克老素抑制地塞米松诱导的MC3T3-E1成骨细胞凋亡

The protective effect of Klotho on apoptosis of MC3T3-E1 osteoblasts induced by dexamethasone

目的探究体外转染克老素(Klotho,KL)基因对地塞米松(dexamethason,DEX)诱导的MC3T3-E1成骨细胞凋亡的影响。方法用携带KL基因的重组腺病毒(Ad-KL)及携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP)感染细胞,并建立DEX诱导凋亡的细胞模型。运用荧光倒置显微镜观察重组腺病毒的转染效果,qPCR与Western blot法分析KL mRNA和蛋白表达情况;CCK-8法检测不同浓度DEX作用于MC3T3-E1细胞后的存活率及各研究组细胞的生存情况;流式细胞仪分析各研究组细胞的凋亡率;qPCR与Western blot分析凋亡标志物的mRNA、蛋白表达;免疫荧光分析caspase-9的荧光蛋白表达情况。结果重组腺病毒成功感染MC3T3-E1成骨细胞,KL组和KL+DEX组的KL mRNA与蛋白表达量较非转染组明显升高。CCK-8法检测出DEX的适宜干预浓度为2.0 mmol·L^(-1)。DEX组、GFP+DEX组较其余组细胞存活率明显降低、凋亡率明显增加,Bax、Bcl-2的mRNA与蛋白表达分别表现出增加和降低趋势;KL组细胞存活率、凋亡率,以及Bcl-2、Bax mRNA与蛋白表达较DEX组及KL+DEX组均明显改善。结论大剂量DEX的运用能够诱发MC3T3-E1成骨细胞凋亡,上调KL表达具有抵抗DEX诱导成骨细胞凋亡的作用,提示上调KL表达可改善糖皮质激素诱导性骨质疏松,为大剂量使用糖皮质激素所致医源性骨质疏松的治疗提供新的靶点。

Aim To explore the effect of Klotho(KL)gene transfection on the apoptosis of MC3T3-E1 osteoblasts induced by dexamethasone(DEX).Methods MC3T3-E1 osteoblasts were transfected by recombinant adenovirus containing KL gene(Ad-KL)and recombinant adenovirus containing green fluorescent protein(GFP)gene(Ad-GFP).The apoptosis model was constructed.The transfection efficiency of Ad-KL and Ad-GFP in cells were observed using inverted fluorescent microscope,and the level of KL mRNA and protein was detected by qPCR and Western blot,respectively.The cell viability after different concentrations of DEX acting on the cells and the viability of every research group were determined by cell counting kit-8(CCK-8)assay.The apoptotic rate was evaluated by flow cytometry.The level of mRNA and protein was analyzed by qPCR and Western blot,respectively.The level of caspase-9 protein was detected by immunofluorenscence assay.Results Cells were transfected by Ad-KL and Ad-GFP successfully.KL group and KL+DEX group had higher level of KL mRNA and protein than that in other groups.The optimum concentration of DEX was 2.0 mmol·L-1.When DEX acting on the cells,the cells viability decreased and apoptotic rate increased obviously in DEX group and GFP+DEX group.The level of Bax mRNA and protein presented a upward trend in DEX group and GFP+DEX group,while the level of Bcl-2 mRNA and protein was opposite.But after KL transfecting MC3T3-E1 osteoblasts,the markers described above in KL group had more dramatic improvement than in DEX group and KL+DEX group.Conclusions High-dosage DEX can induce the apoptosis of MC3T3-E1 osteoblasts,and the pro-apoptosis effect of high-dosage DEX in MC3T3-E1 osteoblasts can be suppressed by up-regulating KL gene expression level,suggesting that the glucocorticoid-induced osteoporosis might be improved by up-regulating KL gene expression level,and it may be a new target for the treatment of latrogenic osteoporosis induced by high-dosage glucocorticoid in clinic.