Peng Jianqiang, Yi Zhixin, Wu Mingxin, Huang Aijun, Lin Kun, Jin Song, Li Liangping, Huang Sheng, Luo Jiaquan, Zou Xuenong
Department of Spine Surgery, the Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen Guangdong, 518033, P. R. China.
Department of Orthopedics, the Third People's Hospital of Huizhou.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2016 Sep 8;30(9):1146-1152. doi: 10.7507/1002-1892.20160234.
To explore the effect of HO-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice.
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 HO, 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 HO-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by HO or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by HO) and C (stimulated by HO), 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 HO), and experimental group (adding the supernatant stimulated by HO). 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).
The results of MTS and SOD assay showed that the appropriate concentration and action time of HO-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 (<0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (<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 (<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 (<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 (<0.05).
The appropriate concentration and action time of HO-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The HO-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.
探讨经血红素加氧酶（HO）激活的RAW264.7巨噬细胞对小鼠MC3T3-E1细胞迁移、增殖及成骨基因表达的影响。
将MC3T3-E1细胞和RAW264.7细胞培养至第7代。用0、25、50和100 μmol/L的HO刺激RAW264.7巨噬细胞，在刺激后1、3和6小时用MTS法检测细胞增殖率，在刺激后1小时用超氧化物歧化酶（SOD）检测试剂盒检测SOD含量。得出HO激活RAW264.7的合适浓度及作用时间。在24小时收集经HO刺激或未刺激的RAW264.7巨噬细胞的上清液。然后，将上清液用于培养B组（未用HO刺激）和C组（用HO刺激）的MC3T3-E1细胞，A组用DMEM作为对照。在12和24小时通过细胞划痕试验检测MC3T3-E1细胞的迁移情况，在24、48和72小时通过MTS法检测MC3T3-E1细胞的增殖情况。空白对照组仅用完全培养基培养MC3T3-E1细胞，阳性组用含50 μg/mL维生素C + 10 nmol/Lβ-甘油磷酸钠的完全培养基培养，正常对照组（加入未用HO刺激的上清液），实验组（加入用HO刺激的上清液）。在3、7和14天，用逆转录聚合酶链反应（RT-PCR）检测碱性磷酸酶（ALP）、Runx2、骨桥蛋白（OPN）、骨钙素（OC）、骨涎蛋白（BSP）和I型胶原（COL-I）的成骨相关mRNA表达。
MTS和SOD检测结果显示，HO激活RAW264.7巨噬细胞的合适浓度和作用时间分别为25 μmol/L和1小时。MTS法检测显示，B组和C组MC3T3-E1细胞的增殖率显著高于A组（<0.05），B组高于C组，且在第2和3天两组间有显著差异（<0.05）。细胞划痕试验表明，在12小时时，B组和C组MC3T3-E1细胞的迁移速度显著快于A组，C组快于B组（<0.05）；在24小时时，B组和C组所有划痕部位均可见许多迁移细胞。与阳性对照组相比，实验组在第7和14天ALP、Runx2、OC和BSP的mRNA表达显著下调（<0.05）。与空白对照组相比，实验组在第7和14天OPN和COL-I的mRNA表达显著下调（<0.05）。
HO激活RAW264.7巨噬细胞的合适浓度和作用时间分别为25 μmol/L和1小时。经HO激活的RAW2
