摘要
目的建立衣霉素诱导成骨细胞内质网应激的模型,探讨胃饥饿素(ghrelin)对衣霉素诱导成骨细胞内质网应激的影响。方法选取小鼠成骨细胞MC3T3-E1为研究对象,(1)分别将不同浓度的衣霉素(0、0.5、1、1.5μg/mL)加入成骨细胞培养基中,分别孵育24h后,采用CCK-8法检测细胞增殖活性;二氯二氢荧光素-乙酰乙酸酯探针(DCFH-DA)检测胞内活性氧(ROS)的含量;real-time quantitative PCR(qRT-PCR)检测各组细胞内内质网应激相关标志性基因BIP、CHOP、caspase-12 mRNA的表达。最后选择衣霉素作用最敏感浓度1.5μg/mL,建立成骨细胞内质网应激的模型。(2)观察ghrelin对成骨细胞内质网应激的影响。分别用不同浓度的ghrelin(0、10^-11、10^-9、10^-7mmol/L)预处理成骨细胞4h后,加入1.5μg/mL的衣霉素诱导成骨细胞内质网应激。培养结束后,利用上述方法检测细胞增殖活性、胞内ROS的含量、内质网应激相关标志性基因的表达。结果与对照组相比,不同浓度的衣霉素干预成骨细胞24h后,细胞增殖和存活率呈浓度依赖性明显降低。1.0μg/mL和1.5μg/mL的衣霉素干预细胞24h后细胞增殖和存活率的降低有统计学意义(P<0.05),而0.5μg/mL的衣霉素干预后无统计学意义(P>0.05);与对照组相比,胞内ROS的含量随浓度的增加逐渐增加(P<0.05);qRT-PCR结果显示,与对照组相比,不同浓度衣霉素干预细胞后CHOP mRNA的表达均有明显的提高且均有统计学意义(P<0.05),而BIP、caspase-12 mRNA的表达只在1.0μg/mL和1.5μg/mL衣霉素干预后有统计学意义(P<0.05),0.5μg/mL衣霉素干预后无统计学意义(P>0.05);(2)与单纯1.5μg/mL衣霉素相比,用不同浓度的ghrelin预处理成骨细胞4h后再加1.5μg/mL衣霉素,发现细胞增殖和存活率随ghrelin浓度增高而增加,10^-9、10^-7mmol/L ghrelin预处理后有统计学意义(P<0.05),而10^-11mmol/L ghrelin预处理后无统计学意义(P>0.05);与单纯1.5μg/mL衣霉素相比,不同浓度的
Objective To establish models of tunicamycin-induced endoplasmic reticulum stress( ERS) and to explore the effect of ghrelin on osteoblasts of tunicamycin-induced ERS. Methods MC3T3-E1 cells were chosen for the study.( 1) MC3T3-E1 osteoblasts cultured in vitro were treated with different concentrations of tunicamycin( 0,0. 5,1. 0,1. 5μg/mL) for 24 h. CCK-8 assay was used to determine cell viability. DCFH-DA probe was used to detect intracellular ROS level. Real-time quantitative PCR was used to measure the expression of BIP,CHOP,caspase-12 mRNA in each group. Finally,the most sensitive concentration of tunicamycin( 1. 5 μg/mL) was chosen to establish the ERS model.( 2) The effect of ghrelin on osteoblasts under ERS was observed. MC3T3-E1 osteoblasts cultured in vitro were pretreated with different concentrations of ghrelin( 0,10^-11,10^-9,10^-7 mmol/L) for 4 h. Then the cells were treated with 1. 5 μg/mL tunicamycin to establish ERS model. After the completion of the culture,the cell proliferation activity,the content of intracellular ROS,and the expression of ERS-related marker genes were detected using the above method. Results Compared with the control group,the osteoblast viability and proliferation decreased in a dose-dependent manner after treated with different concentrations of tunicamycin for 24 h. The statistical significance appeared in cells treated with 1. 0 μg/mL and 1. 5 μg/mL of tunicamycin( P<0. 05),but not in those with 0. 5 μg/mL of tunicamycin( P>0. 05). Compared with the control group,the content of intracellular ROS increased in a dose-dependent manner( P<0. 05). qRTPCR result showed that the expressions of CHOP mRNA in all concentration of tunicamycin-additioned cells were higher than those in the control group( P<0. 05). The expressions of BIP and caspase-12 mRNA were statistically significant only in cells with 1. 0μg/mL and 1. 5 μg/mL of tunicamycin( P<0. 05),but not in cells with 0. 5 μg/mL of tunicamycin( P>0. 05).( 2) Compared with cells with addition of 1. 5 μg/mL tunicamyc
作者
刘焕娜
张砚华
徐丽丽
朱晓琳
李华建
王春芝
杨乃龙
LIU Huanna;ZHANG Yanhua;XU Lili;ZHU Xiaolin;LI Huajian;WANG Chunzhi;YANG Nailong(Department of Endocrinology,the Affiliated Hospital of Qingdao University,Qingdao 266000,China;Department of Internal Medicine,Central Hospital of Licang District,Qingdao 266041,China;Department of Geriatrics,Qingdao Municipal Hospital,Qingdao 266071,China;Department of Neurosurgery,Haiyang People’s Hospital,Yantai 265100,China;Department of Endocrinology,Haiyang People’s Hospital,Yantai 265100,Shandong,China)
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2019年第7期913-919,共7页
Chinese Journal of Osteoporosis
关键词
成骨细胞
胃饥饿素
内质网应激
骨质疏松症
osteoblast cells
ghrelin
endoplasmic reticulum stress
osteoporosis
