Animal Experiment Of CM-DIL Labeled New Zealand Rabbit Bone Marrow Mesenchymal Stem Cells Combined With Bladder Acellular Matrix For Bladder Repair

Objective(1)Feasibility of CM-DIL-labeled bone marrow mesenchymal stem cells(BMSCs),and observe the survival of pre-labeled bone marrow mesenchymal stem cells and bladder acellular matrix(BAMG)to ensure transplantation to experimental animals.The survival effect of seed cells in the body.(2)Whether bone marrow mesenchymal stem cells survive and migrate in the body,whether they participate in the reconstruction of bladder tissue defects.(3)To evaluate the effects of different seed cell transplantation pathways on the repair of defective bladder tissue.(4)To observe the expression of IL-2 and IL-4 in bladder tissue of each group.Methods New Zealand rabbit bone marrow mesenchymal stem cells were isolated and cultured to prepare rabbit bladder acellular matrix.The p3 generation bone marrow mesenchymal stem cells were collected and cultured for flow cytometry.BMSCs were labeled with CM-DIL.The experimental rabbits were divided into the following four groups:intravenous BMSCs combined with BAMG to repair the bladder group,local injection of BMSCs combined with BAMG to repair the bladder group,BAMG alone for repairing the bladder group,and the same area of the bladder wall were directly sutured.The bladder defect model was successfully established and cultured.After seven weeks,the animals were sacrificed and the bladder was removed.The bladder repair sites were taken and divided into two parts.The frozen sections were used to observe the coloration and tissue structure of the stained BMSCs,and the bladder tissue was reconstructed by immunohistochemistry.The expression of skin(AE1/AE3 mAb),smooth muscle(?-SMA mAb)and blood vessels(CD31 mAb)was used to measure the repair effect of the newborn bladder,and the other part was used to detect the anti-inflammatory factor IL-4 by RT-qPCR.And changes in the expression of the pro-inflammatory factor IL-2.Results(1)CM-DIL successfully labeled rabbit bone marrow mesenchymal stem cells in vitro.The fluorescent labeling rate was 93.7% when the labeling concentration was 4 mg/L.The CCK-8 method showed that this concentration had no significant effect on cell viability.(2)Two groups of animals injected with BMSCs labeled with CM-DIL were able to find chromogenic BMSCs in the repaired bladder tissue after 7 weeks,and the color development effects were satisfactory,and the number of chromogenic cells in the repaired tissue of the locally injected BMSCs group was significantly higher.In the intravenous group(p <0.05).(3)Return of HE staining results: The bladder structure of the BAMG group was only visible,but the smooth muscle tissue was disordered,and the blood vessels and nerves were scarce.The three-layer structure of the bladder was formed in the BMSCs group and the BMSCs group,and the nerve,blood vessel and smooth muscle tissues were observed in the adventitia.The urothelium(AE1/AE3 mAb),smooth muscle(?-SMA mAb)and vascular(CD31 mAb)of the repaired bladder tissue were well expressed,and the IOD of the three indicators determined by semi-quantitative analysis The values(positive expression area)were local injection of BMSCs group > intravenous BMSCs group > simple repair BAMG group(p<0.05).(4)In the seventh week after operation,the concentrations of IL-2 and IL-4 in the bladder tissue were all repaired in the BAMG group > intravenous BMSCs group > local injection BMSCs group(p<0.05).Conclusion(1)It is feasible to use CM-DIL as a tracer of BMSCs,which can provide stable fluorescence expression to seed cells without harming cell activity.(2)Intravenous CM-DIL-BMSCs homing in experimental rabbits,and the number of tissue-injected CM-DIL-BMSCs colonized the damaged tissues was more than that in the intravenous group.(3)Compared with the BAMG-repaired bladder,the experimental tissue of the rabbits injected with BMSCs and the BMSCs group was close to the normal rabbit bladder structure,and there were abundant urothelium,smooth muscle and vascular tissue.(4)Transplantation of rabbit BMSCs can reduce the levels of IL-2 and IL-4 in bladder tissue and promote the repair of damaged parts.