髓系及非髓系TLR4对糖尿病视网膜病变的影响的研究

发布时间：2018-05-02 11:53

本文选题：Toll样受体4 + 骨髓移植　；参考：《华中科技大学》2012年硕士论文

【摘要】：【目的】探讨髓系及非髓系细胞来源的Toll样受体4（toll-like receptor4，TLR4）在糖尿病视网膜的表达及对糖尿病视网膜病变（Diabetic retinopathy，DR）的影响。【方法】取12周龄的雄性TLR4基因突变小鼠C3H/HeJ（Mut）及相应野生型小鼠C3H/HeN(WT)，制备骨髓移植嵌合体模型，用两种纯合体小鼠作自身移植对照（按骨髓供体至受体分组记为WT/Mut, Mut/WT，WT/WT, Mut/Mut），一月后用链脲佐菌素（STZ,200mg/Kg）建立糖尿病动物模型，另取未做处理小鼠做空白对照（记为N组），于糖尿病模型建成一个月、两个月、四个月后取材，电镜观察小鼠视网膜组织超微结构的变化，免疫组化染色观察细胞间粘附分子1(ICAM-1)在视网膜组织的表达，RT-PCR检测视网膜TLR4及巨噬细胞炎性蛋白2（MIP-2）mRNA的表达，，ELISA检测血清中MIP-2、肿瘤坏死因子a（TNF-a）的浓度并测血清丙二醛（MDA）的浓度。【结果】超微电镜显示，糖尿病一月时小鼠视网膜组织即有神经节细胞及神经纤维水肿，核周间隙增宽，核固缩，胞浆颜色变淡,两月时毛细血管内皮细胞胞浆线粒体水肿，嵴变短，管腔狭窄，周细胞水肿,各模型组间损伤程度相比，以WT/WT组最重，Mut/Mut组最轻，WT/Mut组重于Mut/WT组；免疫组化定位ICAM-1显示，ICAM-1染色在空白组视网膜内颗粒层及节细胞层呈微黄色，糖尿病一月时视网膜神经节细胞呈棕黄色阳性表达，两月时染色呈褐色强阳性表达，并且新生毛细血管管壁呈阳性表达，各模型组间表达强度相比，以WT/WT组强于Mut/Mut组，WT/Mut组强于Mut/WT组；视网膜组织RT-PCR结果显示，各模型组TLR4及MIP-2的mRNA的表达在糖尿病一月、两月、四月时依次上调，各模型组间相比，mRNA的表达由高至低依次为WT/WT、WT/Mut、Mut/WT、Mut/Mut组（P0.05）；血清ELISA结果显示，各模型组TNF-a、MIP-2及MDA浓度在糖尿病一月、两月、四月时依次上升，均高于空白对照组，各模型组间相比，浓度由高至低依次为WT/WT、WT/Mut、Mut/WT、Mut/Mut组（P0.05）。【结论】糖尿病视网膜病变随病程发展而加重，神经病变早于微血管病变；TLR4基因突变减轻糖尿病小鼠视网膜病变；髓系及非髓系来源的TLR4基因表达产物均对DR的病程发展有影响；并且髓系来源的TLR4基因表达产物相对于内皮系来源的表达产物对DR的影响更为显著，主要表现在超微结构受损等方面。通过我们的实验证明，这种差异是由于不同细胞来源的TLR4表达不同，以至于对下游炎症因子TNF-a、MIP-2、ICAM-1及MDA的表达量及其活性的促进作用产生显著性差异而造成的。
[Abstract]:[objective] to investigate the expression of Toll receptor 4(toll-like receptor 4 (TLR4) in diabetic retina and its effect on diabetic retinopathy. [methods] the chimerism model of bone marrow transplantation was established in 12-week-old male TLR4 mutant mice (C3H / HeJ mutant) and corresponding wild-type mice C3H / HeNWTG. Two homozygous mice were used as self-transplant controls (WT-Mut, Mut-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT-WT@@ After the diabetic model was established for one month, two months and four months later, the ultrastructure of the retina of the mice was observed by electron microscope. Immunohistochemical staining was used to detect the expression of ICAM-1) in retinal tissue. The expression of TLR4 and macrophage inflammatory protein (2(MIP-2)mRNA) in retina was detected by RT-PCR. Serum MIP-2, TNF-a and malondialdehyde (MDA) were detected by Elisa. [results] ultrastructural electron microscopy showed that there were edema of ganglion cells and nerve fibers in the retina of mice at 1 month after diabetes mellitus, the space around the nucleus widened, the nucleus became pyknosis, the color of the cytoplasm became light, and the mitochondria of the endothelial cells of the capillaries became mitochondria edema at two months. Cristae became shorter, lumen narrow, pericyte edema, injury degree of each model group, WT/WT group was heavier than Mut/WT group, ICAM-1 staining showed that ICAM-1 staining was yellowish in retinal granular layer and ganglion cell layer in blank group. The expression of retinal ganglion cells was brownish positive in one month and brown strong in two months, and the positive expression of neocapillary wall was positive. The intensity of expression in each model group was higher than that in the control group. The results of RT-PCR in retina tissue showed that the mRNA expression of TLR4 and MIP-2 in each model group was up-regulated in January, two months and four months after diabetes mellitus. The results of serum ELISA showed that the concentrations of TNF-a MIP-2 and MDA in each model group increased in turn from high to low in January, two months and four months after diabetes mellitus, and were higher than those in blank control group. The order of concentration from high to low is the WTR / WTR MutR / MutN / Mut group (P0.05). [conclusion] Diabetic retinopathy is aggravated with the course of disease, neuropathy is earlier than microangiopathy and TLR4 gene mutation can alleviate diabetic retinopathy in mice. The expression products of TLR4 gene from medullary and non-medullary systems had an effect on the development of Dr, and the expression products of TLR4 gene from medullary system were more significant than those from endothelial cells. The main manifestation is the ultrastructure damage and so on. Our experiments show that this difference is due to the different expression of TLR4 from different cell sources, so that there is a significant difference in the expression and activity of ICAM-1 and MDA in the downstream inflammatory factor TNF-afi-MIP-2.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

【参考文献】