纳米微囊-血管内皮生长因子复合体对创伤组织中Bcl-2及CD34表达的影响  

Effects of nanosphere-vascular endothelial growth factor complex on the expression of Bcl-2 and CD34 in wounded tissue

作　　者：彭湃[1] 杨力[1] 宋保强[1] 夏文森[1] 张卫民[2] 

机构地区：[1]解放军第四军医大学西京医院整形科,陕西省西安市710032 [2]解放军第四二一医院消化科,广东省广州市510318

出　　处：《中国组织工程研究与临床康复》2007年第18期3568-3572,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:观察纳米微囊-血管内皮生长因子复合体对创伤组织Bcl-2及CD34表达的影响,探讨其促进损伤组织修复的作用机制。方法:实验于2004-10/2005-06在西京医院整形外科实验室完成。实验材料:血管内皮生长因子的真核表达载体与10mmol/L纳米微囊50μL涡旋振荡混匀,静置30min,制备纳米微囊-血管内皮生长因子复合体。另设纳米微囊-空载质粒(不含血管内皮生长因子基因)为对照。实验分组:选择健康新西兰白兔25只,构建兔耳慢性难愈性创面模型。另在环切部位的近端腹侧皮肤制备一个直径6mm的圆形创面,作为非缺血对照。转基因组(一侧兔耳慢性创面滴加纳米微囊-血管内皮生长因子复合体100μL)、慢性创面组(另一侧兔耳慢性创面滴加纳米微囊-空载质粒100μL)、非缺血创面组(双侧兔耳非缺血创面滴加纳米微囊-空载质粒100μL)。于术后1,3,7,10,14d,每个时间点麻醉后处死5只动物,以每一创面为中心,切取1cm×1cm正方形组织块(含兔耳全层)。实验评估:①Bcl-2免疫组织化学染色观察兔耳创面组织中阳性反应细胞情况。②创面微血管计数采用抗CD34单克隆抗体免疫组化染色,孤立的棕色血管内皮细胞或细胞簇代表1条单独的微血管。③术后14d细胞外基质特殊染色观察细胞外基质的变化。结果:①兔耳创面组织中阳性反应细胞:Bcl-2阳性信号表达于创面内皮细胞、炎细胞及成纤维细胞胞浆中,正常皮肤及创缘表皮层会出现弱阳性表达。转基因组与非缺血创面组阳性细胞表达及胞浆阳性信号强于慢性创面组,术后3d时差异明显。②创面微血管计数:术后1～14d各组微血管数量逐渐增加,其中转基因组数量最多,慢性创面组数量最少。术后7,10,14d转基因组及非缺血创面组微血管计数高于慢性创面组[以术后7d为例,分别为(20±7),(18±5),(12±4)条/200倍视野],差异有显著性意义(P<0.05)。③细胞外基质�AIM. To study the effects of nanosphere complexes-mediated vascular endothelial growth factor （VEGF） on the expression of Bcl-2 and CD34 in wounded tissue, and investigate its mechanisms of improving the repair of wound tissue. METHODS： Experiments were completed in the laboratory of Department of Plastic Surgery, Xijing Hospital between October 2004 and June 2005. Materials： Nanosphere-VEGF complex was prepared by mixing eukaryote expression vector of VEGF with 50 μL suspension of nanosphere （10 mmol/L） by vortex shaking, and then were standing of 30 minutes. And nanosphere-idler （without VEGF） was set.as controls. Groups： A total of 25 health New-Zealand albino rabbits were selected to establish a model of chronic wound （CW） in rabbit ear. And a round wound with anteroposterior diameter of 6 mm in the ventriskin at the proximal end of circumcised site for non-ischemia wound （NW） control. Rabbits were divided into CW＋VEGF （exerted 100 μL nanosphere-VEGF complex on one side of CW in ear of each rabbit）, CW （exerted 100 μL nanosphere-idler on the other side）, NW （exerted 100 μL nanosphere-idler on bilateral NWs of each rabbit）. Five animals were killed post anesthesia at 1, 3, 7, 10, 14 days postoperatively to get a square architecture by 1 cmxl cm （full-thickness of ear）, with the center of wounded tissue. Assessment： （1）Positive cells in wound tissue of rabbit ears were observed with Bcl-2 immunohistochemistry dying. （2）Microvessel count was performed with anti-CD34 monoclonal antibory immunohistochemistry dying, considering the brown vascular endothelial cell or cytocluster as one independent microvessel. （3） Change of extracellular matrix was determined by using special staining at 14 days postoperatively. RESULTS： （1） Positive cells in wound tissues of rabbit inflammatory cells and fibroblasts, while Bcl-2 was less ears： Bcl-2 was positive in cytoplasm of endothelial cells, positive in that of normal skin and cuticular layer around the woun

关 键 词：创面愈合 基因治疗 血管内皮生长因子 纳米微囊 生物材料 

分 类 号：R318.08[医药卫生—生物医学工程]