骨髓单个核细胞局部移植预防缺血性胆道狭窄的实验研究

Experimental study on bone marrow mononuclear cells on vWF and VEGF expression in ischemic bile duct tissue

目的探讨骨髓单个核细胞局部移植对缺血胆管组织血管再生的促进作用机制，观察对胆道缺血性病变的预防和治疗效果。方法将30只清洁级昆明小白鼠随机分为3组：①缺血胆管骨髓单个核细胞移植组（A组），②缺血胆管PBS注射组（B组），③对照组（C组），每组10只。3组均于胆囊管汇入肝总管处向下解剖一段长约0．5cm完全游离的胆总管。A组和B组用2枚显微血管夹钳夹游离胆总管的两端，90min后取出血管夹（C组不钳夹胆管）。3组均游离大网膜一束约0．3cm×0．3cm×0．5cm，将其环绕并固定于游离的胆总管。A组将BrdU示踪剂标记的同种系小鼠骨髓单个核细胞悬液均匀注射于环绕的大网膜上，B组和C组仅注射等量磷酸盐缓冲液。术后21d再开腹行胆道造影。取包绕胆管的大网膜和胆管组织制作石蜡切片。用兔抗Brdu单克隆抗体（NeoMarkers，USA）和标有异硫氰酸荧光素（FITC）的山羊抗兔IgG进行免疫组织化学染色和免疫荧光染色（SABC法），观察缺血胆管局部骨髓单个核细胞的分布与分化。分别用第Ⅷ因子相关抗原的抗体和VEGF抗体进行免疫组织化学染色，检测毛细血管密度。结果术后第21天，A组狭窄率为40％，明显低于B组（100％），C组无胆管狭窄发生；A组骨髓单个核细胞集中分布于缺血胆管周围，其中一些已分化成血管内皮细胞，毛细血管密度明显高于B组和C组（P〈0．01）。结论移植到缺血胆管局部的骨髓单个核细胞，在缺血微环境的诱导下，可分化成血管内皮细胞，并通过促进新生毛细血管的形成，改善缺血胆管血供，明显降低胆管狭窄和梗阻的发生率。

Objective To investigate the effect of BM-MNCs implantation on neovascularization in mouse model of ischemic bile duct. Methods An animal model of ischemic biliary stenosis was established with clamping manipulation. The animals were divided into the BM-MNCs implantation group, control group and normal group, and there were 10 mice in each group. Mouse BM-MNCs were isolated from femur using density gradient centrifugation. BM-MNCs or PBS were injected into 8 points of bile duct tissue in three groups respectively （25 ul/point）. On the 21th day after operation, eholangiography was done. Differentiation of the engrafted cells, capillaries density, cytokine expression （VEGF） in the bile duct were analyzed by immunohistochemical staining. Results The engrafted cells could differentiate into endothelial cells. Stricture rate in the implantation group was 40%. It was significantly lower than that in the control group （100%）. The expression of VEGF in the implantation group was significantly higher than that in the control group or the normal group. Conclusion The implantationof BM-MNCs can induce neovascularization by increasing the expression of VEGF in the ischemic bile duct and differentiate angio-endotheliocytes. It could remarkably improve blood supply of ischemic bile duct to prevent or slow biliary ischemic stricture.