肝细胞生长因子基因转染骨髓间充质干细胞异体移植修复烧伤创面

Bone marrow mesenchymal stem cells transfected with hepatocyte growth factor gene for repair of burn wound after allotransplantation

背景:经携带人肝细胞生长因子的重组腺病毒(adenoviral vector mediated human hepatocyte growth factor,Ad-HGF)修饰的骨髓间充质干细胞创面移植后,可分化为"修复细胞",并能够在创面持续释放肝细胞生长因子。目的:观察Ad-HGF基因转染的骨髓间充质干细胞异体移植对大鼠烧伤创面愈合的影响。设计、时间及地点:随机对照动物观察,于2006-02/2007-03在解放军兰州军区兰州总医院临床实验科和烧伤整形科实验室完成。材料:Wistar大鼠40只,由解放军兰州军区兰州总医院动物实验科提供。Ad-HGF与携带绿色荧光蛋白的重组腺病毒(adenoviral vector mediated human hepatocyte growth factor,Ad-GFP)颗粒均由哈小琴博士惠赠。方法:取雄鼠10只,Percoll密度梯度离心法体外分离培养骨髓间充质干细胞,并行Ad-HGF转染。取雌鼠30只,随机分为未转染细胞组、Ad-HGF转染细胞组、Ad-GFP转染细胞组、Ad-HGF对照组、假伤组,6只/组。前4组大鼠复制背部深Ⅱ度烧伤模型,并分别于创面真皮层下方多点注射对应的细胞悬液或病毒悬液1mL;假伤组大鼠背部剃毛后浸入37℃温水12s模拟烧伤,同法注射等量生理盐水。主要观察指标:对采集的创面标本行组织学观察和羟脯氨酸含量测定,采用免疫组化分析创面肝细胞生长因子的表达。结果:苏木精-伊红染色结果显示,Ad-HGF转染细胞组移植后7d创面表皮化优于其他组,至21d再生表皮明显厚于其他组,并可见真皮钉脚下伸。胶原特殊染色结果显示,Ad-HGF转染细胞组移植后7,21d创面中的胶原排列较其他组整齐。移植后7d,Ad-HGF转染细胞组羟脯氨酸含量显著低于其他组(F=3.216,P<0.01)。移植后21d,Ad-HGF转染细胞组愈合创面中肝细胞生长因子的阳性表达位于真皮层,表达强度明显强于其他组。结论:Ad-HGF基因修饰的大鼠骨髓间充质干细胞异体移植后,对烧伤创面愈合存在一定的促进作用。

BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） with transfection of adenoviral vector mediated human hepatocyte growth factor （Ad-HGF） can differentiate into ＂repair cells＂ after wound transplantation. BMSCs can consecutively release HGF. OBJECTIVE： To explore effects of BMSCs with transfection of Ad-HGF on burn wound healing in rats. DESIGN, TIME AND SETTING： The randomized, controlled animal experiment was performed at the Department of Burns and Plastic Surgery, Department of Clinical Experiment, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from February 2006 to March 2007. MATERIALS： Totally 40 Wistar rats were supplied by Department of Animal Experiment, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA. Ad-HGF and recombinant adenovirus carrying green fluorescent protein （GFP） were gifted by Doctor Ha. METHODS： BMSCs were collected from 10 male Wistar rats and cultured by Percoll density gradient centrifugation method, and transfected with Ad-HGF. A total of 30 female animals were equally and randomly divided into non-transfection MSC group, Ad-HGF MSC group, Ad-GFP MSC group, Ad-HGF control group, and sham wound group. Rats in the non-transfection MSC group, Ad-HGF MSC group, Ad-GFP MSC group, and Ad-HGF control group were made into partial-thickness burn models on the back. 1 mL of cell suspension or viral suspension was injected into the dermis at multiple points. Rats in the sham wound group were subjected to warm water at 37 ℃ for 12 seconds to imitate burn, and injected with an equal volume of saline. MAIN OUTCOME MEASURES： Collected samples were observed by histology to detect hydroxyproline contents. Immunohistochemistry was used to analyze the expression of hepatocyte growth factor. RESULTS： Hematoxylin-eosin staining showed that wound epidermidalization was better in the Ad-HGF MSC group than other groups at 7 days after transplantation, and the regenerated epidermis was significantly thicker than other groups at 21 days, as well as demonstrated that the nails of dermis inserted into the derma of burn wounds. Collagen specific stain confirmed that collagen arranged regularly in the Ad-HGF MSC group at 7 and 21 days. Hydroxyproline contents were significantly lower in＇the Ad-HGF MSC group than other groups at 7 days （F=3.216, P 〈 0.01）. Hepatocyte growth factor was positively expressed in the dermis in the Ad-HGF MSC group, and the expression was higher compared with other groups at 21 days. CONCLUSION： Transplantation of Ad-HGF transfected MSCs have positive effects on the healing of burn wounds.