本文選題：小型豬 + 微創(chuàng)外科��； 參考：《東北農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：小型豬與人在比較醫(yī)學(xué)上同源關(guān)系較近,在解剖學(xué)、生理學(xué)、生物化學(xué)和疾病發(fā)生機(jī)理等方面極其相似,常用作理想的非嚙齒類實(shí)驗(yàn)動(dòng)物模型,如小型豬糖尿病模型、腹壁拉鏈模型、心肌梗死模型等,然而采用微創(chuàng)技術(shù)建立小型豬肝臟損傷模型的研究在國(guó)內(nèi)外尚未見(jiàn)報(bào)道,加之目前科學(xué)研究中對(duì)理想大動(dòng)物肝臟損傷模型的迫切需求,小型豬的實(shí)驗(yàn)用肝臟損傷模型已經(jīng)成為獸醫(yī)學(xué)、比較醫(yī)學(xué)等領(lǐng)域亟待解決的問(wèn)題。因此,本課題將采用腹腔鏡微創(chuàng)外科技術(shù)進(jìn)行小型豬肝臟損傷模型建立的研究,探索腹腔鏡下建立肝臟大部分切除及缺血合并部分切除損傷模型的操作方法,同時(shí)對(duì)模型建立的安全性進(jìn)行監(jiān)測(cè),并與傳統(tǒng)開(kāi)放性手術(shù)進(jìn)行對(duì)比研究,最后在模型基礎(chǔ)上進(jìn)一步探討損傷后肝臟環(huán)境的變化。本試驗(yàn)選用54頭巴馬小型豬為實(shí)驗(yàn)動(dòng)物,首先隨機(jī)選取5頭小型豬,進(jìn)行腹腔鏡下肝臟局部術(shù)野探查和肝臟解剖學(xué)觀察,然后將剩余的小型豬隨機(jī)分為7組,每組7頭,分別為腹腔鏡肝葉大部分切除組、腹腔鏡肝葉大部分切除假手術(shù)對(duì)照組、開(kāi)腹肝葉大部分切除組,腹腔鏡肝臟缺血合并部分切除組、腹腔鏡肝臟部分切除組、腹腔鏡肝臟缺血合并部分切除假手術(shù)對(duì)照組、開(kāi)腹肝臟缺血合并部分切除組。試驗(yàn)過(guò)程中通過(guò)氣腹、建立手術(shù)通路、分離韌帶、解剖第一肝門、貫穿肝實(shí)質(zhì)與止血、右半肝缺血、切斷肝實(shí)質(zhì)、沖洗腹腔、切除肝葉的取出、縫合切口等操作步驟進(jìn)行腹腔鏡下小型豬肝臟損傷模型的建立。模型建立過(guò)程中對(duì)生命體征、循環(huán)系統(tǒng)、呼吸系統(tǒng)、體溫等重要生理指標(biāo)進(jìn)行全程連續(xù)監(jiān)測(cè),并記錄術(shù)中和術(shù)后并發(fā)癥、手術(shù)完成及術(shù)后恢復(fù)情況,術(shù)后30d行腹腔鏡二次探查手術(shù)部位粘連。各組分別于術(shù)前、4 h、1d、3d、7d、14d、30d采取靜脈血,檢測(cè)血常規(guī)和血清總蛋白、谷草轉(zhuǎn)氨酶、r-谷氨酰轉(zhuǎn)移酶、總膽紅素、尿素氮、肌酐及血漿凝血功能;在術(shù)前、術(shù)后即刻、術(shù)后1d、術(shù)后3d、術(shù)后7d和術(shù)后1個(gè)月進(jìn)行腹腔鏡下肝臟活組織檢查,做石蠟切片和HE染色觀察組織病理學(xué)變化,同時(shí)針對(duì)不同模型分別檢測(cè)肝臟組織增殖細(xì)胞核抗原、細(xì)胞周期素D1、腫瘤壞死因子-α、白細(xì)胞介素-6的表達(dá)和組織丙二醛含量、超氧化物歧化酶、過(guò)氧化氫酶活性及Caspase-3活性,統(tǒng)計(jì)并對(duì)比腹腔鏡和開(kāi)腹手術(shù)結(jié)果和血清皮質(zhì)醇、C反應(yīng)蛋白的變化。腹腔鏡肝臟局部術(shù)野探查和解剖學(xué)觀察發(fā)現(xiàn),小型豬肝臟解剖位置、外形結(jié)構(gòu)、韌帶分布、管道系統(tǒng)及肝葉劃分方面與人類近似,其特有的解剖學(xué)結(jié)構(gòu)便于肝臟模型的建立。本試驗(yàn)成功完成了小型豬腹腔鏡下肝臟大部分切除模型和小型豬腹腔鏡下肝臟缺血合并部分切除模型的建立,術(shù)中和術(shù)后各項(xiàng)生理生化指標(biāo)雖有不同程度的波動(dòng),但均未超出小型豬的安全耐受范圍;相對(duì)于開(kāi)腹組,腹腔鏡組的手術(shù)時(shí)間稍長(zhǎng),但手術(shù)切口小,出血量少,術(shù)后腹腔粘連輕,且所有小型豬術(shù)后1d精神狀態(tài)和食欲基本恢復(fù),能進(jìn)食少量流質(zhì)飼料,術(shù)后7-9d套管口和腹壁切口取第一期愈合。模型建立后,小型豬肝臟大部分切除模型肝臟環(huán)境變化主要表現(xiàn)為術(shù)后1d和術(shù)后3d血清AST與術(shù)前和對(duì)照組相比差異極顯著(P0.01),術(shù)后7d差異顯著(0.01P0.05);病理學(xué)觀察可見(jiàn)術(shù)后7d局部肝細(xì)胞腫脹、變性并伴有炎性細(xì)胞浸潤(rùn),肝組織損傷較明顯;術(shù)后1d肝細(xì)胞PCNA表達(dá)水平于達(dá)到高峰,之后逐漸降低。肝臟組織Cyclin D1基因表達(dá)量在肝大部切除后早期表達(dá)明顯升高,術(shù)后1d增高明顯,與對(duì)照組相比差異極顯著(P0.01),一直持續(xù)到術(shù)后3d差異顯著(0.01P0.05)。術(shù)后肝組織TNF-α先升高后降低,在術(shù)后1d迅速升高到最高值,與對(duì)照組相比差異極顯著(P0.01),術(shù)后3d、7d逐漸降低,與對(duì)照組相比差異顯著(0.01P0.05),;IL-6變化幅度較小,術(shù)后1d和3d與對(duì)照組相比差異顯著(0.01P0.05)。肝臟缺血合并部分切除模型肝臟環(huán)境變化主要表現(xiàn)為,與對(duì)照組相比術(shù)后1d和3d血清AST差異極顯著(P0.01),術(shù)后7d差異顯著(0.01P0.05),其它監(jiān)測(cè)時(shí)間點(diǎn)變化不顯著(P0.05)。模型組剩余肝組織術(shù)后1d有廣泛的肝細(xì)胞變性和輕度萎縮,偶見(jiàn)竇狀隙淤血,肝小葉內(nèi)有局部肝細(xì)胞壞死灶或片狀壞死,間質(zhì)中炎性細(xì)胞浸潤(rùn);術(shù)后7d可見(jiàn)局部肝小葉內(nèi)壞死區(qū)域有肝細(xì)胞的增殖,局部匯管區(qū)纖維性結(jié)締組織增生等。與對(duì)照組相比,在術(shù)后即刻丙二醛升高差異極顯著(P0.01),術(shù)中1h和術(shù)后1d肝組織丙二醛變化差異顯著(0.01P0.05),其他監(jiān)測(cè)時(shí)間點(diǎn)變化不顯著(P0.05);在術(shù)后即刻超氧化物歧化酶變化差異極顯著(P0.01),術(shù)后1d變化差異顯著(0.01P0.05),其他監(jiān)測(cè)時(shí)間點(diǎn)變化不顯著(P0.05);在術(shù)后即刻過(guò)氧化氫酶變化差異極顯著(P0.01),術(shù)中1h術(shù)后1d差異顯著(0.01P0.05),其他監(jiān)測(cè)時(shí)間點(diǎn)變化不顯著(P0.05)。在術(shù)后1d肝組織中Caspase-3活性相對(duì)于對(duì)照組變化差異極顯著(P0.01),術(shù)中即刻和術(shù)后3d變化差異顯著(0.01P0.05),其他時(shí)間點(diǎn)變化不顯著(P0.05)。通過(guò)試驗(yàn)結(jié)果,得出以下結(jié)論:(1)運(yùn)用腹腔鏡微創(chuàng)外科技術(shù)成功建立了小型豬肝臟大部分切除模型和小型豬肝臟缺血合并部分切除模型。(2)通過(guò)對(duì)小型豬圍手術(shù)期生命體征、循環(huán)系統(tǒng)、呼吸系統(tǒng)、肝腎功能、凝血功能等綜合監(jiān)測(cè)和評(píng)價(jià),各項(xiàng)生理指標(biāo)均維持在安全范圍之內(nèi),證明了該技術(shù)和操作方法是安全、可行的。(3)與傳統(tǒng)開(kāi)腹手術(shù)建立小型豬肝臟損傷模型相比,腹腔鏡手術(shù)切口小、出血量少、術(shù)后粘連輕,且具有對(duì)動(dòng)物機(jī)體應(yīng)激反應(yīng)小、術(shù)后恢復(fù)較快等微創(chuàng)優(yōu)勢(shì)。(4)在模型建立后,通過(guò)對(duì)小型豬肝臟組織及血清的檢測(cè)與評(píng)估,明確了損傷后肝臟功能、病理學(xué)以及肝臟細(xì)胞增殖、細(xì)胞因子與生長(zhǎng)因子、氧化應(yīng)激反應(yīng)和凋亡的變化規(guī)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.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R575;R-332

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