Experimental Research Of Endothelial Progenitor Cells Transplanted For Rabbit's Urethral Defect Repair

Background: Congenital malformation, trauma, or tumor and other urinary diseases can lead to urethra injury, stricture, or defect. All these should repair or reconstruct the urethra. At the present, there are more than 300 operation manners can be used to treat these urethral damage, but fistula, stricture , diverticulum and other complications after the operation still keep a high incidence rate. The clinical data confirmed that the rate of these complications keep inverse ratio to whether the new urethra have an abundant blood circulation. Now, many experimental research of animal and clinical data indicated that the endothelial progenitor cell (EPC) can help to improve the blood circulation of the ischemia or damaged organism.Endothelial progenitor cell (EPC) is the ancestral cell of endothelial cell, it can be called angioblast, too. It is one of the phases in the stem cells differentiate into mature cells. Research indicated that all of the mononuclear cells of CD34~+ or CD133~+ from vein blood of umbilical cord, distal vessels blood of adult body and bone marrow can differentiate into the cells like endothelia cells in vitro.So these cells are considered to be the original endothelial progenitor cells(EPCs). Further research found out that all the endothelial progenitor cells are from the bone marrow in mankind. Through increase the level of internal VEGF, the damage or ischemia of body can mobilize the endothelial progenitor cells to move and differentiate from the bone marrow to the target tissue. When the endothelial progenitor cells differentiate into endothelial cells and participate in the vascular regeneration, the level of CD133 in these cells gradually weaken and almost disappear finally.There were no reports about endothelial progenitor cells were used in the urethral defect repair whether in inland or overseas.Object: To observe the effect of endothelial progenitor cell (EPC) in improving blood circulation of the new urethra after the repair of urethral defect. Offer a new idea to descend and avoid fistula and other complications after the repair operation of urethral defect, then increase the successful rate of urethral repair only once.Methods: Aseptically extract the bone marrow from the both lower extremities of the rabbit, density gradient centrifugation is used to disbranch and get the mononuclear cells, then culture the cells in vitro and induct them to differentiate into endothelial progenitor cells (EPCs). And the 3rd generation cells will be the spare parts. Male rabbits are the experimental objects, remove the middle part of their urethrae artificially, about 1 cm long, and then we have the model of urethral defect. In the following, repair the urethral defect with human amnion extracellular matrix (HAECM). In the study team, the 3rd generation EPCs are injected in the junction sites and the corresponding site of the subcutaneous tissue; and the control team are treated with isometric culture solution only in the same manner. After the operation, nurse them according to the operation of hypospadias. 4 and 12 weeks after the operation, cut the repaired urethra tissue, and fix them by formalin, then embed the tissue by olefin, then slice up them and colorate by HE. Observe the vascular regeneration in the new urethra, and count the number of the capillary vessels microscopically (enlarge 100 times, chose 20 fields of vision randomly). Eventually calculate the numbers by SPSS10.0—a kind of statistics software, and check whether the difference between the two teams has statistics significance or not.Results:1. The mononuclear cells from the rabbits' bone marrow grow in the manner of keeping close to the wall in vitro, and this process completed just in the first 48 hours. The cells grew slowly in the early 4 days, it seemed to be resting. But the growth obviously accelerated after that, and the grow manner is like cloning. 10 days after the culture, the cells can almost be a layer on the bottom of the culture-bottle, and the cells' shape just like slabstone. Induce the cells by VEGF and bFGF in vitro, then the cells have a cord-like or strip-like form, and the colorate result indicated that the level of CD133 in the cells fell gradually. The surface marker of the cells changed from CD34~+/CD133~+/CD31~+ to CD34~+/CD133~-/CD31~+;2. Both 4 and 12 weeks after the operation, observe the new urethra tissue of both teams in histologically, and the results indicates that the number of the capillary vessels in the new urethra of the study team are more than the control team obviously. 4 weeks later, the number of capillary vessels in one vision which enlarged 100 times are 4.40±0.82 VS 1.30±0.66 , and 12 weeks later, the parallel number are 6.55±0.76 VS 2.50±0.83. Treat the numbers with SPSS10.0, and the result indicates that the difference between both teams has statistics significance whether in 4 weeks or 12 weeks after the operation.(P<0.01).Conclusions:1. The mononuclear cells from rabbit's bone marrow can differentiate to endothelial progenitor cells (EPCs) in vitro;2. Endothelial progenitor cell (EPC) can apparently improve the blood circulation of the new urethra after the urethral repair.