CAJ | 학술논문

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.
배경:부혈소판혈장중함유대량골재생소수적생장인자,차각생장인자적비례시궤체자신형성적,구유량호적협동작용.목적:탐토부혈소판혈장체외유도견골수간충질간세포성골적효과.설계、시간급지점:세포학체외관찰,우2007-06/2008-02재중남대학상아의원중심실험실완성.재료:건강12월령웅성비격견,유중남대학상아의학원실험동물부제공.방법:수집제3대견골수간충질간세포,분위4조:대조조가입표준배양기;성골유도배양기조향배양판공내가입함태우혈청、지새미송、β-감유린산납、유생소C적고당DMEM배양기;부혈소판혈장조근거예실험결과,향배양판내가입함체적분수위6.25%부혈소판혈장적저당DMEM배양기;연합조향배양판내가입함지새미송、β-감유린산납、유생소C、체적분수위6.25%부혈소판혈장적고당DMEM배양기.주요관찰지표:세포내감성린산매활성,면역세포화학염색검측I형효원적표체,개진Yon Kossa염색표기개결절형성정황,RT-PCR검측유도후골개소mRNA적표체.결과:각조감성린산매활성균수유도시간적연장이축점증고,연합조승고폭도최위명현(P<0.05).유도7,14 d후,성골유도배양기조、연합조I형효원균정양성표체,부혈소판혈장조、대조조I형효원시종정음성표체.유도14 d후,성골유도배양기조、연합조가견란원형개결절.유도7,14 d후,대조조여부혈소판혈장조지간골개소mRNA표체수평무명현차이(P>0.05),차2조골개소mRNA표체수평균명현저우성골유도배양기조、연합조(P<0.05);성골유도배양기조골개소mRNA표체수평명현저우연합조(P<0.05).결론:경성골조건배양기유도배양적골수기질간세포,부혈소판혈장능재체외현저유도기성골지표적표체.
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.