【摘要】 目的研究不同时期兔耳增生性瘢痕组织血管生成,探索新的增生性瘢痕防治方法。方法19只日本大耳白兔,体重2.0～2.5kg,制备兔耳增生性瘢痕模型。其中8只于创面上皮化后10、30、60及90d行微血管计数、微循环监测及HE染色观察。另11只选择每只兔的左、右侧耳为实验组及对照组,于上皮化后10d,实验组兔耳瘢痕局部多点注射基因重组血管生成抑制因子1(adenovirus extracellular protein with metalloprotease and thrombospondin1domains,Ad-METH1)重组腺病毒40μL,对照组注射等量空载腺病毒。取1只兔于注射后3d,采用RT-PCR和Western blot方法检测基因转染后瘢痕组织中METH1mRNA和蛋白的表达。余10只兔注射后30d,行两组大体观察、微血管计数及HE染色。结果上皮化后10、30、60及90d瘢痕组织微血管计数分别为(42.37±3.89)、(49.46±4.13)、(33.12±4.34)及(13.24±2.31)支;瘢痕组织微循环灌注分别为(37.75±2.11)、(59.87±6.46)、(44.53±6.14)及(29.21±1.84)PU;上皮化后10～60d微血管计数及血流灌注值明显高于上皮化后90d,差异均有统计学意义(P<0.05)。兔耳创面上皮化后10～30d组织学为瘢痕增生早期和增生期表现;60d时仍为增生期表现,但已出现成熟迹象;90d时大部分瘢痕软化,为成熟期表现。Ad-METH1注射后3d,实验组METH1mRNA及蛋白有较高水平的表达,对照组未检测到靶基因表达;注射Ad-METH1后30d,大体观察:实验组瘢痕颜色接近正常兔耳肤色,质地接近正常;对照组瘢痕明显高出兔耳腹侧皮面,质地坚硬;瘢痕组织微血管计数实验组为(12.38±2.56)支,对照组为(48.12±6.46)支,组间比较差异有统计学意义(P<0.01)。组织学染色显示实验组瘢痕微血管分布较少,成纤维细胞散在,胶原排列有序;对照组见大量成纤维细胞,血管分布丰富,胶原纤维粗大、排列紊乱。结论血管生成与增生性瘢痕的形成有密切关系,血管抑制基因治疗有望成为一种有效的增生性瘢痕防治方法。更多还原

【Abstract】 Objective To investigate the angiogenesis in hypertropic scar tissue of rabbit ears at different periods and to explore a new method to prevent hyperplastic scar. Methods Nineteen Japanese white rabbits(weigthing 2.0-2.5 kg) were made animal models of hypertropic scar of ear. At 10th,30th,60th and 90 days,after epithelization,the microvessel and microcir-culation in hyperplastic scar of 8 rabbits were studied by microcirculation microscope and laser Doppler flowmetry. The other 11 rabbits’ right or left ears were randomly chosen into experimental group and control group. At 10 days after epithelization,40 μL of adenovirus extracellular protein with metalloprotease and thrombospondin 1 domains(Ad-METH1) was injected into tissue of scar along the perimeter of the scar in experimental group. The same volume of empty adenovirus was injected in con-trol group. After 30 days of injection,the gross appearance of 10 rabbits’ ears scar was recorded,the number of microvessel in scar was counted and HE stainning of scar tissue was performed in experimental and control groups. One additional rabbit was used to evaluate the mRNA and protein expression of METH1 by RT-PCR and Western blot after 3 days of injection. R e sults The average number of microvessel at 10,30,60 and 90 days after epithelization was 42.37 ± 3.89,49.46 ± 4.13,33.12± 4.34 and 13.24 ±2.31,respectively;the average value of microcirculatory perfusion at 10,30,60 and 90 days after epithetlization was(37.75 ±2.11),(59.87 ± 6.46),(44.53 ± 6.14) and(29.21 ± 1.84)PU;the density of microvessels and perfusion of microcirculationin scar tissues during proliferative stage(from 10 to 60 days after epithelization) were markedly higher than that dur-ing mature period(90 days after epithelization,P < 0.05).At 10 to 30 days after epithelization,the histoligical features of scar showed early stage of proliferation and proliferative stage appearance;at 60 days after epithelization,it is still in pro-liferative stage,while some of scars were in mature phase;at 90 days after epithelization,the histoligical features of scar were mature period appearance. At 3 days after Ad-METH1 injection,METH1 gene was successfully expressed at both mRNA and protein levels in experimental group,but not in control group. At 30 days after injection,the gross appear-ance observation showed that scars in experimental group were flat and soft with the color close to normal,but scars in control group were obvious and hard. The number of microvessel of scar tissue was 12.38±2.56 in experimental group and 48.12±6.46 in control group,showing statistically significant difference between two groups(P < 0.01). In experi-mental group,HE staining shows that the density of microvessel and the number of fibroblasts were greatly decreased and collagen fibers arranged regularly. In control group,plenty of fibroblasts and abundant microvessels were observed. Thick and tight collagen fibers were seen in the outer layer of dermis with a irregular arrangement. Conclusion The anti-angiogenesis by Ad-METH1 may have a promising application in the prevention of human hyperthropic scar.更多还原