摘要
目的探索氧化应激活化RAW264.7巨噬细胞对MC3T3-E1成骨细胞迁移、增殖及成骨基因表达的影响。方法取MC3T3-E1细胞系及RAW264.7细胞系,分别培养传至第7代进行实验。利用不同浓度(0、25、50、100μmol/L)H_2O_2刺激RAW264.7细胞,MTS检测培养1、3、6 h后细胞增殖率,采用超氧化物歧化酶(superoxide dismutase,SOD)检测试剂盒检测培养1 h时细胞内SOD含量,筛选H2O2引起RAW264.7细胞氧化应激的最佳浓度和作用时间,并对应收集RAW264.7细胞(加或不加H_2O_2处理)24 h上清液。取第7代MC3T3-E1细胞,分别以100μL无血清DMEM培养基(A组)、未氧化应激RAW264.7细胞上清液(B组)、氧化应激RAW264.7细胞上清液(C组)培养,采用MTS法检测细胞增殖情况,行划痕实验检测细胞迁移能力;另取细胞分为4组,空白对照组,以完全培养基培养;阳性对照组,以含50μg/m L维生素C及10 nmol/L的β甘油磷酸钠的完全培养基进行成骨诱导培养;正常对照组,以含50μg/m L维生素C及10 nmol/L的β甘油磷酸钠的完全培养基及未氧化应激RAW264.7细胞上清液培养;实验组,以含50μg/m L维生素C及10 nmol/L的β甘油磷酸钠的完全培养基及氧化应激RAW264.7细胞上清液培养。培养3、7、14 d,RT-PCR检测细胞内成骨相关基因ALP、Runx2、骨桥蛋白(osteopontin,OPN)、Ⅰ型胶原蛋白(collagen typeⅠ,COL-Ⅰ)、骨钙素(osteocalcin,OC)、骨唾液酸蛋白(bone sialoprotein,BSP)表达水平。结果 MTS和SOD检测结果显示:25μmol/L H_2O_2刺激1 h为构建RAW264.7细胞氧化应激模型最佳浓度和作用时间。细胞生长检测显示:1、2、3 d B、C组细胞增殖率显著高于A组(P<0.05),C组低于B组,其中2、3 d时组间比较差异有统计学意义(P<0.05)。划痕实验显示12 h时B、C组MC3T3-E1细胞迁移显著快于A组,C组快于B组,细胞迁移距离比较差异有统计学意义(P<0.05);24 h时B、C组划痕已被细胞爬满。除3 d外,实验组各时间点ALP、Runx2、OC、BSP m RNA相对表
Objective To explore the effect of H_2O_2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice. Methods MC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H_2O_2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase（SOD） content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H_2O_2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H_2O_2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B（not stimulated by H_2O_2） and C（stimulated by H_2O_2）, and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/m L vitamin C ＋ 10 nmol/ L β sodium glycerophosphate in positive group, normal control group（adding the supernatant not stimulated by H_2O_2）, and experimental group（adding the supernatant stimulated by H_2O_2）. At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related m RNA expressions of alkaline phosphatase（ALP）, Runx2, osteopontin（OPN）, osteocalcin（OC）, bone sialoprotein（BSP）, and collagen type I（COL-I）. Results The results of MTS and SOD assay showed that the appropriate concentration and action time of H_2O_2-actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A（P0.05）, in group B than group C, and significant difference was shown between groups at 2 and 3 days（P0.05）. The cell scratch test indicated that the migration
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2016年第9期1146-1152,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(31430030)~~
