Effect Of Raloxifene On The ERS During The Proliferation And Differentiation Of 3T3-E1 Cells

Objective:TO investigate the role and mechanism of selective estrogen receptor（SERM）–raloxifene（Second-generation synthetic non-steroidal benzothiophene compounds）on the proliferation and differentiation of the 3T3-E1 cells.Methods:（1）The osteoblastic progenitor cell 3T3-E1 were cultured,and cell growth morphology was observed under an inverted microscope.（2）During the process,the osteoblastic progenitor cell cultured in logarithmic growth phase,was treated with different concentrations of raloxifene(10^-9mol/l、10^-8mol/l、10^-7mol/l)before treatment of osteogenesis.Somatic cells 3T3-E1,a blank control group（without adding raloxifene）,24hours after the use of CCK8 kit to detect cell proliferation.（3）The osteoblastic progenitor cell 3T3-E1 were cultured in logarithmic growth phase,was treated with different concentrations of raloxifene(10^-9mol/l、10^-8mol/l、10^-7mol/l)before treatment of osteogenesis.Somatic cells 3T3-E1,blank control group（no raloxifene added）,alkaline phosphatase（ALP）activity was measured using an alkaline phosphatase assay kit after 24hours.（4）The osteoblastic progenitor cell 3T3-E1 were cultured to logarithmic growth phase,and raloxifene with a higher proliferative and ALP activity group was added to the different concentration groups in the above-mentioned group,and a blank control group was also set.RNAwas extracted at 24 hours,and the expression of ATF6,eIF2αand IRE-1in the cells were detected by real-time quantitative polymerase chain reaction（qRT-PCR）.（5）According to all the data obtained by the above experimental methods,SPSS19.0statistical analysis software was used to analyze the data,and Graphpad Prism5 mapping software was used for mapping.Results:（1）Observed under the inverted microscope,the osteoblastic progenitor cells 3T3-E1 showed the characteristics of adherent growth,the shape was long spindle,triangular or polygonal irregular shape,each cell is connected by the length of different dendrites.In line with general characteristics of osteoblast cell line growth.（2）The raloxifene group added to the raloxifene group significantly increased the proliferation of the osteoblastic progenitor cell 3T3-E1 compared with the control group（P<0.05）.The increase of raloxifene concentration increased the degree of cell proliferation（P<0.05）,and the highest concentration of raloxifene(10^-7mol/l)promoted the most proliferative capacity.（3）The activity of alkaline phosphatase（ALP）increased after treatment with raloxifene compared with the blank control group（P<0.05）.And with changes in the concentration of raloxifene and change,and high concentrations of raloxifene group(10^-7mol/l)increased alkaline phosphatase（ALP）activity is more obvious.（4）The proliferation and alkaline phosphatase（ALP）activity were higher in the group of the osteoblastic progenitor cell 3T3-E1,and the mRNA expression of ATF6,eIF2αand IRE-1were increased in raloxifene-treated the osteoblastic progenitor cell 3T3-E1（P<0.05）.Conclusion:（1）The osteoblastic progenitor cell 3T3-E1 were observed under the microscope.The cells were irregular and connected through dendrites,which corresponded to the general characteristics of osteoblast cell line growth.（2）Raloxifene can promote the proliferation of the osteoblastic progenitor cell 3T3-E1,and its proliferative capacity increase with the increase of concentration.（3）Raloxifene can promote the osteoblastic progenitor cell 3T3-E1,and the degree of differentiation increases as the concentration increase.（4）Raloxifene may promote the proliferation and differentiation of the osteoblastic progenitor cell 3T3-E1 by up-regulating the expression of the ATF6,eIF2αand IRE-1.