变性胶原体外培养模型的建立

Establishment of in vitro models of denatured collagen

目的探讨不同温度对I型胶原分子二级结构的影响，确定合适的胶原变性温度，研究热变性后胶原纤维排列及三维凝胶性质的改变，比较胶原变性后不同培养环境成纤维细胞形态差异，以建立变性胶原一细胞体外培养模型。方法I型胶原蛋白溶液在不同温度作用后通过蛋白质圆二色光谱仪分析胶原分子二级结构改变。扫描探针显微镜观察胶原变性后纤维结构的改变。制备不同种类三维胶原凝胶并通过气相压力仪检测胶原凝胶断裂模量。将变性后的胶原进行二维包被和三维胶原凝胶制作，倒置相差显微镜及光镜下观察不同培养环境下细胞形态变化。结果温度达到50℃时，I型胶原分子二级结构发生明显改变，在二维胶原包被时可见胶原纤维凝集成团，含变性胶原的三维凝胶断裂模量明显下降。在变性胶原存在环境中培养成纤维细胞，细胞形态均有显著改变。结论经50％作用后I型胶原分子二级结构发生明显改变，含变性胶原的三维凝胶断裂模量明显下降，I型胶原包被及三维凝胶模型培养的成纤维细胞形态明显不同，可作为变性胶原影响细胞生物学活性的体外模型。

Objective To investigate influence of different temperatures on secondary structure of type I collagen, determine the proper temperature for collagen denaturation, observe changes of collagen fi- bre arrangement and three dimensional collagen gel properties after thermal denaturation, compare morpho- logical variation of fibroblasts seeded in mediums with denatured collagen and therefore establish a standard- ized culture model with denatured collagen in vitro. Methods Changes of the secondary structure of type I collagen was measured by circular dichroism speetrameter after the collagen solution had been treated with different temperatures. Changes of the fibre structure after collagen denaturation were observed by scanning probe microscope. Different three-dimensional collagen gels were prepared and break moduli of the collagen gels were determined by gas pressure machine. The denatured collagens were coated on culture plate or mixed into three-dimensional collagen gels. Morphology of the fibroblasts seeded in different culture condi- tions was detected using the inverted phase contrast microscope and the light microscope. Results At 50~C, the secondary structure of typeI collagen changed dramatically. Collagen fibers were aggregated when denatured collagens were coated on culture plate. Break moduli of three-dimensional collagen gels contai- ning denatured collagens had distinct decrease. Morphology of the fibroblasts seeded in mediums with dena- tured collagen were varied significantly. Conclusion Type I collagen reveals an obvious change of its secondary structure at 50 C. Break moduli of three-dimensional collagen gels containing the denatured col- lagens present a great degree of decrease. Coating and three-dimensional collagen gel models containing type I denatured collagen present different influences on morphology of fibroblasts and thus can be taken as in vitro models to investigate effect of the denatured collagen on bioactivity of cells.