【作者】 刘传林；

【导师】 陈西广；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 石鳖的齿舌被发现有磁铁矿沉积以来,齿舌成为生物矿化研究尤其是铁矿化研究的最佳材料,其齿舌中矿化了大量铁化物包括以纳米颗粒形式存在的磁铁矿。磁性纳米颗粒在很多领域有着广泛的应用前景。红条毛肤石鳖是我国沿海潮间带习见的石鳖物种,本文以其齿舌为研究对象,利用光学显微镜、扫描电镜、生物透射电镜、高分辨率透射电镜、超导量子干涉磁强计、X-射线衍射、红外光谱及显微硬度计等技术从多个角度对齿舌主侧齿齿尖的矿化过程及机理开展研究。首先,形态学上对主侧齿齿尖的产生、矿化过程及磁铁矿颗粒的微结构进行研究;其次,磁学上对处于不同矿化段的齿舌段的起始磁化曲线、磁滞回线及磁铁矿含量进行测定,材料学上对处于不同矿化阶段的齿尖显微硬度进行测定;随后从组织化学和组织免疫学上对组织内的铁还原酶及铁蛋白进行定位,最后,从生物化学角度对铁蛋白进行提取纯化。研究结果显示,在长约1.5cm,宽约2～3mm,约有～60排齿列的齿舌上有形态和颜色明显不同于其他齿片的两列主侧齿纵贯其中。主侧齿齿片由形态迥然不同的两部分结构组成——齿尖和齿基,齿尖三尖齿状,齿基长柄状,两部分由连接区结合在一起。不同部段上的主侧齿齿尖在颜色有明显差异,反映了齿舌矿化历程。齿舌大体可分为四个矿化程度不同的部段,矿化程度的轻重使主侧齿齿尖内组分及结构随之发生变化。生成越早的主侧齿齿尖颜色越深,呈亮黑色,矿化程度重;越晚的颜色越浅,呈红棕色,矿化程度轻;新生成的几乎白色,无矿化。齿片的生成由齿舌囊末端生长点细胞群完成,细胞有规则地分布于囊壁四周并沿壁纵向分布,具很强的分裂能力和分泌功能,齿片形成过程中各司其职,有的负责齿舌基膜合成,有的负责齿片形成,有的则负责功能酶分泌。新生齿片齿尖内部大量的有机纤维细丝构成网眼状结构,结构内无矿化物;初矿化齿尖的有机结构内开始出现颗粒状矿化物,有机纤维细丝逐渐成束,矿化物沉积于纤维束结构之中;随矿化程度不断加深,矿化物随之不断地积累于齿尖中,齿尖也不断成熟。齿尖的矿化分为两个途径,即内外途径。齿尖外围齿舌囊上皮细胞形成胞外微突起与齿尖外表面连接形成微突起层,细胞内分泌物可通过突起层不断地将有机质和矿化物元素转运到齿尖内,为外部途径;齿尖后齿面内侧近齿尖与齿基连接处有形如一梳状的结构,齿基内腔通道内上皮细胞可通过梳状结构运送有机物和矿化物到齿尖内部,称谓内途径。齿舌囊上皮细胞内有大量的被膜包裹的含铁蛋白颗粒的聚集体,聚集体的表现形态不一,有完全充满的,有部分填充的,充满的聚集体中还有颗粒密集的和松散的之分,聚集体多椭圆形,直径一般在～600nm。上皮细胞组织中有高铁（三价铁）还原酶存在,此酶对齿尖矿化可能起关键作用。成熟齿尖长（上下轴）～250μm,宽（左右轴）～150μm,厚（前后轴）～100μm,齿基长～400μm,两者间为宽～2μm的连接区。齿尖结构分为内外共三层,表层（外层）由黑色的磁铁矿（Fe₃O₄）包裹,其中部分磁铁矿氧化转化为同样具磁性的磁赤铁矿（γ-Fe₂O₃）,前后齿面的磁铁矿层厚度不同,后齿面最厚处～20μm,前齿面则薄些,最厚处～5μm;磁铁矿层由长条状的磁铁矿构成,条状磁铁矿长达几个微米,宽～100nm,顺齿尖长轴方向排布,由晶体状磁铁矿组成,晶体大小不一,单晶平均大小为～52nm,晶体中有单晶、孪晶和多晶形式,晶体间有层错现象发生,晶体（111）和（022）晶面间距分别为～0.48nm和0.29nm。紧贴磁铁矿层的内侧为砖红色纤铁矿层（α-Fe₂O₃）,厚度～1μm;内芯为矿化程度低的有机基质层,厚～80μm,矿化了少量的钙矿物;齿基矿化成分很少,成分主要是α-几丁质。齿基近两个端点处各有一椭圆形开口,向内连接齿基内腔通道,通道直径～50μm,在齿基前端,通道直达齿尖与齿基连接处下方。矿化结果使齿舌各矿化段（Ⅰ-Ⅳ段）的磁铁矿含量不同,Ⅰ段为零,Ⅱ-Ⅳ段分别为干重的6.83%、16.39%、14.71%,饱和磁化强度分别为6.5 emu/g、15.1 emu/g、14.0 emu/g,相应各段齿尖的饱和磁化强度分别为68.6 emu/g、79.2 emu/g、76.0 emu/g。对整个齿舌而言,磁铁矿含量约为干重的13%,矿物重量的57%,每个齿舌约含10^11-12个磁铁矿颗粒,齿舌中铁元素的89.04%（13%/14.60%）以磁铁矿（或磁性物质）形式存在。相应各段（Ⅱ-Ⅳ）齿尖表面显微硬度分别为124.38 kg/mm²、473.04 kg/mm²、646.80 kg/mm²,以第三、四段成熟齿尖的显微硬度计算,其平均显微硬度为559.92 kg/mm²。齿舌囊内组织提取的铁蛋白每个分子约含铁原子～1000个,Fe/P值～,分子量～340KD,pI为5.0～6.0,分子大小～10nm,蛋白分子由两种亚基构成,分别为25.4KD和34.8KD。结果表明红条毛肤石鳖齿舌主侧齿齿尖的矿化在时空上是连续进行的,矿化有两条途径,齿基有内腔通道,齿舌囊上皮细胞组织中有高铁还原酶和铁蛋白,两者对齿尖矿化极为重要,齿舌的磁铁矿含量较大,磁铁矿以结晶体形式存在矿层内,矿化结果使齿尖具磁性和硬度。更多还原

【Abstract】 Since biogenic magnetite was first found in the teeth of chitons （Polyplacophora, Mollusca）, the radular teeth have been good materials for biomineralization research, particularly for iron mineralization, as massive iron biomierals including magnetite nanoscale particles are deposited in the major lateral teeth, and that magnetic nanoparticles have been widely applied to many fields. The Acanthochiton rubrolineatus Lischke （1873） is one familiar chiton species which lives in the rocky intertidal zones in China. In this article, the process and mechanism of biomineralization of cusp of the major lateral tooth, together with some physical, chemical and material characteristics determinations of the radula were undertaken detailed investigation using a combination of light microscopy, scanning and transmission electron microscopy, superconducting quantum interference device magnetometer, powder X-ray diffraction and Fourier transform infrared spectroscopy. At first, in the morphology aspects, a holistic approach has been adopted that encompasses observations over a range of spatial scales, from whole radula mineralization process to those occurring within individual tooth cusps at various stages of development, also observation of microstructure of magnetite crystals in the magnetite layer. Secondly, in the magnetism aspects, the initial magnetization curves of radular segments and tooth cusps at various stages of development were measured, the hysteresis loops of radular segments were also measured, as a consequence of measurement, the contents of magnetite within the cups and radular segments were calculated; in addition, in the materials science aspects, microhardness of tooth cusps at various stages of development was determined. Thirdly, in the histochemistry and immunohistology aspects, the locations of chiton ferritin and ferric iron reductase in radular sac epithelia were identified. Finally, in the biochemistry aspects, the ferritin was isolated and purified.Investigation results showed that chiton A.rubrolineatus has a length of 1.5cm and width of 2—3mm tongue-like radula which bears about 60 transverse rows of teeth. Among these teeth, two lines of major lateral teeth that are obviously distinguished from others by shape and color distribute along the length of the radula. Major lateral tooth consists of two parts, cusp and tooth base, which are different from each other in shape, and the two parts connect by a joint zone. Cusp presents tricuspid shape, while tooth base presents long handle shape. According to mineralization degree, the radula can be divided into four stages of development （I-IV segments）, and the cusps of major lateral teeth at various stages appear different colors range from white, red brown to shinny black, indicating the process of radula mineralization. Accompanying mineralization development, the cusp undergoes substantial changes in composition and framework. New formed cusp shows white color and no minerals are deposited in; while incipient mineralizing cusp shows red-brown color and minerals starts to be deposited in; for mature cusp, it shows shiny-black color and lots of minerals are deposited in.The formation of new teeth is taken place at the terminal end of radular sac, at which the cells of so-called sac odontoblasts distribute regularly within the sac in three dimensional organization, and they are of strong ability of cell division and secretory functions, cells at different regions of sac perform various roles, some are responsible for formation of radular membrane, some for formation of new teeth, and some for secretion of functional enzymes. Within newly formed cusp, massive organic microfibrils constitute mesh-like framework, in the structure there are no minerals; within incipient mineralizing cusp, organic fibrous filaments represent bundle forms, mineral granules starts to be biomineralized inside the fibres; with development of biomineralization, more of minerals granules are accumulated into the organic matrix and results in high biomierlization of mature cusp. Cusp mineralization has two pathways, namely internal and external pathways. By the external pathway, the superior sac epithelia surrounding the cusp via microvillus-like structure may unceasingly transport organic materials and mineral elements into the cusp; by the internal pathway, the inferior sac epithelia in the stylus canal of tooth base can provide materials via a comb-like structure occurring below the joint zone between the cusp and tooth base. Radular sac epithelium contains numerous membrane bound iron-containing aggregates, these aggregate exhibit various statuses in morphology, some of them are fully filled, and some of them are partly filled; in general, aggregates are oval shaped, and on average are about～600nm in diameter. Immunohistology reaction indicates the iron-containing granules are ferritin nature. Ferric iron reductase exists in membrane of the epithelium, which possibly plays important roles in ironbiomineralization of cusp, also may be responsible for the transformation of magnetite from other phases of iron minerals.The mature cusp is about～250μm in length （vertical axis）,～150μm in width （horizontal axis） and～100μm in thickness （longitudinal axis）; while tooth base is approximately～400μm in length. Cusp may be divides into three layers in structure, the magnetite （Fe3O4） layer （outer layer）, is in different thickness on the posterior and anterior surface respectively, and partial magnetite in the layer transforms to maghemite （γ-Fe2O3） as magnetic as magnetite by oxidation. On the posterior surface, the maximum thickness of magnetite layer reaches approximately～20μm, on the contrary, the maximum thickness is only approximately～5μm; Inside the magnetite layer, magnetite present in a form of lath-shaped pieces that are several microns in length and about～100nm in width, these pieces arrange parallel to the vertical axis direction of cusp and are composed of many magnetite crystals, magnetite crystals vary in size, and mean size is approximately～52nm in diameter, crystals exhibit single, twinned and polycrystalline forms, fault phenomenon also occurs between the crystals, the d-spacing of （111） and （022） in single crystal is～0.48nm and 0.29nm respectively. Adjacent to the magnetite layer is lepidocrocite layer （α-Fe₂O₃）, which is about～1μm in thickness and appears in brick red color; the core mainly is organic matrix, about～80μm thick, in which a few of calcium minerals and so on are mineralized. The tooth base deposits little minerals compared to the cusp, main composition is ofα-chitin in nature. Inside the tooth base, there is a long stylus canal that is about～50μm in diameter, and one apex of the canal directly reaches the site just below the joint zone; two openings are situated on the two ends of the tooth base respectively. For II-IV segments of radula, the saturated magnetization value is 6.5emu/g, 15.1emu/g and 14.0emu/g respectively; while for cusps, the saturated magnetization value is 68.6emu/g, 79.2emu/g and 76.0emu/g respectively. As a result, the magnetite content calculated within each of radula segments （II-IV segments） is 6.83%, 16.39% and 14.71% by dry weight, for the whole radula, magnetite content arrives at approximately 13% by dry weight, 57% by minerals weight, being equal to approximately 1011-12 magnetite crystals, approximately 89.04% iron （13%/14.60%） presents in the magnetite （or other magnetic material） form. Therefore, the value of microhardness of cusps of corresponding segments is 124.38 kg/mm2, 473.04 kg/mm2, 646.80 kg/mm2 respectively, taking account of the cusps within II segment are not fully mature, so the mean value of microhardness of cusps of III-IV segments as whole is calculated to be 559.92 kg/mm2. The chiton ferritin has been isolated and purified from radula sac to electrophoretic purity by ion-exchange chromatography and electrophoresis, and the native ferritin contains 1000 Fe atoms per molecule of protein and considerable amounts of phosphate （Fe/P =30）. The molecule size is 8.2 （±1.0） nm. The ferritin has Mr of 300kDa, pI of 4.2-5.3 and is composed of two subunits Mr 25.4kDa and Mr 34.8kDa. Conclusion is that biomineralization of the cusp of major lateral tooth of chiton A. rubrolineatus is a progressive process which develops temporally and spatially, and mineralization has two pathways, ferric iron reductase and ferritin occurs in radular sac epithelia, they are important for the mineralization. In addition, magnetite content within radula is large, and magnetite presents in crystalline form. Therefore, the biomineralization causes the cusps are of magnetic, and have somemechanical hardness.更多还原