本文選題：Toll樣受體4 + 骨髓移植��； 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：【目的】探討髓系及非髓系細胞來源的Toll樣受體4（toll-like receptor4，TLR4）在糖尿病視網(wǎng)膜的表達及對糖尿病視網(wǎng)膜病變（Diabetic retinopathy，DR）的影響。 【方法】取12周齡的雄性TLR4基因突變小鼠C3H/HeJ（Mut）及相應(yīng)野生型小鼠C3H/HeN(WT)，制備骨髓移植嵌合體模型，用兩種純合體小鼠作自身移植對照（按骨髓供體至受體分組記為WT/Mut, Mut/WT，WT/WT, Mut/Mut），一月后用鏈脲佐菌素（STZ,200mg/Kg）建立糖尿病動物模型，另取未做處理小鼠做空白對照（記為N組），于糖尿病模型建成一個月、兩個月、四個月后取材，電鏡觀察小鼠視網(wǎng)膜組織超微結(jié)構(gòu)的變化，免疫組化染色觀察細胞間粘附分子1(ICAM-1)在視網(wǎng)膜組織的表達，RT-PCR檢測視網(wǎng)膜TLR4及巨噬細胞炎性蛋白2（MIP-2）mRNA的表達，，ELISA檢測血清中MIP-2、腫瘤壞死因子a（TNF-a）的濃度并測血清丙二醛（MDA）的濃度。 【結(jié)果】超微電鏡顯示，糖尿病一月時小鼠視網(wǎng)膜組織即有神經(jīng)節(jié)細胞及神經(jīng)纖維水腫，核周間隙增寬，核固縮，胞漿顏色變淡,兩月時毛細血管內(nèi)皮細胞胞漿線粒體水腫，嵴變短，管腔狹窄，周細胞水腫,各模型組間損傷程度相比，以WT/WT組最重，Mut/Mut組最輕，WT/Mut組重于Mut/WT組；免疫組化定位ICAM-1顯示，ICAM-1染色在空白組視網(wǎng)膜內(nèi)顆粒層及節(jié)細胞層呈微黃色，糖尿病一月時視網(wǎng)膜神經(jīng)節(jié)細胞呈棕黃色陽性表達，兩月時染色呈褐色強陽性表達，并且新生毛細血管管壁呈陽性表達，各模型組間表達強度相比，以WT/WT組強于Mut/Mut組，WT/Mut組強于Mut/WT組；視網(wǎng)膜組織RT-PCR結(jié)果顯示，各模型組TLR4及MIP-2的mRNA的表達在糖尿病一月、兩月、四月時依次上調(diào)，各模型組間相比，mRNA的表達由高至低依次為WT/WT、WT/Mut、Mut/WT、Mut/Mut組（P0.05）；血清ELISA結(jié)果顯示，各模型組TNF-a、MIP-2及MDA濃度在糖尿病一月、兩月、四月時依次上升，均高于空白對照組，各模型組間相比，濃度由高至低依次為WT/WT、WT/Mut、Mut/WT、Mut/Mut組（P0.05）。 【結(jié)論】糖尿病視網(wǎng)膜病變隨病程發(fā)展而加重，神經(jīng)病變早于微血管病變；TLR4基因突變減輕糖尿病小鼠視網(wǎng)膜病變；髓系及非髓系來源的TLR4基因表達產(chǎn)物均對DR的病程發(fā)展有影響；并且髓系來源的TLR4基因表達產(chǎn)物相對于內(nèi)皮系來源的表達產(chǎn)物對DR的影響更為顯著，主要表現(xiàn)在超微結(jié)構(gòu)受損等方面。通過我們的實驗證明，這種差異是由于不同細胞來源的TLR4表達不同，以至于對下游炎癥因子TNF-a、MIP-2、ICAM-1及MDA的表達量及其活性的促進作用產(chǎn)生顯著性差異而造成的。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R774.1

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