Effect Of Silencing Trpv6 Gene On Proliferation, Differentiation And Apoptosis Of Chicken Osteoblasts

Osteoblasts are the main function cells of forming medullary bone in layer hens, which is regulated by Ca2+. Transient receptor potential vanilloid 6 (TRPV6) serves as an important rate-limiting step in facilitating the entry of Ca2+into cells.RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene, which is now commonly used as a tool in biological and biomedical research. Some studies have attempted to investigate the role of TRPV6 in osteoblasts, but mainly were focused on rodents or man, little literature has provided information on TRPV6 in laying hens. Therefore, to investigate the effect of new Ca2+ channel TRPV6 in chicken osteoblast, shRNA targeted against TRPV6 plasmids were transfected into chicken osteoblast in vitro and the effect of TRPV6 on osteoblast proliferation, differentiation and apoptosis was measured to provide theory for the formation of medullary bone.Chicken osteoblasts were cultured and digested with enzymes by twice and then identified with ALP stain and calcified nodules stain. The calvariae were excised from chicken embryos (16 days of age), cut into pieces and then digested the calvariae with 0.1% trypsin and collagenase Ⅱ. Cells were identified with NBT/BCIP staining kit and Alizarin Red. Passage-two chicken osteoblasts were seeded into a 96-well culture dishes and were divided into 3 groups, which were silencing TRPV6 gene group (TRPV6-shRNA plasmids, reagents for transfection and osteoblast), unsilencing TRPV6 gene group (negative-shRNA plasmids, reagents for transfection and osteoblast) and blank control (reagents for transfection and osteoblast), eight replicates for each group. Transfection efficiency was evaluated with fluorescence microscopy. Then cells which were tranfected successfully were incubated for 24 h, 48 h and 72 h. The proliferation rate and differentiation of chicken osteoblasts were detected with the MTT and PNPP methods, respectively. Passage-two osteoblasts with good growth status were chosen and seeded into a 6-well culture dishes (3 groups were divided with the same as MTT assay). The mRNA and the protein expression levels were detected by quantitative real-time RT-PCR analysis and Western blot analysis after 48 h transfection. Apoptosis was assessed with flow cytometry after 72 h transfection.The results indicated that Cells by digested with enzymes were characteristic of osteoblasts which were adherent wall growth, triangular and short spindle under a inverted microscope.80%-90% cells were stained blue-purple with NBT/BCIP ALP staining kit and displayed a dense, round red nodule after identified by Alizarin Red. So these cells were identified as high pure osteoblasts after digestion the calvariae with 0.1% trypsin and collagenase Ⅱ. The results also indicated that the silencing TRPV6 gene group could significantly suppress the expression of TRPV6 at mRNA and protein level compared with the unsilencing TRPV6 gene group which indicated that the silencing TRPV6 gene plasmid can be used for the subsequent trials. The osteoblast numbers and ALP activity of TRPV6-shRNA group were reduced significantly compared with blank group and negative control group (P<0.05). After transfection with TRPV6-shRNA, the apoptotic rate and the mRNA expression levels of apoptosis-related genes (Caspases-3, Caspases-7 and Caspases-9) were increased, especially the mRNA of Caspases-3 is increased significantly (P<0.05).In conclusion:It is showed that silencing TRPV6 gene can inhibit chicken osteoblast proliferation and differentiation and promote chicken osteoblast apoptosis in this study, in turn that TRPV6 chicken can promote chicken osteoblast proliferation and differentiation and inhibit apoptosis.