富血小板血浆诱导犬骨髓间充质干细胞的体外成骨

背景:富血小板血浆中含有大量骨再生所需的生长因子,且各生长因子的比例是机体自身形成的,具有良好的协同作用.目的:探讨富血小板血浆体外诱导犬骨髓间充质干细胞成骨的效果.设计、时间及地点:细胞学体外观察,于2007-06/2008-02在中南大学湘雅医院中心实验室完成.材料:健康12月龄雄性比格犬,由中南大学湘雅医学院实验动物部提供.方法:收集第3代犬骨髓间充质干细胞,分为4组:对照组加入标准培养基;成骨诱导培养基组向培养板孔内加入含胎牛血清、地塞米松、β-甘油磷酸钠、维生素C的高糖DMEM培养基;富血小板血浆组根据预实验结果,向培养板内加入含体积分数为6.25%富血小板血浆的低糖DMEM培养基;联合组向培养板内加入含地塞米松、β-甘油磷酸钠、维生素C、体积分数为6.25%富血小板血浆的高糖DMEM培养基.主要观察指标:细胞内碱性磷酸酶活性,免疫细胞化学染色检测I型胶原的表达,改进Yon Kossa染色标记钙结节形成情况,RT-PCR检测诱导后骨钙素mRNA的表达.结果:各组碱性磷酸酶活性均随诱导时间的延长而逐渐增高,联合组升高幅度最为明显(P<0.05).诱导7,14 d后,成骨诱导培养基组、联合组I型胶原均呈阳性表达,富血小板血浆组、对照组I型胶原始终呈阴性表达.诱导14 d后,成骨诱导培养基组、联合组可见卵圆形钙结节.诱导7,14 d后,对照组与富血小板血浆组之间骨钙素mRNA表达水平无明显差异(P>0.05),此2组骨钙素mRNA表达水平均明显低于成骨诱导培养基组、联合组(P<0.05);成骨诱导培养基组骨钙素mRNA表达水平明显低于联合组(P<0.05).结论:经成骨条件培养基诱导培养的骨髓基质干细胞,富血小板血浆能在体外显著诱导其成骨指标的表达.

Platelet-rich plasma induces osteogenetic activity of canine bone marrow mesenchymal stem cells in vitro

BACKGROUND: Platelet-rich plasma (PRP) contains abundant growth factors that were needed for osteanagenesis. Moreover,the proportion of each growth factor formed by an organism, with good synergism.OBJECTIVE: To explore the influence of PRP on osteogenetic activity of canine bone marrow mesenchymal stem cells (BMSCs) after induction in vitro.DESIGN, TIME AND SETTING: The in vitro cytological experiment was performed at the Central Laboratory of Xiangya Hospital of Central South University from June 2007 to February 2008.MATERIALS: Healthy 12-month male Beagle dogs were supplied by the Experimental Animal Center, Xiangya Medical College,Central South University.METHODS: The 3~(rd) generation BMSCs were collected and divided into 4 groups. BMSCs in the control group were incubated in standard medium. BMSCs in the osteogenetic induction medium group were incubated in high-glucose DMEM containing fetal calf serum, dexamethasone, beta-sodium glycerophosphate and vitamin C. BMSCs in the PRP group were incubated in low-glucose DMEM containing 6.25% PRP. BMSCs in the combination group were incubated in high-glucose DMEM containing 6.25% PRP, dexamethasone, beta-sodium glycerophosphate, and vitamin C.MAIN OUTCOME MEASURES: Alkaline phosphatase activities were measured. Expression of collagen type I was examined by immunocytochemical staining. Calcium tuberoses were labeled using modified Von Kossa staining. Expression of osteocalcin mRNA was examined by RT-PCR.RESULTS: Levels of alkaline phosphates of all groups became increased along with time. The alkaline phosphates level of combination group was strongest (P < 0.05). Following 7 and 14 days of induction, type I collagen expressed positively in the osteogenetic induction medium and combination groups, but negatively in the PRP and control groups. Following 14 days,formation of calcium nodules were found in the osteogenetic induction medium and combination groups. Following 7 and 14 days,expression of osteocalcin mRNA were similar between the control and PRP groups (P > 0.05), which was significantly lower than the osteogenetic induction medium and combination groups (P < 0.05). Expression of osteocalcin mRNA was significantly lower in the osteogenetic induction medium group compared with the combination group (P < 0.05).CONCLUSION: PRP gel can effectively promote osteoblastic effect of BMSCs after induction in vitro following induction in osteogenetic medium.