摘要
目的探索低水平的钙对成釉细胞生物学特性的影响。方法用标准DMEM培养基培养大鼠原代成釉细胞,培养5 d后,RT-PCR及免疫组化对其进行鉴定,然后分别加入含Ca^(2+)0、0.6、1.2 mmoL/L及100 mL/L胎牛血清的不同钙浓度DMEM培养基。48 h后倒置显微镜下观察细胞形态变化,MTT观察细胞增殖活力,Annexin V-PI观察细胞凋亡情况,Real time-PCR检测细胞釉原蛋白及激肽释放酶-4(KLK4)的mRNA表达水平。结果标准DMEM培养基培养5 d后,细胞呈铺路样排列,RT-PCR显示细胞釉原蛋白、KLK4均有表达;免疫组化显示多数细胞釉原蛋白染色阳性。不同浓度钙干预48 h后,1.2 mmoL/L Ca^(2+)组部分细胞较0 mmoL/L和0.6 mmoL/L Ca^(2+)组胞核密度高、透光性差、高柱状细胞数目多。随着培养基中Ca^(2+)浓度降低,MTT显示成釉细胞增殖活性增加(P<0.01);Annexin V-PI检测显示凋亡细胞百分率降低,1.2 mmoL/L Ca^(2+)组与0 mmoL/L Ca^(2+)组间差异具有统计学意义(P<0.05);Real time-PCR显示细胞釉原蛋白及KLK4 mRNA表达水平均降低,差异具有统计学意义(P<0.01)。结论钙缺乏可能抑制成釉细胞分化,进而影响釉质矿化成熟。
Objective To explore the effect of low levels of calcium on the biological characteristics of ameloblasts. Methods Rat primary ameloblasts were cultured in standard DMEM medium. After five days they were identified by RT-PCR and immunohistochemistry. Then 0, 0.6 and 1.2 mmoL/L Ca^(2+) and 100 mL/L fetal bovine serum were added into DMEM medium without calcium. After 48 hours, the cell morphology was observed by inverted microscope. The proliferation and apoptosis of cells were separately examined by MTT and AnnexinV-PI. Real-time quantitative PCR was used to detect the expression levels of amelogenin and KLK4 mRNA. Results After Five days in standard DMEM medium, the cells were shaped like the paving pattern. RT-PCR showed that both amelogenin and KLK4 were expressed in the cells. Immunohistochemical staining showed that most cells had positive staining for amelogenin. After 48 hours of calcium intervention, some cells in 1.2 mmoL/L Ca^(2+) group had higher nuclear density and poor light transmittance, and more high columnar cells could be observed in 1.2 mmoL/L Ca^(2+)group than those in 0 and 0.6 mmoL/L Ca^(2+)groups.With the decrease in calcium concentration in the medium,MTT showed that the proliferation activity of ameloblasts reduced(P <0.01).Annexin V-PI showed that the percentage of apoptotic cells decreased,and there was a significant difference between 1.2 mmoL/L and 0 mmoL/L Ca^(2+)groups(P<0.05).Real time-PCR showed that the expressions of amelogenin and KLK4 mRNA reduced(P<0.01).Conclusion Low-level calcium may inhibit the differentiation of ameloblasts,thereby affecting the formation of enamel mineralization.
作者
王永刚
阮建平
周静
田剑刚
黄瑞哲
高江红
WANG Yonggang;RUAN Jianping;ZHOU Jing;TIAN Jiangang;HUANG Ruizhe;GAO Jianghong(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;Department of Stomatology,Dongying People’s Hospital,Dongying 257091,China)
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2021年第2期257-261,266,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.81602812)
中国中央高校基本科研业务费(No.xjj2016105)。
关键词
成釉细胞
增殖
凋亡
分化
钙
ameloblast
proliferation
apoptosis
differentiation
calcium
