| [Objective]1. To investigate pathological changes of intestinal barrier disfuction in rats by inducing intestinal ischemia and reperfusion injuries (I/R I) model.2. To isolate, culture and purify bone marrow-derived mesenchymal stem cells(MSCs) of rats, and identify MSCs according to suffice markers.3. To observe the protective effects of MSCs on intestinal barrier by transplanting MSCs to the rats of intestinal barrier disfuction.[Methods]1. Intestinal ischemia and reperfusion injuries model was induced by clamping and unclamping the arteria mesenterica cranialis (AMC) in rats. Male Sprague-Dawley(SD) rats, ranging from 250g to 350g, anesthetized by ketamine, were fixed on the operating plat. Open the obdominal cavity from the anterior median line, find the AMC and block blood flow for 45 min with artery clamp. The rats were killed after 24 h to do histopathologic examination of small intestine. Sham operation was performed in control group rats.2. Whole bone marrow adherent culture was chosen to culture MSCs. Whole bone marrow was collected from healthy adult rats in sterility condition. Both lower limbs of the rats were removed and femur medullary cavities were flushed with Dulbecco's modification of Eagle's medium(DMEM), low glucose until the diaphysis became white. The douche was centrifuged for 5 min at 1000 rpm, then removed the supernatant, and resuspended the deposition with DMEM-low glucose,10%~15% fetal bovine serum(FBS) and antibiotics. The cells suspension was innoculated in 75cm2 cell culture flask and cultured in the incubator maintained at 37℃in a saturated humidity atmosphere with 5% CO2. Growth and adherence of MSCs was obseved every day. Medium was changed every 3 days and non-adherent cells were removed concomitently. MSCs were subcultured at a ratio of 1:2 or 1:3 according to the density. After passage 3, surface markers were detected with flow cytometry analyses(FACS). The harvested MSCs were resuspended with phosphate buffered saline (PBS) and than separated into 7 tubes with each tube about 1×106 cells. CD34, CD45, CD90 and CD29 antigen on the surface of MSCs were detected.3. MSCs were obtained from male SD rats and subcultured, proliferated and purified as described above. Intestinal ischemia and reperfusion injuries model was induced in male SD rats as described yet. The MSCs of passage 3 were stained with 4,6-diamidino-2-phenylindole (DAPI) and observed DAPI positive cells with fluorescent microscope. The stained cells were harvested and infused into the rats by caudal vein with each rat 1×106 cells 60 min after I/R I model induction. The control group rats were injected with NS of same volume. The rats were killed 6h, 24h and 72h respectively after injection. Serum IL-6,IL-10,TNF-α,MDA and SOD level were detected. Part of small intestine were for fast frozen to observe DAPI positive cells with fluorescent microscope; part were for HE stain to observe the injuries of intestinal musoca and part for immunohistochemical test to observe gereration of musoca cells.[Results]1. Mesenteric arteries pulsation dispeared and small intestine turned gray and crimpled after clmaped AMC. About 40 min latter, intestine turned flatulent and darken. Intestine returned red and enterokinesia recover after unclamping AMC. Histopathological changes were observed in intesinal tissues. Intestinal mucous of most specimen were damaged, and neutrophilic leukocyte infiltrated into interstitial tissues. Even worse, damage of glandular organs in propria lamina and atrophy of intestinal mucosa was observed. The Chiu'damage grades of small intestine were 54.83±4.26.2. Adherent cells were seen when primarily cultured for 24h and the shapes were fusiform and polygon. Seventy hours latter, the number of adherent cells became larger and larger. MSCs showed a fibroblast-like shape and at the 9th day showed whirlpool-like distribution. Cells were purified by changing the medium and subculturing. In passage 3, MSCs had uniform shapes and membrane spread. FACS showed that the rate of CD29+MSCs was 98.6%, and that of CD90+MSCs was 99.6%. MSCs were not express CD34 and CD45. The positive rates were 0.56% and 0.89% respectively.3. Serum levels of IL-6 and TNF-αwere both significantly lower in MSCs group than in NS group at 6h and 24h of MSCs injection (P<0.05), but IL-10 was higher(P<0.05). There were significant differences between the two groups at 6h, 24h and 72h of serum MDA level(P<0.05) and at 24h of serum SOD level(P<0.01). DAPI positive cells could be seen at intestinal tissue of MSCs group rats with fluorescent microscope. Pathological and immunohistochemical results showed that the necrosis of entorocyte was more gently at 6h and 24h and the gereration was obvious observed at 24h and 72h in MSCs group(P<0.05).[Conclusion]1. The intestine I/R I model of intestinal barrier disfuntion is reliable with good reproducibility and can be used for research.2. Adherent culture of whole bone marrow can gain highly purified MSCs. Purity of P3 MSCs is over 95%. FACS results show that MSCs are positive for CD29 and CD90 and negative for CD45 and CD34.3. Intestinal I/R I could lead to systemic inflammation response in rats, which was serious at 24h after reperfusion but alleviated much at 72h. Transplanted MSCs could transfer to and settle at injuried intestine and showed the beneficial effects on intetinal I/R I. The results demonstrated that MSCs had the ability to regular immune response induced by intestinal I/R injuries and alleviate intestinal immune damages by inhibiting over-releasing of inflammatary cytokines and promoting secreting of anti-inflammatary cytokines and clearance of oxygen free radicles. Simultaneously, MSCs played an important role in accelerating generation of entorocyte and repairing of intestinal mucosa, thus to maintain the integrity of intestinal barrier. |
