摘要
背景:成骨生长肽在体内外细胞培养中具有明显的促成骨活性,这种促成骨作用的分子机制还不清楚,现已通过人工方法成功制备了合成成骨生长肽。目的:探讨合成成骨生长肽对体外培养的人骨髓间充质干细胞向成骨方向分化的作用。设计、时间及地点:基因和蛋白水平的细胞学体外观察,于2006-07/2007-12在福建省医学科学研究所基因工程实验室及复旦大学附属中山医院中心实验室完成。材料:骨髓标本来源于福建省立医院骨一科收治的男性髂骨植骨患者,合成成骨生长肽由中国科学院上海生命科学研究院生物化学与细胞生物学研究所崔大敷教授惠赠,ERK1/2抑制剂U0126为美国CST公司产品。方法:密度梯度离心法体外分离培养人骨髓间充质干细胞,单纯矿化液组加入由10mmol/Lβ-甘油磷酸钠、50mg/LL-抗坏血酸组成的矿化液;10-7,10-9,10-11mol/L合成成骨生长肽组分别加入对应浓度的合成成骨生长肽,再加入矿化液;常规培养组加入含体积分数为0.1胎牛血清的低糖DMEM。主要观察指标:倒置显微镜观察细胞形态变化,RT-PCR法和免疫组织化学染色检测成骨标志物的表达,Western-blot法分析合成成骨生长肽对ERK1/2信号通路的影响,观察U0126对合成成骨生长肽作用的干预。结果:加入合成成骨生长肽后,骨髓间充质干细胞体积增大,突起增多,呈多角形,胞浆含有较多颗粒状物质,出现致密细胞结节。合成成骨生长肽在mRNA和蛋白水平均能促进成骨标志物碱性磷酸酶、Ⅰ型胶原、骨钙素的表达,定量分析结果显示10-9mol/L合成成骨生长肽促成骨作用最强。加入合成成骨生长肽后能够引起ERK1/2信号通路的持续性激活,经U0126预处理后几乎完全阻断了合成成骨生长肽对骨髓间充质干细胞的促成骨作用。结论:合成成骨生长肽可明显促进人骨髓间充质干细胞向成骨方向的转化,在10-9mol/L浓度下促成骨能力最强,其促成骨作用可能是通过激活MAPK家族ERK1/2途径实现的。
BACKGROUND： Osteogenic growth peptide has significantly promotion on osteogenesis activity in vitro or vivo cell culture. The molecular mechanism of this promotion is still unclear. Synthetic osteogenic growth peptide is successfully artificially prepared. OBJECTIVE： To investigate the effect of the synthetic osteogenic growth peptide on the differentiation of in vitro cultured human bone marrow mesenchymal stem cells （BMSCs） into osteoblasts. DESIGN, TIME AND SETTING： The cytology in vitro study in gene and protein levels was performed at the Laboratory of Gene Engineering, Institute of Medical Science of Fujian Province, and Central Laboratory, Zhongshan Hospital, Fudan University from July 2006 to December 2007. MATERIALS： Bone marrow samples were obtained from male lilac bone graft patients at the First Department of Orthopaedics, Fujian Provincial Hospital. Synthetic osteogenic growth peptide was presented by Professor Cui from the Institute of Biochemistry and Cytobiology, Shanghai Academy of Life Science, Chinese Academy of Science. Extracellular signal-regulated kinase1/2 （ERK1/2） inhibitor U0126 was purchased from CST, USA. METHODS： Human BMSCs were harvested and incubated in vitro by the density gradient centdfugation. BMSCs in the simple mineralized solution group were incubated in mineralized solution, supplemented with 10 mmol/L .β -sodium glycerophosphate and 50 mg/L L-antiscorbic acid. BMSCs in the 10^-7, 10^-9, 10^-11 mol/L synthetic osteogenic growth peptide groups were separately treated with a corresponding concentration of synthetic osteogenic growth peptide, and then with mineralized solution. BMSCs in the conventional culture group were subjected to low-glucose DMEM containing 0.1 volume fraction fetal bovine serum. MAIN OUTCOME MEASURES： Morphological changes were observed under an inverted microscope. Osteogenesis marker expression was measured using reverse transcription-polymerase chain reaction and immunohistochemistry. Effects of synthetic osteogenic growth peptide on ERK1/2 signal pathway were analyzed utilizing Western-blot. Influence of U0126 on synthetic osteogenic growth peptide was observed. RESULTS： Following synthetic osteogenic growth peptide was added, BMSCs with many processes became large, polygonal. Plenty of particle substance and dense nodule were found in cytoplasm. Synthetic osteogenic growth peptide mRNA or protein could enhance the expression of alkaline phosphatase, typeⅠ college and osteocalcin. Quantitative analysis showed that 10^-9 mol/L synthetic osteogenic growth peptide had the strongest osteogenesis. Synthetic osteogenic growth peptide treatment could cause persistent activation of ERK1/2 signal pathway. U0126 pretreatment nearly completely blocked the osteogenesis promotion of synthetic osteogenic growth peptide on BMSCs. CONCLUSION： Synthetic osteogenic growth peptide can significantly enhance the osteogenic transformation of human BMSCs. At 10-9 mol/L, osteogenesis promotion effect is the strongest, and this effect is probably mediated by the ERK1/2 signaling pathway.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第6期1053-1058,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
福建省科技厅青年人才创新项目(2006F3031)~~
