磷酸化收钙素多肽抑制草酸钙结晶的动力学研究
发布时间：2018-08-07 09:48
【摘要】：生物矿化是指生物体在有机基质的参与和调控下,形成无机固相矿物的过程。此过程中蛋白分子的添加,可以有效的促进或者抑制生物的无机化进程。本研究首先通过凝胶法合成了可用于原子力显微镜(AFM)实验的一水合草酸钙(COM)晶体,再通过体外合成不同磷酸化程度的收钙素(CS)多肽片段,结合钙离子电极检测及原位原子力显微镜(AFM)等物理化学表征手段,模拟了收钙素对COM晶体成核及表面生长的影响,得到的主要结果如下:(1)选用硅胶和膨润土作为培养介质,凝胶法合成COM晶体结果表明,在硅胶体系中Ca2+和C2O42-浓度为0.5 mol·L-1,室温培养60天;膨润土体系中,Ca2+和C2O42-浓度为0.75 mol·L-1室温培养90天和浓度为0.5 mol·L-1,62℃培养60天条件下合成的COM晶体结构稳定,表面平整,尺寸合适(晶面宽度不小于20μm),基本满足AFM下的样品实验要求。(2)在近生理条件下(σCOM=1.62,I=0.15 mol·L-1,p H=7.0,25°C),借助钙电极监控的方法,研究在不同本底电解质(KCl或Na Cl)下,含有14个氨基酸残基的CS多肽(14-mer CS)对COM成核和生长的影响。结果表明,在KCl作为本底电解质的溶液体系中,COM成核速度高于Na Cl中,添加非磷酸化和含有两个磷酸化位点的收钙素蛋白CS-2和(2p)CS-2后,抑制效果随着添加浓度的升高,成核所需的诱导时间显著延长,且(2p)CS-2对COM的成核抑制效果更为明显。通过扫描电镜及XRD等对成核后的晶体形貌及结晶性进行检测,结果表明CS-2和(2p)CS-2对成核晶体形貌均有影响,且(2p)CS-2可以明显削弱晶体结晶性,并有参与成键的可能性。(3)借助原位原子力显微镜,观测在不同本底电解质的过饱和溶液(σCOM=0.816,I=0.15 mol·L-1,p H 7.0,25°C)中,磷酸化CS-2多肽对COM(-101)和(010)面台阶生长的影响,结果表明,以Na Cl作为本底电解质时,Na Cl对COM晶面台阶的生长具有干扰作用,不利于添加CS多肽后数据的测量,因此在AFM实验中选用KCl调节溶液的离子强度。CS-2和(2p)CS-2阻止了台阶的移动,随着多肽浓度的升高,台阶生长生长速度降低。CS-2对COM(-101)面的抑制效果强于(2p)CS-2,且在相对较高浓度下(≥30 nmol·L-1),台阶生长完全停滞。对于COM(010)面,(2p)CS-2抑制作用较强,尤其对(010)面的[0-2-1]方向。因此,肽段的组成及空间结构决定着它们在不同晶面不同方向台阶上的选择性吸附。
[Abstract]:Biomineralization is the process of the formation of inorganic solid minerals under the participation and regulation of organic matrix. In this process, the addition of protein molecules can effectively promote or inhibit the inorganic process of organisms. In this study, calcium oxalate monohydrate (COM) crystals, which can be used in atomic force microscope (AFM) (AFM) experiments, were first synthesized by gel method, and then different levels of phosphorylation of cadherin (CS) polypeptides were synthesized in vitro. The effects of cadherin on the nucleation and surface growth of COM crystal were simulated by means of calcium ion electrode detection and in situ atomic force microscopy (AFM). The main results were as follows: (1) silica gel and bentonite were used as culture medium. The results of COM crystal synthesis by gel method showed that the concentration of Ca2 and C _ 2O _ 4 _ 2- was 0.5 mol L ~ (-1) in silica gel system and cultured at room temperature for 60 days. In bentonite system, the crystal structure of COM was stable and the surface was smooth when the concentration of Ca 2 and C 2O 4 2- was 0.75 mol L-1 for 90 days at room temperature and 60 days at 62 鈩，
本文编号：2169640
[Abstract]:Biomineralization is the process of the formation of inorganic solid minerals under the participation and regulation of organic matrix. In this process, the addition of protein molecules can effectively promote or inhibit the inorganic process of organisms. In this study, calcium oxalate monohydrate (COM) crystals, which can be used in atomic force microscope (AFM) (AFM) experiments, were first synthesized by gel method, and then different levels of phosphorylation of cadherin (CS) polypeptides were synthesized in vitro. The effects of cadherin on the nucleation and surface growth of COM crystal were simulated by means of calcium ion electrode detection and in situ atomic force microscopy (AFM). The main results were as follows: (1) silica gel and bentonite were used as culture medium. The results of COM crystal synthesis by gel method showed that the concentration of Ca2 and C _ 2O _ 4 _ 2- was 0.5 mol L ~ (-1) in silica gel system and cultured at room temperature for 60 days. In bentonite system, the crystal structure of COM was stable and the surface was smooth when the concentration of Ca 2 and C 2O 4 2- was 0.75 mol L-1 for 90 days at room temperature and 60 days at 62 鈩，
本文编号：2169640
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