【作者】 李泸平；

【导师】 范应中；

【作者基本信息】 郑州大学 ， 小儿外科学， 2007， 硕士

【摘要】 研究背景：先天畸形、外伤或肿瘤等疾病致使尿道损伤、狭窄或缺损，都需要进行尿道的修复、重建。尽管目前有300多种尿道修复的术式可供选择，但是术后尿瘘、尿道狭窄等并发症的发生率却依然居高不下。临床观察证实，这些并发症的出现几率与术后尿道的血供丰富与否成反比。目前大量的动物实验研究和临床资料均表明，血管内皮祖细胞(EPC)能帮助改善机体缺血部位和损伤部位的血供。血管内皮祖细胞(EPC)是血管内皮细胞的前体细胞，又称为成血管细胞或者血管母细胞(angioblast)，是干细胞分化成熟过程中的一个细胞阶段。研究表明，在脐带静脉血、成体外周血和骨髓中的CD34~+单个核细胞或CD133~+单个核细胞离体培养均能分化为内皮样细胞，从而认定这些细胞即为原始的血管内皮祖细胞(EPC)，进一步的研究发现人脐带血、外周血中的血管内皮祖细胞(EPC)均来自于骨髓。机体的损伤或缺血可以通过引起内源性VEGF的水平升高来动员骨髓中的血管内皮祖细胞(EPC)向靶组织归巢、分化，而且在向成熟血管内皮细胞(endothelial cell，EC)分化、参与靶组织的血管再生过程中对CD133的表达逐渐转弱甚至消失。国内外尚未见到有关血管内皮祖细胞(EPC)移植应用于尿道缺损修复方面的文献报道。研究目的：观察血管内皮祖细胞(EPC)在尿道缺损修复术后改善新尿道组织血液循环的效果。为减少和避免临床上尿道缺损修复术后尿瘘等并发症的出现以及提高一期尿道修复的成功率提供一个新的思路。研究方法：无菌取兔双下肢骨髓，应用密度梯度离心法分离并在体外培养其中单个核细胞层细胞，诱导其向血管内皮祖细胞(EPC)方向分化，将细胞传至第3代后备用。人为去除雄性兔尿道中段长约1cm，制作成兔尿道缺损模型。然后应用制备好的人脱细胞羊膜基质修复尿道缺损。同时，在实验组中，将诱导培养后的第3代细胞点状注射于修复后的尿道吻合口处及其外层的皮下组织中，对照组中应用等量的空白细胞培养液处理，术后按尿道下裂手术后常规护理。然后分别于术后4w、12w切取修复后的尿道组织，10％福尔马林固定，石蜡包埋切片后行HE染色，观察新尿道组织中血管再生情况，并随机计数100倍光镜下20个视野内毛细血管断面的数目，应用SPSS10.0统计学软件进行数据处理，比较实验组与对照组间的差异有无统计学意义。研究结果：1、兔骨髓源单个核细胞在体外呈贴壁生长，48h内即贴壁牢靠。前4d内生长较慢，近乎静止状态，4d后生长明显加速，呈现出克隆样生长；10d后细胞基本长满瓶底，表现出典型的铺路石样改变；经VEGF和bFGF在体外联合诱导培养后，细胞表现出条索状和草束状的排列方式。细胞免疫化学染色显示，目标细胞对CD133的表达水平下调，由具有CD34~+／CD133~+／CD31~+表型的细胞群逐渐分化成具有CD34~+／CD133~-／CD31~+表面特征的细胞群；2、术后4w、12w分别行尿道组织学观察，发现实验组中毛细血管数目明显多于对照组。4w时实验组和对照组的数据对比为：4.40±0.82和1.30±0.66，12w时的数据对比为：6.55±0.76和2.50±0.83。应用SPSS10.0统计学软件分别对两次数据行统计学处理，结果显示两次数据之间的差异均有统计学意义(P＜0.01)。研究结论：1、兔骨髓源单个核细胞在体外能诱导分化成血管内皮祖细胞(EPC)；2、血管内皮祖细胞(EPC)在改善尿道缺损修复术后新尿道血液循环方面效果明显。更多还原

【Abstract】 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.更多还原