The Research On CXCR-4 Gene Transfected Combined With VCAM-1 Antibody Coated To Accelerate MSCs Homing And Their Mechanism Involved In The Impairment Of Experimental IBD Mice Colon

Background:Inflammatory Bowel Disease (IBD) is a nonspecific inflammatory intestinal disease, including Ulcerative Colitis (UC), Crohn’s Disease (CD) and Indeterminate Colitis. Bone marrow mesenchymal stem cells (BMSCs) are a kind of non-hematopoietic stem cells in bone marrow. They have many advantages, for example, multipotential differentiation, immunoregulation, low immunogenicity, accessible and no ethics problems. And they can promote healing of injured IBD intestinal mucosa and rectify the effect of mucosal immune dysfunction. So, they can be applied in the treatment of IBD. In previous studies, we found that the number of intravenous transplantation of BMSCs migrated and planted in diseased colon was low, limiting its therapeutic effect. Stem cell homing behavior involves a variety of chemokines and their receptors, equal participation of adhesion molecules. Currently, Chemokine Receptor-4/Stromal cell-derived Factor-1 (CXCR-4/SDF-1) and Vascular cell adhesion molecule-1/Very newest Antigen-4 (VCAM-1/VLA-4) molecule are confirmed to play an important role in promoting stem cell homing. According to this theory, using CXCR-4 gene transfected and VCAM-1 antibody coated BMSCs can improve the targeting property and high efficiency of the treatment.Objective:By constructing CXCR4-BMSCs, VCAMl-BMSCs and CXCR4-VCAM1-BMSCs to improve the plantation and curative effect of experimental IBD model, and to explore its mechanism, then to provide the theory basis for which way of targeting BMSCs to treat IBD clinically.Contents:1. To study the immune mechanism of BMSCs treatment in damaged intestinal mucosa of experimental IBD model.2. Construct and identify BMSCs, CXCR4-BMSCs, VCAM1-BMSCs and CXCR4-VCAM1-BMSCs, and assess their migration rates in vitro.3. Observe the migration rate and heal of damaged intestinal mucosa in experiment IBD models.4. Explore the mechanisams of targeted BMSCs homing to and repairing intestinal lesions.Methods:1. The serum specimens of our previous study were used. Flow cytometry was applied to detect the levels of Thl related cytokines (IL-2, IFN-y, TNF-a), Th2 related cytokines (IL-4, IL-10) and Th17 related cytokines(IL-6, IL-17).2. The colon tissue specimens of our previous study were used. Real time PCR and Flow cytometry were applied to detect the level of Thl/Th2/Th17 related cytokines.3. Real time PCR and Western Blot were applied to detect the expression of T-bet (Master regulator of Thl), GATA-3 (Master regulator of Th2), ROR-yt (Master regulator of Th17), TGF-P(Treg related cytokine) and Foxp3, Smad2 (Master regulators of Treg).4. BMSCs were derived from bone chip of male BABL/C (2-3 weeks), which labeled MSC group. CXCR4-Lentivirus were transfected to BMSCs to construct CXCR4-BMSCs (labeled C-MSC group). VCAM-1 antibody were coated to BMSCs to construct VCAMI-BMSCs (labeled V-MSC group). VCAM-1 antibody were coated to CXCR4-BMSCs to construct CXCR4-VCAM1-BMSCs (labeled C-V-MSC group). Real time PCR were applied to detect and compare the expression of CXCR-4 in 4 groups; Real time PCR and flow cytometry were applied to to detect and compare the expression of VCAM-1 in 4 groups.5. Morphologies of BMSCs in 4 groups were observed. Growth curves of 4 groups were determined by counting cells. The potentials of BMSCs in 4 groups to differentiate into osteogenic and adipogenic lineages were examined. Flow cytometry was used to determine the surface markers, the cell cycle distributions and the rates of apoptosis of BMSCs in 4 groups. Trypanblau dying was applied to determine cell viability of BMSCs in 4 groups.6. Transwell migration system was used to compare the migration of BMSCs in 4 groups to SDF-1 or FBS in vitro.7. Female BALB/C mice of 6-8weeks were randomly divided into 6 groups. And CM-Dil tracer were labeled into BMSCs in 4 groups. (1) Control group:Normal saline (100μl/per mouse) was used to enema.0.1ml PBS was injected through tail vein on the next day after enema. (2) TNBS group (labeled T group):TNBS 2.0mg/50% alcohol (100μl/per mouse) was used to enema.0.1ml PBS was injected through tail vein on the next day after enema. (3)TNBS-MSC transplant group (labeled MT group):TNBS 2.0mg/50% alcohol (100μl/per mouse) was used to enema.0.1ml PBS containing 1×106 BMSCs was injected through tail vein on the next day after enema. (4)TNBS-C-MSC transplant group (labeled CMT group):TNBS 2.0mg/50% alcohol (100μl/per mouse) was used to enema.0.1ml PBS containing 1×106 CXCR4-BMSCs was injected through tail vein on the next day after enema. (5)TNBS-V-MSC transplant group (labeled VMT group):TNBS 2.0mg/50% alcohol (100μl/per mouse) was used to enema.0.1ml PBS containing 1×106 VCAM1-BMSCs was injected through tail vein on the next day after enema. (6)TNBS-C-V-MSC transplant group(labeled CVMT group):TNBS 2.0mg/50% alcohol (100μl/per mouse) was used to enema.0.1ml PBS containing 1×106 CXCR4-VCAM1-BMSCs was injected through tail vein on the next day after enema. The day of transplantation was labeled D0,1-13 days after transplantation, respectively for D1-13.8. The degree of diseased IBD models and curative effects of different BMSCs were evaluated by general conditions, weight changes, survival rates, disease activity index (DAI) scores, gross colons and their changes of length, and histological scores.9. Three mice in each group were killed, and serum, intestinal tissue, mesenteric lymph nodes and spleen samples were collected on D0、D1、D3、D5、D7、D9、D11、 D13.10. Red fluorescence locations in colon were observed by fluorescence microscopy. The relative expressions of Sex-determining gene (SRY) were detected by Real time PCR.11. IHC was applied to detect the expression of Ki67, eNOS, Claudin-1 and JAM-1.12.Western Blot was applied to detect the expression of Lysozyme, TLR-4, MyD88, TRAF-6, ERK1/2, JNK1/2, MAPK and NF-κB.13. Flow cytometry was applied to detect the expression of VCAM-1 (CD 106).14. Elisa was applied to detect the expression of VLA-4.Results:1. In the experimental IBD group, the levels of pro-inflammatory cytokines IL-2, IFN-y, TNF-a (related to Thl) and IL-6, IL-17 (related to Th17) in serum increased significantly (p<0.05), but in the BMSCs transplantation group significantly decreased (p<0.05). In the experimental IBD group, the levels of anti-inflammatory cytokines IL-4 and IL-10 (related to Th2) didn’t change (p>0.05), but in the BMSCs transplantation group increased significantly (p<0.05).2. In the experimental IBD group, the levels of pro-inflammatory cytokines related to Thl and Th17 in colon increased significantly (p<0.05), but in the BMSCs transplantation group significantly decreased (p<0.05). In the experimental IBD group, the levels of anti-inflammatory cytokines related to Th2 and TGF-β related to Treg didn’t change (p>0.05), but in the BMSCs transplantation group increased significantly (p<0.05).3. In the experimental IBD group, the levels of T-bet (Master regulator of Thl) and ROR-yt (Th17) in colon increased significantly (p<0.05), but in the BMSCs transplantation group significantly decreased (p<0.05). In the experimental IBD group, the levels of GATA-3 (Th2) and Foxp3 (Treg) didn’t change (p>0.05), Smad2 (Treg) decreased (p<0.05), but in the BMSCs transplantation group increased significantly (p<0.05).4. High expression of CXCR-4 was determined after CXCR-4 gene transferred to BMSCs. High expression of VCAM-1 was determined after VCAM-1 antibody coated to BMSCs. High expressions of CXCR-4 and VCAM-1 were determined after VCAM-1 antibody coated to CXCR4-BMSCs, and the expression of CXCR-4 higher than C-MSC group, the expression of VCAM-1 higher than V-MSC group (p<0.05).5. BMSCs in 4 groups were growing as a long spindle and beam pattern. Their growth curves were S-shaped. The ratio of adipogenic differentiation was about 60%. The number of osteogenic differentiation was about 20/per view. CD105+,90+, 11b-and 45-, which are markers of BMSCs, didn’t change.Trypanblau dying showed about 90% cell viability. The above outcomes of BMSCs in 4 groups showed no difference (p>0.05). In the C-MSC group, the copy period in cell cycle increased (p<0.05). In the V-MSC and C-V-MSC group, the apoptosis rate increased (p<0.05).6. The migration experiments in vitro showed that the number of different BMSCs had moved to SDF-1 were C-MSC group> C-V-MSC group> V-MSC group> MSC group (p<0.05), and that to FBS were C-V-MSC group> C-MSC group> V-MSC group> MSC group(p<0.05).7. TNBS induced experimental model, the mental state, body weight and survival rate decreased significantly, DAI and histological score obviously increased, the gross colons were significantly shortened accompanied hyperemia, hemorrhage and narrow, and histopathological found that ulceration, a large number of neutrophil infiltrated in the mucosa and submucosa (p<0.05). But on the second day after transplantation, compared with T group, in the MT, CMT, VMT and CVMT group, clinical symptoms improved, weights and survival rates started to rise, DAI and histological scores decreased, hyperemia, hemorrhage and narrow reduced (p<0.05). In the CVMT group, symptoms, weight recoveries and histopathological changes were faster than other treatment group (p<0.05).8. Red fluorescence was observed in colon of MT, CMT, VMT and CVMT group. SRY gene was detected in the male, MT, CMT, VMT and CVMT group, and the engraftment rate was C-V-MSC group> C-MSC group> V-MSC group> MSC group.9. The expressions of Ki67, eNOS, Claudin-1 and JAM-1 significantly increased in MT, CMT, VMT and CVMT group, especially in C-V-MSC group.10. Western Blot results showed:The relative expressions of TLR-4, MyD88, TRAF-6 and NF-κB in T group (DO) increased, decreased in the MT, CMT, VMT and CVMT groups (D3), especially in the CVMT group with greatest reduction. The expression of Lysozyme decreased in T group, increased in the treatment groups, especially in the CMT group and VMT group with obvious increase. The expression of ERK1/2 and JNK1/2 decreased in T group, and relative expression of MAPK increases. Compared with T group, in the MT, CMT, VMT and CVMT group, the expression of ERK1/2 and JNK1/2 increased, the expression of MAPK decreased, especially in the CVMT group with greatest reduction (p<0.05).11.The expression of VCAM-1 in the control group was 41.75%, in the T group decreased to 18.73%, in the BMSCs transplantation groups increased in different degrees, CVMT group (73.51%)> VMT group (67.10%)> CMT group (66.55%)> MT group (51.42%). The normal colon tissue contains a certain amount of VLA-4. On D3, the amount of VLA-4 in the T group increased (p<0.05), in the BMSCs transplantation group decreased in different degrees, CVMT group> VMT group> CMT group> MT group. On D7, the amount of VLA-4 in all groups returned to normal (p> 0.05).Conclusion:1. This curative effect of BMSCs was primary conducted by down-regulation of both Thl-Thl7-mediated pro-inflammatory responses, and by up-regulation of Th2-regulatory T cells-mediated anti-inflammatory responses. BMSCs also had effect on promoting Thl/Th2 and Thl7/Treg immune balance. This theory played a vital role in the regulation of systemic and local immune state in experimental IBD mice.2. CXCR-4 gene and VCAM-1 protein couldn’t change the biological activities of BMSCs and could be coordinated in expression.3.Migration experiments in vivo and in vitro showed that CXCR-4 and VCAM-1 could increase the mobility of BMSCs, and they could be coordinated to strengthen the migration characteristics of BMSCs.4. The rate of migration was positively related with curative effect in the treatment of experimental IBD by different BMSCs transplantation. BMSCs could predominately inhibit excessive activation of TLR/MyD88/TRAF6/NFκB signaling pathways, to promote intestinal mucosa regeneration, repair, angiogenesis and epithelial barrier reconstruction.5. VCAM-1/VLA-4 and CXCR-4/SDF-1 played a key role in the migration, plantation and anti-inflammatory effects through TLR4/NFκB and JNK/ERK/MAPK signaling pathways.