细胞调控胶原液晶矿化的研究

发布时间：2018-03-26 21:20

本文选题：成骨细胞　切入点：胶原液晶　出处：《暨南大学》2015年硕士论文

【摘要】：天然骨组织是一种具有完美分级结构的、天然的复合材料。在成骨细胞作用下,沿C轴取向生长的纳米羟基磷灰石(Hydroxyapatite,HA)沿着胶原纤维的平行方向有序聚集形成具有特殊的高级自组装结构,从而使天然骨具有优良的抗压或抗张等物理性质及生物性能。在体外成骨细胞的作用下,模拟天然骨成骨的生理环境,从而系统得研究骨组织形成的本质及结构与性能的关系,对设计高性能的仿骨材料具有重要意义。为了模拟天然骨组织的矿化过程,本文以甘油磷酸钙(Glycerol phosphate calucium salt/Calcium Glycerophosphate,GPC)为钙磷源,以I型胶原(Type I collagen,Col I)为大分子基质材料,在碱性磷酸酶(Alkaline phosphatase,ALP)或前体成骨细胞的作用下,通过Sol-Gel相转变矿化方法和细胞诱导矿化的方法,构建了纳米HA/Col复合材料。一方面从仿生角度出发,制备出胶原液晶膜:采用偏光显微镜、扫描电镜和原子力显微镜研究胶原膜的液晶织构和拓扑结构。通过XRD、SEM、TEM观察和EDS等测试对GPC-Col-ALP的矿化体系对综合测评,探究探讨钙磷无机矿物在液晶态胶原基质上沉积的过程和机理。另一方面通过MTT法、电镜观察、细胞骨架荧光染色、骨钙素染色、冯.卡门染色、碱性磷酸酶观察研究GPC矿化体系对小鼠前体成骨细胞(MC-3T3-E1)细胞的增殖、生长形貌及细胞骨架的影响。结果表明,在ALP能够解离GPC上的磷酸酯键,从而使GPC解析出PO43-和Ca2+,在弱碱性和体温的条件下生成羟基磷灰石,胶原具有促进该体系矿化的作用。低浓度的甘油磷酸钙矿化培养基具有促进MC-3T3-E1细胞增殖生长的功能,当其浓度达到一定浓度15mmol/L后抑制细胞的增殖生长。GPC能够促进成骨细胞分泌ALP,一定浓度范围内,随着GPC浓度的增大,ALP的活性增大,GPC在15mmol/L时,ALP的活性最大,随着GPC的浓度增大,ALP的活性逐渐减小。
[Abstract]:Natural bone tissue is a kind of natural composite material with perfect grading structure. Nano-hydroxyapatite HA, grown along C-axis orientation, aggregates in an ordered manner along the parallel direction of collagen fibers to form a special advanced self-assembly structure. So that the natural bone has excellent physical properties and biological properties, such as compressive or tensile resistance. Under the action of osteoblasts in vitro, the physiological environment of natural bone osteogenesis is simulated, and the nature of bone formation and the relationship between structure and performance are systematically studied. In order to simulate the mineralization process of natural bone tissue, the calcium Glycerol phosphate calucium salt/Calcium Glycerate Phosphate (GPC) was used as calcium and phosphorus source and type I collagen type I collagen I collagenesis (Col I) was used as macromolecular matrix material in order to simulate the mineralization process of natural bone tissue. Under the action of alkaline phosphatase Alkaline phosphatase (ALP) or precursor osteoblasts, Sol-Gel phase transformation mineralization and cell induced mineralization were used to construct HA/Col nanocomposites. The preparation of collagen liquid crystal membrane: the liquid crystal texture and topological structure of collagen membrane were studied by polarizing microscope, scanning electron microscope and atomic force microscope. The mineralized system of GPC-Col-ALP was comprehensively evaluated by TEM observation and EDS test. To explore the process and mechanism of calcium phosphate inorganic mineral deposition on liquid crystal collagen matrix. On the other hand, through MTT method, electron microscope observation, cytoskeleton fluorescence staining, osteocalcin staining, von Carmen staining, The effects of GPC mineralization system on the proliferation, morphology and cytoskeleton of mouse precursor osteoblast (MC-3T3-E1) cells were studied by alkaline phosphatase assay. The results showed that ALP could dissociate the phosphate bonds on GPC, so that GPC could dissociate PO43- and Ca2. Hydroxyapatite was formed under the condition of weak alkalinity and body temperature. Collagen promoted the mineralization of the system. The low concentration of calcium glycerophosphate mineralized medium could promote the proliferation and growth of MC-3T3-E1 cells. When the concentration of 15mmol/L reached a certain concentration, inhibiting the proliferation and growth of osteoblasts. GPC could promote the secretion of ALP by osteoblasts. In a certain concentration range, the activity of ALP increased with the increase of GPC concentration. The activity of GPC decreased with the increase of GPC concentration.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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