王文彦,何文雯,刘宇平,桂传枝,王龙,陈莹,邓明芬,南楠,段筱娟,官志忠
WANG Wenyan,HE Wenwen,LIU Yuping,GUI Chuanzhi,WANG Long,CHEN Ying,DENG Mingfen,NAN Nan,DUAN Xiaojuan,GUAN Zhizhong

• 1:贵州医科大学病理学教研室
• 2:贵黔国际总医院病理科
• 3:贵州医科大学
• 4:广州医科大学附属第四医院病理科

摘要(Abstract)：

目的 探讨L-单甲基-精氨酸(L-NMMA)与过量氟化钠(NaF)共培养对小鼠颅顶前成骨(MC3T3E1)细胞中一氧化氮(NO)和诱导型一氧化氮合酶(iNOS)表达的影响。方法 将MC3T3E1成骨细胞分为空白组(只含培养基)、NaF染毒组(0、0.5、1.0、2.0、4.0、8.0 mmol/L)及L-NMMA染毒组(0、5、10、20、40、80 mol/L),采用细胞增殖-毒性检测试剂盒(CCK8)检测细胞存活率，并筛选最佳染氟浓度和最佳L-NMMA用药浓度；再将MC3T3E1成骨细胞分为对照组(0.0 mmol/L NaF)、低氟组(1.0 mmol/L NaF)、高氟组(4.0 mmol/L NaF)、低氟+L-NMMA组(1.0 mmol/L NaF+20μmol/L L-NMMA)、高氟+L-NMMA组(4.0 mmol/L NaF+20μmol/L L-NMMA)、L-NMMA组(20μmol/L L-NMMA),处理24 h,采用硝酸还原酶法检测细胞中NO含量，蛋白免疫印迹法及实时荧光定量PCR法分别检测iNOS蛋白及mRNA表达水平。结果 与对照组相比，1.0、2.0、4.0、8.0 mmol/L NaF染毒组及80μmol/L L-NMMA染毒组MC3T3E1成骨细胞存活率降低(P<0.05),选择1.0 mmol/L和4.0 mmol/LNaF为染氟浓度、20μmol/LL-NMMA用药浓度进行后续实验；与对照组比较，低氟组、高氟组MC3T3E1成骨细胞中NO含量增加(P<0.05),低氟+L-NMMA组中NO含量低于低氟组(P<0.05),高氟+L-NMMA组细胞中NO含量低于高氟组(P<0.05);与对照组相比，高氟组MC3T3E1成骨细胞中iNOS蛋白及mRNA表达水平升高(P<0.05);与高氟组比较，高氟+L-NMMA组细胞中iNOS蛋白及mRNA表达水平降低(P<0.05)。结论 过量氟可致MC3T3E1成骨细胞损伤、iNOS蛋白和mRNA表达增强、细胞中NO含量增加，L-NMMA与氟共培养后可减弱这一效应，提示L-NMMA对过量氟所致MC3T3E1成骨细胞损伤有一定的保护作用。
Objective To investigate the effect of co-cultivation of NG-monomethyl-arginine(L-NMMA) and excessive sodium fluoride(NaF) on the expression of nitric oxide(NO) and inducible nitric oxide synthase(iNOS) in MC3T3E1 cells. Methods MC3T3E1 osteoblasts were divided into blank group(cell culture medium only), NaF exposure group(0, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L) and L-NMMA exposure group(0, 5, 10, 20, 40, 80(mol/L). The cell viability was detected by Cell Counting Kit-8(CCK8), and the optimal fluoride concentration and optimal L-NMMA concentration were screened. MC3T3E1 osteoblasts were then divided into control group(0.0 mmol/L NaF), low-dose fluoride group(1.0 mmol/L NaF), high-lose fluoride group(4.0 mmol/L NaF), low-dose fluoride+L-NMMA group(1.0 mmol/L NaF+20 μmol/L L-NMMA), high-dose fluoride+L-NMMA group(4.0 mmol/L NaF+20 μmol/L L-NMMA), and L-NMMA group(20 μmol/L L-NMMA). The cells in each group were treated for 24 h. The content of NO in cells was detected by nitrate reductase method, and the expression levels of iNOS protein and mRNA were detected by Western blotting and Quantitative real-time PCR. Results Compared with the control group, the survival rate of MC3T3E1 osteoblasts in 1.0, 2.0, 4.0, 8.0 mmol/L NaF exposure group and the 80 μmol/L L-NMMA exposure group decreased(P<0.05). 1.0 mmol/L and 4.0 mmol/L NaF were selected as the fluoride concentration and 20 μmol/L L-NMMA was selected for subsequent experiments. Compared with the control group, the content of NO in MC3T3E1 osteoblasts in the low-dose fluoride group and the high-dose fluoride group increased(P<0.05). The content of NO in the low-dose fluoride+L-NMMA group was lower than that in the low-dose fluoride group(P<0.05), and the content of NO in the high-dose fluoride+L-NMMA group was lower than that in the high-dose fluoride group(P<0.05). Compared with the control group, the expression levels of iNOS protein and mRNA in MC3T3E1 osteoblasts in the high-dose fluoride group were increased(P<0.05). Compared with the high-dose fluoride group, the expression levels of iNOS protein and mRNA in the high-dose fluoride+L-NMMA group were decreased( P<0.05). Conclusions Excessive amount of fluoride exposure can cause MC3T3E1 osteoblast damage, increase iNOS protein and mRNA expression, and increase NO content in cells. L-NMMA can attenuate this effect after co-cultivation with fluoride, suggesting that L-NMMA may have a certain protective effect against MC3T3E1 osteoblast damage caused by excessive fluoride.

关键词(KeyWords)： 氟;诱导型一氧化氮合酶;一氧化氮;L-单甲基-精氨酸;MC3T3E1细胞
fluoride;inducible nitric oxide synthase(iNOS);nitric oxide(NO);NG-monomethyl-arginine(L-NMMA);MC3T3E1 cells

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(U1812403);; 贵阳市科技局基金(筑科合同[2019]9-8号)

作者(Author): 王文彦,何文雯,刘宇平,桂传枝,王龙,陈莹,邓明芬,南楠,段筱娟,官志忠
WANG Wenyan,HE Wenwen,LIU Yuping,GUI Chuanzhi,WANG Long,CHEN Ying,DENG Mingfen,NAN Nan,DUAN Xiaojuan,GUAN Zhizhong

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