| Background Fracture healing is a complex the organism itself repair process, needs a variety of growth factor involved. The fractures with diabetes are very common in the clinic, the delayed union or nonunion rate is a thorny problem faced by clinicians. Studies suggest that the low content of BMP-2 and IGF-I in serum and partial callus tissue is one of the reasons causing the fracture nonunion or delayed unions in diabetes fracture healing process. As the development of stem cell engineering and tissue engineering, bone marrow stromal cells become an important method in studying and treating various orthopaedic disease and its complications. BMP-2 and IGF-I can promote the proliferation and osteoblast differentiation of BMSC. However, we knew little on the proliferation and differentiation effect of BMSC about combining application.Objective To observe the expression of target gene and the proliferation of BMSC transfected by BMP-2 and IGF-1 gene and to further understand biological characteristics of BMSC, and to provide a new method for bone seed cells in cartilage tissue engineering, and to provide the basis for BMSC transplantation in the treatment of diabetes fracture nonunion, delayed union in clinical.Methods Ad-BMP-2 and Ad-IGF-I transfected rat BMSC, according to the transfect situation, the BMSC were divided into 5 groups:the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by BMP-2 (Group C), the group transfected by IGF-I (Group D), and the group transfected both by BMP-2 and IGF-I (Group E).The transfection effect was observed by immunofluorescence after 24h,48h,72h and 96h by transfection, to observe the form of BMSC in different time. Protein expression of BMSC was detected by Western blotting analysis after 24h,48h,72h by transfection. Cell growth curves were obtained by cell counting, MTT assay and flow cytometer to observe the growth and proliferation potential of BMSC.SPSS 16.0 statistical package was used to deal with all the data.There was significant difference when P<0.05.Results (1) Compared with the non-transfected group (Group A) and the group transfected by empty vector (Group B), the form of BMSC of gene carrying groups (Group C, Group D, Group E) was no significant change after early transfected, with the extension of incubation time, polygonal cells increased in gene carrying groups.(2) Green fluorescence was observed in almost all of the cells after transfection, the strongest green fluorescence after 48h in the group transfected both by BMP-2, after 72h in the group transfected by IGF-I and the group transfected both by BMP-2 and IGF-I.The carrying rate was very high in both transfected groups. (3) In the Western blotting analysis, positive signal lane of BMP-2 and IGF-I was observed in transfected group, and protein expression gradually strengthened with times going.(4)Cell growth curves, MTT assay and flow cytometer show that proliferation reached the peak in all groups on the fifth day, and was the strongest in the combined group (P<0.05). The absorbency values of A to E group were 0.324±0.027,0.319±0.017,0.622±0.028,0.626±0.020,0.778±0.031. Flow cytometry show that the proliferative percentage from A to E group were 23.92±3.07,23.51±2.11,34.37±6.85,35.04±1.45,42.56±1.15.Conclusion It is feasible for BMSC to be transfected with BMP-2 and IGF-I gene which was incorporated into adenovirus under high glucose environment, and the protein can be stably expressed in the cultured BMSCs.BMP-2 or IGF-I can promote the proliferation of BMSC cultured in vitro, but the combined has the strongest effect. |
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