兔骨髓源单个核细胞诱导血管内皮祖细胞修复尿道缺损

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1 cm2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1010 L-1，注射于新尿道两端的吻合口处，每处0.1 mL，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0.5 mL；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，4 d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。

Bone marrow mononuclear cells-differentiated vascular endothelial progenitor cells for urethral defect repair in rabbits

BACKGROUND: How to solve the source of material substitute for repair of urethral defect and improve the blood supply of new urethra has become a critical problem in the urethral repair and reconstruction. OBJECTIVE: To investigate the effects of endothelial progenitor cells (EPCs) on improving blood circulation in the new urethra following urethral defect repair. DESIGN, TIME AND SETTING: In vivo tissue engineering experiment, performed at the Laboratory of Department of Surgery, First Affiliated Hospital of Zhengzhou University between January 2006 and February 2008. MATERIALS: Thirteen 3-5-month-old male Japanese rabbits were included for this study. Of them, one was used for preparation of bone marrow mononuclear cells, and the remaining twelve rabbits were divided into EPC repair group (n = 8) and model group (n = 4).METHODS: Under the aseptic condition, bone marrow was taken from the rabbit bilateral anterior superior iliac spine. Mononuclear cells isolated by Percoll method were induced in vitro using medium supplemented with vascular endothelial growth factors (VEGFs) and bovine basic fibroblast growth factors. When covering the whole bottom of culture flask, the mononuclear cells were digested with trypsin for passage. Animal models of urethral defect were developed in the two groups. One piece of aseptic fresh acellular human amnion (1 cm2) was sutured to each defected urethra using 0/6 DG suture for forming urethra. In the EPC repair group, 1010/L passage 3 rabbit bone marrow mononuclear cell suspension was injected to two anastomotic stomas of the new urethra, 0.1 mL for each stoma. The subcutaneous tissue was interruptedly sutured to the formed urethra using 0/6 DG suture. In addition, 0.5 mL bone marrow mononuclear cell suspension was added to the region between each anastomotic stroma and newly repaired urethra. The same procedure was performed in the model group except that bone marrow mononuclear cell suspension was replaced by cell-free medium. At weeks 4 and 12 after surgery, paraffin sections of urethral tissue were made. MAIN OUTCOME MEASURES: Identification of cellular morphology; vascular regeneration of urethral tissue after urethral defect repair. RESULTS: After surgery, rabbit bone marrow mononuclear cells adhesively grew in vitro. Four days later, these cells exhibited rapid clone-like growth. Ten days later, they had typical slabstone-like change, presenting with strip-shaped and bundle-shaped growth. The phenotype of cultured cells gradually turned from CD34+/CD133+/CD31+ to CD34+/CD133-/CD31+. At weeks 4 and 12 after surgery, the number of regenerated blood capillaries in the urethral tissue was significantly greater in the EPC repair group than in the model group (t = -9.034 to 5.985, P < 0.01). CONCLUSION: Rabbit bone marrow mononuclear cells-differentiated EPCs can apparently improve local blood circulation in the urethral defect repair.