本文選題：磷酸化OPN多肽 + 磷酸鈣��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：為了更好的理解植物體內(nèi)草酸鈣的生物礦化過程,本文借助模擬人體病理礦物(如腎結(jié)石)的形成,研究多肽分子對其形成的影響,從而比較植物與動物體內(nèi)生物礦化作用的差異,為探明植物中草酸鈣類晶體的生物礦化機(jī)理提供理論依據(jù)。人體腎結(jié)石中,含鈣結(jié)石是最普遍的形式,約80%的腎結(jié)石由草酸鈣和磷酸鈣組成。尿液中的Osteopontin(OPN)蛋白可抑制草酸鈣和磷酸鈣的結(jié)晶。然而,對于OPN蛋白磷酸化程度和抑制效果之間關(guān)系,及OPN蛋白在納米尺度調(diào)控礦物晶面臺階生長和溶解動力學(xué)機(jī)制的相關(guān)研究較少。本研究主要借助原位原子力顯微鏡,結(jié)合其他體相結(jié)晶技術(shù)及蛋白分析和表征手段,定量分析磷酸鈣和草酸鈣的成核及臺階生長和溶解動力學(xué),得到的主要結(jié)果如下:1.OPN多肽羥基磷灰石(HAP)結(jié)晶的抑制與多肽磷酸化程度及濃度緊密相關(guān)在近生理?xiàng)l件下(p H 7.40,離子強(qiáng)度I=0.15 mol/L),借助電位監(jiān)控的方法,研究含有14個氨基酸殘基的OPN多肽(14-mer OPN)對HAP成核和生長的影響。結(jié)果發(fā)現(xiàn),隨著OPN多肽(156 nmol/L)磷酸化程度的增加,HAP成核所需的誘導(dǎo)時間顯著延長。相同序列的非磷酸化OPN只在其濃度較高(234 nmol/L)時,才能通過延長誘導(dǎo)時間有效抑制HAP的成核。在一定濃度范圍內(nèi)(156 nmol/L),磷酸化修飾的OPN不僅提高了成核期磷酸鈣納米顆粒的穩(wěn)定性,同時還抑制了納米顆粒從無定形相向晶相的轉(zhuǎn)變。這些表明,OPN多肽以磷酸化程度和濃度雙重調(diào)控方式抑制HAP的結(jié)晶。2.磷酸化OPN多肽對二水磷酸氫鈣(DCPD)(010)面臺階生長動力學(xué)和界面能借助原子力顯微鏡,原位探測磷酸化OPN多肽與DCPD(010)面的相互作用時發(fā)現(xiàn),磷酸化OPN多肽專一地吸附[100]Cc方向臺階,從而抑制臺階的移動。經(jīng)典的晶體生長Cabrera-Vermilyea物理模型可很好地解釋在不同過飽和度下,磷酸化OPN多肽對臺階移動的抑制作用。此外,磷酸化OPN多肽側(cè)鏈上的磷酸根通過靜電作用,吸附在[100]Cc臺階改變礦物-水界面能,從而延遲晶面生長過程中臺階的形成。非磷酸化的OPN多肽完全不具備抑制效果。晶面生長動力學(xué)結(jié)果和體相成核結(jié)果一致。進(jìn)一步證實(shí)了磷酸化OPN多肽對臺階動力學(xué)和界面能的雙重控制作用。3.磷酸化OPN多肽顯著抑制草酸鈣的異質(zhì)成核和團(tuán)聚在模擬的酸性尿液生理?xiàng)l件下,借助原子力顯微鏡,原位觀察尿液的兩種組分草酸和OPN多肽對DCPD(010)面[101]Cc,[100]Cc和[101]Cc三個方向臺階溶解的影響。結(jié)果表明磷酸化OPN多肽專一性地抑制[101]Cc方向臺階的移動,進(jìn)而減少鈣離子的釋放,并顯著抑制DCPD表面誘導(dǎo)一水合草酸鈣的成核。這不但體現(xiàn)了礦物溶解再結(jié)晶的過程,同時還展示了天然蛋白對此過程的調(diào)控作用,更加深了我們對結(jié)石形成抑制機(jī)制的理解。本文結(jié)合晶面動力學(xué)和體相結(jié)晶動力學(xué)的方法,著重探討了多肽分子與礦物表面的相互作用。此研究不但揭示了磷酸鈣-草酸鈣病理礦化的動力學(xué)過程,同時還為理解香蕉體內(nèi)生物礦化機(jī)制提供有效線索。
[Abstract]:In order to better understand the biological mineralization process of calcium oxalate in plants, this paper studies the effect of polypeptide molecules on its formation by simulating the formation of human pathological minerals, such as kidney stones, so as to compare the difference of biological mineralization in plants and animals, and provide a theoretical basis for exploring the mechanism of biomineralization of calcium oxalate crystals in plants. Calcium lithiasis is the most common form in human kidney stones. About 80% of the kidney stones are composed of calcium oxalate and calcium phosphate. The Osteopontin (OPN) protein in urine inhibits the crystallization of calcium oxalate and calcium phosphate. However, the relationship between the degree of phosphorylation and inhibition of OPN protein and the regulation of OPN in the nanometer scale regulation of mineral surface step birth In this study, the nucleation and step growth and dissolution kinetics of calcium phosphate and calcium oxalate were quantitatively analyzed by using in situ atomic force microscopy, other body phase crystallization techniques and protein analysis and characterization methods. The main results were as follows: the crystallization of 1.OPN polypeptide hydroxyapatite (HAP) crystallization The inhibition is closely related to the degree and concentration of polypeptides phosphorylation in near physiological conditions (P H 7.40, ionic strength I=0.15 mol/L). By means of potential monitoring, the effect of OPN polypeptide (14-mer OPN) containing 14 amino acid residues (14-mer OPN) on the nucleation and growth of HAP is studied. The results show that with the increase of the phosphorylation of OPN polypeptide (156 nmol/L), HAP formation is found. The non phosphorylated OPN of the same sequence can inhibit the nucleation of HAP by prolonged induction time only when its concentration is higher (234 nmol/L). In a certain concentration range (156 nmol/L), phosphorylated OPN not only improves the stability of calcium phosphate nanoparticles at the nucleation stage, but also inhibits the nano particles at the nucleation stage. The transformation of particles from amorphous phase to crystalline phase shows that OPN peptide inhibits the crystallization of HAP by the dual regulation of phosphorylation and concentration, and the interaction between.2. phosphorylated OPN polypeptide and OPN polyphosphate (DCPD) (DCPD) (DCPD) (010) surface step growth kinetics and interface can be used to detect the interaction of phosphorylated OPN polypeptide and DCPD (010) surface by atomic force microscope. It was found that the phosphorylated OPN polypeptide specifically adsorb the [100]Cc direction step, thus inhibiting the step movement. The classical crystal growth Cabrera-Vermilyea physical model can well explain the inhibition of the phosphorylated OPN polypeptide on the step movement under different supersaturation. In addition, the phosphate on the phosphorylated OPN polypeptide side chain is static by electrostatic action. The adsorption on the [100]Cc step changes the energy of the mineral water boundary, which delays the formation of the steps during the growth of the crystal surface. The non phosphorylated OPN polypeptide has no inhibitory effect. The crystal surface growth kinetics and the body phase nucleation result coincide. Further confirmed the dual control effect of the phosphorylated OPN polypeptide on the step dynamics and the interfacial energy of.3. phosphorus Acidified OPN polypeptide significantly inhibited the heterogenous nucleation and aggregation of calcium oxalate in the simulated acidic urine physiological conditions. The effects of two components of oxalic acid and OPN polypeptide on the dissolution of DCPD (010) surface [101]Cc, [100]Cc and [101]Cc in three directions were observed by atomic force microscopy. The results showed that the phosphorylated OPN polypeptide was specifically inhibited [ The movement of the 101]Cc direction step further reduces the release of calcium ions and significantly inhibits the nucleation of calcium oxalate hydrate on the surface of DCPD. This not only reflects the process of mineral dissolution and recrystallization, but also shows the regulation of the natural protein in this process, and further our understanding of the inhibition mechanism of the formation of stones. This article combines with the crystal surface. The kinetic and crystalline kinetics of the body phase are focused on the interaction between the peptide molecules and the mineral surface. This study not only reveals the kinetic process of calcium phosphate calcium oxalate, but also provides an effective clue to understand the mechanism of biomineralization in banana.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q945

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本文編號：1889842