Effects Of GDF11 And Vitamin D On The Proliferation Of UMR106 Osteoblast-like Cells

BackgroundOsteoporosis is an age-related chronic disease and it is now the sixth most common and frequently-occurring disease in the world.Its main pathological mechanism is the inhibition of the differentiation and proliferation of osteoblasts,which ultimately leads to reduce bone mass and osteoporosis.At present,the treatment of Osteoporosis is focused on inhibiting the function of osteoclasts,such as bisphosphonates,calcitonin,etc.Parathyroid-hormone is the only drug that can enhance the function of osteoblasts.Long-term anti-resorptive therapy leads to excessive inhibition of bone turnover,resulting in the accumulation of old bone,prolonged mineralization of bone matrix,increasing bone fragility and the risk of fracture.Therefore,the discovery of drugs that enhancing the activity of osteoblasts has become a hotspot in the study of Osteoporosis in recent years.GDF11 is a member of the TGF-? superfamily and participates in regulating the development of multiple systems and organs such as skeletal system,nervous system,and the kidney.It is an important regulation of axial development in embryos and also is a hotspot factor in the study of anti-aging in recent years.It can improve the age-related systemic dysfunction such as skeletal muscle injury,muscle atrophy,and so on.The role of GDF11 anti-aging has been questioned with the deepening of research.The studies indicated that exogenous supplementation of GDF11 not only can't improve skeletal muscle injury,even induce osteoporosis.Few studies have been done on the relation of GDF11 and bone metabolism.Some researchers suggest that supplementation of GDF11 can treat senile Osteoporosis,but others think that GDF11 inhibitor is an effective drug for the treatment of senile Osteoporosis.The role of GDF11 in the process of bone formation has not been reported in detail.Vitamin D is an important nutrient to maintain the health of the body.It plays a crucial role in bone development by the activity form 1 ?,25(OH)2D3.It regulates osteoblast differentiation through 1 ?,25(OH)2D3/VDR signal and OPG/RANKL system,but BMP-2/Smads/Runx2 is also one of its regulating pathways.Vitamin D plays a bidirectional regulatory role in the proliferation of osteoblasts,which is mainlyrelated to the level of vitamin D.It is known that vitamin D and calcium can prevent osteoporosis.There is no relevant experimental study on the co-action of GDF11 and vitamin D so far.Part ? Effects of GDF11 on the proliferation of UMR106 cells ObjectiveTo explore the effects of GDF11 on the proliferation of UMR106 cells and the mechanisms.Methods1.UMR106 osteoblast-like cells were cultured and the morphology of the cells was observed.2.The osteoblast-like cells were divided into the control group and the experimental groups.The experiments about GDF11 were divided into four groups: 50ng/m L group,100 ng/m L group,200 ng/m L group and 400 ng/m L group.3.The proliferation of cells were observed through the MTT assay after the cells were cultured at 24,48 and 72 h.4.The change of cell ALP activities were observed through Alkaline Phosphatase kit after the cells were cultured at 24,48 and 72 h.5.The expression of Runx2 m RNA and Osterix m RNA were observed through the qPCR technique after the cells were cultured at 48 h.6.The expression of Runx2 protein was observed through the ELISA kit after the cells were cultured at 48 h.Results1.The shape of adhered cells were round in the early stage.The cells gradually spread and formed in a variety of shapes,such as triangular,polygonal,fusiform,irregular and spindle-shaped.With the prolongation of culture time,the proliferation of cells showed a trend of colony growth,and gradually connected with the adjacent cells,which was similar to the morphological characteristics and growth trend of osteoblasts.2.The OD values of all groups were changed after the cells were cultured at 24,48 and 72h.Compared with the control,the OD values in the 50 ng/m L and 100 ng/m Lgroups were increased(p<0.05),especially the 100 ng/m L group for 48h(p<0.01),and decreased in the 200 ng/m L and 400 ng/m L groups(p<0.05).3.The activities of ALP in each group were changed after the cells were cultured at24,48 and 72 h.Compared with the control,the activities in the 50 ng/m L and 100ng/m L groups were increased(p<0.05),especially the 100 ng/m L group for 48h(p<0.01),and decreased in the 400 ng/m L group(p<0.05).4.The expression of Runx2 m RNA and Osterix m RNA were changed in each group after the cells were cultured at 48 h.Compared with the control,the expression of Runx2 m RNA and Osterix m RNA were increased in the 50 ng/m L and 100ng/m Lgroups(p<0.05);and the expression of Runx2 m RNA were decreased in the 400ng/m L group(p<0.05).5.The expression of Runx2 protein was changed in each group after the cells were cultured at 48 h.Compared with the control,the expression of Runx2 protein were increased in the 50 ng/m L and 100 ng/m L groups(p<0.05)and decreased in the 400ng/m L group(p<0.05).Conclusions1.GDF11 promotes the proliferation of UMR106 osteoblast-like cells.2.GDF11 may play an important role in promoting osteoblast proliferation by activating Runx2 signaling pathway.Part ? Effects of GDF11 and vitamin D on the proliferation of UMR106 cells Objective To explore the effects of GDF11 and vitamin D on the proliferation of UMR106 cells and the mechanisms.Methods1.The osteoblast-like cells were divided into the control group and the experimental group.The experiments about GDF11 and vitamin D were divided into five groups: 50 ng/m L GDF11 group,100 ng/m L GDF11 group,vitamin D(10-6mol/L)group,50 ng/m L GDF11 + vitamin D group and 100 ng/m L GDF11 + vitamin D group.2.The proliferation of cells were observed through the MTT assay after the cells were cultured at 24,48 and 72 h.3.The change of cell ALP activities were observed through Alkaline Phosphatase kit after the cells were cultured at 24,48 and 72 h.4.The expression of Runx2 m RNA and Osterix m RNA were observed through the qPCR technique after the cells were cultured at 24,48 and 72 h.Results1.The OD values of all groups were changed after the cells were cultured at 24,48 and 72h.Compared with the control,vitamin D(24h)and the combination of vitamin D and GDF11 promoted the proliferation of cultured cells(p<0.05).Vitamin D(48 and72h)had no significantly effect on the proliferation of cultured cells.The combination of vitamin D and low concentration of GDF11 was synergistic effect on cell proliferation,while the combination of vitamin D and middle concentration of GDF11 was antagonistic effect on it(p<0.05).2.The activities of ALP in each group were changed after the cells were cultured at24,48 and 72 h.Compared with the control,except for the vitamin D group,the activities of ALP in other group were significantly increased(p<0.05).3.The expression of Runx2 m RNA and Osterix m RNA were changed in each group after the cells were cultured at 24,48 and 72 h.Compared with the control,expect for the vitamin D group and 50 ng/m L GDF11 + vitamin D group,the expression of Runx2 m RNA were significantly increased in other groups,and Osterix m RNA were only highly expressed at 24 and 48h(p<0.05);Compared with the 50 ng/m L GDF11 group,the expression of Osterix m RNA were significantly decreased in 50 ng/m L +vitamin D group(24h)(p<0.05);Compared with 100ng/m L GDF11 group,the expression of Runx2 m RNA were significantly decreased in 100ng/m L + vitamin D group(48h)(p<0.01).Conclusions1.Vitamin D promotes the proliferation of osteoblast-like cells in a short period of time,and may partially or not play a role through the Runx2 signaling pathway.2.The combination of vitamin D and low concentration of GDF11 was a synergisticeffect on the proliferation of osteoblast-like cells,while the combination of vitamin D and high concentration of GDF11 was an antagonistic effect on it.Summary1.GDF11 may play an important role in promoting osteoblast proliferation by activating Runx2 signaling pathway.2.Vitamin D promotes the proliferation of osteoblast-like cells in a short period of time,and may partially or not play a role through activating the Runx2 signaling pathway.3.The combination of vitamin D and low concentration of GDF11 is a synergistic effect on the proliferation of osteoblast-like cells,while the combination of vitamin D and middle concentration of GDF11 is an antagonistic effect on it.