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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 investigate the effect of BMSCs modified with recombinant adenovirus hepatocyte growth factor (Ad-HGF) on wound healing in experimental type 2 diabetic rats. Methods BMSCs from male Wistar rats were isolated by density gradient centrifugation in vitro. BMSCs from passage 3 to passage 5 were transfected with a total of 100 transfection Ad-HGF for later use. Twenty female Wistar rats were injected intraperitoneally with high-fat and high-sugar diet plus low-dose streptozotocin (STZ, 30mg / kg) to establish an experimental type 2 diabetic model and were prepared on both sides of the back A circular wound of 1.5 cm in diameter. According to different treatment methods, the rats were randomly divided into 4 groups with 5 wounds in each group: BMSCs alone group (A group), Ad-HGF alone group (B group) and Ad-HGF modified BMSCs group Group C) and solvent control group (Group D). The wounds were immediately prepared by injecting the corresponding cells, virus suspension and solvent into the wounds at the edge of the wound, with 0.3mL per wound. The wound healing was observed after injection, the healing time was recorded, and the diameter and longitudinal diameter of the wound were measured at 21 days. HE staining was used to observe the tissue healing at 7 and 28 days. The wound healing was detected by enzyme-linked immunosorbent assay Photometric detection of hydroxyproline and advanced glycosylation end products (AGEs) content. Results The general observation showed that the wound healing was basically complete at 21 days in group C with a diameter of 0 and a longitudinal diameter of (0.110 ± 0.024) cm, respectively. However, part of the wounds in group A, B and D were unhealed, and their transverse and longitudinal diameters were A (0.470 ± 0.051) and (0.590 ± 0.041) cm in group B, (0.390 ± 0.042) and (0.480 ± 0.032) cm in group B, 0.700 ± 0.068 and 0.820 ± 0.068 cm in group D There was statistical significance (P <0.05). The healing time of wounds in group A, B, C and D were (28.3 ± 1.9), (25.9 ± 2.3), (20.5 ± 1.9) and (36.6 ± 5.1) days respectively. The difference between group C and other groups was statistically Significance (P <0.05). HE staining showed that compared with other groups, C group showed obvious epithelial cell migration phenomenon on day 7, collagen formed well, and obvious infiltration of inflammatory cells was observed. On the 28th day, the epidermis of the wound surface was flattened and the collagen was reduced, which was rich in newborn Blood vessels. Compared with other groups, the content of hydroxyproline in group C and C at 7 and 14 days after injection significantly increased (P <0.05), while AGEs decreased significantly at 14 and 28 days after injection (P <0.05). Conclusion Ad-HGF modified BMSCs allograft can promote wound healing in diabetic rats.