腺相关病毒载体介导转化生长因子β_3在防治增生性瘢痕愈合中的生物学效应

Biological effects of transforming growth factor beta 3 mediated by adeno-associated virus on hypertrophic scar healing

目的:观察腺相关病毒载体介导转化生长因子β3在防治增生性瘢痕愈合中的作用。方法:实验于2005-07/2006-10在青岛大学医学院创伤骨科研究所和中心实验室完成。①实验方法:通过改进的Morris法建立兔耳增生性瘢痕模型,术后将携带转化生长因子β3的腺相关病毒颗粒转染于增生性瘢痕愈合创面处。②实验评估:观察创面愈合情况,应用反转录-聚合酶链反应和免疫组织化学染色法检测创面愈合组织中转化生长因子β3的表达;应用免疫印迹法检测主胶原Ⅰ,Ⅲ型胶原含量;应用氯胺-T法检测羟脯氨酸含量;评价腺相关病毒载体介导转化生长因子β3在防治增生性瘢痕愈合中的生物学效应。结果:①术后6个月,对照组瘢痕较前仍稍有增生,颜色淡红,质地硬。腺相关病毒载体介导转化生长因子β3转染组愈合创面稍突出皮面,颜色接近肤色,质地接近周围皮肤。②腺相关病毒载体介导转化生长因子β3转染组TGFβ3基因反转录-聚合酶链反应电泳条带亮度明显高于对照组。③腺相关病毒载体介导转化生长因子β3转染组创面愈合组织中羟脯氨酸的含量逐渐降低。至术后3个月,两组创面愈合组织中羟脯氨酸含量始于稳定,腺相关病毒载体介导转化生长因子β3转染组低于对照组(109.57±9.13,285.92±10.63)μg/g(t=2.98,P<0.05)。④免疫印迹检测显示腺相关病毒载体介导转化生长因子β3转染组创面愈合组织中Ⅰ型和Ⅲ型胶原构成比中Ⅲ型胶原的比例高于对照组。结论:①腺相关病毒载体可介导目的基因转化生长因子β3高效转染增生性瘢痕创面并有效防治增生性瘢痕愈合。②其作用可能是通过抑制创面愈合组织中羟脯氨酸的合成以及提高Ⅰ型和Ⅲ型胶原构成比中Ⅲ型胶原的比例实现的。

AIM： To study the biological effects of transforming growth factor β3 （TGF-β3） mediated by adeno-associated virus （AAV） in preventing hypertrophic scar healing. METHODS： The experiment was performed at Orthopedics and Trauma Institute and Central Laboratory, Medical College, Qingdao University from July 2005 to October 2006. （1）The hypertrophic scar model of rabbit ear was established by the modified Morris＇ method and the AAV-TGF β3 virus was transfected into the raw surface. （2）Raw surface healing were observed. TGF β3 expression in raw surface healing was measured by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemical method. Collagen Ⅰ and Ⅱ contents were determined by Western blotting. Hydroxyproline level was detected by chloramines-T method. The biological effects of AAV- TGF β3 in preventing hypertrophic scar healing was evaluated. RESULTS： （1）Carmoisine hard hyperplastic scar appeared in the control group, whereas mildly hypertrophic scar was found and its color and texture were similar to the skin in the transfection group at month 6. （2）The electrophoresis strip was obviously lighter in the transfection group than the control group after RT-PCR. （3）Hydroxyproline content was gradually decreased in the raw surface tissue of the transfection group. Till the 3^rd month, hydroxyproline content of the raw surface healing tissue in the two groups was stable, and was lower in the transfection group than the control group （109.57±9.13,285.92±10.63）μg/g （t =2.98, P 〈 0.05）. （4）The percentage of collagen type Ⅲ in the constituent ratio of collagen type Ⅰ and type Ⅲ was higher in the transfection group than the control group by immunoblotting method. CONCLUSION： （1）The AAV can mediate the target gene TGF-β3 to transfect the raw surface of the hypertrophic scar model in high efficiency and prevent effectively scar healing. （2）The mechanism can be achieved by inhibiting the hydroxyproline synthesis in the raw surface tissues and elevating the percentage of collagen type Ⅲ in the constituent ratio of collagen type Ⅰ and type Ⅲ.