I型胶原对骨髓间充质干细胞黏附、增殖及成骨基因表达的影响
Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells.
作者信息
Liu Gang, Hu Yun-Yu, Zhao Jian-Ning, Wu Su-Jia, Xiong Zhuo, Lu Rong
机构信息
Department of Orthopedics, Nanjing General Hospital, Nanjing Military District, Nanjing, Jiangsu 210001, China.
出版信息
Chin J Traumatol. 2004 Dec;7(6):358-62.
OBJECTIVE
To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs).
METHODS
The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm x 1.2 cm x 2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [(3)H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM).
RESULTS
Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm+/-141 cpm vs. 1797 cpm+/-118 cpm, P=0.017; 8 h, 2311 cpm+/-113 cpm vs. 1891 cpm+/-103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7 th day (1021 cpm+/-159 cpm vs. 451 cpm+/-67 cpm, P=0.002), the 14th day (1472 cpm+/-82 cpm vs. 583 cpm+/-67 cpm, P<0.001) and 21 th day (1728 cpm+/-78 cpm vs. 632 cpm+/-55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21 th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls.
CONCLUSIONS
Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.
目的
研究经I型胶原修饰的多孔聚乳酸-羟基乙酸共聚物（PLGA）对兔骨髓间充质干细胞（MSCs）黏附、增殖及分化的影响。
方法
采用密度梯度离心法从成年兔分离出第三代MSCs，以不同初始浓度接种于尺寸为0.3 cm×1.2 cm×2.0 cm、涂有I型胶原的三维多孔PLGA上，置于含10%胎牛血清的RPMI 1640培养基中培养，同时将MSCs接种于未涂I型胶原的PLGA上作为对照。接种后第7、14和21天，通过测定[(3)H]-TdR掺入率评估细胞的黏附与增殖行为。为检测MSCs的分化情况，采用逆转录-聚合酶链反应（RT-PCR）检测成骨细胞标志物基因骨钙素（OCN）、碱性磷酸酶（ALP）、骨桥蛋白（OPN）mRNA的表达，此外，于第21天通过扫描电子显微镜（SEM）观察细胞形态。
结果
I型胶原促进细胞在PLGA上的黏附。其结果显著高于对照组（6小时，2144 cpm±141 cpm对1797 cpm±118 cpm，P = 0.017；8小时，2311 cpm±113 cpm对1891 cpm±103 cpm，P = 0.01）。接种于涂有I型胶原的PLGA上的细胞在第7天（1021 cpm±159 cpm对451 cpm±67 cpm，P = 0.002）、第14天（1472 cpm±82 cpm对583 cpm±67 cpm，P < 0.001）和第21天（1728 cpm±78 cpm对632 cpm±55 cpm，P < 0.001）的增殖明显高于对照组。在第21天，涂有I型胶原的PLGA上检测到成骨细胞标志物OCN、ALP、OPN mRNA，而对照组未检测到OCN、OPN mRNA。纺锤形和多边形细胞在涂有I型胶原的聚合物上分布良好，而对照组为圆柱形或圆形细胞。
结论
I型胶原可有效促进MSCs在PLGA上的黏附、增殖及分化。
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