| 补肾活血含药血清对成骨细胞经典Wnt/β-Catenin通路的影响研究 |
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Received:August 18, 2014
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| 作者 | Author | 单位 | Unit | E-Mail |
| 许兵 |
XU Bing |
浙江中医药大学附属温州中西医结合医院骨科, 浙江 温州 325000 |
Zhejiang of Fractures Research Institute, Hangzhou 310053, Zhejiang, China |
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| 金红婷 |
JIN Hong-ting |
浙江省骨伤研究所, 浙江 杭州 310053 |
Zhejiang Chinese Medicine Hospital, Hangzhou 310006, Zhejiang, China |
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| 王萧枫 |
WANG Xiao-feng |
浙江中医药大学附属温州中西医结合医院骨科, 浙江 温州 325000 |
Zhejiang of Fractures Research Institute, Hangzhou 310053, Zhejiang, China |
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| 肖鲁伟 |
XIAO Lu-wei |
浙江省骨伤研究所, 浙江 杭州 310053 |
Zhejiang Chinese Medicine Hospital, Hangzhou 310006, Zhejiang, China |
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| 童培建 |
TONG Pei-jian |
浙江省骨伤研究所, 浙江 杭州 310053
浙江省中医院骨伤科, 浙江 杭州 310006 |
Zhejiang Chinese Medicine Hospital, Hangzhou 310006, Zhejiang, China |
johonmark@163.com |
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| 期刊信息：《中国骨伤》2015年28卷，第6期，第553-558页 |
| DOI：10.3969/j.issn.1003-0034.2015.06.017 |
| 基金项目:卫生部科学研究基金(省部共建项目计划)(编号:WKJ2008-2-032);浙江省优秀青年人才基金项目(编号:2014ZQ025); 浙江省科技厅实验动物计划(编号:2012C37087) |
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目的:通过观察补肾活血含药血清对成骨细胞的经典Wnt/β-catenin信号通路的影响, 探究补肾活血颗粒防治骨质疏松症(osteoporosis,OP)的作用机制。
方法:将20只健康SD雌性大鼠随机分为补肾活血组和生理盐水组, 每组10只, 分别给予补肾活血溶液和生理盐水进行灌胃1周, 按照药物含药血清制备方法得到补肾活血含药血清和生理盐水含药血清。采用体外分离乳鼠颅骨按照酶消法培养细胞, 通过瑞氏-姬姆萨(R-J)染色观察细胞形态及碱性磷酸酶(ALP)染色进行成骨细胞的鉴定, 鉴定成功后取第3代成骨细胞分为生理盐水含药血清组、正常培养基组及补肾活血含药血清组, 加入含15%相应的含药血清的培养基培养后检测成骨细胞的ALP含量及增殖率, 观察各组的矿化结节的情况, 采用RT-PCR检测β-catenin、Runx2、Osx mRNA的表达。
结果:培养的细胞经过瑞氏-姬姆萨染色显示胞浆有蓝色颗粒, 细胞核呈紫褐色, 有的可见1～3个核仁, 呈现传代后的成骨细胞的形态, 且ALP染色可见胞浆内含紫红色颗粒, 表明培养的细胞是成骨细胞。补肾活血含药血清组细胞的ALP含量为(6.272±0.131)金氏单位/100 ml, 增殖率为(0.81±0.172)%,与生理盐水含药血清组比较能够明显增强成骨细胞的增殖分化活性(P<0.01);补肾活血含药血清组细胞经过茜素红染色可见4个不同大小的橘红色结节, 最大1.0 cm×1.0 cm,能够明显的促进成骨细胞的矿化功能;RT-PCR检测发现β-catenin、Runx2及Osx mRNA表达量分别为(1.782±0.944)、(1.935±0.994)、(1.610±0.811),与生理盐水组比较有明显的提高(P<0.01),但与正常培养基组比较无明显差异(P>0.05).
结论:补肾活血含药血清能够明显促进成骨细胞的分化增殖及矿化功能, 并且推测激活经典Wnt/β-catenin信号通路可能是补肾活血颗粒防治骨质疏松症的作用机制。 |
| [关键词]:成骨细胞 血清 中草药 |
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| Effects of serum of Bushen Huoxue prescription(补肾活血方) on classic Wnt/β-catenin signaling pathways of osteoblasts |
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Abstract:
Objective:To observe effects of serum Bushen Huoxue prescription(补肾活血方) on classic Wnt/β-catenin signaling pathways of osteoblasts, and explore mechanism of Bushen Huoxue prescription(补肾活血方) for preventing osteoporosis.
Methods:Twenty health female rats were randomly divided into two groups, including Bushen Huoxue(补肾活血方) group and saline group, 10 in each group. Bushen Huoxue (补肾活血方) group and Saline group were gavaged Bushen Huoxue and saline every day for 1 week. Bushenhuoxue containing serum and saline containing serum were got according to methods of serum preparation of drug-containing. The osteoblasts was cultured with neonatal rat skull according to Enzyme Consumer Law, and was identified by Wright-Giemsa staining (R-J) and alkaline phosphatase staining (ALP). The third generation of osteoblasts was divided into three groups, including saline group, normal group, Bushen Huoxue(补肾活血方) group. Each group were added to 15% appropriate medium. ALP activity of osteoblasts and osteoblasts proliferation rate were tested, mineralized nodules were observed, the expression of β-catenin, Runx2, Osx mRNA of osteoblasts were tested by RT-PCR.
Results:There was blue granules in cytoplasm, cell nucleus was flint with 1 to 3 nucleoli showed by R-J staining, morphology of osteoblasts were cultured. ALP staining showed cytoplasm with purple granules, the results showed that the cultured cell was osteoblasts. The content of ALP in Bushen Huoxue(补肾活血方)group was (6.272±0.131) , appreciation rate was (0.81±0.172), and could significantly improve differentiation and proliferation activity of osteoblasts compared with Saline group (P<0.01). There were four different size orange nodules, the Maximun nodule was 1.0×1.0 cm in Bushen Huoxue(补肾活血方)group after Alizarin red staining, the results showed Bushen Huoxue (补肾活血方)group could obviously improve mineralization of osteoblasts. The expression of mRNA ofβ-catenin、Runx2 and Osx in Bushen Huoxue(补肾活血方)group were (1.782±0.944), (1.935±0.994) and (1.610±0.811) by RT-PCR, it was significantly increased compared with saline group (P<0.01), but there was no difference between Bushen Huoxue(补肾活血方)group and normal group (P>0.05).
Conclusion:Bushen Huoxue(补肾活血方)group could obviously promote differentiation, proliferation and mineralization of osteoblasts through activation of Wnt、β-catenin signaling pathway. It suggested that the mechanism of action of Bushen Huoxue(补肾活血方)particle clould prevent osteoporosis through the activation of Wnt、β-catenin signaling pathway. |
| KEYWORDS:Osteoblasts Serum Drugs,Chinese herbal |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 许兵,金红婷,王萧枫,肖鲁伟,童培建.补肾活血含药血清对成骨细胞经典Wnt/β-Catenin通路的影响研究[J].中国骨伤,2015,28(6):553~558 |
| 英文格式: | XU Bing,JIN Hong-ting,WANG Xiao-feng,XIAO Lu-wei,TONG Pei-jian.Effects of serum of Bushen Huoxue prescription(补肾活血方) on classic Wnt/β-catenin signaling pathways of osteoblasts[J].zhongguo gu shang / China J Orthop Trauma ,2015,28(6):553~558 |
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