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背景:如何解决尿道替代修复材料来源和改善新尿道的血液供应已成为尿道修复与重建研究的瓶颈问题。目的:观察血管内皮祖细胞对尿道缺损修复后改善新尿道组织血液循环的效果。设计、时间及地点:组织工程体内实验,于2006-01/2008-02在郑州大学第一附属医院外总实验室完成。材料:3~5月龄雄性日本大耳白兔13只,1只用于制备骨髓源单个核细胞,剩余12只随机分为细胞修复组8只,模型组4只。方法:无菌抽取兔两侧髂前上嵴骨髓,Percoll法贴壁分离培养单个核细胞,加入含VEGF、bFGF的培养基进行体外诱导分化,待细胞长满培养瓶底后胰蛋白酶消化传代。两组兔均建立尿道缺损模型,将脱细胞处理的无菌新鲜人羊膜修剪成1cm2置于尿道缺损处,用0/6DG线将其两端分别与尿道残端连续缝合,形成尿道。细胞修复组将传至第3代的兔骨髓源单个核细胞浓度调整至1010L-1,注射于新尿道两端的吻合口处,每处0.1mL,0/6DG线间断缝合皮下组织覆盖成形后的尿道,并在近吻合口处和新修复尿道之中间处注射细胞悬液,每处0.5mL;模型组在同样位点注射等量的空白细胞培养液。移植后4,12周制作尿道组织石蜡切片。主要观察指标:细胞形态及鉴定结果,修复后尿道组织的血管再生情况。结果:兔骨髓源单个核细胞在体外呈贴壁生长,4d后生长加速,呈克隆样生长;第10天出现典型的铺路石样改变,并迅速表现出条索状、草束状生长形式;所培养的细胞表型由CD34+/CD133+/CD31+逐渐转变成CD34+/CD133-/CD31+。与模型组比较,移植后第4,12周细胞修复组尿道组织内毛细血管再生数量均明显多于模型组(t=-9.034~5.985,P<0.01)。结论:兔骨髓源单个核细胞可在体外诱导分化为血管内皮祖细胞,且血管内皮祖细胞对改善尿道缺损修复后局部血液循环的效果非常明显。
Background: How to solve the problem of urethral replacement repair materials and improve the blood supply of new urethra has become a bottleneck of urethral repair and reconstruction. Objective: To observe the effect of endothelial progenitor cells (VECs) on the blood circulation of new urethra after urethral defect repair. DESIGN, TIME AND SETTING: Tissue engineering in vivo experiments were performed at the General Laboratory of the First Affiliated Hospital of Zhengzhou University from January 2006 to February 2008. MATERIALS: Thirteen male Japanese white rabbits aged 3 to 5 months were used to prepare bone marrow-derived mononuclear cells. The remaining 12 rabbits were randomly divided into 8 cell repair groups and 4 model groups. Methods: Bone marrow of anterior superior iliac crest on both sides of rabbits was aseptically extracted. Percoll method was used to separate and culture mononuclear cells. The medium containing VEGF and bFGF was added into the culture medium for in vitro differentiation. When the cells were full-fledged, trypsin digestion and passage were performed. Two rabbits were established urethral defect model, the acellular fresh human amniotic membrane trimmed into 1cm2 placed urethral defect Department, with 0 / 6DG line at both ends of the urethra were stitched, urethra. Cell repair group will be transferred to the third generation of rabbit bone marrow mononuclear cells concentration adjusted to 1010L-1, injected at the anastomosis at both ends of the new urethra, each 0.1mL, 0 / 6DG line interrupted suture subcutaneous tissue covered after forming Urethra, and near the anastomosis and the middle of the new repair of the urethra injection of cell suspension, each 0.5mL; model group at the same site injection of equal amount of blank cell culture medium. Four and twelve weeks after transplantation, urethral paraffin sections were made. MAIN OUTCOME MEASURES: Cell morphology and identification results, revascularization of urethral tissue after repair. RESULTS: Rabbit bone marrow-derived mononuclear cells grew in adherent growth in vitro. After 4 days, the growth of the mononuclear cells grew in a clonal manner. On the 10th day, the typical paving stone-like cells changed, and the cord-like, The cultured cell phenotype was gradually converted from CD34 + / CD133 + / CD31 + to CD34 + / CD133- / CD31 +. Compared with the model group, the number of capillary vessels in the urethra tissue in the cell repair group at 4 and 12 weeks after transplantation was significantly more than that in the model group (t = -9.034-5.985, P <0.01). CONCLUSION: Rabbit bone marrow-derived mononuclear cells can differentiate into endothelial progenitor cells in vitro and the effect of endothelial progenitor cells on local blood circulation after urethral defect repair is very obvious.