Recombinant human calcitonin in myoblasts promotes the proliferation and differentitation of rat osteoblasts

ABSTRACT

BACKGROUND:

Repeated injections or nasal spray of large doses of calcitonin can effectively prevent postmenopausal osteoporosis. Calcitonin should be taken for a long time.But the use of calcitonin is limited by the need for repeated protein administration, costly production methods and antigenicity. Gene therapy can provide effective economic therapeutic regimen for osteoporosis,and reduce side effect of drugs.

To describe the expression of human calcitonin produced in myoblasts and determine the effects of the recombinant protein on murine osteoblast cells.

A gene-based controlled observational experiment.

Institute of Radiation Medicine,Fudan University.MATERIALS The experiment was carried out at the Institute of Radiation Medicine, Fudan University from December 2005 to June 2006. Ten healthy SD fetal rats were selected from Institute of Radiation Medicine, Fudan University,Human Calcitoninsas monoclonal antibody was purchased from American Santa Cruz Biotechnology Company. L6 myoblast line was provided by Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.

The pcDNA3.0.-hCT liposome transfection mixture (transfection group) and empty vector pcDNA3.0 liposome mixture (control group) were added in the L6 myoblast medium, respectively.The expression and secretion of human calcitonin by myoblast cells were confirmed by enzyme-linked immunosorbent assay (ELISA),Western blot analysis and immunohistochemical analysis.1×10-14,1×10-13,1×10-12 mol/L recombinant human calcitonin and MEM were respectively added in myoblast medium. MAIN OUTCOME

The proliferation and differentiation of rat myoblasts were observed by MTT and alkaline phosphatase (ALP).

Human calcitonin was found by ELISA in the supematant of cell culture. Western blot and immunohistochemical analysis verified that human calcitonin could be expressed stably in myoblasts after transfection. Osteoblast proliferation and ALP activity were higher when recombinant human calcitonin was 1×10-14 and 1×10-13 mol/L than the control group (P＞0.05).It was significantly higher when the concentration was 1×10-12 mol/L than the control group (P＜0.05).

The stable synthesis and secretion of biologically active human calcitonin can be achieved in myoblasts by gene transfection.Recombinant human calcitonin can enhance proliferation and differentiation of osteoblasts.