摘要
多器官功能衰竭（multiple organ failure, MOF）是创伤及感染后最严重的并发症，发病率高，往往由于来势凶猛，病情发展急剧，难以被迄今的器官支持治疗所遏制，故死亡率很高。一般认为MOF起源于持续的、难以控制的炎症反应，立足于全身炎症反应的角度，肠道在MOF的发病中不仅是被损伤的靶器官，更是在应激状态下机体内外环境稳定和全身炎症反应的重要调节者。
肠粘膜含有丰富的肥大细胞，其活化脱颗粒可释放的大量炎症介质，包括TNF-(、组胺、激肽、趋化因子、前列腺素、白三烯、IL-1、6、8、 IFN-γ等。已知TNF-(在MOF发生过程中的细胞因子连锁反应中起核心作用。因此肠粘膜肥大细胞 (Intestinal mucosal mast cells, IMMC) 活化可能参与了MOF的发生。
胃肠道粘膜的神经细胞与神经介质、内分泌细胞与多肽分子、免疫细胞与细胞因子都是机体中数量最集中的部位。胃肠多肽是神经-内分泌-免疫网络中的重要调节因子。已有的少量研究表明，胃肠多肽如P物质（substance P, SP）、生长抑素（somatostatin, SST）、肠血管活性多肽（vasoactive intestinal peptide, VIP）对肠粘膜免疫系统发挥着重要的调节作用，但这些胃肠多肽对IMMC活性的调节目前国内、外研究不多。
由于IMMC体外长期传代培养很困难，因此，目前较多采用新鲜分离的IMMC进行体外研究。根据文献报道的IMMC的分离方法，所获细胞中IMMC纯度仅30%左右。因此，提高所分离IMMC的纯度及细胞的活力十分重要。
目 的
探讨 1) 影响IMMC纯化及细胞活力的因素和最佳条件；2） SP、SST、VIP等胃肠多肽对离体大鼠IMMC释放组胺及脱颗粒的影响；3）MOF时IMMC活性的变化，从粘膜免疫的角度探讨MOF的病理生理机制；4） P物质（substance P, SP）、生长抑素(somatostatin, SST)、肠血管活性肽(vasoactive intestinal polypeptide, VIP) 在MOF状态下对IMMC 活性的调节以及对MOF发展的影响，从粘膜免疫
的角度理解MOF的病理生理机制；5）IMMC上是否具有SST和VIP受体mRNA表达，以确定这些胃肠多肽调节IMMC功能的分子机制
方 法
1、采用胶原酶消化法分离、不连续密度梯度离心纯化正常大鼠IMMC；
2、荧光法测定IMMC细胞内、外及外周血组胺；
3、用透射电镜观察IMMC超微结构；
4、用酵母多糖腹腔注射，制作大鼠MOF模型；
5、用TNF-( ELISA试剂盒测定外周血和大鼠肠道TNF-(水平；
6、用苏木素-伊红染色观察大鼠小肠、肝脏、肾脏和肺脏的组织学改变；
7、采用RT-PCR方法检测IMMC上SST和VIP受体的mRNA表达。
结 果
1、IMMC纯化的最佳条件是：在30%+80%percoll密度梯度中，pH7.4，2~10(C ，渗透压250~300mosmol/Kg ，离心速度2700~3000转/分。在这些条件下，IMMC纯化率可达70%左右，组胺自然释放率为24.8%，细胞能保持良好的活力及超微结构。
2、体外实验中，SP促进大鼠IMMC组胺释放，其效应与SP浓度呈正相关，r=0.983, P<0.01。SP可使IMMC内组胺显著高于对照组，P<0.01。
3、SST显著降低体外实验中IMMC组胺释放率，其效应与SST浓度呈负相关，r= -0.991, P<0.01。但IMMC内组胺含量与对照组比较无明显变化，P(0.05。
4、在体外实验浓度为1(10-5 ~1(10-8 mol/L 范围内，随VIP浓度降低，IMMC组胺释放率呈剂量相关性升高。但继续降低VIP浓度至1(10-9 mol/L时，IMMC组胺释放率降低至自然分泌状态，随着VIP浓度的继续降低，组胺释放无明显变化。
5、大鼠MOF模型建立成功，MOF时大鼠小肠、肝、肾、肺等在病理形态上出现广泛急性炎症改变，功能明显受损。
6、MOF时小肠局部组胺浓度（8.67(1.16 ng/克蛋白）比正常组（11.63(1.97 ng/克蛋白）明显降低，P (0.01；外周血组胺浓度无明显变化，P(0.05；小肠局部
TNF-(浓度较正常明显升高（15.68(1.81 vs 3.18(0.72 pg/克蛋白），P (0.01；外周血TNF-(浓度亦较正常明显升高（12.83(1.10 vs 0 pg/ml），P (0.01。
7、MOF时小肠IMMC数量较正常增加约2倍，出现明显的脱颗粒形态学改变，IMMC的超微结构变化与MOF病理变化一致；MOF恢复期上述指标与正常比较无明显差别。
8、与MOF对照组相比，SP加重MOF大鼠各重要脏器组织学炎症，ALT升高40%~50%、Cr升高45%~50%，PO2降低约40%，各重要器官功能均明显恶化；大鼠IMMC胞浆颗粒数量进一步减少，颗粒包膜相互融合形成的空泡更大、更多，伴肠道组胺及TNF-(释放增加。
9、以20pmol/g体重SST静脉输注后，大鼠各重要器官组织学及器官功能得到明显改善，ALT下降53%、Cr下降60%、PO2升高50%；IMMC脱颗粒现象明显减轻，伴随小肠组胺由8.60(0.50ng/g蛋白升高至14.50(1.08ng/g蛋白，P(0.01。SST剂量降低至0.2pmol/g体重后，大鼠各重要器官组织学及器官功能无改善。
10、以0.2 pmol/g体重的VIP静脉滴注后，MOF大鼠各重要器官组织学及功能均得到明显改善，IMMC趋于稳定，伴肠道组胺及TNF-(释放减少。增大VIP剂量（20 pmol/g体重）后，MOF大鼠各重要器官组织学及功能损伤明显加重，IMMC脱颗粒现象更明显，伴肠道组胺及TNF-(释放增加。
11、IMMC表达了SSTR-1及VIPR-2 mRNA。
结 论
1、三种胃肠多肽在体外可影响I
Multiple organ failure (MOF) is the most serious result induced by trauma and infection. Once established, MOF defies our supportive measures. Mortality ranges from 40% to 100% and is related directly to the number and duration of organ failures. Unfortunately, neither the incidence nor the mortality of the syndrome has improved significantly. MOF is regarded as a continuous inflammatory response that is unable to be controlled. During the development of this systemic inflammatory response syndrome, gut is not only victim organ but also a regulator containing the stability of the inner environment under the condition of stress.
Intestinal mucosa is rich of mast cell, which degranuate when activated and release a number of inflammatory mediators including TNF-(、histamine、tachkinin、prostandinin、IL-1、6、8 and IFN-γ. TNF-( is well known as the key factor in the cytomic cascade of the MOF. Therefore, intestinal mucosal mast cells (IMMC) may be involved in MOF.
Intestinal mucosa is the place plenty of neurocytes and transmitters, endocrinocyte and polypeptides, immunologically competent cells and cytokines. Gut peptides are important regulators in the neuro-endocrine-immune network. Some studies have showed that substance P (SP), somatostatin (SST) and vasoactive intestinal polypeptide (VIP) engage a regulatory role in intestinal mucosal immune system. However, the effects of gut peptides on IMMC especially in the case of MOF remain unclear.
Because long term culture and passage of IMMC in vitro is very difficult, the fresh isolated IMMC was used in this study. Up to now, the purification rate of IMMC is about 30% in most of studies. Therefore, it is essential to get better enriched IMMC.
AIMS
To investigate:
1. the best optimal conditions for the isolation and purification of IMMC
2. the effect of SP, SST and VIP on IMMC in vitro
3. the changes of IMMC reactivity in rats with MOF and pathogenesis of MOF in the view of intestinal immunology
4. the effect of SP, SST and VIP on IMMC of rats with MOF
5. whether IMMC expresses mRNA of receptors for SST and VIP
METHODS
1. IMMC was isolated from rat intestinal mucosa by collegenase digestion.
2. IMMC was purified with percoll unit gravity velocity sedimentation.
3. Histamine level in plasma and intestinal mucosa was measured by fluometery assay.
4. The ultra-microstructure of the IMMC was observed under a transmission electronic microscope.
5. MOF model in rat was established by injection of zymosan.
6. Tumor necrosis factor ( (TNF-() in plasma and intestinal mucosa was determined with a TNF-( ELISA Kit.
7. The tissue sections of essential organs including intestine, liver, kidney and lung were stained with Heamotoxin & Eosine and were studied under a light microscope to understand the pathological alteration.
8. Expression of mRNA of the receptors for SST and VIP was measured by RT-PCR.
RESULTS
1. The best optimal conditions for purification of IMMC include: the gradient of units consisted in 30% and 80% percoll, pH7.4, 2~10(C，osometic pressure 250~300 mosmol/Kg and 2700~3000 rotate per minute. The purification rate was enhanced to 70%. The enriched IMMC remained normal ultra-microstructure and viability. The spontaneous release rate of histamine from IMMC was 22.86±3.22 %.
2. SP significantly increased the histamine release rate of IMMC to 89.98±5.30% in vitro, (P<0.01), which was positively correlated to SP concentration (r = 0.983, P<0.01). In addition, SP remarkably increased the total histamine from IMMC.
3. The inhibitive effect of SST on the histamine release rate of IMMC was negatively correlated to SST concentration. ( r = -0.991, P<0.01).
4. At the concentration from 1(10-5mol/L to 1(10-8mol/L, the higher concentration of VIP was used, the lower histamine release rate was observed (P<0.01). However, the histamine release rate was decreased to spontaneous status by VIP at the concentration of 1(10-9 mol/L and kept in a plateau with the concentration declining.
5. MOF model in
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