血管抑制基因治疗对增生性瘢痕的病理学影响

目的:研究血管抑制基因治疗对兔耳增生性瘢痕的病理学影响。方法:复制兔耳增生性瘢痕,待创面上皮化后10天瘢痕组织局部多点注射基因重组血管抑制剂Ad-METH1(血管生成抑制因子-1,extracellular protein with metalloprotease and thrombospondin1 domains),30天后观察兔耳瘢痕组织形态、超微结构的变化。结果:Ad-METH1注射后30天,兔耳瘢痕组织学染色显示微血管分布明显减少,超微结构显示内皮细胞肿胀、变性,部分血管丧失功能,成纤维细胞增殖及分泌活性明显降低。结论:Ad-METH1对增生性瘢痕的形成有明确的抑制作用,血管抑制基因治疗有望为增生性瘢痕的防治提供新的方法。     

重组血管生成抑制剂Ad-METH-1对兔耳增生性瘢痕的抑制

目的将已经成功构建、携带有METH-1基因的腺病毒表达载体pAdEasy-meth1,转染增生性瘢痕动物模型,研究血管靶向基因治疗对增生性瘢痕的抑制作用.方法复制兔耳增生性瘢痕模型,于创面上皮化后10天,兔耳瘢痕局部注射携带有目的基因METH-1的重组腺病毒颗粒,30天后,用激光多普勒血流仪观察兔耳瘢痕微循环的变化,标本取材,行HE染色、CD34染色、核仁区嗜银颗粒染色,研究分析血管靶向基因治疗对兔耳瘢痕组织血管生成、微循环血流灌注及成纤维细胞增殖的影响.结果与对照组相比,血管靶向治疗30天后,兔耳瘢痕微血管生成、微循环血流灌注及成纤维细胞增殖受到明显抑制.结论在瘢痕形成早期,应用Ad-METH-1进行血管靶向治疗,对兔耳增生性瘢痕的形成,有明显的抑制作用.     

重组血管生成抑制剂Ad-METH-1对兔耳增生性瘢痕的抑制

目的 将已经成功构建、携带有METH-1基因的腺病毒表达载体pAdEasy-meth1,转染增生性瘢痕动物模型,研究血管靶向基因治疗对增生性瘢痕的抑制作用.方法 复制兔耳增生性瘢痕模型,于创面上皮化后10天,兔耳瘢痕局部注射携带有目的基因METH-1的重组腺病毒颗粒,30天后,用激光多普勒血流仪观察兔耳瘢痕微循环的变化,标本取材,行HE染色、CD34染色、核仁区嗜银颗粒染色,研究分析血管靶向基因治疗对兔耳瘢痕组织血管生成、微循环血流灌注及成纤维细胞增殖的影响.结果 与对照组相比,血管靶向治疗30天后,兔耳瘢痕微血管生成、微循环血流灌注及成纤维细胞增殖受到明显抑制.结论 在瘢痕形成早期,应用Ad-METH-1进行血管靶向治疗,对兔耳增生性瘢痕的形成,有明显的抑制作用.     

重组血管生成抑制因子-1对增生性瘢痕组织血管及其相关因子的影响

目的 研究基因转染血管生成抑制对兔耳增生性瘢痕组织血管及其相关因子表达的影响.方法 将基因重组血管抑制剂Ad-METH-1作用于兔耳增生性瘢痕,用微循环显微镜检、组织学染色、免疫组织化学染色等方法,研究Ad-METH-1对兔耳瘢痕组织增生、血管生成及血管内皮细胞生长因子(vascular endothelial cell growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)表达的影响,探讨基因转染血管生成抑制对增生性瘢痕的影响.结果 Ad-METH-1注射后30 d,实验组瘢痕组织微血管计数为12.38±2.56,VEGF阳性细胞百分比为17.64%,bFGF阳性细胞为18.24%;对照组微血管计数为48.12±6.46,VEGF阳性细胞百分比为31.34%,bFGF阳性细胞为28.26%.结果 显示,实验组瘢痕组织微血管计数低于对照组,两组间差异有统计学意义(P＜0.01);实验组瘢痕组织VEGF及bFGF的阳性细胞百分比均低于对照组,两组间差异有统计学意义(P＜0.05).结论 Ad-METH-1对兔耳瘢痕组织增生、血管生成及VEGF、bFGF表达产生了明确的抑制作用,早期行血管抑制治疗可抑制增生性瘢痕的形成.基因转染血管抑制治疗有望成为一种有效的增生性瘢痕防治方法.     

兔耳增生性瘢痕血管生成及腺病毒转染基因重组血管生成抑制因子1

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2.0～2.5 kg,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90 d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10 d,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 thrombospondin 1domains,Ad-METH1) 重组腺病毒40 μL,对照组注射等量空载腺病毒.取 1 只兔于注射后3 d,采用 RT-PCR 和 Westernblot 方法检测基因转染后瘢痕组织中 METH1 mRNA 和蛋白的表达.余 10 只兔注射后30 d,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 90 d 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10～60 d微血管计数及血流灌注值明显高于上皮化后90 d,差异均有统计学意义(P<0.05).兔耳创面上皮化后10～30 d组织学为瘢痕增生早期和增生期表现;60 d时仍为增生期表现,但已出现成熟迹象;90 d时大部分瘢痕软化,为成熟期表现.Ad-METH1 注射后 3 d,实验组 METH1 mRNA 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 30 d,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.     

己基可可碱对兔耳瘢痕组织的作用

目的观察己基可可碱对兔耳增生性瘢痕组织的影响。方法建立兔耳增生性瘢痕动物模型,21d后瘢痕局部注射不同浓度的己基可可碱或生理盐水,49d后观察药物对瘢痕增生指数(hyper trophicindex,HI)的影响及采用图像系统分析瘢痕组织中成纤维细胞数量和胶原含量的变化。结果治疗组中HI与药物浓度呈负相关(P<0.05),治疗组瘢痕组织中成纤维细胞数量与胶原含量均明显减少,呈剂量效应关系;与生理盐水对照组比较,差异有统计学意义(P<0.01)。结论己基可可碱能抑制瘢痕组织中成纤维细胞增殖,并使胶原合成减少,从而抑制兔耳增生性瘢痕组织增生,有望成为防治增生性瘢痕的新药物。     

血管抑制基因对瘢痕组织中VEGF表达的影响

目的 研究血管抑制剂Ad-METH1对兔耳增生性瘢痕组织及其VEGF表达的影响,探讨其作用机制.方法 选取10只健康的日本大耳白兔,将兔耳制作成增生性瘢痕模型后,随机将兔的左右耳分为实验组和对照组,每组10只兔耳.实验组行多点注射重组腺病毒质粒(pAdEasy-meth1),对照组注射空载腺病毒,30d后行成纤维细胞核仁区嗜银颗粒染色(AgNOR)、组织羟脯氨酸含量分析及WesternBlotting分析.结果 AgNOR颗粒计数:实验组为1.86±0.34,对照组为2.53±0.48,两组比较差异有统计学意义(P＜0.05);羟脯氨酸含量:实验组为(4.335±0.156)mg/g,对照组为(5.259±0.169)mg/g,两组比较差异有统计学意义(P＜0.05);VEGF表达的WesternBlotting分析:实验组为0.56±0.15,对照组为0.84±0.23,两组比较差异有统计学意义(P＜0.05).结论 Ad-METH1对兔耳瘢痕组织增生及VEGF的表达有抑制作用,而对早期增生性瘢痕组织中VEGF表达的抑制是其抑制瘢痕增生的可能机制之一.     

鲜地龙液外敷对兔耳增生性瘢痕组织形态学的影响

目的：观察鲜地龙液外敷对兔耳增生性瘢痕组织的影响.方法：选用成年新西兰大耳白兔,建立兔耳增生性瘢痕动物模型,造模术后随机分为空白组、模型组和地龙治疗组和积雪草甙组.各组进行相应的处理50天后,观察鲜地龙液对瘢痕形态及瘢痕增生指数的影响.结果：地龙治疗组与空白组、模型组及积雪草甙组之间相比较,瘢痕增生指数差异有显著性意义（P＜0.05）;光镜显示,明显抑制成纤维细胞增殖以及降低胶原纤维的含量.结论：鲜地龙液外敷可以对兔耳增生性瘢痕组织增生有抑制作用.     

苦参碱对兔耳增生性瘢痕治疗作用的组织形态学研究

目的:研究苦参碱（matrine,Mat）对兔耳增生性瘢痕治疗作用。方法:利用兔耳腹侧面增生性瘢痕模型,采用组织形态学的方法对比研究苦参碱在兔耳增生性瘢痕形成过程中的影响。结果:兔耳腹侧面增生性瘢痕局部注射苦参碱后,按时间段取材;VG和HE染色显示真皮层明显变薄,成纤维细胞数量明显减少,胶原排列趋于一致;12周后,对照组苦参碱组的增生指数、成纤维细胞数密度、胶原密度分别为3.25±0.89/2.15±0.12,4517.5±879.6/3551.2±234.1,39.89±14.12/28.75±10.87。结论:苦参碱能抑制兔耳增生性瘢痕的增殖过程,使瘢痕组织纤维化程度明显减轻。     

Ad-METH1对培养的内皮细胞分泌活性的影响

目的：研究基因重组血管抑制剂Ad-METH1（metalloprotease and thrombospondin1）对培养的内皮细胞分泌活性的影响,探讨血管生成抑制防治增生性瘢痕的可能机制。方法：制备重组血管抑制剂Ad-METH1,转染培养的人脐静脉内皮细胞,通过放射免疫分析法及酶联免疫分析法研究Ad-METH1对内皮细胞分泌内皮素-1（endothelin-1,ET-1）及碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）的影响。结果：基因转染后24h内皮细胞ET-1、bFGF分泌受到明显抑制。结论：Ad-METH1对内皮细胞ET-1、bFGF分泌活性有影响,此是其早期参与抑制增生性瘢痕形成的可能机制之一。     

Adenovirus-mediated METH1 gene expression inhibits hypertrophic scarring in a rabbit ear model

Hypertrophic scarring remains a major problem for patients who have suffered from surgeries or burns. Vascularization plays an important role in the early phase of hypertrophic scarring. Therefore, the inhibition of angiogenesis might be used as a preventive strategy. In this study, we assessed the effect of anti-angiogenesis resulting from adenovirus-mediated METH1 (metalloprotease and thrombospondin1) gene expression on the hypertrophic scar formation in a rabbit ear model of hypertrophic scarring. We first investigated the number of microvessel and microcirculatory perfusion in untreated scars on days 10, 30, 60, and 90 after epithelialization. Then, we examined the effect of anti-angiogenesis by adenovirus-mediated METH1 expression on hypertrophic scar formation by calculating the scar elevation index, counting the microvessel and argyrophilic nucleolar organizer region particle, and detecting the amount of collagen on days 30 and 60 after treatment. We found that untreated scar tissues at the proliferative phase (days 10–60 after epithelialization) had a significantly higher density of microvessel and microcirculatory perfusion than those at the mature phase (day 90 after epithelization) (both p <0.05). On days 30 and 60 after treatment, the hypertrophic scar formation was significantly inhibited in the treatment group. There was significantly reduced scar elevation index, microvessel count, number of argyrophilic nucleolar organizer region, and total collagen content for treated scars. Our results demonstrate that METH1 has a markedly inhibitive effect on the formation of hypertrophic scar, and may thus have a promising application in the prevention of human hyperthropic scars.     

Effect of Sucrose on Collagen Metabolism in Keloid, Hypertrophic Scar, and Granulation Tissue Fibroblast Cultures

Sucrose has been used to treat wounds with excellent results and with minimal abnormal scarring. In this study the effects of sucrose on collagen metabolism in fibroblast culture was evaluated. Sucrose (5.5, 15, or 25 mM) was added to granulation tissue, hypertrophic scar, and keloid fibroblast cultures. mRNA levels and procollagen aminopropeptides for type I and III collagens in cell culture medium were studied. Sucrose decreased mRNA levels for proα1(I) and proα1(III) collagens in fibroblast cultures derived from hypertrophic scar and keloid. In normal granulation tissue fibroblast cultures, 5.5 mM sucrose increased mRNA levels for proα1(I) and proα1(III) collagen, and higher concentrations decreased them. The synthesis of type I collagen decreased dose-dependently in all cell strains, whereas the synthesis of type III collagen decreased only in granulation tissue fibroblasts. To conclude, in vitro high concentrations of sucrose down-regulate both collagen gene expression and synthesis in normal granulation tissue fibroblasts, whereas in fibroblasts derived from abnormal scar sucrose down-regulates only type I collagen gene expression and synthesis, changing the pattern of collagen metabolism toward normal.     

人血管生成抑制分子的分子克隆与真核表达

目的　克隆人血管生成抑制分子 (METH1)全长cDNA ,建立稳定表达人METH1分子的哺乳动物细胞系。方法　RT PCR获取人METH1cDNA ,序列测定后克隆人真核表达载体pCDNA3 0中 ,用脂质体转染入HepG2细胞 ,G4 18筛选出稳定表达细胞系 ,用RT PCR与Westernblot分别检测RNA和蛋白表达水平。结果　RT PCR扩增得到预期大小的目的条带 ,序列分析表明成熟肽编码区与发表序列 [GI∶5 72 5 5 0 5 ]完全一致 ;克隆人pCDNA3 0中得到METH1真核表达载体 ,G4 18筛选 3周后得到稳定表达细胞系 ,RT PCR与Westernblot显示METH1在HepG2细胞中有高水平表达。结论　人METH1全长cDNA成功克隆 ,并在哺乳动物细胞系HepG2中获得稳定表达 ,为进一步研究METH1分子对增生性瘢痕血管形成的作用奠定了理论基础。     

The effect of the angiogenesis inhibitor, angiostatin, on hypertrophic scarring

Introduction: Hypertrophic scarring is commonly seen by plastic surgeons in China. Since the etiology of hypertrophic scarring is still unknown, the only reliable treatment is surgical excision. To understand if angiogenesis plays an important role in the formation of hypertrophic scars, we investigated the effect of angiostatin, a potent angiogenesis inhibitor, on hypertrophic scar formation. Our hypothesis is that angiogenesis is required for increased scar formation and angiogenesis inhibition may be one of the methods that can be used to prevent the formation of hypertrophic scars. Methods: We have developed a reliable model in rabbits that results in hypertrophic scarring by creating a 6mm x 6mm full thickness skin wound on both ears. The cDNA for angiostatin is cloned into the pcDNA 3.1 mammalian expression vector. After the wounds re-epithelialized but prior to excessive scarring, the angiostatin expression vector was injected with Lipofectin 2000 once every two days. The expression of angiostatin was confirmed by RT-PCR. The scar tissue was harvested 14 days after injection and processed for histology and total protein. Histology was examined with routine stains, the amount of collagen deposition in the scar tissue was detected by proline assay, and TGF-β1 and VEGF expression was detected by western blot. Results: Compared to the control injection scar, the injection of angiostatin led to a much more normal-looking of scar in the rabbit ear. The proline assay demonstrated that the injection of the angiostatin expression vector resulted in much less collagen in the scar tissue. Western blot analysis showed there was less TGF-β1 and VEGF protein expression in the treated ear compared to the control. Conclusion: The introduction of a vector over-expression angiostatin can result in the decreased formation of hypertrophic scars in a rabbit ear model. This is corroborated by evidence of decreased collagen deposition, the primary extracellular matrix component of scars. In addition, we demonstrate the decreased expression of τηε pro-fibrosis growth factor, TGF-1, and the potent angiogenic factor, VEGF. These data suggest that angiogenesis inhibitors may have a potential role in the treatment of hypertrophic scarring.     

细菌纤维素对兔耳增生性瘢痕中羟脯氨酸含量的影响

目的：观察细菌纤维素（BC）对兔耳增生性瘢痕的影响。方法：成年新西兰白兔30只,全麻下建立兔耳腹侧增生性瘢痕模型。在术后第21天（创面上皮化）后,根据每只兔耳5个不同瘢痕面随机给予不同处理方式分为5组,分别为3组实验组（贴敷持水性不同的细菌纤维素（BC）膜,A组BC（1：5）、B组BC（1：6）、C组BC（1：8））和2组对照组（贴敷疤痕贴的为阳性对照D组,未贴任何敷料且瘢痕自然生长的为阴性对照E组）。在给予瘢痕面不同处理方式之后的第0天、第14天、第21天、第28天、第42天、第56天分别切取标本行羟脯氨酸（Hpr）含量检测。结果：A、B、C三组瘢痕Hpr含量高于D组但低于E组（P〈0.01）;A、B、C、三组瘢痕Hpr含量分别比较A〉B〉C组（P〈0.05）。结论：兔耳创面愈合后增生性瘢痕贴敷的细菌纤维素抑制了兔耳瘢痕的增生,各组细菌纤维素减轻增生性瘢痕效果比较A组〈B组〈C组。     

Type I and type III procollagen gene expressions in the early phase of ligament healing in rabbits: an in situ hybridization study.

The purpose of this study is to observe type I and type III procollagen gene expressions in the healing ligament using in situ hybridization histochemistry. The rabbit medial collateral ligaments were incised and harvested at 3, 7, 14, and 28 days postoperatively. The healing ligament showed increased expression of both procollagen genes through this period compared with the unoperated ligament. The peak expression level was observed at 7 or 14 days for type I and at 7 days for type III, respectively. The strongest expression of both genes was detected in the scar tissue created between the ends of the old ligament. Although type III procollagen gene expression was observed almost only in the newly created scar tissue, type I procollagen gene was expressed not only in the scar tissue, but also at the ends of the previously normal ligament. These results suggest that type I collagen synthesis begins shortly after ligament injury and occurs at the ends of the injured ligament as well as in the scar tissue, and that type III collagen is largely synthesized in the scar tissue around one week after injury but continues being synthesized for at least four weeks after injury.