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  • Title: [EFFECT OF ACTIVED RAW264.7 INDUCED BY H2O2 ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1].
    Author: Peng J, Yi Z, Wu M, Huang A, Lin K, Jin S, Li L, Huang S, Luo J, Zou X.
    Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2016 Sep 08; 30(9):1146-1152. PubMed ID: 29786373.
    Abstract:
    OBJECTIVE: To explore the effect of H2O2-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 H2O2, 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 H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), 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/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA 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 H2O2-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 (P<0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P<0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P<0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P<0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P<0.05). CONCLUSIONS: The appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes. 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、100 μmol/L）H2O2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H2O2处理）24 h上清液。取第7代MC3T3-E1细胞，分别以100 μL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50 μg/mL 维生素C及10nmol/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50 μg/mL 维生素C及10nmol/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50 μg/mL 维生素C及10nmol/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 H2O2刺激1h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12h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）；24h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 mRNA相对表达量均较阳性对照组明显降低，OPN、COL-ⅠmRNA相对表达量较空白对照组降低，差异有统计学意义（P＜0.05）。. 结论: H2O2构建RAW264.7细胞氧化应激模型的最佳浓度为25μmol/L、作用时间为1 h。氧化应激活化RAW264.7巨噬细胞上清液能促进MC3T3-E1成骨细胞迁移、抑制其增殖及成骨相关基因的表达。.
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