| 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 IMMC2. the effect of SP, SST and VIP on IMMC in vitro3. the changes of IMMC reactivity in rats with MOF and pathogenesis of MOF in the view of intestinal immunology4. the effect of SP, SST and VIP on IMMC of rats with MOF5. whether IMMC expresses mRNA of receptors for SST and VIP METHODS1. 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. RESULTS1. 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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