| Objective:To observe the effects of different concentrations of atorvastatin calcium on theproliferation and differentiation of 3T3-E1 cells cultured in vitro for 24 hours,analyze the relationship between this process and the endoplasmic reticulum stress signal pathway,and explore the therapeutic targets of bone diseases.Method:1.Observe the morphology and growth of 3T3-E1 cells under a microscope.2.3T3-E1 cells were cultured to logarithmic phase,and the control group?without adding atorvastatin calcium?was added.The experimental group was added with different concentrations of atorvastatin calcium?10-88 mol/L,10-77 mol/L,10-66 mol/L?.After 24hours,the effect of atorvastatin calcium on the proliferation ability of 3T3-E1 cells was observed by CCK-8 colorimetry,and the absorbance value was measured.3.3T3-E1 cells were cultured until logarithmic phase,and the control group?without adding atorvastatin calcium?was added.The experimental group was added with different concentrations of atorvastatin calcium(10-8mol/L,10-7mol/L,10-66 mol/L),and after 24hours,ALP activity was measured using an alkaline phosphatase assay kit and absorbance was measured.4.Add 10-66 mol/L atorvastatin calcium,which has the most obvious effect on cell proliferation and differentiation,to 3T3-E1 cells.After intervention for 24 hours,total RNA in cell samples was extracted by Trizol method and real-timefluorescence quantitative polymerization was used.The realtime quantitative polymerase chain reaction?RT-PCR?technique was used to detect the mRNA expression levels of Runx2,Osterix,Bip,PERK and eIF2?,markers of the endoplasmic reticulum stress signaling pathway.5.Statistical analysis:SPSS 21.0 software was used for statistical analysis.Experimental data were expressed as mean±standard deviation?x±s?.One-way analysis of variance was used for each group and two groups were compared using LSD-t test.The difference was considered statistically significant at a value of P<0.05.Result:1.Inverted microscope observation of 3T3-E1 cells cultured in vitro is adherentgrowth state,showing a spindle-shaped,polygonal and irregular shape,some cells protruding protrusions,with osteoblast cell line growth characteristics.2.Different concentrations of atorvastatin calcium?10-88 mol/L,10-77 mol/L,10-66 mol/L?can promote 3T3-E1 cell proliferation?P<0.05?,and with atorvastatin With the increase of calcium intervention concentration,the cell proliferation ability was enhanced?P<0.05?,and the effect of atorvastatin calcium?10-66 mol/L?was most significant in the high concentration group.3.After treatment with different concentrations of atorvastatin calcium(10-88 mol/L,10-7mol/L,10-66 mol/L),the ALP activity in each group increased,and the high concentration group of Atova Statin calcium?10-66 mol/L?increased ALP activity most significantly?P<0.05?.4.The atorvastatin calcium at a final concentration of 10-6mol/L interfered with 3T3-E1cells for 24 hours.RT-PCR results showed that the mRNA expression of Runx2,Osterix,Bip,PERK and eIF2?in 3T3-E1 cells were higher than those in the control group.?P<0.05?.Conclusion:1.Atorvastatin calcium can promote the proliferation of osteoblast precursor cells?3T3-E1?,and in a certain range,with the increase of atorvastatin calcium intervention concentration,the ability to promote cell proliferation ismore obvious.2.Atorvastatin calcium can increase the osteogenic activity of osteoblast precursor cells?3T3-E1?,and the atorvastatin calcium of high concentration group can promote osteogenic activity higher than the relatively low concentration group.3.On mRNA level,atorvastatin calcium can promote the expression of Runx2,Osterix,and participate in bone metabolism.4.On mRNA level,atorvastatin calcium can up-regulate the expression of Bip,PERK,and eIF2?genes that play an important role in the endoplasmic reticulum stress signaling pathway.The mechanism of atorvastatin calcium in promoting proliferation and differentiation of 3T3-E1 cells may be related to endoplasmic reticulum.Stress signal pathway related. |
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