【作者】 张华；

【导师】 王洪斌；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2015， 博士

【摘要】 小型猪与人在比较医学上同源关系较近,在解剖学、生理学、生物化学和疾病发生机理等方面极其相似,常用作理想的非啮齿类实验动物模型,如小型猪糖尿病模型、腹壁拉链模型、心肌梗死模型等,然而采用微创技术建立小型猪肝脏损伤模型的研究在国内外尚未见报道,加之目前科学研究中对理想大动物肝脏损伤模型的迫切需求,小型猪的实验用肝脏损伤模型已经成为兽医学、比较医学等领域亟待解决的问题。因此,本课题将采用腹腔镜微创外科技术进行小型猪肝脏损伤模型建立的研究,探索腹腔镜下建立肝脏大部分切除及缺血合并部分切除损伤模型的操作方法,同时对模型建立的安全性进行监测,并与传统开放性手术进行对比研究,最后在模型基础上进一步探讨损伤后肝脏环境的变化。本试验选用54头巴马小型猪为实验动物,首先随机选取5头小型猪,进行腹腔镜下肝脏局部术野探查和肝脏解剖学观察,然后将剩余的小型猪随机分为7组,每组7头,分别为腹腔镜肝叶大部分切除组、腹腔镜肝叶大部分切除假手术对照组、开腹肝叶大部分切除组,腹腔镜肝脏缺血合并部分切除组、腹腔镜肝脏部分切除组、腹腔镜肝脏缺血合并部分切除假手术对照组、开腹肝脏缺血合并部分切除组。试验过程中通过气腹、建立手术通路、分离韧带、解剖第一肝门、贯穿肝实质与止血、右半肝缺血、切断肝实质、冲洗腹腔、切除肝叶的取出、缝合切口等操作步骤进行腹腔镜下小型猪肝脏损伤模型的建立。模型建立过程中对生命体征、循环系统、呼吸系统、体温等重要生理指标进行全程连续监测,并记录术中和术后并发症、手术完成及术后恢复情况,术后30d行腹腔镜二次探查手术部位粘连。各组分别于术前、4 h、1d、3d、7d、14d、30d采取静脉血,检测血常规和血清总蛋白、谷草转氨酶、r-谷氨酰转移酶、总胆红素、尿素氮、肌酐及血浆凝血功能;在术前、术后即刻、术后1d、术后3d、术后7d和术后1个月进行腹腔镜下肝脏活组织检查,做石蜡切片和HE染色观察组织病理学变化,同时针对不同模型分别检测肝脏组织增殖细胞核抗原、细胞周期素D1、肿瘤坏死因子-α、白细胞介素-6的表达和组织丙二醛含量、超氧化物歧化酶、过氧化氢酶活性及Caspase-3活性,统计并对比腹腔镜和开腹手术结果和血清皮质醇、C反应蛋白的变化。腹腔镜肝脏局部术野探查和解剖学观察发现,小型猪肝脏解剖位置、外形结构、韧带分布、管道系统及肝叶划分方面与人类近似,其特有的解剖学结构便于肝脏模型的建立。本试验成功完成了小型猪腹腔镜下肝脏大部分切除模型和小型猪腹腔镜下肝脏缺血合并部分切除模型的建立,术中和术后各项生理生化指标虽有不同程度的波动,但均未超出小型猪的安全耐受范围;相对于开腹组,腹腔镜组的手术时间稍长,但手术切口小,出血量少,术后腹腔粘连轻,且所有小型猪术后1d精神状态和食欲基本恢复,能进食少量流质饲料,术后7-9d套管口和腹壁切口取第一期愈合。模型建立后,小型猪肝脏大部分切除模型肝脏环境变化主要表现为术后1d和术后3d血清AST与术前和对照组相比差异极显著(P<0.01),术后7d差异显著(0.01<P<0.05);病理学观察可见术后7d局部肝细胞肿胀、变性并伴有炎性细胞浸润,肝组织损伤较明显;术后1d肝细胞PCNA表达水平于达到高峰,之后逐渐降低。肝脏组织Cyclin D1基因表达量在肝大部切除后早期表达明显升高,术后1d增高明显,与对照组相比差异极显著(P<0.01),一直持续到术后3d差异显著(0.01<P<0.05)。术后肝组织TNF-α先升高后降低,在术后1d迅速升高到最高值,与对照组相比差异极显著(P<0.01),术后3d、7d逐渐降低,与对照组相比差异显著(0.01<P<0.05),;IL-6变化幅度较小,术后1d和3d与对照组相比差异显著(0.01<P<0.05)。肝脏缺血合并部分切除模型肝脏环境变化主要表现为,与对照组相比术后1d和3d血清AST差异极显著(P<0.01),术后7d差异显著(0.01<P<0.05),其它监测时间点变化不显著(P>0.05)。模型组剩余肝组织术后1d有广泛的肝细胞变性和轻度萎缩,偶见窦状隙淤血,肝小叶内有局部肝细胞坏死灶或片状坏死,间质中炎性细胞浸润;术后7d可见局部肝小叶内坏死区域有肝细胞的增殖,局部汇管区纤维性结缔组织增生等。与对照组相比,在术后即刻丙二醛升高差异极显著(P<0.01),术中1h和术后1d肝组织丙二醛变化差异显著(0.01<P<0.05),其他监测时间点变化不显著(P>0.05);在术后即刻超氧化物歧化酶变化差异极显著(P<0.01),术后1d变化差异显著(0.01<P<0.05),其他监测时间点变化不显著(P>0.05);在术后即刻过氧化氢酶变化差异极显著(P<0.01),术中1h术后1d差异显著(0.01<P<0.05),其他监测时间点变化不显著(P>0.05)。在术后1d肝组织中Caspase-3活性相对于对照组变化差异极显著(P<0.01),术中即刻和术后3d变化差异显著(0.01<P<0.05),其他时间点变化不显著(P>0.05)。通过试验结果,得出以下结论:(1)运用腹腔镜微创外科技术成功建立了小型猪肝脏大部分切除模型和小型猪肝脏缺血合并部分切除模型。(2)通过对小型猪围手术期生命体征、循环系统、呼吸系统、肝肾功能、凝血功能等综合监测和评价,各项生理指标均维持在安全范围之内,证明了该技术和操作方法是安全、可行的。(3)与传统开腹手术建立小型猪肝脏损伤模型相比,腹腔镜手术切口小、出血量少、术后粘连轻,且具有对动物机体应激反应小、术后恢复较快等微创优势。(4)在模型建立后,通过对小型猪肝脏组织及血清的检测与评估,明确了损伤后肝脏功能、病理学以及肝脏细胞增殖、细胞因子与生长因子、氧化应激反应和凋亡的变化规律。更多还原

【Abstract】 Miniature pig was ideal experimental animal for its similarity with human anatomy, physiology, biochemistry and disease mechanism. The experimental liver injury animal models of miniature pigs were the urgent needed problem to resolve in veterinary medicine and comparative medicine. This topic proceed a pioneering research in the building of liver injury animal models in miniature pigs by laparoscopic minimally invasive surgical technique, and explore the operation method of laparoscopic liver resection and ischemia-reperfusion injury model, and monitoring the changes of the liver environment of the injury. We also compared laparoscopic and open model at the same time, and further to evaluate scientific and minimally invasive advantage of the model, this also lay the foundation for the laparoscopic technique in building miniature pig experimental models.This experiment selects 40 miniature pigs as experimental animals, randomly selected five pigs for laparoscopic abdominal cavity surgical exploration. Then the remaining 35 pigs were randomly divided into 5 groups, each group of seven, respectively, the laparoscopic majority liver resection model group and its control group, the laparoscopic liver ischemia combined resection model group, laparoscopic liver resection group and the control group. By pneumoperitoneum, surgical access, separation of ligaments, the anatomy of the first porta hepatis, throughout the liver parenchyma and hemostasis, right hepatic ischemia, cutting off the liver parenchyma, washing of the abdominal cavity, resection of liver taking out, the liver injury models were established.Intraoperative of vital signs, circulation system, respiratory system and body temperature were entire continuous monitored and record the intraoperative and postoperative complications, operation and postoperative recovery, after 30 d laparoscopic quadratic probe surgical site adhesion. Between groups in preoperative, 4h, 1d, 3d, 7d and 14 d, 30 d take venous blood, detection routine blood and serum total protein, aspertate aminotransferase, r-glutamine transferase, total bilirubin, urea nitrogen, creatinine, and plasma coagulation function; In the preoperative and postoperative immediate, postoperative 1d, 3d, 7d after operation and postoperative 1 month after surgery, laparoscopic liver biopsy were taken, paraffin section and HE staining to observe the histopathological changes, at the same time for different model of liver tissue was detected PCNA and cyclin D1, tumor necrosis factor-alpha and interleukin-6 expression and organization of malondialdehyde content, superoxide dismutase, catalase activity and Caspase-3 activity, statistics and contrast the results of laparoscopy and laparotomy and serum cortisol, the changes of c-reactive protein.Laparoscopic liver partial operative field exploration and anatomical observation found that small liver of pig anatomical location, shape structure, ligament distribution, pipeline system and lobe division and human approximation, its unique anatomical structure to facilitate the liver model.This subject successfully completed the miniature pig most laparoscopic liver resection liver ischemia model and miniature pig laparoscopic combined resection model, intraoperative and postoperative physiological have degree of volatility, but neither is beyond the scope of miniature pigs safety tolerance; Relative to laparotomy, laparoscopic surgery time is a bit long, but the small incision, less blood loss, postoperative abdominal adhesions, and all the miniature pigs have a rapid recovery, postoperative mental status of 1d and appetite to eat a small amount of liquid feed, postoperative 7-9 d the abdominal incision for the first phase of healing.After model establishment, the mainly environmental changes of majority liver resection model are: the postoperative serum AST extremely significant difference compared with the preoperative and the control group(P<0.01) for 1d and 3d after operation, the significant difference(0.01<P<0.05) 7d after operation. Pathology observation are liver cell swelling, degeneration and inflammatory cell infiltration, hepatic tissue damage is obvious7 d after surgery; Postoperative 1d hepatocellular PCNA expression level in peak, then gradually reduced. Amount of liver tissue Cyclin D1 gene expression in liver most early expression increased significantly after resection, postoperative 1d increased significantly(P<0.01), which continues to postoperative 3d(0.01<P<0.05).The postoperative liver tissue TNF alpha reduce after rising first, 1d lifts quickly after surgery to the peak, the extremely significant difference compared with the control group(P<0.01), 3d, 7d after gradually reduce, significant difference(0.01<P<0.05); Small variations in IL-6, 1d and 3d significant difference compared with control subjects(0.01< P <0.05).Liver environment changes of liver ischemia combined resection model are: compared with the control group 1d and 3d postoperative serum AST extremely significant difference(P<0.01), postoperative7 d significant difference(0.01<P<0.05), and other monitoring point in time no significant change(P>0.05).Postoperative 1 d model group remaining liver tissue has a wide range of liver cell degeneration and mild atrophy, occasional sinus gap passive congestion, hepatic lobule with local focal hepatocyte necrosis or patchy necrosis, interstitial inflammatory cell infiltration; Postoperative visual local lobular necrosis area within 7 d liver cell proliferation, local portal area fibrous connective tissue hyperplasia, etc. Compared with control group, in the immediate postoperative malondialdehyde rise significant difference(P <0.01), intraoperative and postoperative 1h,1d liver tissue malondialdehyde changes significant difference(0.01<P<0.05), and other monitoring point in time no significant change(P>0.05); In the immediate postoperative superoxide dismutase changes extremely significant difference(P<0.01), postoperative 1d changes significant difference(0.01<P<0.05), and other monitoring point in time no significant change(P>0.05); In the immediate postoperative catalase extremely significant difference(P<0.01), intraoperative 1 h,1d after significant difference(0.01<P<0.05), and other monitoring point have no significant change(P>0.05). Compared with the control group, Caspase-3 activity in liver tissue changes difference was significant(P<0.01) in postoperative 1d, intraoperative and postoperative immediately 3d change significant difference(0.01<P<0.05), no significant change in other time points(P >0.05). Through the experimental results, the following conclusions were drawn:(1) The use of laparoscopic minimally invasive surgery technology can successfully built the majority liver resection model. ansd liver ischemia combined resection model in miniature pigs(2) Through the comprehensive monitoring of perioperative vital signs, circulation system, respiratory system, liver and kidney function, blood coagulation function, the physiological indexes are maintained in a safe range, proved that the technology and operation method is safe and feasible.(3) Compared with traditional laparotomy, laparoscopic surgery have small incision, less blood loss, postoperative adhesion is light, and the stress response to animals is small, with rapid postoperative recovery and minimally invasive advantage.(4) After model establishment, through the test and evaluation of liver tissue and serum, we clear the changing rule of liver function, pathology, and liver cell proliferation, cytokines and growth factors, oxidative stress and apoptosis after injury.更多还原