透骨消痛颗粒诱导骨髓间充质干细胞向软骨细胞的分化

Granula of Penetrating Bne and Rmoving Pain induce the differentiation of bone marrow mesenchymal stem cells to chondrocytes

背景:研究表明,透骨消痛颗粒通过有效抑制细胞因子、基质金属蛋白酶3、氧自由基的生物效应来延缓关节宏观形态、软骨基质及软骨细胞退变的作用。目的:验证透骨消痛颗粒含药血清促进骨髓间充质干细胞增殖和诱导骨髓间充质干细胞向软骨细胞分化的影响。设计、时间及地点:随机对照动物实验,于2006-12/2007-08在福建中医学院中西医结合研究院分子生物学实验室完成。材料:选用清洁级4周龄SD大鼠80只。透骨消痛颗粒,由巴戟天、杭白芍、青风藤、川芎等组成,由福州屏山药厂制作。方法:取72只SD大鼠随机分为5组,透骨消痛颗粒的水提物高剂量组、水提物中剂量组、水提物低剂量组、醇提物组各16只,空白对照组8只,分别以含14,7,3.5g/(kg?d)药量的水溶液2mL灌胃,对照组以等量生理盐水2mL灌胃,2次/d,连续给药3d后,腹主动脉采血制备血清。从另8只4周龄SD大鼠骨髓中分离出骨髓间充质干细胞,体外传代培养,取第2代骨髓间充质干细胞分为空白对照组﹑软骨诱导剂组及透骨消痛颗粒组,通过透骨消痛颗粒含药血清诱导骨髓间充质干细胞向软骨细胞分化。主要观察指标:采用MTT法检测骨髓间充质干细胞增殖和免疫组织化学检测Ⅱ型胶原在细胞分化过程中的表达。结果:透骨消痛颗粒含药血清各组均能促进骨髓间充质干细胞增殖,与空白对照组相比差异均有非常显著性意义(P<0.01),中剂量药物浓度时达到最大效应(P<0.01),中剂量1h组与2h组相比差异无显著性意义(P>0.05)。透骨消痛颗粒含药血清能增强Ⅱ型胶原在细胞分化过程中的表达,与空白对照组相比差异有非常显著性意义(P<0.01),与软骨诱导剂组相比差异有显著性意义(P<0.05)。结论:透骨消痛颗粒含药血清能有效促进骨髓间充质干细胞增殖和诱导骨髓间充质干细胞向软骨细胞分化。

BACKGROUND： Studies have indicated that the Granula of Penetrating Bone and Removing Pain could delay cataplasis of the articular macroscopy appearance, the cartilage matrix and the cartilage cells by actively suppressing the biological effects of cell factor, matrix metalloproteinase-3 and oxygen free radical. OBJECTIVE： To investigate the effects of the serums containing the Granula of Penetrating Bone and Removing Pain on promoting the proliferation of bone marrow mesenchymal stem cells （BMSCs） and inducing to differentiate from BMSCs to chondrogenic cells phenotype. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed in the laboratory of molecular biology, Integrated Traditional and Western Medicine Institute, Fujian University of Traditional Chinese Medicine between December 2006 and August 2007. MATERIALS： Eighty SD rats aged 4 weeks and of SPF grade. Granula of Penetrating Bone and Removing Pain was produced by Fujian Pingshan Pharmaceutical Factory, comprising mornda root, radix paeoniae alba from Hangzhou of China, orientvine stem, szechwan lovage rhizome. METHODS： The 72 SD rats were randomly divided into 5 groups, i.e. aqueous extract high-dose group of the Granula of Penetrating Bone and Removing Pain （n=16）, aqueous extract midst-dose group （n=16）, aqueous extract low-dose group （n=16）, ethanol extract group （n=16） and control group （n=8）. All animals received intragastric administration of 2 mL water solution of the Granula of Penetrating Bone and Removing Pain 14, 7, 3.5 g/kg.d, the control group were enforced intragastric administration of equivalent dose of physiological saline twice every day. After three days, blood was sampled from abdominal aorta to measure the content of serum. The BMSCs were separated from 8 SD rats？ bone marrow, which were cultivated in vitro, the second generation BMSCs were randomly divided into 3 groups： control group, chondrogenic inductor group and group of the Granula of Penetrating Bone and Removing Pain. BMSCs were induced to differentiate to chondrocytes by the serums containing the Granula of Penetrating Bone and Removing Pain. MAIN OUTCOME MEASURES： Proliferation of the BMSCs was determined by MTT and the expression of collagen Ⅱ in cellular differentiation was assayed by immunohistochemistry. RESULTS： All of the experimental groups which had highly significant difference （P 〈 0.01） compared with the control group could promote the BMSCs to proliferate. The effect of proliferation in the midst-dose group reached the maximal （P 〈 0.01）. There was no significant difference between 1-hour group and 2-hour group at the midst-dose （P 〉 0.05）. The serums of the Granula of Penetrating Bone and Removing Pain was shown to promote the expression of collagen Ⅱ, which were highly significant difference （P 〈 0.01） compared with the control group, and were significantly different （P 〈 0.05） compared with chondrogenic inductor group. CONCLUSION： The serums of the Granula of Penetrating Bone and Removing Pain can promote the BMSCs to proliferate and induce to differentiate from BMSCs to chondrogenic cells phenotype.