Adrenomedullin Gene Transfect And Hypoxia Cultureincrease Bone Marrow Mesenchymal Stem Cells In The Anti-apoptotic Ability

Objective:To investigate the apoptosis and expression of Bax, Bcl-2, Caspase-3 in BMSCs after adrenomedullin(ADM) gene transfection and hypoxia culture.Methods:BMSCs were cultured for 0, 3, 6, 9, 12 h under hypoxia condition. In order to determine the best time of hypoxia,the m RNA expression of Bax, Bcl-2, Caspase-3 were detected by RT-PCR and the protein expression of Bax, Bcl-2, Caspase-3 were detected by western blot; to determine the best ADM concentration, BMSCs were cultured under different concentrations of ADM(control, 1 ng/ml, 10 ng/ml, 100 ng/ml)and hypoxia 6 h,the m RNA expression of Bax, Bcl-2, Caspase-3 were detected by RT-PCR and the protein expression of Bax, Bcl-2, Caspase-3 were detected by western blot; after cultured under hypoxia and serum-free conditions,BMSCs were divided into control group, Ad-Lac Z group, Ad-ADM transfection group, ADM group. The m RNA expression of ADM, ADM receptor(CRLR /RAMP-2)were detected by RT-PCR.The m RNA expression of Bax, Bcl-2, Caspase-3 were detected by RT-PCR and the protein expression of Bax, Bcl-2, Caspase-3 were detected by western blot. Cell apoptosis was detected by TUNEL.Results:1.The infection rate of Ad-ADM increased with MOI in a dos dependant manner.When MOI was 150, the infect rate reached 94%. 2. BMSCs cultured in hypoxia and serum-free medium for 0, 3, 6, 9, 12 h, the expression of Bax and Bcl-2 increased on both m RNA and protein level( P<0.05),Bax / Bcl-2 reached minimum ratio(vs control group P<0.05),the expression of Caspase-3 was lowest(P<0.05).3.BMSCs cultured under different concentraions of adrenomedullin and hypoxia for 6 h, the expression of Bax decreased, Bcl-2 increased,Bax/Bcl-2 ratio decreased,Caspase-3 decreased on both m RNA and protein level(P <0.05).4. After the adrenomedullin gene transfected BMSCs cultured under hypoxia and serum-free conditions for 6h,the m RNA expression of ADM, CRLR, RAMP-2 increased,the m RNA and protein expression of Bax decreased, Bcl-2 increased,Bax/Bcl-2 ratio decreased,Caspase-3 decreased, and cell apoptosis reduced(P<0.05).Conclusion: 1.Ad-ADM have high transfection efficiency on BMSCs. 2. Hypoxia 6h c improved BMSCs in anti-apoptotic ability. 3. Adrenomedullin improved BMSCs in anti-apoptotic ability, and its effect was concentraion-dependent. 4.The adrenomeduilin gene transfected BMSCs inceased the expression of ADM, CRLR, RAMP-2. 5. adrenomedullin gene transfection and hypoxia culture enhanced BMSCs in anti-apoptotic ability.