BMSCs联合携带肝细胞生长因子的重组腺病毒促进糖尿病大鼠创面修复的研究

STUDY ON BONE MARROW MESENCHYMAL STEM CELLS TRANSFECTED WITH ADENOVIRUS HEPATOCYTE GROWTH FACTOR GENE PROMOTING WOUNDS REPAIR IN DIABETIC RATS

目的探讨携带肝细胞生长因子的重组腺病毒(adenovirus hepatocyte growth factor,Ad-HGF)修饰的BMSCs对实验性2型糖尿病大鼠创面修复的作用。方法采用密度梯度离心法体外分离、培养雄性Wistar大鼠BMSCs,取第3～5代BMSCs以转染复数100转染Ad-HGF后备用。取20只雌性Wistar大鼠以高脂、高糖饲料加小剂量链脲菌素(streptozotocin,STZ,30mg/kg)腹腔注射建立实验性2型糖尿病模型,并在大鼠背部两侧分别制备一直径为1.5cm的圆形创面。根据治疗方法不同将大鼠随机分为4组,每组5只10个创面:单纯BMSCs治疗组(A组),单纯Ad-HGF治疗组(B组),Ad-HGF修饰的BMSCs治疗组(C组)及溶剂对照组(D组),创面制备即刻将相应细胞、病毒悬液及溶剂行创面边缘多点注射,每个创面0.3mL。注射后观察创面愈合情况,记录愈合时间,并于21d检测创面的横径和纵径;7、28d行HE染色观察组织愈合情况;3、7、14、28d分别用酶联免疫法和荧光分光光度计检测羟脯氨酸和糖基化终末产物(advanced glycosylation end products,AGEs)的含量。结果大体观察示C组于21d时创面基本愈合,横径和纵径分别为0、(0.110±0.024)cm,而A、B、D组均部分创面未愈,横径和纵径分别为A组(0.470±0.051)、(0.590±0.041)cm,B组(0.390±0.042)、(0.480±0.032)cm,D组(0.700±0.068)、(0.820±0.068)cm,与C组比较差异均有统计学意义(P<0.05)。A、B、C、D组创面愈合时间分别为(28.3±1.9)、(25.9±2.3)、(20.5±1.9)、(36.6±5.1)d,C组与其他各组比较差异均有统计学意义(P<0.05)。创面组织HE染色示与其他各组相比,C组7d时创面可见明显的上皮细胞迁移现象,胶原形成良好,可见明显炎性细胞浸润;28d创面新生表皮平整,胶原减少,有丰富的新生小血管。与其他各组相比,注射后7、14dC组羟脯氨酸含量明显增高(P<0.05),而AGEs含量在注射后14、28d显著下降(P<0.05)。结论 Ad-HGF修饰的BMSCs异体移植可促进糖尿病大鼠创面修复。

Objective To explore the effects of bone marrow mesenchymal stem cells （BMSCs） transfected with adenovirus hepatocyte growth factor （Ad-HGF） on wound repair in diabetic rats. Methods BMSCs from male Wistar rats were isolated by density gradient centrifugation, cultured, and transfected with Ad-HGF. The multiplicity of infection was 100. Diabetic models were established in 20 female Wistar rats by diets in high fat and sugar plus intraperitoneal injection of streptozotocin （30 mg/kg）. Then 2 full-thickness skin wounds （approximately 1.5 cm in diameter） were made on the dorsum. The rats were randomly divided into 4 groups （n=5 rats）. After wounding, the 0.3 mL suspensions of BMSCs （group A）, Ad-HGF （group B）, BMSCs transfected with Ad-HGF （group C）, and PBS （group D） were injected directly into the derma of wounds. The transverse diameter and longitudinal diameter of wound were measured at 21 days after treatment. At 7 days and 28 days after treatment, HE staining was performed to evaluate wound healing. The contents of hydroxyproline and advanced glycosylation end products （AGEs） in the wounds were measured by enzyme linked immunosorbent assay and fluorospectrophotometer, respectively, at 3, 7, 14, and 28 days after treatment. Results At 21 days after treatment, the wounds almost healed in group C, and the transverse diameter and longitudinal diameter were 0 and （0.110 ± 0.024） cm, respectively. But the wounds healed partially in groups A, B, and D, and the transverse diameter and longitudinal diameter were （0.470 ± 0.051） cm and （0.590 ± 0.041） cm, （0.390 ± 0.042） cm and （0.480 ± 0.032） cm, and （0.700 ± 0.068） cm and （0.820 ± 0.068） cm, respectively. There were significant differences in wound healing between group C and groups A, B, and D （P 0.05）. The wound healing time of group C [（20.5 ± 1.9） days] was significantly shorter （P 0.05） than those of groups A, B, and D [（28.3 ± 1.9）, （25.9 ± 2.3）, and （36.6 ± 5.1） days]. At 7 days, the HE staining showed that evident epidermis transportation, collagen formation, and leukocytes infiltration were observed in group C. At 28 days, the HE staining showed that the epidermis in group C was significantly thinner and more regular than those in other groups, and the decreased collagen and many small vessels were observed in group C. The content of hydroxyproline in group C was higher than those in groups A, B, and D at 7 days and 14 days （P 0.05）. The contents of AGEs in group C was lower than those in groups A, B, and D at 14 days and 28 days （P 0.05）. Conclusion Transplantation of BMSCs transfected with Ad-HGF can accelerate the wounds repair in diabetic rats.