【摘要】：研究背景:胰島素樣生長因子結(jié)合蛋白相關(guān)蛋白1(insulin-like growth factor binding protein-related protein 1,IGFBPr P1)是一種分泌性蛋白,具有細(xì)胞增殖、分化、衰老、凋亡及血管形成等多種生物學(xué)活性,廣泛分布于各種正常組織中。轉(zhuǎn)化生長因子β1(transforming growth factor beta 1,TGFβ1)是一種具有多功能和多向作用的非組織特異性細(xì)胞因子,是目前公認(rèn)導(dǎo)致組織器官纖維化病變的一種關(guān)鍵因子,尤其對肝、腎組織具有重要作用。導(dǎo)師課題組前期研究發(fā)現(xiàn)IGFBPr P1具有致肝纖維化作用,且與TGFβ1在肝纖維化發(fā)生發(fā)展過程中存在著互為因果的關(guān)系;此外,還在經(jīng)皮下注射硫代乙酰胺(thioacetamide,TAA)后成功誘導(dǎo)的比格犬肝纖維化模型中發(fā)現(xiàn),表達(dá)在肝組織和血漿中的IGFBPr P1隨著肝纖維化程度的加重而逐漸升高。那么,IGFBPr P1是否像TGFβ1一樣也對腎組織具有作用呢?目前國內(nèi)外尚未見相關(guān)明確報道。為此設(shè)計本課題。首先,在經(jīng)腺病毒介導(dǎo)IGFBPr P1轉(zhuǎn)染的SD大鼠中,研究其對腎組織的影響及部分機(jī)制;其次,在經(jīng)皮下注射TAA的比格犬中,觀察腎組織病理學(xué)改變及IGFBPr P1的變化情況。目的:探索IGFBPr P1是否對SD大鼠和比格犬腎組織具有作用及部分作用機(jī)制。方法:1、健康雄性SD大鼠60只,隨機(jī)分為3組:A組(n=10):經(jīng)尾靜脈注射生理鹽水;B組(n=25):經(jīng)尾靜脈注射腺病毒攜帶的增強(qiáng)型綠色熒光蛋白(Ad-EGFP)4×109pfu/只;C組(n=25):經(jīng)尾靜脈注射腺病毒攜帶的IGFBPr P1(Ad-IGFBPr P1)4×109pfu/只;各組分別于注射后1、2、4、6、9w留取腎組織,經(jīng)10%中性福爾馬林固定后待測。2、健康雄性比格犬18只,隨機(jī)分為3組:D組(n=6):正常飲食飼養(yǎng);E組(n=6):皮下注射TAA 12 mg/kg,每周2次;F組(n=6):皮下注射TAA 12 mg/kg,每周2次,5w起協(xié)同注射IGFBPr P1抗體5μg/kg,每周1次;各組分別于12w末留取腎組織,經(jīng)10%中性福爾馬林固定后待測。HE染色和Sirius red染色觀察SD大鼠和比格犬腎組織形態(tài)結(jié)構(gòu)病理改變和膠原纖維沉積情況;免疫組織化學(xué)染色檢測SD大鼠腎組織IGFBPr P1、白細(xì)胞介素1β(IL-1β)、TGFβ1、纖維連接蛋白(FN)以及比格犬腎組織IGFBPr P1、IL-1β、FN的分布和表達(dá)情況。結(jié)果:1、SD大鼠1.1 HE染色:A組和B組腎小球和腎小管結(jié)構(gòu)完整;C組腺病毒轉(zhuǎn)染后1w和2w出現(xiàn)腎小管上皮細(xì)胞腫脹,管腔擴(kuò)張,伴炎癥細(xì)胞浸潤;4w管腔內(nèi)出現(xiàn)透明管型,6w管腔內(nèi)存在大量透明管型,9w腎間質(zhì)內(nèi)可見纖維增生,個別腎小球甚至出現(xiàn)萎縮壞死。1.2 Sirius red染色:A組和B組均僅在腎小囊壁層可見少量紅色膠原纖維;C組除腎小囊壁層外,腎小球、腎間質(zhì)內(nèi)紅色膠原纖維沉積逐漸增多,9w時明顯增多。與A組、B組和C組1、2、4、6w相比,C組9w膠原纖維含量明顯升高(0.28±0.10),差異具有統(tǒng)計學(xué)意義(P0.05)。1.3免疫組織化學(xué)染色結(jié)果判定:深棕色和棕褐色顆粒為陽性表達(dá)。(1)IGFBPr P1:A組和B組表達(dá)微弱;C組從1w開始,腎小球、腎小管上皮細(xì)胞胞漿陽性表達(dá)顆粒增多,轉(zhuǎn)染2w后達(dá)高峰(4.55±0.78),4w后逐漸減弱,9w表達(dá)甚少;(2)IL-1β:A組和B組幾乎無表達(dá);C組1w腎小球、少量腎小管上皮細(xì)胞胞漿弱陽性表達(dá),隨轉(zhuǎn)染時間延長,陽性表達(dá)的范圍和程度均增加,6w時達(dá)到高峰(1.93±0.15),9w較6w表達(dá)明顯減弱。(3)TGFβ1:A組和B組表達(dá)量極少;C組位于腎小球、腎小管上皮細(xì)胞胞漿的陽性染色顆粒自轉(zhuǎn)染后1w開始增多,2w時明顯增多,9w時達(dá)高峰(3.64±0.71)。(4)FN:A組和B組極少表達(dá);C組1、2、4、6w腎小球、腎間質(zhì)弱陽性表達(dá),9w時陽性表達(dá)明顯增強(qiáng)(0.60±0.07)。2、比格犬2.1 HE染色:D組腎小球、腎小管形態(tài)正常;E組腎小管上皮細(xì)胞出現(xiàn)空泡變性,管腔擴(kuò)張,小管腔內(nèi)多有顆粒管型或透明管型,可見炎癥細(xì)胞浸潤和纖維組織增生;F組病變較E組顯著減輕;2.2 Sirius red染色:D組僅在腎小囊壁層和腎小管基底膜可見少量紅色膠原纖維;E組除以上部位,腎組織間質(zhì)內(nèi)出現(xiàn)較多紅色膠原纖維沉積;F組較E組明顯減輕。E組膠原面積百分比高于D組(0.21±0.05 vs 0.29±0.10,P0.01);F組膠原面積百分比低于E組(0.24±0.08 vs 0.29±0.10,P0.05),但仍高于D組(0.21±0.05 vs 0.24±0.08,P0.05),以上差異均具有統(tǒng)計學(xué)意義。2.3免疫組織化學(xué)染色:結(jié)果判定:深棕色和棕褐色顆粒為陽性表達(dá)。(1)IGFBPr P1:D組微量表達(dá);E組在腎小管上皮細(xì)胞胞漿內(nèi)陽性表達(dá)明顯增強(qiáng);F組較E組表達(dá)減弱。(2)IL-1β:D組幾乎無表達(dá);E組腎小球、腎小管上皮細(xì)胞及腎間質(zhì)內(nèi)棕褐色顆粒增多;F組呈淺棕色顆粒。(3)FN:D組微量表達(dá);E組腎間質(zhì)出現(xiàn)棕褐色染色顆粒;F組染色呈淺棕色。以上指標(biāo)統(tǒng)計分析結(jié)果示:E組的IGFBPr P1、IL-1β、FN陽性表達(dá)均顯著高于D組(0.90±0.12 vs 3.99±0.59,P0.01;0.24±0.02 vs 1.28±0.13,P0.05;0.60±0.07 vs 0.88±0.10,P0.01);F組的IGFBPr P1、IL-1β、FN表達(dá)低于E組(2.15±0.11 vs 3.99±0.59,P0.01;0.47±0.03 vs 1.28±0.13,P0.01;0.71±0.05 vs 0.88±0.10,P0.01),但仍高于D組(0.90±0.12 vs 2.15±0.11,P0.01;0.24±0.02 vs 0.47±0.03,P0.01;0.60±0.07 vs 0.71±0.05,P0.01)。以上差異均具有統(tǒng)計學(xué)意義。結(jié)論:1、IGFBPr P1可導(dǎo)致SD大鼠和比格犬腎組織發(fā)生纖維化病變;2、IGFBPr P1是一種類似于TGFβ1的具有非組織特異性的細(xì)胞因子。
[Abstract]:Background: the insulin like growth factor binding protein related protein 1 (insulin-like growth factor binding protein-related protein 1, IGFBPr P1) is a secretory protein that has many biological activities, such as cell proliferation, differentiation, senescence, apoptosis and angiogenesis, and is widely distributed in various normal tissues. Transforming growth factor beta 1 (TR) Ansforming growth factor beta 1, TGF beta 1) is a non tissue specific cytokine with multifunction and multidirectional action. It is recognized as a key factor leading to tissue and organ fibrosis, especially for liver and kidney tissue. The previous study in tutor project group found that IGFBPr P1 has the effect of liver fibrosis, and it has been found to be an important factor in liver fibrosis. TGF beta 1 has a causal relationship in the development and development of liver fibrosis; in addition, it is also found that IGFBPr P1 expressed in liver tissue and plasma gradually increases with the severity of liver fibrosis in the liver fibrosis model after subcutaneous injection of thioacetamide (thioacetamide, TAA). Then, IGFBPr P1 is gradually increased. Then, IGFBPr P1 Does the TGF beta 1 play a role in the renal tissue as well? There is no clear report at home and abroad. To this end, this topic is designed. First, in SD rats transfected by adenovirus mediated IGFBPr P1, the effect of the renal tissue and some mechanism are studied. Secondly, the pathological changes of renal tissue and I are observed in the Beagle dogs injected with TAA by percutaneous injection. GFBPr P1 changes. Objective: To explore whether IGFBPr P1 has a function and part of action on the renal tissue of SD rats and beagle dogs. Methods: 1, 60 healthy male SD rats were randomly divided into 3 groups: A group (n=10): normal saline injected through the tail vein; B group (n=25): 4 * 1 of enhanced green fluorescent protein (Ad-EGFP) carried by adenovirus via the tail vein. 09pfu/ only; group C (n=25): IGFBPr P1 (Ad-IGFBPr P1) 4 x 109pfu/ carried by the adenovirus via the tail vein; each group was injected 1,2,4,6,9w after injection of the kidney tissue. After 10% neutral Faure Marin fixation,.2 and 18 healthy male beagles were randomly divided into 3 groups: D group (n=6): subcutaneous injection of 12 minorities, each 2 times week, group F (n=6): subcutaneous injection of TAA 12 mg/kg, 2 times a week, 5W with IGFBPr P1 antibody 5 mu, 1 times a week; each group left the kidney tissue at the end of 12W. After 10% neutral formalin fixation, the morphological and pathological changes of renal tissue and the deposition of collagen fibrils in the renal tissue of rats and beagles were observed by.HE staining and Sirius red. The distribution and expression of renal tissue IGFBPr P1, interleukin 1 beta (IL-1 beta), TGF beta 1, fibronectin (FN) and IGFBPr P1, IL-1 beta and FN in kidney tissue of beagle dogs were detected by immunohistochemical staining. Results: 1, SD rats were stained with 1.1 HE: glomerular and renal tubules were intact. The epithelial cells were swollen, the lumen dilated and the inflammatory cells infiltrated, the transparent tube type appeared in the 4W lumen, a large number of hyaline tubes were found in the 6W lumen, fibrous hyperplasia in the 9W renal interstitium, and even atrophy necrosis of the glomeruli in the individual glomeruli and.1.2 Sirius red staining: the A group and the B group were only a small amount of red collagen fibers in the small capsule wall of the kidneys, and the C group except the renal capsule wall. The accumulation of red collagen fibers in the glomeruli and renal interstitium increased gradually and increased obviously in 9W. Compared with group A, group B and C group 1,2,4,6w, the content of 9W collagen fibers in group C increased significantly (0.28 + 0.10). The difference was statistically significant (P0.05).1.3 immunohistochemical staining results: Dark Brown and brown brown granules were positive. (1) IGFBPr P1:A. The expression of group and B group was weak, and in group C from 1W, glomerular and renal tubular epithelial cell cytoplasmic positive expression particles increased, after transfection of 2W to peak (4.55 + 0.78), 4W gradually weakened, 9W expression was little, (2) IL-1 beta: A group and B group almost no expression, C group 1W glomeruli, a small number of renal tubular epithelial cell cytoplasm weak positive expression, as the transfection time prolonged, positive positive. Positive The range and degree of expression increased, 6W reached a peak (1.93 + 0.15), and the expression of 9W decreased obviously than that of 6W. (3) the expression of TGF beta 1:A group and B group was very few; C group was located in the glomerulus, and the positive staining particles in the cytoplasm of the renal tubular epithelial cells began to increase, the 2W increased obviously, and the 9W reached the peak (3.64 + 0.71). (4) FN:A group and B group were rarely expressed. Group 1,2,4,6w glomeruli, weak positive expression of renal interstitium, 9W positive expression increased significantly (0.60 + 0.07).2, Beagle Dog 2.1 HE staining: D group glomeruli, renal tubule morphology normal; E group of renal tubular epithelial cell vacuoles degeneration, dilation of the lumen, small tubule or transparent tube type, inflammatory cell infiltration and fibrous tissue hyperplasia; F Group lesions were significantly less than those in the E group; 2.2 Sirius red staining: D group only found a small amount of red collagen fibers in the wall of the renal vesicles and the basement membrane of the renal tubules; in group E, there were more red collagen fibrils in the interstitial tissue of the kidneys, and in the F group, the ratio of collagen area to the.E group was significantly higher than that of the D group (0.21 + 0.05 vs 0.29 + 0.10, P0.01). The percentage of collagen area was lower than that in group E (0.24 + 0.08 vs 0.29 + 0.10, P0.05), but still higher than that in group D (0.21 + 0.05 vs 0.24 + 0.08, P0.05). The above differences were statistically significant.2.3 immunohistochemical staining: the result was that deep brown and brown brown granules were positive. (1) the micro expression of IGFBPr P1:D group; E group in the renal tubular epithelial cell cytoplasm. The expression of positive expression was obviously enhanced in group F than that in group E. (2) IL-1 beta: group D was almost no expression, E group glomeruli, renal tubule epithelial cells and renal interstitial brown granules increased; group F was light brown granules. (3) FN:D group was expressed in group FN:D; E group renal interstitium appeared Brown stained granules; F group staining was light brown. The statistical analysis of the above index showed E: E analysis results showed E: E The positive expressions of IGFBPr P1, IL-1 beta and FN were significantly higher than those in the D group (0.90 + 0.12 vs 3.99 + 0.59, P0.01; 0.24 + 0.02 vs 1.28 + 0.13, P0.05; 0.60 + 0.07 vs 0.88 + 0.10, P0.01). In group D (0.90 + 0.12 vs 2.15 + 0.11, P0.01; 0.24 + 0.02 vs 0.47 + 0.03, P0.01; 0.60 + 0.07 vs 0.71 + 0.05, P0.01). All the above differences were statistically significant. Conclusion: 1, IGFBPr P1 can lead to fibrosis in SD rats and beagle renal tissues; IGFBPr P1 is a kind of non tissue specific cytokine similar to that of beta.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692

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