本文選題：小型豬 + 腹腔鏡手術(shù)　； 參考：《東北農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：近年來小型豬作為比較醫(yī)學(xué)的模型動物受到了越來越多學(xué)者的關(guān)注,在醫(yī)學(xué)各個領(lǐng)域被廣泛應(yīng)用,由于其與人類的高度同源性,作為模型動物有著不可替代的優(yōu)勢。多年來對于腎衰的動物模型研究局限于鼠或兔等實驗動物,與其相比小型豬的體型與結(jié)構(gòu)與人類更為相似且同源性較高,同時與傳統(tǒng)手術(shù)相比應(yīng)用微創(chuàng)技術(shù)建立模型更大限度的降低了影響因素,然而應(yīng)用微創(chuàng)外科技術(shù)建立小型豬慢性腎衰模型在國內(nèi)外仍未見報道,建立小型豬的實驗用腎衰模型及腎衰模型動物的研究現(xiàn)已成為獸醫(yī)學(xué)及比較醫(yī)學(xué)等領(lǐng)域迫切需要解決的問題。因此,本實驗應(yīng)用腹腔鏡技術(shù)建立小型豬慢性腎衰模型,并檢測腎衰模型的病理及代謝變化,分析RAAS系統(tǒng)(腎素-血管緊張素-醛固酮系統(tǒng))在腎衰發(fā)展中的作用。實驗將廣西巴馬小型豬分為3組,每組5頭,共15頭小型豬,分別為2/3組、3/4組、5/6組,比較各組間差異,模型建立共需要進(jìn)行兩次腹腔鏡下手術(shù)。第一次手術(shù)2/3組進(jìn)行腹腔鏡下1/3左腎切除術(shù),3/4組進(jìn)行腹腔鏡下1/2左腎切除術(shù),5/6組進(jìn)行腹腔鏡下2/3左腎切除術(shù),通過建立氣腹、安置套管、腹腔鏡下阻斷腎動靜脈、按比例切除部分腎組織、開放腎臟動靜脈及關(guān)閉腹腔等操作步驟完成手術(shù);一周后所有動物進(jìn)行腹腔鏡下右側(cè)全腎切除術(shù)。二次手術(shù)后進(jìn)行模型動物的監(jiān)測,術(shù)前、術(shù)后3 d及每周進(jìn)行包括體溫、心率、血細(xì)胞檢查、腎功能的檢測,以此篩選出最安全、有效、實用的小型豬腎衰模型。篩選出的最佳腎衰模型進(jìn)行腎臟微環(huán)境的觀察(RF組),另外選取5頭小型豬進(jìn)行最佳腎衰模型的建立,在二次手術(shù)術(shù)前、術(shù)后3 d及術(shù)后每周進(jìn)行新型腎功能標(biāo)志物(NAGL、CLU、OPN、Kim-1、CysC及β2-微球蛋白)的血液、尿液水平檢測,術(shù)后2 w、4w、6w、8w、10w對模型動物開腹取剩余腎臟組織進(jìn)行腎功能標(biāo)志物基因、凋亡基因、轉(zhuǎn)化生長因子TGF-β基因檢測以及腎臟病理學(xué)檢查。應(yīng)用最佳腎衰模型進(jìn)行RAAS系統(tǒng)干預(yù)實驗,選取27頭小型豬,共分為3組,每組9頭,分別為ACEI組(飼喂卡托普利)、ARB組(飼喂氯沙坦)、SARA組(飼喂螺內(nèi)酯),其中每組5頭在模型建立后術(shù)前、術(shù)后3 d及每周進(jìn)行包括體溫、心率、血細(xì)胞檢查、腎功能檢測、腎功能標(biāo)志物水平檢測;剩余每組4頭進(jìn)行術(shù)后開腹取剩余腎臟組織進(jìn)行腎功能標(biāo)志物基因、凋亡基因、轉(zhuǎn)化生長因子TGF-β基因檢測以及腎臟病理學(xué)檢查,并與RF組比較差異。本實驗首次成功使用腹腔鏡技術(shù)完成了2/3、3/4、5/6比例的腎臟切除,進(jìn)行大部分腎臟切除后的小型豬呈現(xiàn)慢性腎衰竭狀態(tài),術(shù)后到12 w之間心率、體溫均有不同程度的下降,組間無顯著差異;腎功能檢測發(fā)現(xiàn)術(shù)后血肌酐、尿素氮呈持續(xù)升高狀態(tài),其中肌酐與術(shù)前相比在術(shù)后2 w產(chǎn)生極顯著差異(P0.01),而尿素氮在4 w開始產(chǎn)生極顯著差異(P0.01);5/6組腎功能降低速度顯著快于其他兩組,在5 w后顯著高于2/3和3/4組(P0.05)。不同比例的腎切除建立腎衰模型中,5/6組腎功能持續(xù)降低,雖然2/3組和3/4組與其趨勢相似,但進(jìn)展緩慢,沒有腎臟功能持續(xù)惡化的顯著趨勢,因此本研究中5/6腎臟切除作為最優(yōu)的小型豬腎衰模型。在腎衰竭模型的病理學(xué)研究中發(fā)現(xiàn)腎臟環(huán)境主要變化為,在術(shù)后2 w、4 w以腎小管損傷為主要病變,可見腎小管擴(kuò)張、原有上皮細(xì)胞水腫、部分腎組織萎縮、異物多核巨細(xì)胞等病變,6 w之后則主要以再生、增生為主要病變,可見腎小管再生、新生上皮細(xì)胞、炎細(xì)胞浸潤、纖維組織及膠原增生等,有些可見蛋白管型。腎損傷標(biāo)志物中CysC、NGAL、OPN和β2-MG反應(yīng)快、變化幅度大,在術(shù)后一周產(chǎn)生極顯著差異(P0.01),并且在尿液檢測中差異更大;組織中NGAL mRNA含量在2 w時與術(shù)前相比就升高30多倍,CysC mRNA也升高10多倍,其他表達(dá)情況與腎損傷情況相同,相比其他檢測腎功能指標(biāo),CysC和NGAL反應(yīng)迅速、變化靈敏,在腎損傷早期就發(fā)生顯著升高,對腎功能的預(yù)后發(fā)展起到更好的指示作用。在干預(yù)小型豬腎衰模型RAAS系統(tǒng)的實驗中,ACEI組的小型豬平均8 w前死亡,在相同時間點血肌酐、尿素氮、CysC及NGAL的檢測中發(fā)現(xiàn)與RF組動物相比較高,腎功能降低迅速,加劇了腎衰的進(jìn)展;而ARB組和SARA組與RF組相比均有良好效果,延緩了腎衰的發(fā)展,其中SARA組在6 w時血清CysC含量顯著低于RF組(P0.05),在7 w時血清ALD含量顯著低于RF組(P0.05),8 w后肌酐尿素氮水平顯著低于RF組(P0.05),ARB組的肌酐、尿素氮水平在9 w后觀察到與RF組的顯著差異(P0.05),在9 w后ALD與RF組出現(xiàn)顯著差異(P0.05);在組織學(xué)檢查中發(fā)現(xiàn)ARB和SARA能夠延緩纖維增生的進(jìn)展,抑制了TGF-β的表達(dá)。通過本次腎衰模型的建立與研究得出以下結(jié)論:1.成功應(yīng)用腹腔鏡技術(shù)建立了小型豬腎衰竭模型,具有重復(fù)性高,易于復(fù)制,術(shù)后腎功能逐漸減退的特點。2.通過大部分比例腎臟切除建立小型豬腎衰模型是可行的,相比于5/6腎臟切除,2/3和3/4腎臟切除的小型豬腎衰竭進(jìn)展緩慢,5/6腎臟切除是最佳的小型豬腎衰模型。3.5/6切除法建立腎衰模型時,6 w前主要病理損傷體現(xiàn)為腎小管擴(kuò)張與壞死,而6 w后則體現(xiàn)為明顯的纖維性增生和上皮細(xì)胞再生。CysC與NGAL與傳統(tǒng)腎功能指標(biāo)相比,反應(yīng)速度快、靈敏度高,能夠更早的提示腎功能變化,與腎功相關(guān)性更好,具有更好的診斷意義。4.RAAS系統(tǒng)參與了腎臟損傷過程,腎衰時ALD的表達(dá)顯著升高;ARB與SARA的應(yīng)用對腎衰模型起積極治療及預(yù)防的作用,顯著地抑制了腎衰時ALD的表達(dá),延緩了腎功能惡化和纖維化的進(jìn)展;而ACEI的應(yīng)用加劇了腎衰進(jìn)展,使腎臟功能迅速惡化。
[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.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R692.5;R-332

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