Research Of Mesenchymal Stem Cells Transplantation In Inflammatory Bowel Disease’s Therapy

Background: Bone marrow mesenchymal stem cells (BMMSCs), a kind of bonemarrow-derived non-hematopoietic stem cells, have become the ideal cells inregeneration medicine because of their self-renewal, multi-directionaldifferentiation, low immunogenicity and immunomodulation characteristics andwidely applied in ischemic or traumatic disease, organ transplantation, andanti-immunological rejection, with some initial promising outcomes.Inflammatory Bowel Disease (IBD), including Ulcerative Colitis (UC) andCrohn’s Disease (CD)， is a chronic inflammatory disorder. Thus, in theory,BMMSCs transplantation can repair gastrointestinal inflammatory injury.Objective:To study the effect and mechanism of cultured GFP-BMMSCs on therepair of injured large intestinal mucosa of IBD models.Methods:(1) GFP-BMMSCs from male mice were cultured in vitro withcompact bone culture method (A) and whole bone marrow adherent culturemethod (B), respectively, and amplified. Their natural growth curves werecompared by MTT assay. Their surface antigens were detected by flow cytometryand induced differentiations of adipocytes and osteocytes were observed.(2) Fifty-four healthy female BALB/C mice of6-8weeks old were randomlydivided into control group, TNBS-PBS group and TNBS-MSC transplantationgroup (18in each group). Mice in control group were given normal saline forenema and underwent GFP-BMMSCs transplantation through intravenousinjection of0.1mL PBS containing GFP-BMMSCs (1×106/each) the next day afterenema, those in TNBS-PBS group were given100μL TNBS (2.0mg/50%)alcohol for enema and injected with0.1mL PBS not containing GFP-BMMSCsthrough intravenous injection the next day after modeling, and those inTNBS-MSC group were given100μL TNBS (2.0mg/50%) alcohol for enema and injected with0.1mL PBS containing GFP-BMMSCs (1×106/each) throughintravenous injection the next day after modeling. The animals in each group werekilled and intestinal tissue samples were taken on days1,2,3,5,7, and9afterGFP-BMMSCs transplantation.(3)The degree of IBD models and treatment effect of BMMSCs were evaluated bygeneral condition, change of weight, survival rate, disease activity index (DAI)score, gross colon, its length and histological score. Location of transplantedGFP-BMMSCs in colon was observed by fluorescence microscopy.Sex—determining gene (SRY) on Y chromosome was detected by PCR.Expression levels of Ki67and LGR-5were measured by immunohistochemistry.Results：(1)MSCs have the characteristics of adherent growth, Most of them werespindle, oval and polygonal in shape, and arranged in fasciculate, whirl andradiate forms when they were fused. The amplification speed of MSCs was faster,the purity of MSCs was higher, and the natural growth curve of MSCs was clearerafter cultured with method A than after cultured with method B (P<0.05). Thethird passage of MSCs cultured with method A showed a rather good homogeneitywith their surface antigen CD90positively expressed and their surface antigensCD11b and CD45negatively expressed. No significant difference was found indifferentiation of the third passage of GFP-BMMSCs cultured with methods A andB into adipocytes and osteocytes (P>0.05). The biological nature ofGFP-BMMSCs was stable and the expression level of GFP did not attenuate after5passages.（2）InTNBS induced IBD model, the generalsituation is worse, the bodyweightand the survival rate decreased obviously, disease activity index (DAI) scoresignificantly increased, the gross colons were significantly shortened andhistological score both rose. Pathologic examination revealed that the constrictionand ulceration of colon were severer and the infiltration of massive neutrophils,lymphocytes, phagocytes and fibrocytes in mucosa and submucous layer was more significant in TNBS-PBS group and TNBS-MSC transplantation group thanin control group (P<0.05). But on2days after MSCs transplantation,theseconditions aboved have significantly improved.Moreover,on the48-h after MSCstransplantation,the green fluorescence MSCs were found scattered in the colon ofIBD models that underwent MSCs transplantation,while these were not detectedin the control group at the corresponding points.The SRY gene was detected in thecolon of IBD models that underwent transplantation of MSCs derived from malemice9days after transplantation,while it was not detected in the control gruop.（3）Immunohistochemistry staining demonstrated that the expression levels ofKi67and LGR-5were higher in mouse IBD model after MSCs transplantationthan before MSCs transplantation.Conclusion:(1) More GFP-BMMSCs with a high concentration can be obtainedafter cultured with method A than with method B after primary or first passage.Their purity is higher, their cell cycle is shorter, and their biological activity ismore stable after cultured with method A than with method B. Their fluorescenceof GFP-BMMSCs does not attenuate after several passages and can thus be usedas a cell label in vivo.(2) GFP-BMMSCs transplantation through tail vein can belocalized in the injured colorectum of IBD model, thus promoting its mucosarepair and alleviating the disease．(3) MSCs may accelerate intestinal woundhealing and the mechanism maybe that MSCs could not only differentiate intointestinal epithelium by themselves but also promote instinct intestinal stem cells’differentiation.