BMSCs成骨分化的干预效应血小板裂解液对大鼠  

INTERVENTIONAL EFFECT OF PLATELET LYSATE ON OSTEOGENIC DIFFERENTIATION OF BMSCs IN RATS

作　　者：宋会平[1] 王志强[2] 赵亚平[1] 陈学英[1] 张育敏[1] 李宝兴[1] 

机构地区：[1]山西省医用组织库 [2]唐山市第二医院创伤科

出　　处：《中国修复重建外科杂志》2008年第6期737-741,共5页Chinese Journal of Reparative and Reconstructive Surgery

摘　　要：目的观察血小板裂解液(platelet lysate,PL)对大鼠BMSCs成骨诱导分化的干预效应。方法成年健康清洁级Wistar大鼠24只,体重250～300 g,雌雄不限。取16只大鼠心内采血,采用3次离心结合反复冻融法制备PL,ELISA法测定PL中PDGF、TGF-β1、IGF-1和VEGF含量。另取8只大鼠,采用全骨髓分离培养法扩增传代BM SCs,取第4代细胞按1×104/cm2接种行成骨诱导培养。按诱导培养液的不同,实验分为3组,A、B组基础诱导培养基中分别含终浓度为5%和1%的PL,C组仅含基础诱导培养基。倒置相差显微镜动态观察各组细胞形态变化及生长状况;诱导培养7 d,各组行ALP染色;诱导培养2、8、10 d,ALP/总蛋白含量确定ALP活性;诱导培养20 d,茜素红染色观察矿化结节形成并定量分析。结果PL中PDGF、TGF-β1、IGF-1和VEGF含量分别为(300±30)、(140±25)、(80±35)和(70±20)pg/mL。倒置相差显微镜观察,各组BMSCs形态逐渐发生改变,出现类成骨细胞样形态特征;A组细胞形态变化不及B、C组明显,但细胞增殖较快;20 d A组细胞仍密集生长,ECM中形成的矿物沉积显著少于B、C组。诱导培养7 d,A组细胞胞浆中有少量颗粒,ALP染色呈弱阳性;B、C组细胞胞浆中有大量棕褐色颗粒,ALP染色呈强阳性。诱导培养2、8、10 d,ALP活性定量分析示A组显著低于B组和C组(P<0.05)。诱导培养20 d,茜素红染色显示A、B、C组钙结节数量分别为(7.67±1.10)、(12.87±0.81)和(15.59±1.25)个;矿化结节面积分别为(161 778.70±44 550.80)、(337 349.70±56 083.24)和(415 921.70±71 725.39)像素;A组均明显低于B组和C组(P<0.05),B、C组间比较差异无统计学意义(P>0.05)。结论PL是多种生长因子的承载体系,以剂量依赖方式抑制BMSCs成骨诱导中ALP活性和矿化结节形成。Objective To explore the interventlonal effect of platelet lysate （PL） on osteogenic differentiation of BMSCs by induction in rats in vitro. Methods Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, male or female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A （5% PL of final concentration in basic induction medium）, B （1% PL of final concentration in basic induction medium）, and C （no presence of PL in basic induction medium as a control）. The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining （7 days） and ALP/TP （2, 8, 12 days） of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular martrix was examined with Alizarin red staining which provided quantitative analysis of mineral deposits. Results ELISA assay showed that the content ofPDGF, TGF-β1, IGF-1 and VEGF in PL reached （300 ± 30）, （140 ± 25）, （80 ± 35）, （70 ± 20） pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from splndle-shape to square- or polygon-shape as the morphorlogical type of osteoblast-1 ike cells at 7 days. The cells in group A showed slower shape changes but higher proliferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense growth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells of group A showed smaller amoun

关 键 词：血小板裂解液 生长因子 BMSCS 细胞分化 大鼠 

分 类 号：R96[医药卫生—药理学]