Objective To evaluate the transfection efficiency and expression level of hepatocyte growth factor (HGF) by transfecting a recombinant adenovirus carrying HGF gene (Ad-HGF) into bone marrow mesenchymal stem cells (BMSCs) and to explore the effect of the expression supernatant on BMSCs in vitro so as to lay a foundation for the manufacture of gene medicine which expresses efficient cell factors. Methods Rat BMSCs were isolated using Percoll density gradient method and cultured according to the adherent property of BMSCs. The expression of c-Met was detected by immunohistochemical examination. BMSCs were infected with a recombinant adenovirus carrying green fluorescent protein gene (Ad-GFP) at multipl icity of infection (MOI, 0, 25, 50, 100, and 200 pfu/cell). To select an optimal MOI, the transfection efficiency and the degree of cell damage were assayed by flow cytometry and MTT, respectively, at 48 hours after transfecting. The expression of HGF in BMSCs transfected with optimal MOI Ad-HGF was measured with ELISA assay. MTT method was used to evaluate the prol iferation effect of HGF expression supernatant on BMSCs. Results Immunohistochemical staining showed that BMSCs expressed c-Met receptor for HGF. At 48 hours after transfecting with different MOI Ad-GFP (0, 25, 50, 100, and 200 pfu/cell), the transfection efficiencies were 0.34% ± 0.04%, 40.72% ± 0.81%, 61.72% ± 1.04%, 85.33% ± 0.83%, and 17.91% ± 0.63%, respectively; and the highest transfection efficiency was observed at 100 pfu/cell MOI. The cell damage was obviously observed when MOI was 200 pfu/cell. The expression of HGF in BMSCs reached the highest level after being transfected with 100 pfu/cell MOI Ad-HGF for 48 hours. The expression product could stimulate the prol iferation of BMSCs. The prol iferation of BMSCs gradually rose with the increase of HGF protein, and reached the highest level at 10% (320 pg). Conclusion BMSCs can be transfected efficiently with Ad-HGF and express HGF protein, which stimulates the prol iferation of BMSCs. It suggests that BMSCs is an ideal repair cells with gene vector.
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 multi pl icity of infection was 100. Diabetic models were establ ished in 20 female Wistar rats by diets in high fat and sugar plus intraperitoneal injection ofstreptozotocin (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 heal ing. The contents of hydroxyprol ine and advanced glycosylation end products (AGEs) in the wounds were measured by enzyme l inked 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 heal ing between group C and groups A, B, and D (P lt; 0.05). The wound heal ing time of group C [(20.5 ± 1.9) days] was significantly shorter (P lt; 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 hydroxyprol ine in group C was higher than those in groups A, B, and D at 7 days and 14 days (P lt; 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 lt; 0.05). Conclusion Transplantation of BMSCs transfected with Ad-HGF can accelerate the wounds repair in diabetic rats.