补肾活血方诱导大鼠骨髓间充质干细胞成骨分化的实验研究
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摘要
目的观察补肾活血方对大鼠骨髓间充质干细胞(BMSCs)向成骨细胞分化的情况,从而为其治疗骨质疏松症提供细胞生物学研究依据。方法 SPF健康雌性SD大鼠,采用密度梯度离心法分离骨髓间质干细胞,差速贴壁法进行纯化,细胞培养至第3代(P3),应用流式细胞术鉴定BMSCs纯度并分选纯化细胞,纯化过的细胞经补肾活血方诱导1天,3天和7天,在不同时间点进行碱性磷酸酶(ALP)染色鉴定成骨细胞,经茜素红染色鉴定钙结节。结果 P3含有CD44的细胞数占总数的96. 3%,CD29的细胞数占总数的77. 1%,说明培养的细胞中骨髓间充质干细胞的纯度较高,经补肾活血方诱导后可见细胞由梭型变成铺路石样,体积增大,细胞成簇状生长。经碱性磷酸酶鉴定可见胞浆着蓝色,有典型成骨细胞的表达。经茜素红染色,可见成簇的细胞呈红色钙结节样。结论补肾活血方的作用靶点可能是通过促进BMSCs向成骨的分化,从而起到防治骨质疏松的作用。
Objective To observe the differentiation of bone marrow mesenchymal stem cells( BMSCs) into osteoblasts in rats,and to provide a basis for the treatment of osteoporosis. Methods Female SD rats,SPF health using density gradient centrifugation separation of bone marrow mesenchymal stem cells,differential sticking wall method to purify,cell culture to third-generation( P3),application of flow cytometry and purity identification of BMSCs cells separation and purification,purification of the cells by kidney invigorate the circulation induced by 1 day,3 and 7 days,at different time points for alkaline phosphatase( ALP) staining identified osteoblast,alizarin red staining identified the formation of calcium nodules. Results P3 contains cells number accounts for 96. 3% of the total number of CD44,CD29 77. 1% of the total number of cells of high purity,visible after inducing by Bushen Huoxue recipe the circulation of the induced cells changed from fusiform to paving stone,sample,size,into clusters of cell growth. The cytoplasmic blue was identified by alkaline phosphatase,and the expression of typical osteoblasts was found. The cells in the clusters were red with red calcium nodules. Conclusion The action target of invigorating kidney and activating blood may be to promote the differentiation of BMSCs to osteoblasts,thus preventing osteoporosis.
Objective To observe the differentiation of bone marrow mesenchymal stem cells( BMSCs) into osteoblasts in rats,and to provide a basis for the treatment of osteoporosis. Methods Female SD rats,SPF health using density gradient centrifugation separation of bone marrow mesenchymal stem cells,differential sticking wall method to purify,cell culture to third-generation( P3),application of flow cytometry and purity identification of BMSCs cells separation and purification,purification of the cells by kidney invigorate the circulation induced by 1 day,3 and 7 days,at different time points for alkaline phosphatase( ALP) staining identified osteoblast,alizarin red staining identified the formation of calcium nodules. Results P3 contains cells number accounts for 96. 3% of the total number of CD44,CD29 77. 1% of the total number of cells of high purity,visible after inducing by Bushen Huoxue recipe the circulation of the induced cells changed from fusiform to paving stone,sample,size,into clusters of cell growth. The cytoplasmic blue was identified by alkaline phosphatase,and the expression of typical osteoblasts was found. The cells in the clusters were red with red calcium nodules. Conclusion The action target of invigorating kidney and activating blood may be to promote the differentiation of BMSCs to osteoblasts,thus preventing osteoporosis.
引文
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[18]鲍悦,高久堂,孙佳明,等.中药鹿角胶的研究进展[J].杏林中医药,2016,36(2):173.
[19]王明仁,吴纯标,周晓红.骨髓基质干细胞成骨-成脂分化及补肾中药对其影响的研究进展[J].中国骨质疏松杂志,20(10):1240.
[20]张虎林,张自强,许宏斌,等.不同剂量骨碎补对兔骨髓间充质干细胞增殖分化的影响[J].中国组织工程研究,2014,18(41):6649.
[21]卞玉群,李超,樊巧玲.补肾方药诱导BMSCs骨向分化的研究述评[J].中医学报,2014,29(5):692.
[2]徐晓东,郑洪新.肾虚与骨质疏松症[J].辽宁中医药大学学报,2007,9(4):45.
[3] Turner B,Drudge-Coates L,Ali S,et al. Osteonecrosis of he jawin patients receiving bone-targeted therapies:an overview-part I[J]. Urol Nurs,2016,36:111.
[4]郭秋菊,眭承志.血瘀与老年性骨质疏松症关系的研究进展[J].河北中医,2009,2(2):306.
[5]张波,杨传东,史耀勋,等.骨质疏松症(肾虚血瘀证)与骨吸收标志物的相关性研究[J].中国医药指南,2013,11(7):279.
[6]蔡加琴,邓力. Hedgehog信号通路对MSCs的调控[J].中国修复重建外科杂志,2010,24(8):993.
[7]刘斌钰,李宁毅,樊功为,等.大鼠骨髓间充质干细胞的培养鉴定及向成骨细胞诱导分化研究[J].齐鲁医学杂志,2007,22(3):215.
[8]滕瑞军,肖明霞,卫荣.维、汉族绝经后骨质疏松症体重指数、骨密度与中医证型分析[J].新疆医科大学学报,2013,36(12):1705.
[9]卫荣,薛黎,刘海龙,等.维、汉妇女绝经后骨质疏松症中医证型及症候特征分析[J].新疆医科大学学报,2013,36(12):1697.
[10]刘海江,王小平,林娟,等.淫羊藿苷和黄芪苷Ⅰ对骨髓基质细胞增殖及成骨能力的影响[J].中药材,2006,29(10):1062.
[11]成魁,葛宝丰,甄平,等.口服淫羊藿苷可提高大鼠的峰值骨密度和骨质量[J].中国骨质疏松杂志,2014,20(2):120.
[12] Ma HP,Ming LG,Ge BF,et al. Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro[J]. J Cell Biochem,2011,112(3):916.
[13]马小妮,葛宝丰,陈克明,等.淫羊藿苷调节成骨细胞骨形成和破骨细胞骨吸收的机制[J].中国医学科学院学报,2013,35(4):432.
[14]武丽南,陆榕,谷元,等.梓醇在大鼠体内的药代动力学和生物利用度研究[J].中国临床药理学与治疗学,2012,17(2):126.
[15]张春芳,吴珊,彭金咏,等.薯蓣皂苷通过上调Lrp5、β-catenin表达促进成骨细胞MC3T3-E1增殖、分化[J].中国药理学通报,2013,29(9):1255.
[16]翟远坤,牛银波,潘亚磊,等.柚皮苷对体外培养乳鼠颅骨成骨细胞增殖和分化成熟的影响[J].中国中药杂志,2012,38(1):105.
[17]李念虎,徐展望.柚皮苷促进成骨细胞分化并有效改善卵巢切除所致的骨质疏松[J].中国骨质疏松杂志,2013,19(8):777.
[18]鲍悦,高久堂,孙佳明,等.中药鹿角胶的研究进展[J].杏林中医药,2016,36(2):173.
[19]王明仁,吴纯标,周晓红.骨髓基质干细胞成骨-成脂分化及补肾中药对其影响的研究进展[J].中国骨质疏松杂志,20(10):1240.
[20]张虎林,张自强,许宏斌,等.不同剂量骨碎补对兔骨髓间充质干细胞增殖分化的影响[J].中国组织工程研究,2014,18(41):6649.
[21]卞玉群,李超,樊巧玲.补肾方药诱导BMSCs骨向分化的研究述评[J].中医学报,2014,29(5):692.