微创外科技术建立小型猪肾衰竭模型及干预RAAS系统的研究

发布时间：2018-04-19 18:15

本文选题：小型猪 + 腹腔镜手术　；参考：《东北农业大学》2016年博士论文

【摘要】：近年来小型猪作为比较医学的模型动物受到了越来越多学者的关注,在医学各个领域被广泛应用,由于其与人类的高度同源性,作为模型动物有着不可替代的优势。多年来对于肾衰的动物模型研究局限于鼠或兔等实验动物,与其相比小型猪的体型与结构与人类更为相似且同源性较高,同时与传统手术相比应用微创技术建立模型更大限度的降低了影响因素,然而应用微创外科技术建立小型猪慢性肾衰模型在国内外仍未见报道,建立小型猪的实验用肾衰模型及肾衰模型动物的研究现已成为兽医学及比较医学等领域迫切需要解决的问题。因此,本实验应用腹腔镜技术建立小型猪慢性肾衰模型,并检测肾衰模型的病理及代谢变化,分析RAAS系统(肾素-血管紧张素-醛固酮系统)在肾衰发展中的作用。实验将广西巴马小型猪分为3组,每组5头,共15头小型猪,分别为2/3组、3/4组、5/6组,比较各组间差异,模型建立共需要进行两次腹腔镜下手术。第一次手术2/3组进行腹腔镜下1/3左肾切除术,3/4组进行腹腔镜下1/2左肾切除术,5/6组进行腹腔镜下2/3左肾切除术,通过建立气腹、安置套管、腹腔镜下阻断肾动静脉、按比例切除部分肾组织、开放肾脏动静脉及关闭腹腔等操作步骤完成手术;一周后所有动物进行腹腔镜下右侧全肾切除术。二次手术后进行模型动物的监测,术前、术后3 d及每周进行包括体温、心率、血细胞检查、肾功能的检测,以此筛选出最安全、有效、实用的小型猪肾衰模型。筛选出的最佳肾衰模型进行肾脏微环境的观察(RF组),另外选取5头小型猪进行最佳肾衰模型的建立,在二次手术术前、术后3 d及术后每周进行新型肾功能标志物(NAGL、CLU、OPN、Kim-1、CysC及β2-微球蛋白)的血液、尿液水平检测,术后2 w、4w、6w、8w、10w对模型动物开腹取剩余肾脏组织进行肾功能标志物基因、凋亡基因、转化生长因子TGF-β基因检测以及肾脏病理学检查。应用最佳肾衰模型进行RAAS系统干预实验,选取27头小型猪,共分为3组,每组9头,分别为ACEI组(饲喂卡托普利)、ARB组(饲喂氯沙坦)、SARA组(饲喂螺内酯),其中每组5头在模型建立后术前、术后3 d及每周进行包括体温、心率、血细胞检查、肾功能检测、肾功能标志物水平检测;剩余每组4头进行术后开腹取剩余肾脏组织进行肾功能标志物基因、凋亡基因、转化生长因子TGF-β基因检测以及肾脏病理学检查,并与RF组比较差异。本实验首次成功使用腹腔镜技术完成了2/3、3/4、5/6比例的肾脏切除,进行大部分肾脏切除后的小型猪呈现慢性肾衰竭状态,术后到12 w之间心率、体温均有不同程度的下降,组间无显著差异;肾功能检测发现术后血肌酐、尿素氮呈持续升高状态,其中肌酐与术前相比在术后2 w产生极显著差异(P0.01),而尿素氮在4 w开始产生极显著差异(P0.01);5/6组肾功能降低速度显著快于其他两组,在5 w后显著高于2/3和3/4组(P0.05)。不同比例的肾切除建立肾衰模型中,5/6组肾功能持续降低,虽然2/3组和3/4组与其趋势相似,但进展缓慢,没有肾脏功能持续恶化的显著趋势,因此本研究中5/6肾脏切除作为最优的小型猪肾衰模型。在肾衰竭模型的病理学研究中发现肾脏环境主要变化为,在术后2 w、4 w以肾小管损伤为主要病变,可见肾小管扩张、原有上皮细胞水肿、部分肾组织萎缩、异物多核巨细胞等病变,6 w之后则主要以再生、增生为主要病变,可见肾小管再生、新生上皮细胞、炎细胞浸润、纤维组织及胶原增生等,有些可见蛋白管型。肾损伤标志物中CysC、NGAL、OPN和β2-MG反应快、变化幅度大,在术后一周产生极显著差异(P0.01),并且在尿液检测中差异更大;组织中NGAL mRNA含量在2 w时与术前相比就升高30多倍,CysC mRNA也升高10多倍,其他表达情况与肾损伤情况相同,相比其他检测肾功能指标,CysC和NGAL反应迅速、变化灵敏,在肾损伤早期就发生显著升高,对肾功能的预后发展起到更好的指示作用。在干预小型猪肾衰模型RAAS系统的实验中,ACEI组的小型猪平均8 w前死亡,在相同时间点血肌酐、尿素氮、CysC及NGAL的检测中发现与RF组动物相比较高,肾功能降低迅速,加剧了肾衰的进展;而ARB组和SARA组与RF组相比均有良好效果,延缓了肾衰的发展,其中SARA组在6 w时血清CysC含量显著低于RF组(P0.05),在7 w时血清ALD含量显著低于RF组(P0.05),8 w后肌酐尿素氮水平显著低于RF组(P0.05),ARB组的肌酐、尿素氮水平在9 w后观察到与RF组的显著差异(P0.05),在9 w后ALD与RF组出现显著差异(P0.05);在组织学检查中发现ARB和SARA能够延缓纤维增生的进展,抑制了TGF-β的表达。通过本次肾衰模型的建立与研究得出以下结论:1.成功应用腹腔镜技术建立了小型猪肾衰竭模型,具有重复性高,易于复制,术后肾功能逐渐减退的特点。2.通过大部分比例肾脏切除建立小型猪肾衰模型是可行的,相比于5/6肾脏切除,2/3和3/4肾脏切除的小型猪肾衰竭进展缓慢,5/6肾脏切除是最佳的小型猪肾衰模型。3.5/6切除法建立肾衰模型时,6 w前主要病理损伤体现为肾小管扩张与坏死,而6 w后则体现为明显的纤维性增生和上皮细胞再生。CysC与NGAL与传统肾功能指标相比,反应速度快、灵敏度高,能够更早的提示肾功能变化,与肾功相关性更好,具有更好的诊断意义。4.RAAS系统参与了肾脏损伤过程,肾衰时ALD的表达显著升高;ARB与SARA的应用对肾衰模型起积极治疗及预防的作用,显著地抑制了肾衰时ALD的表达,延缓了肾功能恶化和纤维化的进展;而ACEI的应用加剧了肾衰进展,使肾脏功能迅速恶化。
[Abstract]:As a model animal of comparative medicine, small pigs have attracted more and more attention in recent years and are widely used in various fields of medicine. Because of their high homology with humans, they have an irreplaceable advantage as model animals. For years, the animal model of kidney failure is limited to mice and rabbits. The shape and structure of the pig are more similar and more homologous to humans. At the same time, the use of minimally invasive techniques to establish a model compared with traditional surgery reduces the influence factors. However, the application of minimally invasive surgery to establish a miniature pig model of chronic renal failure is still not reported at home and abroad. The establishment of renal failure model and renal failure in small pigs is established. The study of model animals has now become an urgent problem to be solved in the fields of veterinary medicine and comparative medicine. Therefore, this experiment uses laparoscopy to establish chronic renal failure model of miniature pigs, and to detect the pathological and metabolic changes of renal failure model, and to analyze the role of the RAAS system (renin angiotensin aldosterone system) in the development of renal failure. The Guangxi Bama miniature pigs were divided into 3 groups, with 5 heads in each group and 15 small pigs in group 2/3, 3/4 and 5/6. The difference between each group was compared. The model established a total of two laparoscopic operations. The first operation in group 2/3 was performed by laparoscopic 1/3 left nephrectomy, the 3/4 group underwent laparoscopic 1/2 left nephrectomy, and 5/6 group performed 2/3 under laparoscopy. The left nephrectomy was performed by setting up the pneumoperitoneum, placing the cannula, blocking the renal arteriovenous, partial nephrectomy, opening the renal arteriovenous and closing the abdominal cavity. All the animals underwent laparoscopic right total nephrectomy after one week. After two operations, the model animals were monitored, 3 D before and after the operation, and Each week included body temperature, heart rate, blood cell examination, and renal function test to screen out the most safe, effective and practical miniature pig kidney failure model. The best renal failure model was selected to observe the renal microenvironment (group RF), and 5 small pigs were selected for the establishment of the best renal failure model, before two operations, 3 D postoperative and postoperative. Blood and urine levels of new renal function markers (NAGL, CLU, OPN, Kim-1, CysC, and beta 2- microglobulin) were performed weekly, and 2 W, 4W, 6W, 8W, and 10W on the model animals were used for kidney function marker gene, apoptosis gene, transforming growth factor TGF- beta gene detection and renal pathological examination. The best renal failure was applied. The model was conducted by RAAS system intervention experiment. 27 small pigs were divided into 3 groups, with 9 heads in group ACEI (feeding Kato Pury), group ARB (fed with Losartan) and group SARA (feeding spironolactone), of which 5 heads in each group were established before the model, 3 d after operation, heart rate, blood cell examination, renal function test, and renal function markers. The remaining kidney tissues of 4 heads of each group were tested for kidney function markers, apoptotic genes, transforming growth factor TGF- beta gene detection and renal pathological examination, and compared with the RF group. The first successful laparoscopic technique was successfully used to complete the nephrectomy with the 2/3,3/4,5/6 ratio in the large part of the experiment. The minitype pig after nephrectomy showed chronic renal failure, and the heart rate was between 12 W after operation and the body temperature decreased in varying degrees. There was no significant difference between the groups. The renal function test found the blood creatinine and urea nitrogen after the operation, and the creatinine was significantly different from the 2 W after the operation (P0.01), while the urea nitrogen was in 4 W. There was a very significant difference (P0.01) at the beginning, and the rate of renal function reduction in group 5/6 was significantly faster than that in the other two groups, after 5 W, significantly higher than that in the 2/3 and 3/4 group (P0.05). In the renal failure model of different proportions of nephrectomy, the renal function of group 5/6 decreased continuously, although the 2/3 and 3/4 groups were similar to the trend, but the progress was slow and no significant renal function deteriorated significantly. In this study, 5/6 nephrectomy was used as the best renal failure model in miniature pigs. In the pathological study of renal failure model, the major changes in renal environment were found. 2 W after operation, 4 W with renal tubular injury as the main lesion, renal tubular dilatation, oedema of the original epithelial cells, partial renal atrophy, and foreign body multinuclear giant cell diseases, 6 After W, the main lesions were regeneration, proliferation, renal tubular regeneration, neonatal epithelial cells, inflammatory cells infiltration, fibrous tissue and collagen hyperplasia, and some of the protein tubules were visible. The changes of CysC, NGAL, OPN and beta 2-MG in renal damage markers were rapid and significant difference (P0.01) in one week after the operation (P0.01), and in urine test. The NGAL mRNA content in the tissue was more than 30 times higher than that before the operation at 2 W, and the CysC mRNA increased more than 10 times. The other expression was the same as that of the renal injury. Compared with other renal function indexes, the reaction of CysC and NGAL was rapid and sensitive, and it increased significantly in the early stage of renal injury, and was better for the prognosis of renal function. In the experiment of interfering with the RAAS system of the miniature pig kidney failure model, the small pigs in the ACEI group died before 8 W, and the blood creatinine, urea nitrogen, CysC and NGAL at the same time point were found to be higher than those in the RF group, and the renal function decreased rapidly, which aggravated the progression of renal failure, while the ARB group and the SARA group had a good effect compared with the RF group. The serum CysC content in SARA group was significantly lower than that in group RF (P0.05) at 6 W, and the serum ALD content was significantly lower than that of group RF (P0.05) at 7 W, and after 8 W, the level of creatinine and urea nitrogen was significantly lower than that of RF group (P0.05), and the level of urea nitrogen in the group was significantly different from that of the group after 9. Differences (P0.05); in histological examination, ARB and SARA were found to delay the progression of fibrous hyperplasia and inhibit the expression of TGF- beta. Through the establishment and study of this renal failure model, the following conclusions were obtained: 1. the successful application of laparoscopy to establish a miniature pig kidney failure model, with high reproducibility, easy to replicate, and gradually diminish renal function after operation Characteristics.2. is feasible to establish a miniature pig kidney failure model through most proportional nephrectomy. Compared with 5/6 nephrectomy, renal failure in small pigs with 2/3 and 3/4 nephrectomy is progressed slowly. 5/6 nephrectomy is the best miniature pig kidney failure model by.3.5/6 resection to establish renal failure model, and the main pathological damage before 6 W is renal tubular dilatation. Necrosis, and 6 W after the obvious fibrotic hyperplasia and epithelial cells regeneration.CysC and NGAL compared with the traditional renal function index, the reaction speed is fast, the sensitivity is high, the renal function can be changed earlier, the correlation with renal function is better, and the diagnostic significance of.4.RAAS system is involved in the process of renal injury, and the expression of ALD in the renal failure is obvious. The application of ARB and SARA plays a positive role in the treatment and prevention of renal failure, which significantly inhibits the expression of ALD in the renal failure and delays the progression of renal function deterioration and fibrosis, and the application of ACEI exacerbates the progression of renal failure and exacerbates the renal function.

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692.5;R-332

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