腹腔镜技术建立小型猪肝损伤模型及肝脏环境变化的研究

发布时间：2018-04-17 23:24

本文选题：小型猪 + 微创外科　；参考：《东北农业大学》2015年博士论文

【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(P0.01),术后7d差异显著(0.01P0.05);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(P0.01),一直持续到术后3d差异显著(0.01P0.05)。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(P0.01),术后3d、7d逐渐降低,与对照组相比差异显著(0.01P0.05),;IL-6变化幅度较小,术后1d和3d与对照组相比差异显著(0.01P0.05)。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(P0.01),术后7d差异显著(0.01P0.05),其它监测时间点变化不显著(P0.05)。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(P0.01),术中1h和术后1d肝组织丙二醛变化差异显著(0.01P0.05),其他监测时间点变化不显著(P0.05);在术后即刻超氧化物歧化酶变化差异极显著(P0.01),术后1d变化差异显著(0.01P0.05),其他监测时间点变化不显著(P0.05);在术后即刻过氧化氢酶变化差异极显著(P0.01),术中1h术后1d差异显著(0.01P0.05),其他监测时间点变化不显著(P0.05)。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(P0.01),术中即刻和术后3d变化差异显著(0.01P0.05),其他时间点变化不显著(P0.05)。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。
[Abstract]:Mini pig and human in comparative medicine is homology in between, in anatomy, physiology, biochemistry and disease mechanism and other aspects are very similar, non rodent animal models are commonly used as the ideal, such as miniature pig model of diabetes, abdominal wall zipper model, myocardial infarction model, however, to set up a small pig liver research injury model by minimally invasive techniques at home and abroad has not been reported, in addition to the ideal animal models of liver injury and the urgent needs of scientific research at present, pig experimental model of liver injury has become the urgent field of veterinary science, comparative medicine and other problems. Therefore, this issue will be used to study the establishment of laparoscopic minimally invasive surgical techniques small pig liver injury model, explore the establishment of liver resection and partial resection operation method with ischemia injury model by laparoscopy, while the model set up The monitoring of safety, and compared with the traditional open surgery, finally to further explore the changes of liver injury in the environment on the basis of the model. This experiment selects 54 Bama miniature pigs as experimental animal, we randomly selected 5 pigs, laparoscopic liver resection and exploration of local liver anatomy the pigs were divided into 7 groups and then the remaining 7 pigs in each group, respectively, laparoscopic partial hepatectomy group, laparoscopic partial hepatectomy in sham control group, liver hepatectomy group, laparoscopic liver blood deficiency combined with partial resection group, laparoscopic liver resection group, laparoscopic resection of hepatic ischemia with false operation control group, hepatic ischemia combined with open resection group. During the test by establishing pneumoperitoneum, surgical pathway, separation of ligament, anatomy of hepatic portal, through liver parenchyma and hemostasis, right Half liver ischemia, cut the liver parenchyma, peritoneal lavage, remove the hepatic resection, suture of incision operation procedures of laparoscopic mini pig liver injury model. The vital signs, circulatory system, respiratory system in the process of model building, the important physiological indexes of body temperature in the whole continuous monitoring and recording, intraoperative and postoperative complications, and postoperative recovery of surgery, postoperative 30d laparoscopic surgical exploration site adhesion. Two rats in each group were preoperative, 4 h, 1D, 3D, 7d, 14d, 30d to take venous blood, routine blood test and serum total protein, aspartate aminotransferase, r- glutamyl transferase, total bilirubin. Urea nitrogen, creatinine and plasma coagulation; before operation, immediately after operation, postoperative 1D, postoperative 3D, 7d after operation and 1 months after operation of laparoscopic liver biopsy, made paraffin section and HE staining to observe the pathological changes, at the same time according to different model Don't detect liver tissue proliferating cell nuclear antigen, cyclin D1, tumor necrosis factor alpha, interleukin -6 expression and the content of malondialdehyde, superoxide dismutase, catalase activity and Caspase-3 activity, statistics and comparison of laparoscopic and open surgery and the changes of serum cortisol, C-reactive protein C found laparoscopic. Local hepatic surgical field exploration and anatomical observation of liver, porcine anatomical location, shape structure, ligament distribution, pipeline system and liver division and human anatomy established approximation, its unique structure for liver model. This experiment successfully completed laparoscopic liver resection in most pig liver ischemia combined with partial resection model and model laparoscopic miniature pig, physiological and biochemical indexes of the intraoperative and postoperative have different degree of volatility, but did not exceed safety tolerance in miniature pigs Range; compared to the laparotomy group, laparoscopic surgery group time is slightly longer, but a small incision, less bleeding, postoperative abdominal adhesion and recovery of 1D light, all the basic spirit and appetite of mini pigs after eating a small amount of liquid feed, postoperative 7-9d casing mouth and abdominal incision healing. The first model after the establishment of miniature pig liver resection model of liver environmental changes mainly for the 3D of serum AST and postoperative 1D and postoperative and control group significantly (P0.01), 7d after operation were significantly difference (0.01P0.05); pathological observation showed 7d after partial liver cell swelling, degeneration and inflammatory cell infiltration, liver tissue injury is obvious; postoperative 1D expression level of PCNA in the peak, then decreased gradually. The expression of Cyclin in liver D1 gene expression level was significantly higher in the early stage of liver after hepatectomy, postoperative 1D increased significantly, compared with the control group Significant differences (P0.01), until 3D after operation was significantly different (0.01P0.05). The liver tissue increased after surgery and TNF- alpha decrease in postoperative 1D increased rapidly to the maximum value, compared with the control group significantly (P0.01), postoperative 3D, 7d decreased, compared with control group (0.01P0.05; IL-6), significant changes to a lesser extent, postoperative 1D and 3D have significant difference with the control group (0.01P0.05). The liver ischemia combined with partial resection of liver model environmental changes mainly as compared with the control group after 1D and 3D serum AST significantly (P0.01), 7d (0.01P0.05 after operation were significantly difference), other monitoring points did not change significantly (P0.05). The model group the remaining liver tissue after 1D has extensive degeneration of liver cells and mild atrophy, occasionally sinusoidal congestion, liver lobules or local necrosis of liver cells focal necrosis, interstitial inflammatory cell infiltration; postoperative 7d visible local small liver The area of necrosis liver cell proliferation, local periportal fibrous connective tissue proliferation. Compared with the control group, in the immediate postoperative MDA increased significantly (P0.01), the difference of intraoperative 1H and postoperative 1D liver tissue malondialdehyde (0.01P0.05), the other was monitoring time points did not change significantly (P0.05); in the immediate postoperative SOD changed significantly (P0.01), the difference of 1D change significantly after surgery (0.01P0.05) and other monitoring points did not change significantly (P0.05); a significant difference in the immediate postoperative changes of catalase (P0.01), 1h after operation in 1D were significant difference (0.01P0.05). Other monitoring points did not change significantly (P0.05). The activity of Caspase-3 in liver tissue after operation in 1D group was significantly difference compared with the control (P0.01), the difference of 3D change immediately and after surgery was significantly (0.01P0.05), the other time points did not change significantly (P0.05). Through the test The results, draw the following conclusions: (1) successfully established the liver resection model and small pig pig liver ischemia combined with partial hepatectomy model using laparoscopic minimally invasive surgical techniques. (2) based on the mini pig perioperative vital signs, circulatory system, respiratory system, liver and kidney function, blood coagulation function, comprehensive monitoring and evaluation. Various physiological indexes are maintained in a safe range, prove the technique and method of operation is safe and feasible. (3) compared with the traditional open surgery to set up a small pig liver injury model, laparoscopic surgery of small incision, less bleeding, less postoperative adhesions, and has little reaction to the animal body stress, fast minimally invasive the advantage of recovery after operation. (4) in the model, through the detection and evaluation of porcine liver tissue and serum, the liver function after injury, pathology and liver cell proliferation, cytokine and growth factor The change of oxidative stress and apoptosis.

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R575;R-332

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