摘要
目的探讨艾司氯胺酮对成骨细胞增殖分化的作用及其机制。方法将从C57BL/6雄性小鼠中提取并诱导转化而成的成骨细胞置于不同浓度(0,6.25,12.5,25,50,100,200μmol/L)的N-甲基-D-天冬氨酸(NMDA)中,采用CCK-8细胞毒性实验检测不同时间点(24,48,72,96 h)成骨细胞的活性,确定对增殖分化无毒性的NMDA浓度区间。将诱导分化的成骨细胞分为3组:对照组、艾司氯胺酮(Ket)组以及Ket+NMDA组,干预24,48,72 h采用荧光定量PCR(qPCR)法分别检测各组成骨细胞Runt相关转录因子2(Runx2)、成骨细胞特异性转录因子Osterix mRNA的表达情况;采用酶联免疫吸附法(ELISA)测定各组成骨细胞B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关联的X基因(Bax)和脑源性神经营养因子(BDNF)相关蛋白的表达情况。结果CCK-8细胞毒性实验结果显示,在同一时间点,0~200μmol/L的NMDA对成骨细胞活性的影响差异无统计学意义(P>0.05);在一定浓度下,NMDA干预不同时间对成骨细胞活性的影响差异无统计学意义(P>0.05)。qPCR实验结果显示,与对照组相比,Ket组和Ket+NMDA组Runx2和Osterix表达均升高(P<0.01);与Ket组相比,Ket+NMDA组Runx2和Osterix表达下降(P<0.01)。ELISA结果显示,与对照组相比,Ket组Bcl-2蛋白及BDNF蛋白的表达量升高(P<0.05),Bax蛋白的表达量下降(P<0.05);与Ket组相比,Ket+NMDA组Bcl-2蛋白及BDNF蛋白的表达量下降(P<0.05),Bax蛋白的表达量升高(P<0.05)。结论艾司氯胺酮可能通过阻断N-甲基-D-天冬氨酸受体(NMDAR)引起BDNF表达增加,进而促进抗凋亡蛋白的表达同时抑制凋亡蛋白的表达,最终对成骨细胞的增殖分化起到促进作用。
Objective To explore the effect and its mechanism of esketamine on the proliferation and differentiation of osteoblasts.Methods Osteoblasts were extracted and transformed from C57BL/6 male mice.The toxicity of different concentrations(0,6.25,12.5,25,50,100,200μmol/L)of N-methyl-D-aspartic acid(NMDA)for 24,48,72,96 h to osteoblasts was detected by cell counting kit-8(CCK-8)method to screen the appropriate concentration.The induced osteoblasts were divided into three groups:control group,esketamine(Ket)group and Ket+NMDA group.After 24,48,72 h intervention,the mRNA expression levels of Runt associated transcription factor 2(Runx2)and the osteoblast specific transcription factor Osterix in each group were detected by real-time fluorescence quantitative PCR(qPCR),and the expression levels of B-lymphoblastoma-2 gene(Bcl-2),Bcl-2-associated X gene(Bax)and brain-derived neurotrophic factor(BDNF)proteins in osteoblasts in each group were determined by enzyme-linked immunosorbent assay(ELISA).Results CCK-8 results showed that 0-200μmol/L NMDA had no obvious toxicity to osteoblast proliferation at the same time point(P>0.05)and there was no significant difference in the activity of osteoblasts after NMDA intervention for different time(P>0.05).The qPCR results showed that the expression levels of Runx2 and Osterix in Ket group and Ket+NMDA group were higher than those in control group(P<0.01)and the expression levels of Runx2 and Osterix in Ket+NMDA group were lower than those in Ket group(P<0.01).ELISA results showed that the expression levels of Bcl-2 protein and BDNF protein in Ket group were higher than those in control group,while the expression level of Bax protein was lower(all P<0.05).Compared with Ket group,the expression levels of Bcl-2 protein and BDNF protein were decreased and Bax protein was increased in Ket+NMDA group(all P<0.05).Conclusion Esketamine may promote the proliferation and differentiation of mouse osteoblasts by blocking N-methyl-D-aspartate receptor(NMDAR),which increases BDNF expression,thus increasing the expression of anti-apoptotic proteins while inhibiting the expression of apoptotic proteins.
作者
郑马强
马智聪
ZHENG Maqiang;MA Zhicong(Department of Anesthesiology,Shanxi Medical University,Taiyuan 030001,China;Department of Anesthesiology,Second Hospital of Shanxi Medical University)
出处
《山西医科大学学报》
CAS
2024年第1期85-90,共6页
Journal of Shanxi Medical University
基金
山西省卫生健康委科研课题项目(2023023)。
