大鼠弥漫性脑损伤后小肠黏膜超微结构与黏膜上皮细胞凋亡的动态变化

Dynamic changes of ultrastructure and epithelial cell apoptosis in intestinal mucosa of rats after diffuse brain injury

目的:观察弥漫性脑损伤后大鼠肠黏膜超微结构和上皮细胞凋亡的动态变化,分析二者在肠黏膜屏障功能障碍中的作用。方法:实验于2003-12/2005-12在华北煤炭医学院附属医院动物实验室进行。将雄性Wistar大鼠以随机数字表法分成对照组和伤后1,2,4,8,12,24,48,72,168h共9个时相组,每组15只。采用Marmarou脑损伤动物模型法对损伤组致大鼠重型弥漫性脑损伤,对照组只切开头皮,不行落体致伤,而后缝合切口。严格依照1,2,4,8,12,24,48,72,168h时间点,依次对各组大鼠回盲部近端2cm处切取直径为1mm小肠组织圈。对照组术后立即处死,处理同实验组。透射电镜下比较对照组和致伤组大鼠肠黏膜超微结构的改变,观察伤后小肠黏膜超微结构的动态变化。原位末端标记(TUNEL)法计数各组凋亡细胞的发生率。结果:实验大鼠全部进入结果分析,无脱失。①对照组大鼠肠黏膜上皮细胞微绒毛高而密,排列整齐,细胞间紧密连接完整,线粒体等细胞器结构正常。致伤组微绒毛短而稀,不规则,可见紧密连接开放,线粒体肿胀,内嵴断裂。②TUNEL法显示凋亡细胞胞核为棕褐色,固缩呈断片状,主要位于绒毛顶部,且随致伤时间的延长有由上向下发展的趋势。伤后1h细胞凋亡计数较对照组差异无显著性意义[(2.47±1.26),(2.16±0.83)个/100个细胞,P>0.05],伤后2h直至168h凋亡细胞数较对照组均显著增加[(6.17±2.23),(13.79±2.99),(16.68±2.91),(21.74±3.75),(16.16±2.63),(14.05±3.06),(16.26±2.90),(7.32±2.50)个/100个细胞,P<0.01]。结论:弥漫性脑损伤后,肠黏膜超微结构的改变包括紧密连接开放等是肠黏膜屏障功能障碍的形态学基础,黏膜上皮细胞凋亡过度在肠黏膜屏障功能障碍发生中可能起重要作用。

AIM： To explore the effects on intestinal muco.sal barrier dysfuction of rats after diffuse brain injury by studying the dynamic changes of ultrastructure in the intestinal mucosa and apeptosis of endothelial cell. METHODS： The experiment was conducted in animal laboratory of the affiliated Hospital of North China Coal Medical College between December 2003 and December 2005. Totally 150 male Wistar rats were randomly divided into control group and 9 injury groups at 1, 2, 4, 8, 12, 24, 48, 72 and 168 hours after injury with 15 rats in each group. Animal models of diffuse brain injury were established by Marmarou method, and the rats in the control group were only subjected to incision of the scalp without falling injury, followed by suture. The tissue ring of small intestine 2 cm from the proximal of ileocecal junction of rats in the injury groups were cut off at 1, 2, 4, 8, 12, 24, 48, 72 and 168 hours after injury, respectively. The rats in the control group were immediately killed after incision. Changes of intestinal ultrastucture of the control and injury groups were compared with the transmission electron microscope to observe the dynamic changes of ultrastucture. The number of epithelial apeptotic cells of each group were observed and TUNEL method. RESULTS： All the rats were involved in the result analysis without loss. ①The microvilli of intestinal mucosa in the control group were high and dense, arranged in order and tight junction was intact, and structures of mitochondria and other cell organs were normal. But in the injury groups, the micrevilli were irregular, short and sparse, tight junction was open and mitochondria were swollen with crista collapsed. ②TUNEL method showed that the nucleus of the apeptotic cells were brown, the pyknosis was lamellar, mainly located at the top of microvilli, and prone to down with the prolonging of injured time. The number of apeptotic cells at 1 hour had no significant difference compared with the control group [（2.47±1.26）, （2.16±0.83） cells/100 cells, P 〉 0.05], the apeptotic cells began to in- crease from the 24 hour to the 168^th hour [（6.17±2.23）, （13.79±2.99）, （16.68±2.91）, （21.74±3.75）, （16.16±2.63）, （14.05±3.06）, （16.26±2.90）, （7.32±2.50） cells/100 cells, P 〈 0.01]. CONCLUSION： Changes of ultrastructure including opened tight junction are the morphological basis of intestinal mucosal barrier dysfunction after diffuse brain injury. Excessive apeptosis of intestinal epithelial cells might play the impertant role in the development of intestinal mucosal barrier dysfunction after diffuse brain injury.