Effects of recombinant retroviral vector pLNCX2-human calcitonin gene-related peptide α on proliferation and osteogenic activity of human bone marrow mesenchymal stem cells

Objective To infect human bone marrow mesenchymal stem cells (hBMSCs) with recombinant retrovirus vector carrying human calcitonin gene-related peptide α (hCGRPα) gene in vitro,and observe its effects on the proliferation and osteogenic activity of hBMSCs.Methods The autologous primary hBMSCs were obtaind with Ficoll lymphocyte separation medium by gradient centrifugation,passaged and preserved.A recombinant retrovirus vector pLNCX2 carrying hCGRPα gene was constructed and infected into second generation of hBMSCs.The hBMSCs were divided into three groups:(1) experimental group:the pLNCX2-hCGRPα-infected hBMSCs; (2) empty vector group:empty vector pLNCX2-infected hBMSCs; (3) normal control group:cultured hBMSCs not given special treatment.At day 7,the mRNA and protein expression of hCGRPα was examined by using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively.The cell proliferation was determined by using methyl thiazoleterazolium (MTT) assay during 1 to 7 days.At day 14,the osteogenic activity of BMSCs was detected by using alkaline phosphatase (ALP) activity and osteocalcin in the cell culture medium.At day 7,the content of laminin (LN) was detected.Results Both the mRNA and protein expression of hCGRPα in hBMSCs was detectable in experimental group,but undetectable in empty vector group and normal control group.There was a higher cell proliferation rate in experimental group than in empty vector group (P ＜0.05) and normal control group (P ＜ 0.05).The ALP activity values,content of LN and osteocalcin in the cell culture medium were (119.33 ± 11.95) U/100 mg protein,(12.11 ± 1.56) μg/L and (1.123 ±0.115) μg/L in experimental group,which were significantly higher than those in empty vector group [(66.16 ±6.82) U/100 mg protein,(7.32 ±0.98) μg/L,(0.602 ±0.097) μg/L] and normal control group [(67.73 ± 7.31) U/100 mg protein,(6.46 ± 1.41) μg/L,(0.498 ± 0.109) μg/L] (P ＜ 0.05 for all),but there was no significant difference between empty vector group and normal control group (P ＞ 0.05).Conclusion The recombinant retrovirus hCGRPα was successfully transferred into hBMSCs.The hCGRPα gene could be expressed in hBMSCs,promote the cell proliferation and its osteogenic activity. Key words: Recombinant retrovirus;  Calcitonin gene-related peptide;  Bone marrow mesenchymal stem cells;  Cell proliferation;  Osteogenic activity