The Research Of Over-expression Of CXCR4on Mesenchymal Stem Cells Protect Against Ulcerative Colitis And Synergistic Effect Of Compound Sophorae Decoction

Part I Construction and Identification of Lentiviral Vector Overexpressing Chemokine Receptor4Objective:To construct a lentiviral vector overexpressing chemokine receptor4(CXCR4) gene, and examine its ability to express the CXCR4gene in vitro.Methods:The primer of CXCR4gene was designed and then CXCR4gene fragments were amplified by polymerase chain reaction (PCR); The GV208lentiviral vectors were enzyme digestion by Age I and inserted into the full length CXCR4gene fragments through the ligase; Positive cloning CXCR4-GV208vectors were identified through PCR; According to the packaging kit manual, lentiviral vectors containing CXCR4gene were cotransfected293T cells to package lentivirus. The virus titer was determined by real-time quantitative polymerase chain reaction(RT-qPCR); The recombinant lentiviruses were cotransfected into293T cells and the expression of GEP was observed.Results:CXCR4gene was amplified and connected to GV208lentivirus vectors successfully. The recombinant plasmid was confirmed correct by PCR identification and DNA sequencing. The expression of GFP could be observed after recombinant lentiviruses were cotransfected into293T cells. The overexpressing viruses were obtained and the titer of concentrated virus was2.0×108TU/ml.Conclusion:The lentiviral vector over-expressing CXCR4gene was successfully constructed, which provided a experimental foundation for further studies on the functions of CXCR4. Part II SDF-la/CXCR4Axis Facilitates BMSCs homing toward Injured Colon in Experimental Colitis and synergistic effect of Compound Sophorae decoctionAIM:To investigate the role of stromal cell-derived factor-la(SDF-la)/chemokine receptor4(CXCR4) axis in the therapeutic effects of bone marrow-derived mesenchymal stem cells(BMSCs) for2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis rats.METHODS:BMSCs were isolated from Sprague-Dawley(SD) rats and identified by flow cytometry. Lentivirus transfection was applied to over-express CXCR4/GFP (Ad-CXCR4-BMSCs) or null/GFP (Ad-GFP-BMSCs), western blot was applied to detect the protein expression of CXCR4in BMSCs. Forty Sprague-Dawley rats were randomly divided into five groups(n=8):control group, model group, Ad-GFP-BMSCs group, Ad-CXCR4-BMSCs group and bigeminy group. Experimental colitis was induced by TNBS and treated by intravenous administration; bigeminy group was gavaged with Compound Sophorae decoction. One week after cell therapy, the colons were harvested. The expressions of GFP, CXCR4and SDF-la in colons were measured by western blot and immunofluorescence.RESULTS:The cell viability was approximately90%and eighty percent of BMSCs steadily deliver the GFP protein after lentivirus transfection. Comparison with control group, the protein expression of SDF-1α was distinctly increased in injured colon regions. One week after intravenous administration and drug therapy, Immunofluorescence technique showed that GFP protein was sporadically expressed in Ad-GFP-BMSCs-treated colon, but extensively expressed in Ad-CXCR4-BMSCs-treated colon and bigeminy group. Similar results of GFP protein levels in the colon tissue were also observed by western blot. Compared to Ad-GFP-BMSCs group, the protein expression of GFP was observably increased in the Ad-CXCR4-BMSCs group(P<0.05). Furthermore, compared to Ad-CXCR4-BMSCs group, the protein expression of GFP was observably increased in the bigeminy group(P<0.05).CONCLUSION:The findings illuminate that SDF-1α/CXCR4axis plays a crucial role in BMSCs migration toward injured colon, which may provide an attractive target for BMSCs-based therapies in UC. Moreover, Compound Sophorae decoction may be contribute to the migration. Part Ⅲ Over-expression of CXCR4on mesenchymal stem cells protect against experimental colitisObjective:The present study was designed to investigate the role of SDF-la/CXCR4axis in the therapeutic effects of lentivirus-preconditioned BMSCs for2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis rats and and synergistic effect of Compound Sophorae decoction.METHODS:BMSCs were isolated from Sprague-Dawley(SD) rats and identified by flow cytometry. Lentivirus transfection was applied to over-express CXCR4/GFP (Ad-CXCR4-BMSCs) or null/GFP (Ad-GFP-BMSCs), Thirty-two Sprague-Dawley rats were randomly divided into five groups(n=8):control group, model group, Ad-GFP-BMSCs group, Ad-CXCR4-BMSCs group and bigeminy group. During the whole experiment, the disease activity index (DAI) was recorded. On day1, colitis groups were induced by the TNBS administration. On day4, rats were grouped to receive various treatments by tail vein injections and bigeminy group was gavaged with Compound Sophorae decoction. On day12, animals were anesthetized and submitted to a laparotomy under sterile technique. The entire colon was removed and colon length was measured. Then the distal colon was opened longitudinally, slightly cleaned in physiological saline for faecal residues removal, and tissue samples were harvested and analyzed for various studies.Results:One week after intravenous administration, Ad-GFP-BMSCs failed to colonize in the inflamed colon and had no beneficial effect in TNBS-induced colitis. Instead, Ad-CXCR4-BMSCs signally ameliorated both clinical and microanatomical severity of colitis. The therapeutic effect of Ad-CXCR4-BMSCs was mediated by the suppression of pro-inflammatory cytokines and STAT3phosphorylation in injured colon. Conclusion:Our data indicated that over-expression CXCR4led to enhance in vivo mobilization and engraftment of BMSCs into inflamed colon where these cells can function as an anti-inflammatory and immunomodulatory component of the immune system in TNBS-induced colitis.