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背景:糖尿病患者溃疡创面难愈合,目前认为是由糖尿病患者创面微循环障碍以及内源性生长因子含量降低等因素所造成。目的:观察腺相关病毒介导转化生长因子β1(AAV-TGFβ1)和血管内皮生长因子(AAV-VEGF)联合转基因治疗促进糖尿病皮肤溃疡愈合的生物学效应。设计:随机对照动物实验。单位:青岛大学医学院和青岛大学医学院附属医院。材料:实验于2004-07/2006-01在青岛大学医学院附属医院妇科实验室进行。选用成年健康大白兔24只,单纯随机分为联合转基因组和对照组各12只。方法:①采用耳缘静脉注入四氧嘧啶(130mg/kg)建立糖尿病模型,并通过手术方法建立溃疡创面。②联合转基因组创面局部浸润注射AAV-TGFβ1和AAV-VEGF(9×106/mL病毒颗粒);对照组注射生理盐水。主要观察指标:①术后1个月,通过聚合酶链反应检测愈合组织中转化生长因子β1和血管内皮生长因子基因的转录水平。②术后3周,用微循环显微镜计数创缘毛细血管密度值。③术后6个月通过蛋白凝胶电泳和半干电转移法对Ⅰ、Ⅲ型胶原进行分离及免疫印迹检测。④术后1d,1,2,3周,1,2,3,4,5,6个月时测定溃疡愈合组织中胶原含量。⑤术后观测创面愈合厚度、颜色、质地。结果:24只兔全部进入结果分析。①联合转基因组转化生长因子β1和血管内皮生长因子基因转录表达增多。②联合转基因组创缘毛细血管密度值高于对照组[(19.18±3.56),(6.43±1.52)个/mm2,P<0.05]。③联合转基因组愈合组织中胶原含量均增多(P<0.05),而且Ⅰ型和Ⅲ型胶原构成比中Ⅰ型胶原的比例增加。④在转染后1d,联合转基因组与对照组溃疡愈合组织中的胶原含量接近(P>0.05)。此后,联合转基因组胶原含量均高于对照组(P<0.05)。⑤对照组溃疡愈合明显滞后,愈合质量差。结论:AAV-TGFβ1和AAV-VEGF可联合高效转染糖尿病兔耳皮肤溃疡创面,可使溃疡组织中毛细血管密度明显增多,愈合组织中Ⅰ型和Ⅲ型胶原构成比中Ⅰ型胶原的比例明显提高,并有效地促进溃疡愈合。
BACKGROUND: Ulcerous wounds in patients with diabetes are difficult to heal and are currently believed to be caused by factors such as reduced microcirculation in wounds and decreased levels of endogenous growth factors in diabetic patients. OBJECTIVE: To observe the biological effects of adeno-associated virus-mediated transforming growth factor-β1 (AAV-TGFβ1) and vascular endothelial growth factor (AAV-VEGF) combined with transgene therapy on the healing of diabetic skin ulcers. Design: Randomized controlled animal experiments. Unit: Qingdao University Medical College and Qingdao University Medical College Hospital. MATERIALS: The experiment was performed at Gynecological Laboratory of Affiliated Hospital of Qingdao University Medical College from July 2004 to January 2006. Twenty-four adult healthy rabbits were randomly divided into 12 groups, each of which were randomly divided into the combined transgenic group and the control group. Methods: ① Allo-pyrimidine (130mg / kg) was injected into the ear vein to establish a diabetic model and ulcer wounds were established surgically. (2) AAV-TGFβ1 and AAV-VEGF (9 × 106 / mL virus particles) were infiltrated locally into the wounds of the transgenic group. The control group was injected with normal saline. MAIN OUTCOME MEASURES: ① Transcript levels of transforming growth factor β1 and vascular endothelial growth factor in healing tissue were detected by polymerase chain reaction 1 month after surgery. ② Three weeks after operation, the capillary density of the wounds was counted with a microcirculation microscope. ③ Six months after operation, type Ⅰ and type Ⅲ collagen were separated and detected by Western blotting using protein gel electrophoresis and semi-dry electrotransfer method. ④ At 1, 2, 3 weeks, 1, 2, 3, 4, 5 and 6 months after operation, the content of collagen in ulcer healing tissue was measured. ⑤ postoperative wound healing thickness, color, texture. Results: All 24 rabbits entered the result analysis. ① The transgene expression of transforming growth factor β1 and vascular endothelial growth factor increased in the combined transgenic group. ② The transgene group had higher capillary density value than the control group [(19.18 ± 3.56), (6.43 ± 1.52) / mm2, P <0.05]. (3) Collagen content in the combined transgenic group increased (P <0.05), and the proportion of Type I and Type III collagen in type Ⅰ collagen increased. ④ At 1 day after transfection, the collagen content in ulcer healing tissues of the combined transgenic and control groups was similar (P> 0.05). Since then, the collagen content of the combined transgenic group was higher than that of the control group (P <0.05). ⑤ ulcer healing in the control group was significantly delayed, poor quality of healing. CONCLUSION: AAV-TGFβ1 and AAV-VEGF can be combined with high-efficiency transfection of rabbit skin ulcer wounds on the ear, which can significantly increase the capillary density in the ulcer tissue. The proportion of type I and type III collagen in the healing tissue is more than that of type I collagen Improve, and effectively promote ulcer healing.