雷帕霉素对UUO大鼠细胞自噬及肾小管间质纤维化的影响

发布时间：2018-04-24 00:33

本文选题：肾间质纤维化 + 单侧输尿管梗阻（unilateral　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：肾小管间质纤维化是由各种慢性进展性肾脏疾病不可避免的最终结局。肾间质纤维化的发生发展过程复杂，此过程涉及肾小管间质的损伤，炎性细胞的浸润，多种炎症介质的产生，纤维母细胞的增殖，细胞外基质积聚等过程，多种细胞因子参与其中。最近几年里，自噬在肾脏中的作用逐渐为人们所关注，一系列研究表明，自噬与多种肾脏疾病相关。既往研究显示，自噬功能增强通常对肾脏功能保护有益，然而，在某些情形下，过度的自噬可能造成细胞凋亡或死亡。近年来对肾脏有关自噬方面的研究多集中肾小球上皮细胞、肾脏系膜细胞方面，对于肾小管细胞的研究多限于缺血-再灌注损伤、药物性肾损害,而对于梗阻性肾病与自噬的相关研究较少。目前普遍认为，自噬的诱导由哺乳动物雷帕霉素靶蛋白（mTOR）所调控，mTOR是一种非典型的丝氨酸激酶，在进化上十分保守，广泛存在于各种哺乳动物中，可调节细胞增殖、生长以及自噬。当细胞处于营养充足或缺少应激信号的状态时，mTOR激活，自噬受到抑制。然而，一旦处于营养缺乏或者是应激状态，mTOR活性下降或受到抑制，自噬进程启动。细胞饥饿时诱导自噬的目的在于降解细胞内蛋白质，最终获得氨基酸再度为细胞供应新的能量来源。当mTOR活性下降时，自噬相关蛋白（Atg）13去磷酸化，允许其与另一个Atg形成活化聚合物Atg1。该聚合物启动了自噬体膜的形成，然后自噬体膜延伸包裹待降解的内容物。该膜的延展包括了多种Atg蛋白，其中最重要的是蛋白轻链（LC）3，LC3表达水平被普遍地用作自噬标记物，广泛地应用于免疫组化等实验方法中。转化生长因子β1(Transforming growth factor, TGF-β1)被认为是肾小管间质纤维化病理进程中的一个中心媒介。它可以促进细胞外基质成份大量生成，促进炎症细胞的活化和趋化活动，介导肾小管上皮细胞向间充质细胞转化，以及导致肾小球硬化及小管间质纤维化，最终发展至终末期肾衰竭。雷帕霉素，哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）信号转导通路抑制剂，是一种由链球菌属产生的大环内酯类抗免疫抗生素，目前主要应用于移植后抗排异反应。近年来研究发现，该物质在抑制肾间质纤维化方面有所成效，这可能与自噬有关。但目前尚未有研究证明雷帕霉素对梗阻性肾损伤中细胞自噬有何种程度的影响，梗阻后自噬水平与时间的变化有着何种关联，以及其与肾脏纤维化之间的关系。因此，本实验拟通过对SD大鼠建立单侧输尿管梗阻(unilateral urethralobstruction, UUO)模型，应用雷帕霉素进行干预，结合免疫组织化学等手段，观察不同实验组中，TGF-β1、LC3、mTOR表达随时间所发生的变化，探讨雷帕霉素对肾间质纤维化的潜在保护作用，探索肾间质纤维化治疗的新靶点，为肾间质纤维化的临床防治探索新的可能方向。方法：健康雄性SD大鼠，，SPF级，体重（180±20）克，54只，经过1周适应性喂养后，进行随机分组如下：1、对照组(A组)；2、UUO模型组(B组)；3、雷帕霉素治疗组(C组)；每组各18只。分别于术后第3、7、14天随机选取6只大鼠取出其梗阻侧肾脏组织，分别为D3、D7、D14组，每组各6只。手术过程：B、C组大鼠给予10%水合氯醛（0.1ml/kg）腹腔注射+2%盐酸利多卡因皮下麻醉。备皮消毒铺单后，给予左侧腹部纵形切口，暴露左肾下极后，沿之寻找左侧输尿管，在左侧输尿管中上1/3交界处的上下2处分别用丝线结扎，并从中上1/3交界处剪断，以避免逆行感染；而A组除了不结扎、剪断输尿管外，其余过程与B、C二组相同。给药：从手术前1天开始，给予各组大鼠每天灌胃1次，每周复查体重，根据体重变化调整给药量；治疗组以雷帕霉素2mg/kg/d剂量灌胃；A组及B组以等容的0.9%NaCl溶液替代雷帕霉素溶液灌胃。实验期间不限制食水。术后第3天、7天、14天，分别从A、B、C组中随机选取6只大鼠取出梗阻肾，4%多聚甲醛固定，石蜡包埋。之后HE染色、Masson染色，以观察各组大鼠梗阻肾组织病理变化，并采用免疫组织化学方法测定TGF-β1、mTOR及LC3表达水平，应用图像分析系统进行半定量分析。所有实验数据用SPSS20.0统计分析软件进行统计学处理，以均数±标准差(x±ｓ)表示统计结果，应用单因素方差分析对数据进行分析， P0.05表示结果有统计学意义。结果：光镜下HE染色：A组，未见明显异常；B组，在术后第3天，可见少量炎症细胞在肾间质局部浸润，肾小管有轻度扩张；第7天，可见肾间质大量炎症细胞浸润，肾间质宽度明显增加，肾小管明显扩张，肾小管上皮细胞变性、坏死；第14天，可见较多肾小管萎缩，肾小管基底膜有弥漫性增厚、皱缩；C组，术后肾小管间质病变随梗阻时间延长进行性加重，其程度较同时相B组的病变程度轻，但仍明显重于A组。Masson染色：A组，未见明显异常；B组，在术后第3天可见肾小管有轻度扩张，胶原纤维较A组有所增加；术后第7天及第14天，肾小管扩张程度进行性加重，胶原纤维显著增多，间质纤维化现象明显；C组，术后肾小管扩张程度、胶原纤维表达水平随梗阻时间延长进行性加重、增多，其较同时相B组的病变程度稍轻，但仍明显终于A组。免疫组织化学结果：(1)TGF-β1： A组，肾小管上皮细胞中可见少量表达； B组，肾小管上皮细胞中可见表达，随着梗阻时间的延长，其表达水平呈进行性升高，且与A组同时相相比较均有统计学差异(P0.05)； C组，TGF-β1的表达水平与同期B组表达水平相比较均有显著下降(P0.05)，但仍高于同期A组中(P0.05)的表达水平。⑵LC3:A组，存在基础水平表达； B组，第3天时肾小管上皮细胞中出现广泛水平的表达，较A组显著增加(P0.05)，第7天时，其表达水平较第3天有所下降(P0.05)，第14天，其表达水平下降接近至基础水平；C组，第3天时肾小管上皮细胞中出现广泛水平的表达，且较同时期B组表达水平增高(P0.05)，第7天时有所下降，但仍高于同时期B组之表达水平(P0.05)，第14天时进一步下降，表达水平仍高于同时期B组(P0.05)。⑶mTOR：A组，在肾小管上皮细胞中有一定水平的表达；B组，第3天时，表达水平较A组显著下降(P0.05)，第7天时表达水平较第3天升高(P0.05)，第14天时其表达水平进一步升高(P0.05)；C组，第3天时，其表达水平较A组显著下降(P0.05)，较B组同期有所下降，第7、14天时表达水平较第3天时上升(P0.05)，且随着梗阻时间延长进行性增高；同时相表达水平较B组同期下降。结论：（1）梗阻早期肾小管上皮细胞自噬水平显著增加，晚期显著减少，mTOR表达趋势与之相反。提示梗阻早期肾小管细胞受到轻度损伤时，可能通过抑制mTOR，增强了自噬活性对抗因梗阻所致的损伤，从而自我保护，而梗阻晚期，肾小管细胞损伤过于严重失代偿，mTOR抑制解除，自噬活性减弱，自我保护作用失能。（2）雷帕霉素干预后，整体自噬表达水平升高，mTOR表水平下降，TGF-β1表达明显减少，提示雷帕霉素可能通过阻断mTOR途径，增强自噬活性，进而减轻单侧输尿管梗阻大鼠肾小管间质纤维化的程度。
[Abstract]:Objective: renal tubulointerstitial fibrosis is an inevitable final outcome of various chronic progressive renal diseases. The process of renal interstitial fibrosis is complicated. This process involves renal tubulointerstitial damage, inflammatory cell infiltration, various inflammatory mediators, fibroblast proliferation, extracellular matrix accumulation and so on. Cytokines are involved in it.
In recent years, the role of autophagy in the kidney has gradually been concerned. A series of studies have shown that autophagy is associated with a variety of renal diseases. Previous studies have shown that enhanced autophagy is usually beneficial to renal function protection. However, in some cases, excessive autophagy can cause apoptosis or death. In recent years, the kidney is related to the kidney. Many studies focus on glomerular epithelial cells, and in the mesangial cells of the kidney, the study of renal tubular cells is limited to ischemia reperfusion injury, drug induced renal damage, and less research on obstructive nephropathy and autophagy.
It is widely believed that the induction of autophagy is regulated by the mammalian rapamycin target protein (mTOR). MTOR is an atypical serine kinase, which is very conservative in evolution and widely exists in various mammals. It can regulate cell proliferation, growth and autophagy. When cells are in a state of sufficient nutrition or lack of stress signals, mTOR stimulated. Living, autophagy is inhibited. However, once the nutritional deficiency or stress state is in the state of stress, the activity of mTOR decreases or is inhibited and the autophagy process starts. The purpose of inducing autophagy during the starvation of cells is to degrade the intracellular protein and eventually obtain the amino acid to supply a new source of energy for the cells.
When the mTOR activity drops, the autophagy related protein (Atg) 13 dephosphorylates, allowing it to form an activated polymer with another Atg Atg1. that starts the formation of the autophagic membrane, and then the autophagic membrane extends to the content of the content to be degraded. The extension of the membrane includes a variety of Atg proteins, the most important of which is the protein light chain (LC) 3, and the LC3 expression of water. Ping is widely used as a marker of autophagy, and is widely used in immunohistochemistry and other experimental methods.
Transforming growth factor beta 1 (Transforming growth factor, TGF- beta 1) is considered to be a central medium in the pathological process of renal tubulointerstitial fibrosis. It can promote the production of extracellular matrix components, promote the activation and chemotaxis of inflammatory cells, mediate the transformation of renal tubular epithelial cells into mesenchymal cells, and lead to glomerular hard. Chemotherapy and tubulointerstitial fibrosis eventually develop to end-stage renal failure.
Rapamycin, a mammalian target of rapamycin (mTOR) signal transduction pathway inhibitor, is a macrolide resistant immuno antibiotic produced by the genus Streptococcus, and is currently mainly used for anti rejection after transplantation. In recent years, it has been found to be effective in inhibiting renal interstitial fibrosis. This may be associated with autophagy, but there has been no study on the extent of the effect of rapamycin on autophagy in the obstructive renal injury, what is the association between the level of autophagy after obstruction and the change of time, and the relationship with renal fibrosis. Therefore, this experiment is to establish unilateral ureteral obstruction in SD rats (Unila Teral urethralobstruction, UUO) model, using rapamycin to intervene, combined with immunohistochemistry, to observe the changes in the expression of TGF- beta 1, LC3, mTOR in different experimental groups, explore the potential protection of rapamycin for renal interstitial fibrosis, explore the new target of renal interstitial fibrosis, as the renal interstitial fiber. The clinical prevention and treatment of vitamin D is a new possible direction.
Methods: healthy male SD rats, SPF grade, weight (180 + 20) g, 54 rats, after 1 weeks of adaptive feeding, were randomly divided into groups as follows: 1, control group (group A), 2, UUO model group (B group), 3, rapamycin group (C group), 18 rats in each group. After 3,7,14 day, 6 rats were randomly selected to take out the obstructed renal tissue, D3 respectively. D7, group D14, each group of 6. Operation process: B, group C rats were given 10% chloral chloral (0.1ml/kg) intraperitoneal injection of lidocaine hydrochloride subcutaneous anesthesia. After preparation of skin disinfection sheet, the left abdomen was given a longitudinal incision and left lower left kidney was exposed to the left ureter, and the 2 upper and lower parts of the upper and lower 1/3 junction in the left ureter were made of silk thread, respectively. Ligation and cut off from the middle and upper 1/3 junction to avoid retrograde infection; while group A, in addition to non ligature and cut off the ureter, the rest of the process was the same as that of group B, C two. Administration: from 1 days before the operation, the rats were given 1 times a day, the weight was retuned to the body weight, and the dosage of rapamycin 2mg/kg/d was administered to the treatment group. In group A and group B, the equal volume 0.9%NaCl solution was replaced by rapamycin solution. During the experiment, water was not restricted. Third days, 7 days, 14 days after the operation, 6 rats were randomly selected from the group A, B, and C to take out the obstructed kidneys, 4% polyformaldehyde fixed, and paraffin embedded. Then HE staining, Masson staining, in order to observe the pathological changes of the renal tissue of the rats in each group, and take the observation of the pathological changes of the rats' obstructive kidneys. The expression level of TGF- beta 1, mTOR and LC3 was measured by immunohistochemical method. Semi quantitative analysis was carried out by the image analysis system. All the experimental data were statistically processed with SPSS20.0 statistical analysis software. The statistical results were expressed with the mean number of standard deviation (x + s), and the data were analyzed by single factor square analysis. The P0.05 results showed that the results were unified. The significance of learning.
Results: HE staining under light microscope: no obvious abnormality was found in group A. In group B, a small amount of inflammatory cells were infiltrated in the renal interstitium and slight dilatation of renal tubules on the third day after operation; on the seventh day, a large number of inflammatory cells in the renal interstitium were infiltrated, the width of the renal interstitium was significantly increased, the renal tubules were dilated, the renal tubular epithelial cells denatured and necrotic; fourteenth days, More renal tubules were atrophied and the basement membrane of renal tubule was diffuse thickening and crinkling. In group C, the renal tubulointerstitial lesions were aggravated with the prolongation of the obstruction time. The degree of renal tubulointerstitial lesions was lighter than that of group B, but it was still significantly heavier in group A than in group A. In group A, there was no obvious abnormality; in group B, the renal tubules were slightly enlarged at the third day after operation. After seventh and 14 days after operation, the dilatation degree of renal tubules was aggravated, the collagen fiber increased significantly and the interstitial fibrosis was obvious. In group C, the degree of renal tubule dilation and the expression of collagen fiber increased with the prolongation of the obstruction time, and the degree of pathological changes in group B was slightly lighter than that of group B at the same time. But still obviously finally A group. Immunohistochemical results: (1) TGF- beta 1:A group, renal tubular epithelial cells can be seen a small amount of expression, B group, renal tubular epithelial cells can be seen in the expression, with the prolongation of the obstruction time, the expression level is progressive, and compared with the A group is statistically significant (P0.05); C group, TGF- beta 1 The level of B was significantly lower than that in the same period of the same period (P0.05), but still higher than the expression level of the A group (P0.05) in the same period. (2) LC3:A group, there was a basic level of expression; in group B, there was a wide level expression in the renal tubular epithelial cells at third days, which was significantly increased in the A group (P0.05). At the seventh day, the expression level was lower than that of the third day. (P0.05), on the fourteenth day, the decrease of expression level was close to the base level; in group C, the expression of renal tubular epithelial cells was widely expressed in third days, and the expression level of B group was higher than that of the same period (P0.05), and decreased at seventh days, but it was still higher than that of B group at the same time (P0.05). The expression level was further lower than that in the same period, and the expression level was still higher than that at the same time. In group B (P0.05). (3) mTOR:A, there was a certain level of expression in the renal tubular epithelial cells; in group B, the expression level was significantly lower than that in the A group (P0.05) at third days (P0.05), the expression level was higher than that of the third day (P0.05), and the expression level was further increased at fourteenth days (P0.05), and in the C group, the expression level was significantly lower than that of the A group (P0.05), compared with B in the A group (P0.05), compared with B, and B. At the same time, the expression level of the group decreased at the 7,14 day (P0.05) and increased with the prolongation of the obstruction time, and the expression level of the phase was lower than that of the B group at the same time.
Conclusion: (1) the autophagy level of renal tubular epithelial cells increased significantly in early stage of obstruction, significantly decreased in late stage, and the opposite of mTOR expression. It suggests that the early obstruction of renal tubule cells may enhance the autophagy activity against the obstruction caused by obstruction during the early stage of obstruction of renal tubule, and thus protect the renal tubule cells in the late stage of the obstruction, and the renal tubule cells are late. The damage was too serious, mTOR was relieved, autophagic activity was weakened, and the self protective effect was impaired. (2) after rapamycin, the level of autophagy increased, the level of mTOR decreased, and the expression of TGF- beta 1 decreased significantly. It suggested that rapamycin may enhance autophagic activity by blocking the mTOR pathway, and thus alleviated the kidney of unilateral ureteral obstruction in rats. The degree of tubulointerstitial fibrosis.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R693;R692.6

【共引文献】