IGFBPrP1对大鼠和比格犬肾组织的影响及意义
发布时间：2018-07-21 18:20
【摘要】：研究背景:胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding protein-related protein 1,IGFBPr P1)是一种分泌性蛋白,具有细胞增殖、分化、衰老、凋亡及血管形成等多种生物学活性,广泛分布于各种正常组织中。转化生长因子β1(transforming growth factor beta 1,TGFβ1)是一种具有多功能和多向作用的非组织特异性细胞因子,是目前公认导致组织器官纤维化病变的一种关键因子,尤其对肝、肾组织具有重要作用。导师课题组前期研究发现IGFBPr P1具有致肝纤维化作用,且与TGFβ1在肝纤维化发生发展过程中存在着互为因果的关系;此外,还在经皮下注射硫代乙酰胺(thioacetamide,TAA)后成功诱导的比格犬肝纤维化模型中发现,表达在肝组织和血浆中的IGFBPr P1随着肝纤维化程度的加重而逐渐升高。那么,IGFBPr P1是否像TGFβ1一样也对肾组织具有作用呢?目前国内外尚未见相关明确报道。为此设计本课题。首先,在经腺病毒介导IGFBPr P1转染的SD大鼠中,研究其对肾组织的影响及部分机制;其次,在经皮下注射TAA的比格犬中,观察肾组织病理学改变及IGFBPr P1的变化情况。目的:探索IGFBPr P1是否对SD大鼠和比格犬肾组织具有作用及部分作用机制。方法:1、健康雄性SD大鼠60只,随机分为3组:A组(n=10):经尾静脉注射生理盐水;B组(n=25):经尾静脉注射腺病毒携带的增强型绿色荧光蛋白(Ad-EGFP)4×109pfu/只;C组(n=25):经尾静脉注射腺病毒携带的IGFBPr P1(Ad-IGFBPr P1)4×109pfu/只;各组分别于注射后1、2、4、6、9w留取肾组织,经10%中性福尔马林固定后待测。2、健康雄性比格犬18只,随机分为3组:D组(n=6):正常饮食饲养;E组(n=6):皮下注射TAA 12 mg/kg,每周2次;F组(n=6):皮下注射TAA 12 mg/kg,每周2次,5w起协同注射IGFBPr P1抗体5μg/kg,每周1次;各组分别于12w末留取肾组织,经10%中性福尔马林固定后待测。HE染色和Sirius red染色观察SD大鼠和比格犬肾组织形态结构病理改变和胶原纤维沉积情况;免疫组织化学染色检测SD大鼠肾组织IGFBPr P1、白细胞介素1β(IL-1β)、TGFβ1、纤维连接蛋白(FN)以及比格犬肾组织IGFBPr P1、IL-1β、FN的分布和表达情况。结果:1、SD大鼠1.1 HE染色:A组和B组肾小球和肾小管结构完整;C组腺病毒转染后1w和2w出现肾小管上皮细胞肿胀,管腔扩张,伴炎症细胞浸润;4w管腔内出现透明管型,6w管腔内存在大量透明管型,9w肾间质内可见纤维增生,个别肾小球甚至出现萎缩坏死。1.2 Sirius red染色:A组和B组均仅在肾小囊壁层可见少量红色胶原纤维;C组除肾小囊壁层外,肾小球、肾间质内红色胶原纤维沉积逐渐增多,9w时明显增多。与A组、B组和C组1、2、4、6w相比,C组9w胶原纤维含量明显升高(0.28±0.10),差异具有统计学意义(P0.05)。1.3免疫组织化学染色结果判定:深棕色和棕褐色颗粒为阳性表达。(1)IGFBPr P1:A组和B组表达微弱;C组从1w开始,肾小球、肾小管上皮细胞胞浆阳性表达颗粒增多,转染2w后达高峰(4.55±0.78),4w后逐渐减弱,9w表达甚少;(2)IL-1β:A组和B组几乎无表达;C组1w肾小球、少量肾小管上皮细胞胞浆弱阳性表达,随转染时间延长,阳性表达的范围和程度均增加,6w时达到高峰(1.93±0.15),9w较6w表达明显减弱。(3)TGFβ1:A组和B组表达量极少;C组位于肾小球、肾小管上皮细胞胞浆的阳性染色颗粒自转染后1w开始增多,2w时明显增多,9w时达高峰(3.64±0.71)。(4)FN:A组和B组极少表达;C组1、2、4、6w肾小球、肾间质弱阳性表达,9w时阳性表达明显增强(0.60±0.07)。2、比格犬2.1 HE染色:D组肾小球、肾小管形态正常;E组肾小管上皮细胞出现空泡变性,管腔扩张,小管腔内多有颗粒管型或透明管型,可见炎症细胞浸润和纤维组织增生;F组病变较E组显著减轻;2.2 Sirius red染色:D组仅在肾小囊壁层和肾小管基底膜可见少量红色胶原纤维;E组除以上部位,肾组织间质内出现较多红色胶原纤维沉积;F组较E组明显减轻。E组胶原面积百分比高于D组(0.21±0.05 vs 0.29±0.10,P0.01);F组胶原面积百分比低于E组(0.24±0.08 vs 0.29±0.10,P0.05),但仍高于D组(0.21±0.05 vs 0.24±0.08,P0.05),以上差异均具有统计学意义。2.3免疫组织化学染色:结果判定:深棕色和棕褐色颗粒为阳性表达。(1)IGFBPr P1:D组微量表达;E组在肾小管上皮细胞胞浆内阳性表达明显增强;F组较E组表达减弱。(2)IL-1β:D组几乎无表达;E组肾小球、肾小管上皮细胞及肾间质内棕褐色颗粒增多;F组呈浅棕色颗粒。(3)FN:D组微量表达;E组肾间质出现棕褐色染色颗粒;F组染色呈浅棕色。以上指标统计分析结果示:E组的IGFBPr P1、IL-1β、FN阳性表达均显著高于D组(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组的IGFBPr P1、IL-1β、FN表达低于E组(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组(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)。以上差异均具有统计学意义。结论:1、IGFBPr P1可导致SD大鼠和比格犬肾组织发生纤维化病变;2、IGFBPr P1是一种类似于TGFβ1的具有非组织特异性的细胞因子。
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
本文编号：2136383
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
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