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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 observe the effect of platelet lysate (PL) on osteogenic differentiation induced by BMSCs in rats. Methods Twenty-four adult healthy Wistar rats, weighing 250-300 g, were male and female. Blood was drawn from the hearts of 16 rats, PL was prepared by centrifugation three times and repeated freeze-thawing. The contents of PDGF, TGF-β1, IGF-1 and VEGF in PL were measured by ELISA. Another 8 rats were used to amplify the passage BM SCs by whole bone marrow isolation and culture. The fourth passage cells were inoculated into osteogenic medium at a dose of 1 × 10 4 / cm 2. According to the different inducing culture medium, the experiment was divided into three groups. The basal induction medium of group A and group B contained 5% and 1% of PL respectively, while group C contained only basic induction medium. ALP staining was performed in each group for 7 days. ALP / ALP / total protein contents were determined at 2, 8 and 10 days after induction, and the ALP activity was determined by inverted phase contrast microscope. After induction for 20 days, alizarin Red staining to observe the formation of mineralized nodules and quantitative analysis. Results The levels of PDGF, TGF-β1, IGF-1 and VEGF in PL were (300 ± 30), (140 ± 25), (80 ± 35) and (70 ± 20) pg / mL, respectively. Inverted phase contrast microscope, morphological changes of BMSCs gradually appeared in each group, and osteoblast-like morphological features appeared. The morphological changes of cells in group A were less than those in groups B and C, but the cells proliferated rapidly. Cells in group A remained densely growing on day 20 and ECM In the formation of mineral deposition was significantly less than the B, C group. After induction for 7 days, a small amount of granules were found in the cytoplasm of group A, and ALP staining was weakly positive. There were a large number of brown granules in cytoplasm of group B and C, and ALP staining was strongly positive. After induction for 2, 8, and 10 d, the ALP activity of group A was significantly lower than that of group B and group C (P <0.05). Alizarin red staining showed that the number of calcium nodules in group A, B and C were (7.67 ± 1.10), (12.87 ± 0.81) and (15.59 ± 1.25), respectively. The area of mineralized nodules were (161 778.70 ± 44 550.80), (337 349.70 ± 56 083.24) and (415 921.70 ± 71 725.39) pixels in group A were significantly lower than those in group B and C (P <0.05). There was no significant difference between group B and C Significance (P> 0.05). Conclusion PL is a carrier system of various growth factors and inhibits the ALP activity and mineralized nodule formation induced by osteogenic induction of BMSCs in a dose-dependent manner.