An Experimental Study Of Treating Rats With Short Bowel Syndrome By Intestinal Side Population Cells Transplantation

Objective:Asubsetofstemcellsorprogenitorcellsinadulttissuehasbeenidentifiedas side-population cells (SP cells). A recently study indicated that SP cells isolatedfrom mouse are highly enriched for stem cells. In this study, we isolated SP cells fromintestine of fetal Wistar rat and analyzed their properties to provide evidence forfurther study of their potential therapeutic effects in rat model.Methods:Individual cells from whole mucosa of fetal Wistar small bowel werestained with Hoechst 33342 and propidium iodide and then sorted usingfluorescence-activated cell sorting. Total RNA and protein was isolated from sortedfractions and used for real-time reverse-transcription polymerase chain reaction andWestern-blot to evaluate the expression of the intestinal stem-cell marker, Musashi-1.Results:IntestineinfetalWistarratcontainaviablepopulation ofcellsthatshowstheSP phenotype and is sensitive to verapamil. These cells are 9.2±0.37-fold (p<0.01)enriched for Musashi-1 mRNA compared with intact tissue and 45.7±0.45-fold(P<0.001) compared with non-SP cells. These cells are also enriched for Musashi-1protein.Conclusion: The SP fraction enriched in fetal Wistar small intestinal epithelial cellshas similar characteristics to mouse SP fraction. Part2:AnExperimentalStudyofTreatingRats WithShort Bowel SyndromebyIntestine SidePopulation Cells TransplantationObjective: We evaluated the therapeutic effect of intestinal SPcells transplanted intorats with short bowel syndrome.Methods:JuvenileWistar ratsunderwenta75%smallintestinalresection.Ratswererandomized to experiment group and control group. After 5 days, all rats wereaccepted 300cGy total body radiation. About 5×105 intestinal SP cells were injectedto the intestine submucosa of the experimental group in 12 hours. The same volumesaline was injected into the intestine submucosa of the control group. After 4 monthD- xylose absorption, nitrogen balance and fat balance were investigated. Then allanimals were sacrificed, intestinal morphology (including measurements of length,area, and weight) and histology were evaluated.Result: There are significant difference in body weight(280±23g,252±21g),intestinal length(39.0±9.8cm, 26.5±6.7cm), D-xylose absorption(0.17±0.07%,0.10±0.04%), nitrogen balance(0.53%±0.14%, 0.38%±0.17%) and fat balance(0.98±0.01%,0.86±0.08%) between experiment group and control group.Conclusion: Intestine SP cells transplantation can improve absorbabilityof rats withshort bowel syndrome.Part 3:15N-LeucineUsedAs Tracers to Measure IntestinalAbsorptionObjective: To evaluate the absorptive function of the intestine after side populationcells transplantation by using nonradioactive isotope labeled L-15N Leucine.Methods:After L-15N Leucine (0.25g/kg) was intragastric administration, theenrichment of L-15N Leucine in serum of all rats (including experiment group,control group and normal group )was measured in different intervals(0.25h, 0.5h, 1h, 2h). The area under the curve was calculated.Result: The peak value of the absorption was at 0.5 hour after intragastricadministration in all rats. The relative absorption rate of L-15N Leucine was 70%and 37% in experiment group and control group compared with normal group.Conclusion: Intestinal SPcell transplantationcan improveabsorption of 15N-Leucinein small bowel syndrome rats.Part4:Detectionof Donor CellsinIntestineof Recipient RatsbyDigoxigenin in SituHybridizationObjective:todetectthedonorcellsinintestineofrecipientratsbyDigoxigenininsituhybridization.Methods:Miceweresacrificed in4monthaftertransplantationandsectionsof12μmthickness were used for analysis.Result:Ychromatosomewasdetectedinallrecipientofexperimentgroup.Conclusion: Regeneration of intestinal epithelia with donor-derived cells shows apotential clinical application of intestinal SP cells for short bowel syndrome.