沙格列汀对MC3T3-E1细胞增生及CHOP基因、PERK基因表达的影响

Effects of saxagliptin on proliferation of MC3T3-E1 cells and expression of CHOP and PERK genes

目的探讨沙格列汀对成骨前体细胞MC3T3-E1细胞增生及影响生长停滞DNA损伤(growth arrest and DNA damage)的CHOP(C/EBP homologous protein)基因、蛋白激酶R样内质网激酶(protein kinase R like endoplasmic reticulum kinase,PERK)基因表达的影响。方法常规购置成骨MC3T3-E1细胞,置于α-MEM培养基(α-minimum essential medium,α-MEM)中培养、传代。取第3代对数生长的细胞分为4组。正常对照组加入α-MEM培养基培养液,A组加入10^(-9)mol/L沙格列汀培养液,B组加入10^(-8)mol/L沙格列汀培养液,C组加入10-7 mol/L沙格列汀培养液,连续培养24 h,采用微板比色法,以细胞计数试剂盒(cell counting kit-8,CCK-8)检测细胞增生率,采用流式细胞术测定细胞凋亡率;采用酶联免疫吸附试验测定各组细胞碱性磷酸酶(alkaline phosphatase,ALP)水平;对参与内质网应激的CHOP、PERK基因进行扩增,并采用实时荧光定量PCR检测基因的表达。结果A组、B组、C组加入沙格列汀干预后细胞形态正常,细胞数量较多,且呈剂量依赖性,5μmol/L沙格列汀干预细胞数量最多;A组、B组、C组细胞增生能力(0.976±0.062、1.028±0.026、1.052±0.044)、细胞凋亡能力(6.34±1.42、5.61±0.94、4.26±0.62)%及ALP水平(0.13±0.06、0.23±0.08、0.34±0.10)呈剂量依赖性,且不同组间差异有统计意义(P<0.05);C组CHOP、PERK mRNA水平(0.193±0.02、0.180±0.025)均低于A组(0.490±0.026、0.578±0.122)和B组(0.293±0.053、0.312±0.087)(P<0.05);B组CHOP、PERK mRNA水平低于A组(P<0.05)。结论沙格列汀用于成骨前体细胞MC3T3-E1细胞能促进细胞增生,且呈浓度依赖性,可能与降低内质网应激相关CHOP、PERK基因调控有关。

Objective To investigate the effects of saxagliptin on the proliferation and growth arrest DNA damage(CHOP)gene,expression of protein kinase R like endoplasmic reticulum kinase(PERK)gene of osteoblast precursor MC3T3-E1 cells.Methods Osteogenic MC3T3-E1 cells were routinely cultured and passaged inα-MEM medium.The third passage of logarithmically grown cells were divided into four groups.The normal control group was added withα-minimum essential medium(α-MEM)medium,group A was added with 10^(-9)mol/L saxagliptin medium,and group B was added with 10^(-8)mol/L saxagliptin culture medium,group C was added with 10^(-7)mol/L saxagliptin medium.The cells were cultured continuously for 24 hours,and the cell proliferation rate was detected by microplate colorimetry cell counting kit-8(CCK-8),and flow cytometry was used to detect the cell proliferation rate.Cell apoptosis rate was determined by cytometry;Alkaline phosphatase(ALP)levels in each group were determined by enzyme-linked immunosorbent assay.CHOP and PERK genes involved in endoplasmic reticulum stress were amplified,and real-time quantitative PCR was used to detect gene expression.Results After the intervention of saxagliptin,the cells in groups A,B,and C were normal in morphology,and the number of cells was increased in a dose-dependent manner,with the highest number of cells in 5μmol/L saxagliptin group.The proliferation ability(0.976±0.062,1.028±0.026,1.052±0.044),apoptosis ability(6.34±1.42,5.61±0.94,4.26±0.62)%and ALP level(0.13±0.06,0.23±0.08,0.34±0.10)were dose-dependent and statistically among different between groups(P<0.05).CHOP and PERK mRNA levels in group C(0.193±0.02,0.180±0.025)were lower than those in group A(0.490±0.026,0.578±0.122)and group B(0.293±0.053,0.312±0.087)(P<0.05).CHOP and PERK mRNA levels in group B were lower than those in group A(P<0.05).Conclusion Saxagliptin can promote cell proliferation in MC3T3-E1 in a concentration-dependent manner,which may be related to down-regulation of endoplasmic reticulum stress-related CHOP and PERK genes.