陇中损伤散提取物对成骨细胞增殖影响的血清药理学方法的建立及验证  

Establishment and Validation of Seropharmacology Method for Investigating Effects of Longzhong Sunshang Powder Extract on Proliferation of Osteoblast

作　　者：李强[1] 卢娟[2] 李盛华[3] 叶丙霖[3] 

机构地区：[1]湖北医药学院附属人民医院骨科,湖北十堰442000 [2]湖北医药学院基础医学院药理教研室,湖北十堰442000 [3]甘肃省中医院,甘肃兰州730050

出　　处：《湖北医药学院学报》2015年第3期241-245,249,F0003,共7页Journal of Hubei University of Medicine

基　　金：甘肃省教育厅科研资助项目(034B-04)

摘　　要：目的:建立并验证陇中损伤散提取物(extract of Longzhong sunshang powder,LSE)对成骨细胞增殖影响的血清药理学研究方法。方法:用LSE末次给药后颈总动脉采血制备含药血清,按照LSE不同给药剂量、给药时间、采血时间分组,分别用含不同浓度LSE的血清培养胎鼠成骨细胞(osteoblast,OB)。采用MTT法检测细胞增殖能力,倒置相差显微镜观察OB形态,Von kossa染色法观察OB的矿化结节,以碱性磷酸酶活性、矿化结节数、基质钙含量、Ⅰ型胶原免疫组化等指标与空白对照组进行对比分析。结果:等剂量组(给药剂量),5 d组(给药时间),1h组(采血时间)的OD值,与对照组比较有显著性差异(P<0.05);不同浓度含药血清组的OD值:10%组>15%组>5%组,组间比较有显著性差异(P<0.05);LSE可显著增加OB的碱性磷酸酶染色阳性细胞数、矿化结节数、基质钙含量(P<0.01)以及上调Ⅰ型胶原表达(P<0.05)。结论:LSE以等剂量(1 g/kg)灌胃给药5 d,末次给药1h后采血制备血清,以10%浓度与OB细胞共培养,是促进胎鼠成骨细胞增殖的优选方案。Objective To establish and validate a serum pharmacology method for investigating effects of Longzhong sunshang powder extract（ LSE） on the proliferation of osteoblast（ OB）. Methods The blood were collected from carotid artery of rats after the final administration,and prepared for LSE serum. Based on the different dosage,administration times and blood collection time,the fetal rat osteoblasts were cultured with different concentration of LSE serum,respectively. The proliferation of osteoblasts was detected by MTT method,and the morphology of osteoblasts was observed with inverted phase contrast microscope,and the mineralization tubercle was observed by Von Kossa staining method. The activity of alkalinity phosphoric acid enzyme（ ALP）,number of mineralization tubercle,content of matrix calcium,and result of type Ⅰ collagen immunohistochemistry were comparatively analyzed with control group. Results The OD value of osteoblast in group that cultured with equal dosage of LSE serum,in group that cultured with LSE serum collecting after administrating for 5 days,and in group that cultured with LSE serum collecting after administrating for 1 hour were all significantly higher than those of corresponding control groups（ P 0. 05）. The OD value of osteoblasts cultured with 10% LSE group was significantly bigger than that of 15% LSE group,the value of 15% LSE group was significantly bigger than that of 5% LSE group（ P 0. 05）. LSE could obviously increase the numbers of positive cells staining by ALP and mineralization tubercles,contents of matrix calcium（ P 0. 01）,meanwhile,upregulate the expression of type Ⅰ collagen（ P 0. 05）. Conclusion The optimized program for enhancing the proliferation of osteoblast is as follow,administrating with dose of 1g / kg LSE to rats for 5 days,collecting blood from common carotid artery at 1 hour after the final administration,then culturing the osteoblasts with 10% LSE serum.

关 键 词：陇中损伤散 提取物 成骨细胞 增殖 血清药理学 

分 类 号：R285.5[医药卫生—中药学]