牙生物磷灰石结合肽的淘选及生物矿化的初步研究

发布时间：2018-03-21 05:00

本文选题：生物磷灰石结合肽　切入点：生物矿化　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：无机物表面识别基因工程肽是一类来源于随机肽展示系统的无机物。通常这类生物活性分子能够识别一些无机物的表面,并被广泛运用于药物传递、医学成像以及组织工程等领域。根据其结合底物的种类不同,这类小分子肽可以被广义地分为三大类。其中矿物结合肽能够识别并结合于矿物类无机物的表面,并可能在相应矿物的矿化过程中起到活性调控分子的作用。本文首次提出经过适当处理的牙体硬组织为模版,结合噬菌体展示技术淘选获取牙生物磷灰石的结合肽,并在体外矿化实验中验证上述结合肽影响钙磷沉积的活性。本研究的第一部分,首先通过平行和垂直牙冠长轴的方向切割制备牙冠纵切面和横切面,然后利用扫描电镜观察样本表面形貌。样本在温和条件下进行热碱水解处理以降低表面的有机成分含量。利用高能量微区X线衍射技术及扫描电镜技术相结合对处理前后的样本进行微观形貌和晶体学性能的分析。结果提示用以下的方式制样是获得稳定淘选用模版的可行方法：选用年轻恒牙为样本以减少牙间差异所致的影响；统一在唇侧牙冠中部取样以减少牙内变异所致的影响；近釉牙本质界处牙釉质和牙本质在制备模版时应予以排除因为孔隙和有机物含量较高；温和的碱处理不仅可以减少牙体硬组织样本表面的有机质含量,还能去除因制样受损所导致的晶体排列取向紊乱的表层。在论文的第二部分,淘选模版被用于噬菌体展示的环肽系统进行结合肽的淘选。在底物结合、洗脱和结合肽收集等标准淘选程序中,分别使用单纯化学洗脱和化学-物理复合洗脱两种不同的方式收集结合肽。从第三轮和第四轮中挑选单克隆噬菌体进行结合力分析。每种方法挑选出三十个单克隆分别计算噬菌体回收／投入比值,选择具有较强结合力的单克隆进行测序分析。统计结果发现,两种不同洗脱方式对于得到强结合力噬菌体结合肽来说并没有统计学意义上的差异(P0.05)。总计有十四条不同的展示肽序列被认为是具有较强结合力的,其中有两个单克隆噬菌体展示的是同一条序列。在论文的第三部分中,十四条含有不同展示肽序列的单克隆噬菌体储液被添加进微孔板凝胶系统以检验其影响矿化的活性。绝大多数单克隆在凝胶系统中抑制钙/磷的沉积的程度和WM13之间没有统计学差异(P0.05)。但MR414和MR401添加剂组的终末产物量介于阴性对照和WM13之间(P0.05)。因此对这两种噬菌体上所展示的序列进行体外合成并分别验证合成环肽的牙釉质亲和力。荧光素标记的MR414(-CMPQVMPMC-)和MR401(-CMPQVMPMC-)均能结合于牙釉质并使样本表面发出明暗相间的荧光。而在微孔板凝胶矿化实验中,合成环肽MR414(-CMPQVMPMC-)显现出对凝胶中钙/磷沉积量的抑制具有浓度效应。相比之下MR401(-CMPQVMPMC-)则没有上述的浓度效应。模拟体液矿化实验中,MR401(-CMPQVMPMC-)添加剂组沉积物为针状晶体为主混有少量的片状晶体的混合体,且晶体有沿c轴择优取向排列的趋势；MR414(-CMPQVMPMC-)添加剂组沉积物为针状和片状间杂的混合体且晶体以随机取向排列为主。
[Abstract]:Surface recognition of inorganic genetic engineering peptide is a kind of random peptide display from the inorganic surface system. Usually this kind of bioactive molecules can identify some inorganic compounds, and are widely used in medical imaging, drug delivery, tissue engineering and other fields. According to the combination of different types of this kind of substrate, small molecule peptide is broadly divided into three categories. The mineral binding peptides can recognize and bind to the surface of the inorganic mineral, and may play a role in the regulation of the activity of molecular minerals in the corresponding mineralization process. This paper first dental hard tissue after proper treatment is put forward as a template, show binding peptide technology acquisition panning teeth the phage biological apatite, and mineralization in vitro experiments verify the binding peptides influence the activity of calcium and phosphorus deposition.
The first part of this study, first through the parallel and perpendicular to the axis of the crown cutting preparation of crown longitudinal section and transverse section, and then observe the sample surface morphology by scanning electron microscope. The samples were hot alkali hydrolysis treatment under mild conditions to reduce the content of organic compounds. The analysis and surface by high energy micro area x-ray diffraction technique and scanning the electron microscopic technique the morphology and crystallographic properties of the combination of the samples before and after treatment. Results in the following way is to obtain a stable sample selection method: Amoy feasible template selection for the sample of young permanent teeth to reduce the impact caused by differences between teeth; uniform in the labial crown central sampling to reduce the impact caused by the variation of teeth; near the enamel dentinal junction enamel and dentin should be excluded because of the high porosity and organic matter content in the preparation of template; alkali treatment can not only moderate To reduce the content of organic matter on the surface of the hard tissue samples, the surface of the crystal arrangement disorder caused by the damage of the sample can also be removed.
In the second part, panning the phage display template is used for peptide binding peptide panning system. In substrate binding, binding peptide elution and collection standard panning procedure, receiving binding peptide using a simple chemical elution and chemical physical compound eluted in two different ways. From the third and fourth rounds in the selection of phage binding analysis. Each method selected thirty monoclonal phage recovery / input ratio were calculated, with strong binding force of the monoclonal selection were sequenced. The results showed that two different elution method to get strong bonding force phage displayed peptide was not statistically significant difference (P0.05).. a total of fourteen different display peptide sequence is considered to have strong binding force, of which there are two monoclonal phage display the same sequences.
In the third part of this thesis, fourteen different peptide sequences containing monoclonal phage display liquid was added to microtiter plate gel system to test the effects of mineralization activity. The vast majority of monoclonal in gel system between WM13 and degree of inhibition of calcium / phosphorus deposition had no statistical difference (P0.05). But the MR414 and MR401 the additive group of end product weight between the negative control and WM13 (P0.05). So the sequence of the two phage display on the in vitro synthesis and verification of the synthesis of cyclic peptide affinity. Enamel fluorescein labeled MR414 (-CMPQVMPMC-) and MR401 (-CMPQVMPMC-) were combined with fluorescence in enamel and the sample surface issued white light. While in the microplate gel mineralization experiment, synthetic cyclic peptide MR414 (-CMPQVMPMC-) has shown effect on inhibition of gel concentration of calcium / phosphorus deposition. Compared to MR401 (-C MPQVMPMC-) no concentration effect. The simulated body fluid mineralization experiment, MR401 (-CMPQVMPMC-) mixture additive group sediment is needle like crystal mixed with a small amount of flake crystals, and the crystals along the c axis preferred orientation of the trend; MR414 (-CMPQVMPMC-) additive group is acicular and flaky sediment mixed mixed the crystal body and arranged in a random orientation.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R783.1

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