CN114133441B - 一种促进牙本质矿化的活性短肽及其应用 - Google Patents
一种促进牙本质矿化的活性短肽及其应用 Download PDFInfo
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- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
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Abstract
本发明公开一种促进牙本质矿化的活性短肽及其应用,活性短肽的结构式如下:(S)x(E)y。其中,x为1~8的整数,y为1~8的整数。本发明的活性短肽是由丝氨酸与谷氨酸构成,序列短小且仅含两种氨基酸,可以诱导促进羟基磷灰石晶体的生长。此外,本发明的活性短肽不仅具有牙本质吸附功能,而且与脱矿牙本质具有较强的亲和力,进而可以促进脱矿牙本质再矿化;而且具有成本低、合成简单、生物相容性好等优点,在龋齿、牙本质敏感等口腔临床常见疾病中应用前景可观,如应用于修复因为龋病暴露的牙本质小管并显著改善牙本质敏感等方面中。
Description
技术领域
本发明涉及牙科领域,具体涉及一种促进牙本质矿化的活性短肽及其应用。
背景技术
牙齿和骨骼是人体重要的的硬组织,其本质上是矿物质,大部分为无机成分,主要由羟基磷灰石Ca10(PO4)6(OH)2晶体构成;有机成分则包含胶原蛋白与非胶原蛋白。硬组织的体内生物矿化过程十分复杂,胶原蛋白提供一个细胞外基质矿化的框架,羟基磷灰石晶体的成核与生长过程则主要由以SIBLING蛋白家族为代表的、带负电荷的富含酸性氨基酸(谷氨酸、天冬氨酸、磷酸化丝氨酸)的非胶原蛋白调控。受参与调控生物矿化过程的酸性细胞外基质蛋白的启发,我们设计合成了细胞外基质中的酸性非胶原蛋白的功能片段衍生肽,由丝氨酸(S)与谷氨酸(E)构成,在生物相容性及细胞毒性表现良好,体内可被蛋白酶分解为小分子氨基酸,副作用小,且具有成本低、易合成等优点。
龋齿为口腔专业常见的疾病,由细菌产酸等因素而导致牙齿脱矿,牙体硬组织的过度脱矿会导致大面积的牙体缺损,而目前生物硬组织的缺损无法在生理条件下实现再生。促进脱矿牙釉质及牙本质的再矿化,是防治早起龋齿的重要方法。因此,基于对生物矿化的研究,设计合成促进矿化并对牙本质有再矿化作用的小分子多肽,对于牙体缺损的再矿化有着实际的治疗意义。
另外,牙本质敏感也为患者常见的口腔症状,表现为牙齿一过性的冷热敏感。目前研究主要认为导致牙本质敏感的主要原因为牙本质小管的开放。目前主流的治疗手段为在牙齿表面涂抹使用含氟的材料,以形成氟化钙沉淀隔离并封闭牙本质小管。而目前氟化物再矿化对牙本质缺乏亲和力,且具有过量使用导致氟中毒的安全隐患。因此,设计一种牙本质亲和力强,具有促进牙本质再矿化作用的且安全高效的小分子多肽具有重要意义。
发明内容
本发明是为了解决上述问题而进行的,目的在于提供一种促进牙本质矿化的活性短肽及其应用。
本发明提供了一种促进牙本质矿化的活性短肽,具有这样的特征,活性短肽的结构如下:
(S)x(E)y
其中,x为1~8的整数,y为1~8的整数。
在本发明提供的一种促进牙本质矿化的活性短肽中,还可以具有这样的特征:其中,x为1,y为1。
本发明还提供了一种促进牙本质矿化的活性短肽在促进牙本质矿化方面的应用。
在本发明提供的一种促进牙本质矿化的活性短肽在促进牙本质矿化方面的应用中,还可以具有这样的特征:其中,应用为体外矿化测试,包括以下过程:在浓度为10mM的HEPES缓冲液(pH=7.4)溶解冻干活性短肽,使活性短肽溶液溶度达到500μg/mL,在活性短肽溶液中加入浓度为2.7mM和1.35mM的CaCl2和K2HPO4得到混合液,混匀后取1mL混合液添加至放置有12mm直径玻璃爬片的24孔板中,用封口膜包好,将24孔板置于25℃的恒温培养箱中48h后捞出,65℃烘箱烘干4h,在离子溅射仪中喷金。
在本发明提供的一种促进牙本质矿化的活性短肽在促进牙本质矿化方面的应用中,还可以具有这样的特征:其中,应用为对牙本质的吸附力测试,包括以下过程:将收集的离体牙并制成150μm厚度的牙本质薄片,用砂纸进行打磨抛光并使用37%磷酸酸蚀30s,在酸蚀表面滴加浓度为1mg/mL的异硫氰酸荧光素-活性短肽(FITC-SE)并作用10min后用去离子水冲洗。
在本发明提供的一种促进牙本质矿化的活性短肽在促进牙本质矿化方面的应用中,还可以具有这样的特征:其中,应用为脱矿牙本质再矿化测试,包括以下过程:将收集的离体牙横截面磨片表面进行酸蚀,酸蚀后滴加浓度为1mg/mL的活性短肽作用30min,去离子水冲洗后浸泡在模拟体液8天,每天更换模拟体液,将离体牙片烘干后再喷金。
发明的作用与效果
本发明提供了一种促进牙本质矿化的活性短肽及其应用,活性短肽的结构为:(S)x(E)y。其中,x为1~8的整数,y为1~8的整数。本发明的活性短肽是由S与E构成,序列短小且仅含两种氨基酸,可以诱导促进羟基磷灰石晶体的生长。
此外,本发明的活性短肽不仅具有牙本质吸附功能,而且与脱矿牙本质具有较强的亲和力,进而可以促进脱矿牙本质再矿化;而且具有成本低、合成简单、生物相容性好等优点,在龋齿、牙本质敏感等口腔临床常见疾病中应用前景可观,如应用于修复因为龋病暴露的牙本质小管并显著改善牙本质敏感等方面中。
附图说明
图1是本发明的实施例2中体外矿化产物扫描电子显微镜图;
图2是本发明的实施例2中透射电子显微镜形貌图及选区电子衍射图;
图3是本发明的实施例2中活性短肽对牙本质吸附力的荧光图;以及
图4是本发明的实施例2中模拟脱矿牙本质在短肽作用下的再矿化图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,以下实施例结合附图和实施例中的试验例对本发明的技术方案作具体阐述。
<实施例1>
本实施例中的促进牙本质矿化的活性短肽序列短小,且仅含两种氨基酸,其氨基酸构成来源于体内硬组织细胞外基质中调节矿化的关键酸性蛋白的序列启发。牙本质基质蛋白1(DMP1)、牙本质涎磷蛋白(DSPP)等SIBLING蛋白家族在蛋白质序列上有共同特征,富含酸性氨基酸(天冬氨酸与谷氨酸)及磷酸化丝氨酸。因此通过分析矿化关键蛋白质的序列,在本实施例中提出一种促进牙本质矿化的活性短肽。
本实施例提供了一种促进牙本质矿化的活性短肽,活性短肽的结构如下:
(S)x(E)y
其中,x为1,y为1。
活性短肽的合成方法包括以下步骤:
步骤1,称取50mg树脂放入反应器,加入二氯甲烷(DCM)溶胀0.5h,然后抽掉DCM,加入100mg的S和100mg的二异丙基乙胺(DIEA),加入二甲基甲酰胺(DMF)、DCM溶解以及过量DIEA,以N2鼓泡反应60min,然后加入250mg MEOH,反应0.5h,抽掉反应液,用DMF和MEOH洗净。
步骤2,往反应器中加入100mg的E、100mg的1-羟基-苯并-三氯唑四甲基六氟磷酸盐(HBTU)以及DCM,N2鼓泡反应0.5h,洗掉液体,茚三酮检测,然后用吡啶和乙酸酐封端,最后洗净,加入适量的脱帽液去除9-芴甲氧羰基保护基(Fmoc),洗净,茚三酮检测。
步骤3,将树脂用氮气吹干后从反应柱中取下,倒入烧瓶中,然后往烧瓶中加切割液,震荡,滤掉树脂后得到滤液。
其中,切割液和树脂的比例为10ml:1g,切割液的组成为95%三氟乙酸(TFA)、2%乙二硫醇、2%三异丙基硅烷以及1%水。
步骤4,向滤液中加入***,析出粗产物,然后离心,清洗即可得到活性短肽序列的粗产物。
步骤5,利用高效液相色谱将粗产物提纯至95%纯度,得到纯化后的活性短肽。
步骤6,纯化后的活性短肽放入冻干机中进行浓缩,冻干成白色粉末。
<实施例2>
本实施例是对实施例1中的促进牙本质矿化的活性短肽的性能测试应用和结果。
1.体外矿化实验
称量并在浓度为10mM的HEPES缓冲液(pH=7.4)溶解冻干活性短肽,使活性短肽溶度达到500μg/mL,在上述溶液中加入浓度为2.7mM和1.35mM的CaCl2和K2HPO4,混匀后取1mL上述液体添加至放置有12mm直径玻璃爬片的24孔板中,用封口膜包好,将24孔板置于25℃的恒温培养箱中48h。
将矿化48h后的爬片捞出,65℃烘箱烘干4h,在离子溅射仪中喷金后在扫描电子显微镜下选择合适的探头距离以及最佳倍数进行观察。
将矿化产物收集至离心管中,离心并使用无水乙醇重复多次以洗去表面附着多肽,使用无水乙醇重悬并在超声洗涤,将矿物滴加在铜网上风干后进行SEM形貌拍摄以及选区电子衍射。
图1是本实施例中体外矿化产物扫描电子显微镜图。
如图1所示,经活性短肽处理组处理后,体外矿化产物尺寸显著增大,大小由1μm左右增长至100μm及10μm级别,对照组呈现球状磷酸钙产物,处理组晶体表面呈板状晶型。
图2是本实施例中透射电子显微镜形貌图及选区电子衍射图。
如图2所示,对照组与处理组的体外矿化产物选区电子衍射图谱均对应羟基磷灰石晶体的衍射环,说明此矿化体系中形成的磷酸钙固体晶型为羟基磷灰石,均呈板状晶型。且处理组晶体大小较对照组更大,与图1相对应。
2.活性短肽对牙本质的吸附力实验
将收集的离体牙并制成150μm厚度的牙本质薄片,用砂纸进行打磨抛光并使用37%磷酸酸蚀30s,在酸蚀表面滴加浓度为1mg/mL的FITC-SE并作用10min后用去离子水冲洗,使用激光共聚焦显微镜拍摄。
图3是本实施例中活性短肽对牙本质吸附力的荧光图。
如图3所示,短肽对牙本质表面有较强的吸附功能。
3.脱矿牙本质再矿化实验
将收集的离体牙横截面磨片表面进行酸蚀,酸蚀后滴加浓度为1mg/mL的活性短肽作用30min,去离子水冲洗后浸泡在模拟体液8天,每天更换模拟体液。将离体牙片烘干后在喷金并在扫描电子显微镜下进行观察并拍摄。
图4是本实施例中模拟脱矿牙本质在短肽作用下的再矿化图。
如图4所示,本发明中的活性短肽对于脱矿牙本质的再矿化有更好的效果,牙本质小管堵塞率增加。
实施例的作用与效果
本发明提供了一种促进牙本质矿化的活性短肽及其应用,活性短肽的结构为:(S)x(E)y。其中,x为1~8的整数,y为1~8的整数。本实施例中的活性短肽是由S与E构成,序列短小且仅含两种氨基酸,可以诱导促进羟基磷灰石晶体的生长。
此外,本实施例中的活性短肽不仅具有牙本质吸附功能,而且与脱矿牙本质具有较强的亲和力,进而可以促进脱矿牙本质再矿化;而且具有成本低、合成简单、生物相容性好等优点,在龋齿、牙本质敏感等口腔临床常见疾病中应用前景可观,如应用于修复暴露的牙本质小管改善牙本质敏感等方面中。
上述实施方式为本发明的优选案例,并不用来限制本发明的保护范围。
Claims (4)
1.一种活性短肽在制备促进牙本质矿化的产品中的应用,其特征在于:
所述活性短肽的结构如下:
(丝氨酸)x(谷氨酸)y
其中,x为1,y为1。
2.根据权利要求1所述的应用,其特征在于:
所述活性短肽的体外矿化测试包括以下过程:
在浓度为10mM的HEPES缓冲液pH=7.4溶解冻干活性短肽,使活性短肽溶液溶度达到500μg/mL,在所述活性短肽溶液中加入浓度为2.7mM和1.35mM的CaCl2和K2HPO4得到混合液,混匀后取1mL所述混合液添加至放置有12mm直径玻璃爬片的24孔板中,用封口膜包好,将24孔板置于25℃的恒温培养箱中48h后捞出,65℃烘箱烘干4h,在离子溅射仪中喷金。
3.根据权利要求1所述的应用,其特征在于:
所述活性短肽的对牙本质的吸附力测试包括以下过程:
将收集的离体牙制成150μm厚度的牙本质薄片,用砂纸进行打磨抛光并使用37%磷酸酸蚀30s,在酸蚀表面滴加浓度为1mg/mL的异硫氰酸荧光素-活性短肽并作用10min后用去离子水冲洗。
4.根据权利要求1所述的应用,其特征在于:
所述活性短肽的脱矿牙本质再矿化测试包括以下过程:
将收集的离体牙横截面磨片表面进行酸蚀,酸蚀后滴加浓度为1mg/mL的所述活性短肽作用30min,去离子水冲洗后浸泡在模拟体液8天,每天更换模拟体液,将离体牙片烘干后再喷金。
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