家兔的Oddi括约肌功能异常模型制作及其组织内NOS和VIP变化的意义

发布时间：2018-04-15 09:01

  本文选题：胆囊切除术 + 迷走神经干切断术　； 参考：《中国医科大学》2008年博士论文

【摘要】： 前言 围绕在胆总管壶腹和胆胰管末端的括约肌,统称为Oddi括约肌(sphincter ofOddi,SO)。正常情况下,SO在维持胆道压力、促进胆汁排泄以及防止十二指肠液和胰液返流方面起重要作用。目前,国内外学者已经认识到SO异常包括SO功能异常(sphincter of Oddi dysfunction,SOD)和SO解剖形态异常两个部分,前者可能不合并形态的异常变化,而后者可能在形态异常的基础上并没有表现出功能异常。但是,SO测压(sphincter of Oddi manometry,SOM)是公认的检测SO功能异常的金标准。我们采用多种手术方法制作SOD的模型,并应用SOM的方法观察实验组家兔模型的各项压力指标,同时与对照组的正常家兔压力指标比较,以便判断模型制作是否成功。此外,我们应用免疫组化链酶亲和素-过氧化物酶法(SABC法)观察各组家兔SO和十二指肠乳头中一氧化氮合酶(Nitric oxidesynthase,NOS)和血管活性肠肽(Vasoactive intestinal polypeptide,VIP)的变化,探讨VIP和一氧化氮(Nitric oxide,NO)在SOD发病机制中的作用。 论文一 材料 1、分组方法 将77只成年家兔随机分为正常对照组8只、胆囊切除组12只、迷走神经干切断组8只、胆总管不完全结扎组11只、SO不完全结扎组9只、胆囊不完全结扎组9只、胆管注入胰酶组12只、SO注射酒精组8只,雄性40只,雌性37只。 2、仪器和药品 ①三通道测压管(长2米,外径1.7mm,3个侧孔径0.5mm,间隔2mm.) ②PC polygram HR高分辨,多通道胃肠功能测定仪 ③低顺应性水灌注系统 ④20%氨基甲酸乙酯;95%酒精;动物胰酶制剂(粉剂) 方法 1、手术操作 氨基甲酸乙酯(1.0g/kg)静脉注射麻醉,全部实验组均经腹中线开腹,且彻底止血后,行两层关腹。(1)对照组:仅行开关腹术;(2)胆囊切除组:结扎胆囊管和胆囊动脉,逆行切除胆囊;(3)迷走神经干切断组:于腹段食管两侧游离出左,右迷走神经干,用丝线将其悬吊,各切除约1cm的神经干;(4)胆总管不完全结扎组:于十二指肠乳头开口处对侧缘纵行切开十二指肠约1cm,将无菌硬膜外插管逆行插入胆总管,钝性分离胆总管周围组织,4~#丝线绕过胆总管,将胆总管结扎在硬膜外插管上,然后,拔出插管,缝合十二指肠切口;(5)SO不完全结扎组:如上述切开十二指肠,应用可吸收线横行缝扎乳头开口,观察仍有少量胆汁流出,缝合十二指肠切口;(6)胆囊不完全结扎组:牵拉肝脏于腹外,显露胆囊,缝合针带丝线绕过胆囊体中部,结扎闭合约50%横断面积;(7)胆管注入胰酶组:如上述切开十二指肠,将无菌硬膜外插管逆行插入胆总管,关闭十二指肠切口,并固定插管在肠壁上,缝合腹膜及腹肌,同时再次固定插管于腹肌上,钝性分离出一皮下隧道至家兔后颈部,插管沿道至后颈部切口,再次固定插管,关闭腹部外皮。将配制10%动物胰酶2ml自插管逆行注入胆总管,1hr和2hr后,分别再注入2ml胰酶,每次注入后均闭管10min;(8)SO注射酒精组:如上述切开十二指肠,乳头周围2mm内均匀四个点各注射95%酒精0.1ml,缝合十二指肠切口。对照组和实验组均于术前12hr和当日禁食不禁水,除胆管注入胰酶组于注完胰酶24hr后测压外,其余各组均于术后1周进行压力测定。 2、测压步骤 氨基甲酸乙酯(1.0g/kg)麻醉满意后,经原切口进腹腔,在十二指肠乳头开口的对侧缘,纵行切开十二指肠约1cm,暴露乳头。将高分辨,多通道胃肠功能测定仪接通三通道测压管,应用低顺应性水灌注系统,以十二指肠压力为曲线零点标定后,经乳头将测压管逆行插入胆总管。观察曲线,确定插入胆总管后,稳定30sec,进行测压3-5 min,然后逐渐撤拉导管达SO处,直视下并结合电脑显示曲线于高压力区处可确定测压管侧孔位于括约肌最佳位置。稳定30 sec后进行压力测定3-5 min,记录压力曲线以待分析。 3、观察指标 SO基础压;SO收缩幅度;SO收缩频率;SO收缩间期;胆总管压力 4、数据处理 本实验计算的压力指标均为计量资料,计量数据以均数±标准差((?)±s)表示。应用spss11.5统计分析软件,采用单因素方差分析的方法行各实验组与对照组比较。P＜0.05为差异显著,P＜0.01为差异非常显著。 结果 1、胆囊切除组家兔SO功能变化 SO基础压较对照组显著升高,平均为36.61±8.60mmHg(p＜0.01) 2、迷走神经切断干组家兔SO功能变化 SO基础压较对照组显著升高,平均为28.54±7.66mmHg(p＜0.05) 3、胆总管不完全结扎组家兔SO功能变化 SO基础压较对照组均显著升高,平均为28.45±7.02mmHg(p＜0.05) 4、其余各组家兔SO各项压力指标较对照组均无显著变化。 结论 家兔分别行胆囊切除、迷走神经干切断、胆总管不完全结扎处理后,可以引起SO运动功能改变。但是,仅是部分压力指标的变化。 材料 1、分组方法 同实验一方法,共8组。将上述家兔测压后,耳缘静脉注射5-10ml空气处死后,迅速取出十二指肠乳头及Oddi括约肌,放到4%多聚甲醛内固定。 2、仪器和药品 ①一抗:血管活性肠肽抗体,一氧化氮合酶抗体;二抗:山羊抗兔IgG ②5%BSA封闭液,SABC,复合消化液,DAB显色试剂盒 ③C-7070/BX41采集图像系统 ④MetaMorph显微图像分析系统 方法 (1)新鲜组织经4%多聚甲醛固定24hr,常规石蜡包埋,5岬连续切片,65℃烤25min;(2)常规脱蜡至水;(3)30%H_2O_2灭活内源性活化酶,浸泡10min后,蒸馏水洗3次;(4)热修复抗原:将切片浸入0.01M枸橼酸盐缓冲液(PH6.0),微波炉加热至沸腾后断电,间隔10min后,反复2次。冷却后PBS洗涤2次;(5)滴加5%BSA封闭液,室温20min。甩去多余液体,不洗;(6)滴加适当稀释的一抗兔IgG,4℃过夜,PBS洗涤;(7)滴加二抗生物素化山羊抗兔IgG,37℃孵育20min,PBS洗;(8)0.05%DAB显色,镜下控制反应时间,流水冲洗终止反应;(9)苏木素轻度复染,脱水,透明,树脂封片。(10)阴性对照用PBS代替一抗,其余步骤相同。 1、结果判断 观察NOS和VIP时,均以SO平滑肌细胞、乳头粘膜柱状上皮细胞和粘膜下结缔组织细胞的胞核蓝色,胞质棕黄色染色为阳性。每张切片选择5个视野,用40×物镜观察采集图像。对于每张图像,我们应用MetaMorph/C-7070/BX41显微图像分析系统测定其阳性区域的积分光密度值。结果记录在表格中,准备统计学分析。 2、统计处理 本实验计算的积分光密度值为计量资料,计量数据以均数±标准差((?)±s)表示。应用spss11.5统计分析软件,采用单因素方差分析的方法行各实验组与对照组比较。P＜0.05为差异显著,P＜0.01为差异非常显著。 结果 1、Oddi括约肌中NOS和VIP的积分光密度值的变化 ①迷走神经干切断组:NOS和VIP的积分光密度值均较对照组显著升高,分别为34.04±27.24(P＜0.01)和45.82±36.35(P＜0.01)。 ②胆总管不完全结扎组:NOS和VIP的积分光密度值较对照组显著升高,分别为29.40±15.99(P＜0.05)和53.06±61.24(P＜0.01)。 ③胆囊不完全结扎组:NOS和VIP的积分光密度值均较对照组显著升高, 分别为53.72±47.74(P＜0.01)和71.95±62.34(P＜0.01)。 ④胆管注入胰酶组:NOS的积分光密度值较对照组显著升高,分别为27.36±24.85(P＜0.05)。 ⑤其余各组家兔Oddi括约肌中NOS和VIP的积分光密度值较对照组均无显著变化。 2、乳头粘膜层中NOS和VIP的积分光密度值的变化 胆囊不完全结扎组,NOS积分光密度值较对照组有显著升高,为29.78±13.08(P＜0.05)。其余各组较对照组均无显著变化。 3、乳头粘膜下层和粘膜肌层中NOS和VIP的积分光密度值变化 胆囊不完全结扎组和胆管注入胰酶组NOS均较对照组显著升高,分别为81.28±47.39(P＜0.01)和75.47±73.11(P＜0.05)。胆总管不完全结扎组VIP较对照组显著升高,为129.09±182.35(P＜0.01),其余各组较对照组均无显著变化。 结论 家兔分别行迷走神经干切断、胆总管不完全结扎、胆囊不完全结扎或胆管注入胰酶处理后,其SO平滑肌、乳头粘膜层、乳头粘膜下层及肌层中的NOS和(或)VIP的数量发生变化,且均呈增多趋势。
[Abstract]:Preface
Around the end of the tube in the common bile duct ampulla and biliary pancreatic sphincter, referred to as Oddi sphincter (sphincter ofOddi, SO). Under normal circumstances, SO in the maintenance of biliary pressure, promote bile excretion and prevent duodenal and pancreatic juice back flow plays an important role. At present, domestic and foreign scholars have been aware of abnormal SO including SO function abnormal (sphincter of Oddi dysfunction, SOD SO) and anatomic abnormalities of two parts form, the abnormal changes of the former may not be combined form, the latter may be based on morphological abnormalities and showed no abnormalities. However, SO (sphincter of Oddi manometry, pressure SOM) is the gold standard accepted detection of SO dysfunction. We use a variety of methods to build SOD model, and using the method of SOM the pressure observation index of rabbit model of experimental group, while the control group in the normal rabbit pressure index, in order to judge The success of model making. In addition, we used immunohistochemical streptavidin peroxidase method (SABC method) of all rabbits SO and duodenal papilla synthase (Nitric oxidesynthase, NOS) and vasoactive intestinal peptide (Vasoactive intestinal, polypeptide, VIP) changes of VIP and nitric oxide (Nitric oxide, NO) in the role of SOD in pathogenesis.
Paper 1
Material Science
1, grouping method
77 adult rabbits were randomly divided into normal control group 8, cholecystectomy group 12, vagotomy Group 8, bile duct ligation incomplete group 11, SO group 9 incomplete ligation, gallbladder incomplete ligation group 9, bile duct injection pancreatin group 12, SO group of 8 alcohol injection only, male 40, female 37.
2, instruments and drugs
(1) three channel pressure tube (2 meters long, outer diameter 1.7mm, 3 side aperture 0.5mm, interval 2mm.)
PC Polygram HR high resolution, multichannel gastrointestinal function measuring instrument
Low compliance water perfusion system
(4) 20% ethyl carbamate; 95% alcohol; animal pancreatin (powder)
Method
1, operation
Ethyl carbamate (1.0g/kg) intravenous anesthesia, the experimental group were all abdominal midline laparotomy, and complete hemostasis, two layer closed abdomen. (1) control group: only switch abdominal surgery; (2) cholecystectomy group: ligation of the cystic duct and the cystic artery, retrograde cholecystectomy; (3) the vagus nerve cut off the stem group in the abdominal segment of esophagus on both sides of the free left and right vagus nerve, with the wire suspension, the removal of about 1cm neural stem; (4) common bile duct ligation group: incomplete in the duodenal papilla at the opening on the side edge of the duodenal longitudinal incision about 1cm, there will be no epidural catheterization retrograde insertion of bile bacteria duct, blunt dissection around the bile duct, 4~# silk around the common bile duct, common bile duct ligation in the epidural catheter, then pull the plug, suture the duodenal incision; (5) SO incomplete ligation group: cutdown the duodenum, application of absorbable suture line with the papilla, observe still A small amount of bile flow, suture the duodenal incision; (6) gallbladder incomplete ligation group: traction in the abdominal liver, gallbladder exposed, suture needle with thread around the central body of gallbladder, ligation of closed about 50% cross-sectional area; (7) bile duct injection pancreatin group: cutdown the duodenum, the sterile epidural cannula inserted retrograde bile duct, closed duodenal incision and fixed intubation in the intestinal wall, closure of peritoneum and abdominal muscles, and again fixed to facilitate intubation, blunt dissection of a subcutaneous tunnel to rabbit neck, along the road to the neck incision after intubation, intubation again fixed, closed abdominal skin. The preparation of 10% animal trypsin 2ml since retrograde intubation injected into the common bile duct, 1hr and 2hr, respectively, and then injected into 2ml pancreatin, close the pipe 10min after each injection; (8) SO alcohol injection group: cutdown the duodenum, even four points around the nipple 2mm within the injection of 95% ethanol 0.1ml, ten suture Two fingers intestinal incision. The control group and the experimental group were all fasted before 12hr and the day of the experiment. Except for the bile duct injected into the pancreatin group, the pressure was measured at 1 weeks after the injection of trypsin 24hr.
2, the step of pressure measurement
Ethyl carbamate (1.0g/kg) after the anesthesia satisfaction, through the original incision into the abdominal cavity, the side edge of the opening of duodenal papilla, longitudinal incision of duodenal papilla. The exposure of about 1cm, high resolution, instrument on three channel multi channel pressure tube determination of gastrointestinal function, using low compliance water perfusion system, the duodenal pressure curve of zero after calibration, the nipple will pressure tube retrograde into the common bile duct. The observation curve, determine the insertion of common bile duct after 30sec were stable, pressure 3-5 min, then gradually withdraw pulling catheter of SO, and combined with the computer display under the open curve in high pressure can be determined at the pressure tube side hole located in the best position of sphincter pressure measurement. 3-5 min were stable after 30 sec analysis, record pressure curve for.
3, observation index
SO base pressure; SO contraction amplitude; SO contraction frequency; SO contraction interval; choledochal pressure
4, data processing
The pressure index calculation for measurement data, measurement data to mean + standard deviation ((?) + s). Using SPSS11.5 statistical analysis software, using the method of single factor analysis of variance for each experimental group compared with control group.P < 0.05, P < 0.01 for the difference, difference is very significant.
Result
1, the changes of SO function in the cholecystectomy group
The base pressure of SO was significantly higher than that of the control group, with an average of 36.61 8.60mmHg (P < 0.01).
2, the changes of SO function in the rabbits with vagotomy
The base pressure of SO was significantly higher than that of the control group, with an average of 28.54 7.66mmHg (P < 0.05).
3, the changes of SO function in the rabbits with incomplete ligation of common bile duct
The base pressure of SO was significantly higher than that of the control group, with an average of 28.45 7.02mmHg (P < 0.05).
4, there was no significant change in the pressure index of SO in the rest of the rabbits compared with the control group.
conclusion
Cholecystectomy, vagotomy, and incomplete ligation of common bile duct can cause changes in motor function of SO in rabbits.
Material Science
1, grouping method
There were 8 groups in the same experiment method. After the above rabbits were given the 5-10ml pressure, the duodenal papilla and Oddi sphincter were quickly removed and placed into 4% paraformaldehyde.
2, instruments and drugs
1 antibody: vasoactive intestinal peptide antibody, nitric oxide synthase antibody; two anti: Goat anti rabbit IgG
(2) 5%BSA sealing solution, SABC, compound digestive juice, DAB chromogenic Kit
C-7070/BX41 acquisition image system
MetaMorph microscopic image analysis system
Method
(1)鏂伴矞缁勭粐缁，

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