M细胞在急性胰腺炎肠黏膜屏障中的作用

Role of microfold cell in intestinal barrier after acute pancreatitis in rats

目的明确M细胞在急性胰腺炎时细胞因子TNF—α、IL-18的表达情况，探索M细胞在肠黏膜屏障功能中的可能作用。方法SD大鼠，随机分为急性胰腺炎6、12、24、48h组及对照组，每组10只，建立大鼠急性胰腺炎模型，收集标本。检测血清淀粉酶、谷丙转氨酶的变化，鲎试剂法检测血清内毒素变化，ELISA法检测血清内细胞因子TNF—α、IL-18的变化，检测肠系膜淋巴结细菌移位情况，观察回肠黏膜病理形态学改变，LCM、RT-PCR法检测M细胞中TNF-α mRNA、IL-18mRNA的表达，免疫荧光双重染色法观察M细胞中TNF-α、IL-18的表达。结果（1）血淀粉酶在胰腺炎6、12、24h组升高，48h组下降。（2）血清内毒素在急性胰腺炎12、24、48h组升高，6h组无明显升高，肠系膜淋巴结细菌移位菌落数在急性胰腺炎各时间点均升高。（3）血清TNF—α在急性胰腺炎组6、12、24h组升高，48h组下降到正常水平，血清IL-18在胰腺炎各时间点均升高，在24h达到最高水平。（4）病理检查发现，在急性胰腺炎组随着时间延长回肠黏膜水肿逐渐加重。（5）免疫荧光双重染色结果显示，在急性胰腺炎组的各时间段M细胞TNF-α蛋白表达均为阳性，在6h、12h和24h组M细胞内的IL-18蛋白表达呈明显阳性，对照组阴性。（6）在急性胰腺炎组各时间TNF-α mRNA的表达量均高于对照组。从术后6h开始升高并达到一个平台期，在12h和24h维持在较高水平，到术后48hTNF-α mRNA的表达量较前下降但仍高于对照组。在6h、12hIL～18mRNA的表达升高，48h下降。结论在急性胰腺炎，肠黏膜屏障功能受到不同程度损害，M细胞合成TNF-α、IL-18增加，M细胞可能在肠道黏膜免疫反应中发挥一定作用，但还需进一步研究。

Objective To investigate TNF-α and IL-18 expression levels in microfold cells in rat＇ s intestine after acute pancreatitis to evaluate the role of microfold cells （M cells） in intestinal barrier. Methods Fifty SD rats were randomized into the acute pancreatitis 6 h group, 12 h group, 24 h group, 48 h group and control group. The model of acute pancreatitis was established. Serum, peyer, s patch, ileum, and mesenteric lymph nodes （MLN） were harvested. Serum amylase, glutamate pyruvate transaminse, total bilirubin, endotoxin, TNF-α, and IL-18 were detected. Pathologic changes of the ileal mucous were observed. M cells were obtained with laser capture mierodissection and the expression of TNF-α mRNA and IL-18 mRNA in M cells were observed with RT-PCR. The expression of TNF-α and IL-18 was determined with immunofluorescence dual staining. Result 1）The level of serum amylase in pancreatitis increased obviously in 6,12,24 h group, but decreased in 48 h group. 2） Endotoxin in blood serum obviously increased in 12, 24, 48 hours after acute pancreatitis. The number of colony of bacterium aversion in MLN increased in acute pancreatitis. 3）TNF-α in blood serum obviously increased in 6, 12, 24 h groups in acute pancreatitis, and then decreased to normal level in 48 h. IL-18 in blood serum increased in acute pancreatitis. 4）Edema was observed in ileal mucus in acute pancreatitis. 5）The immunofluorescence double staining showed that TNF-α and IL 18protein expressed in M cell of all groups of acute pancreatitis but not in control group. 6）The level of TNFa mRNA and IL 18 mRNA expression was significantly higher in acute pancreatitis groups than in the control group. Conclusion Intestinal barrier is damaged in acute pancreatitis. M cells can excrete TNF-α and IL-18 in acute pancreatitis and may play important roles in intestinal mucosal immune.