【作者】 杨德鸿； 金大地； 陈建庭； 景宗森； 武大林； 丁红；

【Author】 Dehong Yang, Dadi Jin, Jianting Chen, et al.Department of Orthopeadic Surgery, Nan Fang Hospital,The First Military Medical University, Guangzhou 510515

【机构】 广州第一军医大学南方医院骨科； 广州第一军医大学南方医院外科中心实验室；

【摘要】 目的:探讨THFβ对PDGF受体α亚型的调节作用及其意义.方法:1.取4～7月龄流产胎儿颅骨,0.5%胶原酶Ⅱ消化,收集细胞于CO₂孵厢内作瓶内单层培养.2.将细胞以2×10⁴/孔种入24孔板,分成3个实验组:（1）对照组:含1%FBS的培养液继续培养.（2）PDGF-AA组:加入PDGF-AA（40ng/ml）培养24h,然后倒掉培养液,PBS洗三遍,含1%FBS的培养液继续培养24h.（3）TGFβ组:加入TGFβ（4ng/ml）因子培养24h,其余操作第2组.每隔4h从上述各实验组取8孔行细胞计数,第2、3组分别和第1组作团体t检验.然后计算每4h细胞增加百分数（PCI）,PCI（%）=(N_i-N_i-1)/N_i-1×100%,N_i代表第i次计数时的细胞量.以PCI为数值轴,时间为分类轴绘细胞增殖曲线图.3.将细胞以2×10⁴ /孔种入24孔板,分成 2个实验组:（1）TGFβ→PDGF-AA组:TGFβ加入培养体系,24h后换入PDGF-AA,继续培养24h.（2）PDGF-AA组:1%FBS培养细胞24h后加入PDGF-AA.细胞计数同二（一）,两组作团体t检验,然后计算PCI和绘细胞增殖曲线图.4.TGFβ培养细胞24h,流式细胞技术检测成骨细胞膜上PDGFR-α的表达.结果:（1）当PDGF-AA和TGFβ加入培养体系,PCI值分别提高了48.2%和22.4%,撤除两因子16h后,PCI恢复到因子加入前的水平.（2）与单纯PDGF-AA培养相比较,TGFβ预先培养24h的成骨细胞再受到PDGF-AA的刺激时,PCI值上升速度明显减慢.（3）TGFβ使PDGFR-α的荧光分布曲线右移48%,标记百分率下降20%.结论:TGFβ通过抑制PDGFR-α的表达削弱了PDGF-AA的促人成骨细胞增殖作用.成骨细胞在含1%FBS的培养液中几乎不能增殖,细胞仅维持生存.当PDGF-AA或TGFβ加入培养体系,细胞的增殖能力明显提高,撤除因子后细胞增殖能力明显下降,表明PDGF-AA或TGFβ均具有促增殖活性.本实验进一步观察到尽管TGFβ预先培养的成骨细胞受到PDGF-AA的刺激后,细胞的增殖能力逐步提高,但未经TGFβ培养的成骨细胞在接受PDGF-AA的刺激后,细胞增殖前一组（P<0.05）.上述提示经过TGFβ培养的成骨细胞,可能发生了某种结构和功能的改变,使细胞对PDGF-AA刺激的反应性下降,引起PDGF-AA的促增殖效应的降低.成骨细胞的细胞膜PDGFR-α是PDGF-AA的唯一高亲和力受体,在人类正常成骨细胞,TGFβ引起PDGFR-α下降.PDGFR-α量的下降势必导致PDGF-AA促成骨细胞增殖能力的削弱.PTGFβ抑制PDGF-AA的促增殖作用体现了因子之间的制衡,这一机制使成骨细胞的增殖处于正常范围之内.随着对PDGF、TGFβ等众多生长因子相互作用和内在联系的深入探讨,人们可更透彻地认识生长因子在各种生理和病理过程中的地位和作用,从而更有利于骨组织代谢紊乱性疾病的诊断和治疗更多还原

【Abstract】 Objective:To investigate the action of which TGF modulated the expression of PDGFR-a on human osteoblasts and discuss its significance. Methods: (l) The osteoblasts were isolated from human fetal calvaria. (2) to count the percentage of cell increase (PCI) in every 4 hours, in order to demonstrate the ability of osteoblasts replication affected by adding or removing PDGF-AA and TGF.(3) The osteoblasts cultured with TGF for 24 hours and then PDGF - AA for 24 hours, the ability of cells replication was showed by PCI too. ( 3) The osteoblast cultured wihth TGF for 24 hours, the PDGFR-a of the cells were measrued by Fluoroimmunoassay. Results: (1) PCI was elevated by 48.2% and 22.4% after PDGF - AA and TGF added into the medium for 24 hours respectively, and PCI was come down after removing these two cytokines. (2) In contrast to incubated only with PDGF-AA, the cells preincubated with TGF for 24 hours and then accepted the stimulation of PDGF-AA, PCI was elevated slowly. (3) TGF downregulated the expression of the PDGFR-a by 20% .Conclusinon: TGFp can downregulated the mito-genesis of PDGF - AA by decreasing the expression of PDGFR-a.更多还原