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| 健骨颗粒含药血清对体外破骨细胞CA Ⅱ、CK、MMP- 9mRNA表达的影响 |
| Effect of Jiangu granule containing serum on the mRNA expression of CA 11/ CK/ MMP-9 in osteoclasts in vitro |
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| DOI：10.3969/j.issn.1006.7108.2015.12.003 |
| 中文关键词: 中药；健骨颗粒 骨质疏松 破骨细胞；骨吸收；II型碳酸酐酶；组织蛋白酶K 基质金属蛋白酶-9 |
| 英文关键词::Chinese medicine Jiangu Granule Osteoporosis Osteoclast Bone resorption Carbonic Anhydrsae II Cathepsin K Matrix metalioproteinases-9 |
| 基金项目:国家自然科学基金课题（81173282）；福建省科技厅资助课题（2015Y0069) |
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| 中文摘要: |
| 目的 通过观察补肾健脾中药健骨颗粒含药血清对体外破骨细胞CAⅡ、CK、MMP-9mRNA表达的影响，进一步揭示健骨颗粒有效防治绝经后骨质疏松症的作用机制。方法 用M-CSF和RANKL诱导破骨前体细胞RAW264.7分化为成熟破骨细胞，alamar Blue法检测破骨细胞血清干预的最佳浓度，于破骨细胞培养第7天开始干预，分别用25%浓度的健骨颗粒含药血清和25%浓度的生理盐水血清干预，培养48小时后抽提总RNA，采用逆转录-聚合酶链反应（RT-PCR)、实时荧光定量SYBR GREEN法检测CAⅡ、CK、MMP-9mRNA表达,并取骨片行甲苯胺蓝染色,计算分析骨陷窝数和骨吸收面积，同时运用氧化显色法检测破骨细胞培养液中TRAP含量，ELISA法检测骨磨片培养液中CTX含量。结果 健骨颗粒含药血清组破骨细胞CA Ⅱ、 CK、MMP-9mRNA表达水平明显低于生理盐水血清组（P <0. 05)，含药血清组骨磨片的骨陷窝数和骨吸收面积以及TRAP和 CTx含量均低于生理盐水血清组（P<0.05)。结论 健骨颗粒能有效抑制破骨细胞CAII、CK、MMP-9mRNA表达，降低破骨细胞功能活性，抑制破骨细胞对骨基质的分解，从而抑制骨吸收。 |
| 英文摘要: |
| Objective To observe the effect of Jiangu granule containing serum on the mRNA expression of CA II, CK, and MMP-9 in osteoclasts, and to reveal the mechanism of Jiangu granule in prevention and treatment of postmenopausal osteoporosis. Methods Mature osteoclasts were induced from osteoclast precursor RAW264. 7 cells with M-CSF and RANKL. The optimal concentration of serum intervention was detected with Aiamar Blue method. On the seventh day of culture, the cells were treated with 25% normal saline containing serum or 25% Jiangu granule containing serum, respectively. After 48 hours, total RNA was extracted and then reversely transcripted using RT-PCR. Real-time fluorescence quantitative SYBR GREEN method was applied to detect CA II, CK, and MMP-9 mRNA expression. Toluidine blue staining bone chip was used to analyze bone resorption lacuna and area. Oxidation color method was used to detect TRAP content in culture fluid. ELISA method was used to detect CTx content in culture bone grinding tablets. Results CA II, CK，and MMP-9 mRNA expression levels in Jiangu granule group were significantly lower than those in normal saline group (P<0. 05). Bone resorption lacuna and area on grinding tablets and levels of TRAP and CTx in Jiangu granule group were lower than those in normal saline group (P < 0. 05). Conclusion Jiangu granule inhibits CA II，CK, and MMP -9 mRNA expression in osteoclasts effectively, reduces functional activity of osteoclasts, and inhibits resorption of bone matrix by osteoclasts. |
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