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Published on: July 30, 2018
[Cloning and expression of insulin-like growth factor-1 platelet-derived growth factor-AA, and tumor growth factor
Da-di Jin1, Dong-bin Qu, De-hong Yang
1Department of Orthopedic and Spinal Surgery, Nanfang Hospital, Guangzhou 510515, China.
Objective:
To establish recombinant adenovirus highly expressing genes of insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-AA (PDGF-AA), and tumor growth factor beta1 (TGF beta 1) to be used in gene therapy.
Methods:
Normal human osteoblasts were obtained from cancellous bone of lilium left from bone grafting and then cultured. Total RAN was extracted and IGF-1, PDGF-AA, and TGF beta 1 cDNAs was obtained by RT-PCR. Vector p-Shuttle containing these cNDAs was cloned into adenovirus and then the recombinant adenovirus was transfected into HEK293 cells. Recombinant adenovirus containing reporter gene LacZ was used in control group. Western blotting was used to detect the expression of these growth factors. Human osteoblasts were cultured and transfected with recombinant adenovirus. MTT method was used to detect the proliferation of the cells. Paranitrophenol method was used to examine the activity of alkaline phosphatase (ALP) in osteoblasts.
Results:
Expression of IGF-1, PDGF-AA, and TGF beta 1 cDNA and expression of IGF-1, PDGF-AA, and TGF beta 1 proteins were found in HEK293 cells transfected with the recombinant adenovirus and not in the control HEK293 cells. The proliferation and ALP activity of osteoblasts transfected with the recombinant virus were significantly increased in comparison with those of the control osteoblasts (all P < 0.010). Immunohistochemical staining showed significant brown particles in the osteoblasts transfected with the recombinant virus and none in the control osteoblasts.
Conclusion:
The recombinant adenovirus thus constructed expresses the proteins of several growth factors with bioactivity in human cells and can be used as a satisfactory gene tool.
