矢车菊素-3-葡萄糖苷对人原代成骨细胞迁移能力的影响及其可能机制
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摘要
目的观察矢车菊素-3-葡萄糖苷(C3G)对人原代成骨细胞迁移能力的影响,并探讨其可能机制。方法剪取股骨头置换术患者的松质骨组织,使用胶原酶消化法提取原代成骨细胞,在诱导矿化后使用瑞氏-姬姆萨复合染色和茜素红染色对成骨细胞进行形态学鉴定。将获得的成骨细胞分为C3G组和对照组。C3G组使用含100μmol/L的C3G的无血清培养基培养,对照组常规培养。采用划痕实验观测两组细胞的迁移能力,分别于划痕后0、6、24、54 h在倒置显微镜下选取适宜区域进行拍照,观察两组划痕愈合能力。采用实时荧光定量PCR法检测两组细胞中的骨桥蛋白(OPN)mRNA。结果划痕后3、6、24、54 h,对照组细胞不断向划痕处迁移,C3G组迁移速度明显减慢,划痕愈合时间延长。对照组、C3G组细胞中OPN mRNA相对表达量分别为1.04±0.28、0.26±0.22,C3G组细胞中OPN mRNA相对表达量低于对照组(P<0.05)。结论 C3G在体外能抑制人成骨细胞迁移,其机制可能与OPN mRNA表达下调有关。
Objective To investigate the effects of cyanidin-3-glucoside(C3 G) on the migration of human primary osteoblasts and to explore its possible mechanism. Methods The primary osteoblasts of human femoral head cancellous bone were prepared by modified collagenase digestion method, and the prepared cells were identified by Wright's-Giemsa staining and alizarin red staining after mineralization induction. The obtained osteoblasts were divided into the C3 G group and control group. The osteoblasts in the C3 G group were cultured in a serum-free medium containing 100 μmol/L C3 G, and the osteoblasts in the control group were routinely cultured. Scratch test was used to observe the migration ability of cells in the two groups. The appropriate areas were selected under the inverted microscope at 0, 6, 24, and 54 h after scratching to observe the healing ability of the two groups. Real-time fluorescent quantitative PCR was used to detect osteopontin(OPN) mRNA in cells of the two groups. Results At 3, 6, 24, and 54 h after scratching, the cells in the control group continued to migrate to the scratches. The speed of migration in the C3 G group slowed down and the healing time of the scratches was prolonged. The expression levels of OPN mRNA in the control group and C3 G group were 1.04±0.28 and 0.26±0.22, respectively. The relative expression of OPN mRNA in the C3 G group was lower than that in the control group(P<0.05). Conclusions C3 G can inhibit the migration of human osteoblasts in vitro, and its mechanism may be related to the down-regulation of OPN mRNA expression.
Objective To investigate the effects of cyanidin-3-glucoside(C3 G) on the migration of human primary osteoblasts and to explore its possible mechanism. Methods The primary osteoblasts of human femoral head cancellous bone were prepared by modified collagenase digestion method, and the prepared cells were identified by Wright's-Giemsa staining and alizarin red staining after mineralization induction. The obtained osteoblasts were divided into the C3 G group and control group. The osteoblasts in the C3 G group were cultured in a serum-free medium containing 100 μmol/L C3 G, and the osteoblasts in the control group were routinely cultured. Scratch test was used to observe the migration ability of cells in the two groups. The appropriate areas were selected under the inverted microscope at 0, 6, 24, and 54 h after scratching to observe the healing ability of the two groups. Real-time fluorescent quantitative PCR was used to detect osteopontin(OPN) mRNA in cells of the two groups. Results At 3, 6, 24, and 54 h after scratching, the cells in the control group continued to migrate to the scratches. The speed of migration in the C3 G group slowed down and the healing time of the scratches was prolonged. The expression levels of OPN mRNA in the control group and C3 G group were 1.04±0.28 and 0.26±0.22, respectively. The relative expression of OPN mRNA in the C3 G group was lower than that in the control group(P<0.05). Conclusions C3 G can inhibit the migration of human osteoblasts in vitro, and its mechanism may be related to the down-regulation of OPN mRNA expression.
引文
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[11] Perrien DS, Brown EC, Aronson J, et al. Immunohistochemical study of osteopontin expression during distraction osteogenesis in the rat[J]. J Histochem Cytochem, 2016,50(4):567-574.
[12] Burton LJ, Smith BA, Smith BN, et al. Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells[J]. CARCIN, 2015,36(9):1019-1027.
[13] Luo LP, Han B, Yu XP, et al. Anti-metastasis activity of black rice anthocyanins against breast cancer: analyses using an ErbB2 positive breast cancer cell line and tumoral xenograft model[J]. Asian Pac J Cancer Prev, 2014,15(15):6219-6225.
[14] Li L, Zhang M, Zhang S, et al. Preparation and antioxidant activity of ethyl-linked anthocyanin-flavanol pigments from model wine solutions[J]. Molecules, 2018,23(5):1066.
[15] Ramachandran S, Kwon K, Shin S, et al. Regulatory role of osteopontin in malignant transformation of endometrial cancer[J]. Mol Biol Rep, 2013,40(5):3623-3629.
[16] Qin H, Wang R, Wei G, et al. Overexpression of osteopontin promotes cell proliferation and migration in human nasopharyngeal carcinoma and is associated with poor prognosis[J]. Eur Arch Otorhinolaryngol, 2018,275(2):525-534.
[17] Shi L, Shi L, Wang X, et al. Regulatory roles of osteopontin in production of monocyte-origin MCP-1[J]. Cell Transplant, 2018,27(8):1185-1194.
[18] Raheja LF, Genetos DC, Yellowley CE. Hypoxic osteocytes recruit human MSCs through an OPN/CD44-mediated pathway[J]. Biochem Biophys Res Commun, 2008,366(4):1061-1066.
[19] Saki F, Sheikhi A, Omrani G, et al. Evaluation of bone mineral density in children with type Ⅰ diabetes mellitus and relationship to serum levels of osteopontin[J]. Drug Res, 2017,67(9):527-533.
[20] Liu J, Liu Q, Wan Y, et al. Osteopontin promotes the progression of gastric cancer through the NF-κB pathway regulated by the MAPK and PI3K[J]. Int J Oncol, 2014,45(1):282-290.
[2] Pantan R, Tocharus J, Phatsara M, et al. Synergistic effect of atorvastatin and cyanidin-3-glucoside against angiotensin II-mediated vascular smooth muscle cell proliferation and migration through MAPK and PI3K/Akt pathways[J]. Arch Pharm Res, 2016,342(2):104-112.
[3] Jiang X, Shen T, Tang X, et al. Cyanidin-3-O-β-glucoside combined with its metabolite protocatechuic acid attenuated the activation of mice hepatic stellate cells[J]. Food Funct, 2017,8(8):2945-2957.
[4] Xu M, Bower KA, Wang S, et al. Cyanidin-3-Glucoside inhibits ethanol-induced invasion of breast cancer cells overexpressing ErbB2[J]. Mol Cancer, 2010,9(1):285.
[5] Xu L, Huang S, Hou Y, et al. Sox11-modified mesenchymal stem cells (MSCs) accelerate bone fracture healing: Sox11 regulates differentiation and migration of MSCs[J]. FASEB J, 2015,29(4):1143-1152.
[6] Cassidy A, Bertoia M, Chiuve S, et al. Habitual intake of anthocyanins and flavanones and risk of cardiovascular disease in men[J]. Am J Clin Nutr, 2016,1(104):587-594.
[7] Olivas-Aguirre F, Rodrigo-Garcia J, Martinez-Ruiz N, et al. Cyanidin-3-O-glucoside: physical-chemistry, foodomics and health effects[J]. Molecules, 2016,21(9):1264.
[8] Jang WS, Seo CR, Jang HH, et al. Black rice (Oryza sativa L.) extracts induce osteoblast differentiation and protect against bone loss in ovariectomized rats[J]. Food Funct, 2015,6(1):265-275.
[9] Dudaric L, Fuzinac-Smojver A, Muhvic D, et al. The role of polyphenols on bone metabolism in osteoporosis[J]. Food Res Int, 2015,77(1):290-298.
[10] Cheng J, Zhou L, Liu Q, et al. Cyanidin Chloride inhibits ovariectomy-induced osteoporosis by suppressing RANKL-mediated osteoclastogenesis and associated signaling pathways[J]. J Cell Physiol, 2017,233(3):2502-2512.
[11] Perrien DS, Brown EC, Aronson J, et al. Immunohistochemical study of osteopontin expression during distraction osteogenesis in the rat[J]. J Histochem Cytochem, 2016,50(4):567-574.
[12] Burton LJ, Smith BA, Smith BN, et al. Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells[J]. CARCIN, 2015,36(9):1019-1027.
[13] Luo LP, Han B, Yu XP, et al. Anti-metastasis activity of black rice anthocyanins against breast cancer: analyses using an ErbB2 positive breast cancer cell line and tumoral xenograft model[J]. Asian Pac J Cancer Prev, 2014,15(15):6219-6225.
[14] Li L, Zhang M, Zhang S, et al. Preparation and antioxidant activity of ethyl-linked anthocyanin-flavanol pigments from model wine solutions[J]. Molecules, 2018,23(5):1066.
[15] Ramachandran S, Kwon K, Shin S, et al. Regulatory role of osteopontin in malignant transformation of endometrial cancer[J]. Mol Biol Rep, 2013,40(5):3623-3629.
[16] Qin H, Wang R, Wei G, et al. Overexpression of osteopontin promotes cell proliferation and migration in human nasopharyngeal carcinoma and is associated with poor prognosis[J]. Eur Arch Otorhinolaryngol, 2018,275(2):525-534.
[17] Shi L, Shi L, Wang X, et al. Regulatory roles of osteopontin in production of monocyte-origin MCP-1[J]. Cell Transplant, 2018,27(8):1185-1194.
[18] Raheja LF, Genetos DC, Yellowley CE. Hypoxic osteocytes recruit human MSCs through an OPN/CD44-mediated pathway[J]. Biochem Biophys Res Commun, 2008,366(4):1061-1066.
[19] Saki F, Sheikhi A, Omrani G, et al. Evaluation of bone mineral density in children with type Ⅰ diabetes mellitus and relationship to serum levels of osteopontin[J]. Drug Res, 2017,67(9):527-533.
[20] Liu J, Liu Q, Wan Y, et al. Osteopontin promotes the progression of gastric cancer through the NF-κB pathway regulated by the MAPK and PI3K[J]. Int J Oncol, 2014,45(1):282-290.
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