论文部分内容阅读
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CAⅡ、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。
OBJECTIVE: To observe the effect of Jiangu Granule containing serum of Bushen Jianpi granule on expression of CAⅡ, CK and MMP-9mRNA in osteoclasts in vitro and further reveal the mechanism of Jiangu granules in preventing and treating postmenopausal osteoporosis. Methods The osteoclast precursor RAW264.7 cells were induced to differentiate into mature osteoclasts by M-CSF and RANKL. The optimal concentration of osteoclasts serum was determined by alamar Blue assay. Intervention was initiated on day 7 of osteoclast culture. Total RNA was extracted after 48 hours of culture by using 25% serum with Jiangu Granule and 25% normal saline. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR (SYBR GREEN) The expression of CAⅡ, CK and MMP-9mRNA were detected by immunohistochemical staining. Toluidine blue staining was used to determine the number of lacunae and bone resorption area. The content of TRAP in osteoclast culture medium was detected by oxidative chromogenic assay. CTx content in grinding media. Results The expression levels of CAⅡ, CK and MMP-9 mRNA in osteoclasts of Jiangu Granule-containing serum group were significantly lower than those in saline group (P <0.05). The number of osteoclasts and bone resorption area in osteoporosis tablets of drug- TRAP and CTx contents were lower than those of saline group (P <0.05). Conclusion Jiangu granules can effectively inhibit the expression of CA Ⅱ, CK and MMP-9 mRNA in osteoclasts, decrease the functional activity of osteoclasts, inhibit the decomposition of osteoclasts on bone matrix, and inhibit bone resorption.