CAJ | 학술논문

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-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.
목적 탐토불동온도대Ⅰ형효원분자이급결구적영향,학정합괄적효원변성온도,연구열변성후효원섬유배렬급삼유응효성질적개변,비교효원변성후불동배양배경성섬유세포형태차이,이건립변성효원-세포체외배양모형. 방법 Ⅰ형효원단백용액재불동온도작용후통과단백질원이색광보의분석효원분자이급결구개변.소묘탐침현미경관찰효원변성후섬유결구적개변.제비불동충류삼유효원응효병통과기상압력의검측효원응효단렬모량.장변성후적효원진행이유포피화삼유효원응효제작,도치상차현미경급광경하관찰불동배양배경하세포형태변화. 결과 온도체도50℃시,Ⅰ형효원분자이급결구발생명현개변,재이유효원포피시가견효원섬유응집성단,함변성효원적삼유응효단렬모량명현하강.재변성효원존재배경중배양성섬유세포,세포형태균유현저개변. 결론 경50℃작용후Ⅰ형효원분자이급결구발생명현개변,함변성효원적삼유응효단렬모량명현하강,Ⅰ형효원포피급삼유응효모형배양적성섬유세포형태명현불동,가작위변성효원영향세포생물학활성적체외모형.
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 fibre arrangement and three dimensional collagen gel properties after thermal denaturation,compare morphological variation of fibroblasts seeded in mediums with denatured collagen and therefore establish a standardized culture model with denatured collagen in vitro.Methods Changes of the secondary structure of type Ⅰ collagen was measured by circular dichroism spectrameter 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 conditions was detected using the inverted phase contrast microscope and the light microscope.Results At 50℃,the secondary structure of type Ⅰ collagen changed dramatically.Collagen fibers were aggregated when denatured collagens were coated on culture plate.Break moduli of three-dimensional collagen gels containing denatured collagens had distinct decrease.Morphology of the fibroblasts seeded in mediums with denatured collagen were varied significantly.Conclusion Type Ⅰ collagen reveals an obvious change of its secondary structure at 50 ℃.Break moduli of three-dimensional collagen gels containing the denatured collagens present a great degree of decrease.Coatiug 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.