急性胰腺炎病情與腸屏障功能損害相關(guān)性的實(shí)驗(yàn)研究

1、急性胰腺炎病情與腸屏障功能損害相關(guān)性的實(shí)驗(yàn)研究         08-11-08 13:47:00     編輯：studa20                   作者：劉麗艷，李玉明，王永兵，顧留根，楊其昌，陶國(guó)華，葛熙【摘要】  目的：探討急性胰腺炎病情與腸屏障功能(IBF)損害的相

2、關(guān)性及干預(yù)治療后腸屏障功能損傷程度的改善情況。方法：日本大耳白兔36只，隨機(jī)分為對(duì)照組A ，輕型胰腺炎組B，重型胰腺炎組C，重型胰腺炎干預(yù)組D，后3組采用胰管結(jié)扎聯(lián)合膽汁注射的方法制成兔SAP模型。造模后3、6、12小時(shí)、第1、3、7天取血測(cè)定淀粉酶的含量及測(cè)血清腫瘤壞死因子及白介素6水平及其變化。實(shí)驗(yàn)第7天處死動(dòng)物收集胰腺、肝臟、肺、小腸組織，觀察其組織病理學(xué)并行胰腺，小腸病理評(píng)分，制備小腸組織勻漿測(cè)定TNF-、IL-6、丙二醛水平。結(jié)果：B組、C組、D組實(shí)驗(yàn)前、后淀粉酶有顯著差異；A組實(shí)驗(yàn)前、后淀粉酶差異無(wú)統(tǒng)計(jì)學(xué)意義。C組與B組、D組之間比較，淀粉酶、TNF-、IL-6、小腸勻漿中TNF-

3、、IL-6、MDA差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。B組、D組之間比較差異無(wú)統(tǒng)計(jì)學(xué)意義。胰腺、肺、肝，小腸的病理學(xué)改變以C組為甚，光鏡下觀察小腸黏膜損傷嚴(yán)重，D組比C組病理?yè)p害明顯減輕，B組僅輕度損傷。結(jié)論：重型胰腺炎腸道黏膜損傷及IBF障礙較輕型胰腺炎明顯，重型胰腺炎給予藥物干預(yù)后腸道黏膜損傷程度及胰腺炎病情明顯低于重型胰腺炎，說(shuō)明早期治療干預(yù)能改善IBF，有助于胰腺炎的治療。 【關(guān)鍵詞】  急性胰腺炎；腸屏障功能；腫瘤壞死因子-；白細(xì)胞介素-6；丙二醛     Abstract  Objective: To investig

4、ate the correlation between the condition of acute pancreatitis and the intestinal mucosal barrier dysfunction and the improvement in the intestinal injury degree after intervention therapy. Methods: Thirty-six experimental rabbits were randomly divided into four groups, sham-operation(A)group, mild

5、 acute pancreatitis(B) group, severe acute pancreatitis(C)group, and intervention on severe acute pancreatitis(D)group. AP model was induced by ligating pancreatic duct and injecting bile. The changes of serum concentration of amylase, TNF- and IL-6 of acute pancreatitis  were observed after 3,

6、 6, 12 hour and 1, 3, 7 day. Specimens of tissues from pancreas, liver, lung and intestine were taken at the 7th day, and pathological changes of these tissues were observed. The pancreas and intestine were evaluated pathologically. The leveles of TNF-、IL-6、and MDA in intestine were determined. Resu

7、lts: The content of amylase, TNF- and IL-6 in serum and TNF-and MDA in intestine had significant differences in group B, C, and D before and after experiment. There were significant differences among group C and groups B and D, but There was no distinct difference between group B and group D, while

8、A group showed no distinct difference. Pathological changes of pancreas, liver, lung and intestine in group C were much greater than in groups A, B and C. The injury of intestinal mucous membrane in group C was serious, the injury in group D was less than in group C and group B was only  mildly

9、 injured. Conclusion: The injury of intestinal mucous membrane and intestinal barrier dysfunction  in severe acute pancreatitis was more obvious than in mild acute pancreatitis. After intervention the injury of intestinal mucous membrane and illness decreased significantly than before intervent

10、ion. This showed that earlier intervention, improving intestinal function, was beneficial for the treatment of pancreatitis.    Key words   Acute pancreatitis；Intestinal barrier function；Tumor necrosis factor alpha；Interleukin-6；Malondialdehyde急性胰腺炎是一種消化系統(tǒng)常見疾病，發(fā)生率為4/10萬(wàn)，近年有增

11、加趨勢(shì)1。重癥急性胰腺炎（severe acute pancreatitis，SAP）起病急、發(fā)展迅速、并發(fā)癥多，早期即可發(fā)生全身炎癥反應(yīng)綜合征（systimic inflammational response synd rome, SIRS）、多器官功能衰竭（multiple organ dysfunction syndrome，MODS）。雖然現(xiàn)在對(duì)SAP 的治療較以前有了很大的進(jìn)展，但病死率仍高達(dá)10%30%24。    已知SAP時(shí)存在有IBF損傷，但I(xiàn)BF障礙對(duì)SAP是促發(fā)因素還是病程發(fā)展的結(jié)果，SAP的病情嚴(yán)重程度與IBF損傷的相關(guān)性，以及如何檢測(cè)IB

12、F的損傷尚有一些不明確之處。本實(shí)驗(yàn)?zāi)康脑谟谔接慖BF與胰腺炎的病情相關(guān)性，從而有助于早期預(yù)防及盡早和盡可能保護(hù)腸屏障，減輕SAP的病情，縮短病程，減低死亡率。    1   材料及方法    1.1   實(shí)驗(yàn)動(dòng)物   日本大耳白兔36只，雄性，體重2.83.5 kg ，南通大學(xué)實(shí)驗(yàn)動(dòng)物中心提供，其中造模27只，對(duì)照9只。    1.2   實(shí)驗(yàn)材料和設(shè)備   戊巴比妥由南通大學(xué)提供,用蒸溜水配成3%的濃度。

13、日本Olympus BX50光學(xué)顯微鏡及北航圖像處理系統(tǒng)；電腦微量輸液泵YBS-購(gòu)自上海電表廠。    1.3   實(shí)驗(yàn)動(dòng)物分組及模型的制作   36只大耳白兔隨機(jī)分為4組：空白對(duì)照組（A組），輕型胰腺炎(MAP)組（B組），重型胰腺炎(SAP)組（C組），重型胰腺炎干預(yù)組（D組），每組各9只。實(shí)驗(yàn)前兔禁食1214小時(shí)，但不禁水。以3%戊巴比妥經(jīng)耳緣靜脈麻醉（30 mg/kg）?？煽匦許AP模型制作方法參照本院實(shí)驗(yàn)組的操作規(guī)范執(zhí)行5。（1）B組胰膽管結(jié)扎留置導(dǎo)管引流，動(dòng)脈夾夾閉胰膽管引流管，術(shù)后5分鐘后松夾引流，制成輕癥胰腺

14、炎模型；（2）C組用2 ml注射器從膽總管引流管抽取膽汁(0.8 ml/kg)注入胰膽管引流管，動(dòng)脈夾夾閉胰膽管引流管，術(shù)后30分鐘后松夾引流，制成重癥胰腺炎模型；（3）D組造模的方法基本同C組；（4）A組打開腹腔翻動(dòng)胰十二指腸后關(guān)腹。    1.4   術(shù)后處理   B、C、D組于成模后第1 天經(jīng)中央靜脈注入腸外營(yíng)養(yǎng)液, 每日補(bǔ)液量為100 ml/kg,分2次給予。D組于成模后3小時(shí)給予空腸營(yíng)養(yǎng)，9小時(shí)后注入腸毒清，（沿空腸造瘺管注入百普利50 ml/d，腸毒清20 ml/d），后每日間隔9小時(shí)分別注入百普利、腸毒清各1次

15、（百普利由鈕迪希亞公司提供，腸毒清由本院制劑室提供）。組術(shù)后禁食6小時(shí)后正常喂養(yǎng)。    1.5   檢測(cè)方法    1.5.1   標(biāo)本采集   經(jīng)耳緣靜脈，分別于造模前，造模后3小時(shí)、6小時(shí)、12小時(shí)、第1天、第3天、第7天、采血2 ml，在離心機(jī)上以3000 r/min離心10分鐘，取上清液。-20冰箱保存待檢測(cè)血淀粉酶、用ELISA法測(cè)定TNF-、IL-6水平。死亡或成活1周后處死，無(wú)菌條件下取胰腺、肺、肝及小腸，5%甲醛固定后做常規(guī)病理切片，經(jīng)HE染色觀察其組織病理學(xué)改變，并行胰腺病理評(píng)分6，小腸Chiu氏評(píng)分。取距回盲10 cm處小腸制備組織勻漿用放射免疫法及硫代巴比妥法測(cè)定小腸勻漿中TNF-、IL-6及丙二醛（MDA）。    1.5.2   腸組織勻漿的制備   冰浴下將切取的小腸縱行剖開，在預(yù)冷的0.01 mol/L PBS中漂洗，濾紙吸干，準(zhǔn)確稱取1g小腸組織，放入10 ml的小燒杯，加入預(yù)冷的勻漿介質(zhì)6 ml，用眼科小剪盡快剪碎組織后倒入玻璃勻漿器，再用3 ml勻漿介質(zhì)沖洗殘留在燒杯中的碎組織，一起倒入勻漿管中，充分研碎，以制成10組織勻漿，在