cytokine. One step proves that 10W STZ-DM rats can be used as animal models of early BDR.
The expression profiles of miRNAs in the retina of the BDR rat model of the early 3. were significantly different than that in normal controls. In these miRNAs, some of these expressions were gradually increasing with the development of the disease course, and some of them decreased with the development of the disease. These differentially expressed miRNAs may be targeted to many genes closely related to the development of DR. MiRNAs may play an important regulatory role in the development of DR.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R774.1

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