暴发性肝衰竭小鼠细菌侵袭肠黏膜方式的研究

The study on bacteria invading the intestinal mucosa barrier in mice with fulminant hepatic failure

目的通过研究细菌侵袭肠黏膜屏障的方式，探讨暴发性肝衰竭（FHF）并发自发性腹膜炎（SBP）的机制。方法取240只雄性BALB／c小鼠，分为等渗盐水（NS）组（40只）、脂多糖（LPS）组（40只）、氨基半乳糖（GaIN）组（40只）、FHF模型组（120只）。分别腹腔注射相同体积的NS、LPS（10μg／kg）、GaIN（800mg／kg）、LPS（10μg／kg）／GalN（800mg／kg）。注射处理后，分别于2、6、9、12h和24h处死小鼠（每个时间点处死8只小鼠）。实验小鼠均在相应时间点摘取眼球，留取血清，并断头处死动物，留肝脏及大肠组织标本。用全自动生物化学分析仪检测ALT；对肝组织和大肠组织进行HE染色检测；透射电镜观察大肠黏膜超微结构及细菌侵袭肠黏膜的方式。用SPSS13．0统计软件进行数据分析，两组间ALT水平的分析采用Mann—WhitneyU检验。结果FHF模型组ALT水平、肝组织病理学检测结果及病死率和临床表现均符合FHF的诊断标准。4组小鼠注射处理后9h，HE染色发现大肠组织仅有轻微水肿及少量炎性细胞浸润，此时，透射电镜下观察发现FHF模型组肠上皮细胞微绒毛断裂、脱落、变短，紧密连接（TJs）不完整，细胞器变化明显，HE染色发现FHF模型组肝脏呈成片的出血性坏死，残存的肝细胞肿胀，出血坏死区见较多炎性细胞浸润，但NS组、LPS组、GaIN组肝脏组织病理形态及大肠黏膜超微结构变化不明显。FHF模型组注射处理后6～9h，细菌以胞饮的形式穿人肠壁，细菌穿入肠壁区域的肠道黏膜绒毛脱落，TJs出现断裂，注射处理后12h发现穿入的细菌以囊胞的形式存在。结论LPS（10μg／kg）／GAIN（800mg／kg）联合注射建立的FHF小鼠模型是成功的。FHF时，肠黏膜TJs的断裂可能为肠道内细菌进入肠黏膜提供了条件，TJS的断裂可能是FHF并发SBP的原因之一。

Objective To explore the mechanism of fulminate hepatic failure （FHF） complicated with spontaneous peritonitis （SBP） through the research of bacteria invading the intestinal mucosa barrier. Methods 240 BalB/c male mice were divided into four groups as isotonic NS group （n = 40）, lipopolysaccharide （LPS） group （n = 40）, galactosamine （GalN） group （n = 40） and FHF model group （n -- 120）. Each mouse received same volume ofNS, LPS （10μg/kg）, GaiN （800 mg/kg） or LPS （10μg/kg）/GalN （800 mg/kg） intraperitoneal injection according to its group. 8 mice were executed at 2, 6, 9, 12 and 24 hours after injection, respectively, and the liver and intestinal tissue samples were taken at the same time. ALT was measured by automatic biochemical analyzer and was compared between groups using Mann-Whitney U test. Liver and intestinal tissue received HE staining. The ultrastructure of intestinal mucosa and the method by which bacteria invaded the intestinal mucosa were observed by transmission electron microscopy. All data were analyzed by SPSS 13.0 statistic software. Results ALT level, results of hepatic pathology, mortality and clinical manifestations of mice in the FHF model group met the diagnostic criteria of FHF. Intestinal tissue was found with slight edema and little inflammatory cells infiltration through HE staining in all the 4 groups of mice 9 hours after injection. Microvilli were found broken, shed and shorten in the intestinal epithelial cells with incomplete tight junction （TJs） and obviously changed organelles in the FHF model group of mice observed by transmission electron microscope. Mass hemorrhagic necrosis of liver cells with remnant liver cells swelling and many inflammatory cells infiltration by HE staining in the FHF model group. But the changes in hepatic pathology and intestinal mucosa ultrastructure were not so obvious in the mice of NS, LPS and GaIN groups. Bacteria penetrated the intestinal wall by pinocytosis 6-9 hours after injection in the FHF model group, the microvilli were broken off and TJs turned rupture in the areas that the bacteria penetrated. The bacteria were found in the form of cyst 12 hours after injection. Conclusions LPS （10 mg/kg）/GalN （800 mg/kg） combined injection was successful in establishing the FHF mice model. The rupture of TJs may provide conditions for intestinal bacteria to penetrate the intestinal mucosa in FHF. Rupture of TJs may be one of the reasons why FHF was complicated with SBP.