钙离子调控KLK4表达对成釉细胞生长的影响  

Effect of calcium ion regulating KLK4 expression on the growth of ameloblast

作　　者：刘晓静 高美丽[2] 阮建平[3] LIU Xiaojing;GAO Meili;RUAN Jianping(Department of Stomatology,the First Hospital of Yulin,Yulin 719000,China;Key Laboratory of Biomedical Information Engineering of Ministry of Education,School of Life Science and Technology,Xi'an Jiaotong University,Xi'an 710049,China;Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases&Department of Preventive Dentistry,College of Stomatology,Xi’an Jiaotong University,Xi’an 710004,China)

机构地区：[1]榆林市第一医院口腔科,陕西榆林719000 [2]西安交通大学生命科学与技术学院,生物医学信息工程教育部重点实验室,陕西西安710049 [3]陕西省牙颌面疾病临床研究中心,西安交通大学口腔医院口腔预防保健科,陕西西安710004

出　　处：《口腔疾病防治》2024年第10期746-755,共10页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：陕西省重点研发计划项目(2023-YBSF-659);榆林市科技计划项目(YF-2021-44)。

摘　　要：目的探讨钙离子对成釉细胞中激肽释放酶4(kallikrein-4,KLK4)表达及细胞生长的影响,为钙离子促进牙釉质正常矿化提供实验依据。方法采用不同浓度CaCl_(2)(0、2.0、2.5、3.0、3.5 mmol/L)处理成釉细胞株ALC(ameloblast-lineage cell)24 h、48 h,q RT-PCR和Western blot检测KLK4 mRNA和蛋白表达水平;CCK-8检测细胞相对活力;流式细胞术、Hoechst 33342染色检测钙离子对细胞周期和细胞凋亡的影响;Western blot检测葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)的蛋白表达水平。结果与对照组(0 mmol/L CaCl_(2))相比,2.5、3.0、3.5 mmol/L CaCl_(2)处理细胞24 h后,KLK4 mRNA表达上升(P<0.05),2.0、2.5、3.0、3.5 mmol/L CaCl_(2)处理细胞24 h后,KLK4蛋白表达上升(P<0.05);3.0、3.5 mmol/L CaCl_(2)处理细胞48 h后,KLK4 mRNA和蛋白表达上升(P<0.05)。与对照组相比,2.0、2.5、3.0 mmol/L CaCl_(2)处理ALC细胞24 h、48 h后,细胞活力增加(P<0.05),其中2.5 mmol/L CaCl_(2)组中细胞活力最高。Hoechst 33342染色结果显示,3.0、3.5 mmol/L CaCl_(2)促使ALC细胞发生凋亡。流式细胞仪检测结果显示,与0、2.0、2.5、3.0 mmol/L CaCl_(2)组相比,3.5 mmol/L CaCl_(2)处理ALC细胞24 h后,G2/M期细胞比例增加,细胞凋亡率上升(P<0.05)。3.0、3.5 mmol/L CaCl_(2)处理细胞24 h后,与对照组相比,GRP78蛋白表达下降(P<0.05);2.5 mmol/L CaCl_(2)处理细胞48 h后,与对照组相比,GRP78蛋白表达下降(P<0.05)。结论钙离子促进ALC细胞中KLK4表达上升、细胞活力增加,但较高浓度的钙离子可使ALC细胞的G2/M期阻滞,诱发ALC细胞凋亡,降低凋亡相关蛋白GRP78的表达。Objective To investigate the effect of calcium ions on the expression of kallikrein-4(KLK4)and cell growth of ameloblast,and to provide an experimental basis for calcium ion promoting normal mineralization of enamel.Methods ALC cells were treated with 0,2.0,2.5,3.0,and 3.5 mmol/L CaCl_(2) for 24 and 48 h.KLK4 expression was analyzed by qRT-PCR and Western blot analysis.The viability of ALC cells was determined by using CCK-8.AnnexinV-FITC/PI dual staining combined with flow cytometry and Hoechst 33342 staining were used to detect the ALC cell cycle and cell apoptosis.The protein expression level of glucose-regulated protein 78(GRP78)was measured by Western blot analysis.Results After 24 h of treatment with 2.5,3.0,and 3.5 mmol/L CaCl_(2),the expression of KLK4 mRNA was increased(P<0.05),and after 24 h of treatment with 2.0,2.5,3.0,and 3.5 mmol/L CaCl_(2),the expression of KLK4 protein was increased(P<0.05).After 48 h of treatment with 3.0 mmol/L and 3.5 mmol/L CaCl_(2),the expression of KLK4 mRNA and protein was increased(P<0.05).Compared with the control group,the viability of ALC cells was increased after 24 and 48 h of treatment with 2.0,2.5,and 3.0 mmol/L CaCl_(2)(P<0.05),and the highest cell viability was observed with 2.5 mmol/L CaCl_(2).Hoechst 33342 staining results showed that 3.0 mmol/L and 3.5 mmol/L CaCl_(2) may promote apoptosis in ALC cells.Flow cytometry showed that the proportion of G2/M phase cells and the apoptosis rate increased after 3.5 mmol/L CaCl_(2) treatment for 24 h(P<0.05),compared with the 0,2.0,2.5,and 3.0 mmol/L CaCl_(2) groups.After 24 h of treatment with 3.0 mmol/L and 3.5 mmol/L CaCl_(2),the expression of GRP78 protein was reduced(P<0.05),and after 48 h of treatment with 2.5 mmol/L CaCl_(2),the expression of GRP78 protein was reduced(P<0.05).Conclusion Calcium ions can promote the increase of KLK4 expression and cell viability in ALC cells,but a higher concentration of calcium ions can block the G2/M phase of ALC cells,thus inducing apoptosis of ALC cells and reducing the expressi

关 键 词：成釉细胞 ALC细胞 钙离子 激肽释放酶4 细胞生长 细胞活力 细胞周期 细胞凋亡 葡萄糖调节蛋白78 

分 类 号：R78[医药卫生—口腔医学]