CN114369115A - 一对对PrP106-126聚集有抑制作用的手性磷脂 - Google Patents
一对对PrP106-126聚集有抑制作用的手性磷脂 Download PDFInfo
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Abstract
本发明公开了一种一对对PrP106‑126聚集有抑制作用的手性氨基酸修饰的磷脂分子,具体应用方式为将手性氨基酸修饰的磷脂分子加工成单层手性磷脂囊泡，与PrP106‑126单体和寡聚体共同孵育，然后使用全功能微孔板检测仪观察PrP106‑126的聚集和纤维化情况。此手性磷脂不仅具有良好的生物相容性，并且对PrP106‑126单体和寡聚体的聚集和纤维化也有显著的抑制效果。
Description
技术领域
本发明涉及生物化学和分析技术领域。具体涉及一对对PrP106-126聚集有抑制作用的的手性磷脂。
背景技术
朊蛋白是一种同时可以在动物和人类中引起中枢神经系统变性的具有传染性的蛋白颗粒，由于其具有较长的潜伏期和较高的致死率，使得公共疾病的防控变得异常艰难。最新的研究表明朊蛋白疾病的致病机理同样适用于其他神经退行性疾病，如阿兹海默等。朊蛋白疾病的主要病理特征是正常的PrPC转变为错误折叠的PrPSc,其中PrP106-126被证实是朊蛋白疾病发生很重要的一个片段。因此，抑制PrP106-126错误折叠或聚集是解决这类朊蛋白病的关键所在。
越来越多的证据表明，细胞膜表面的组分是引起疾病的关键。细胞膜是由磷脂双分子层组成的，表面有很多手性氨基酸，因此研究磷脂的分子手性对抑制PrP106-126聚集的影响是必要的。
发明内容
本发明发现手性磷脂囊泡能够强烈抑制与朊蛋白疾病密切相关的PrP106-126单体和寡聚体的聚集和纤维化过程，该对手性磷脂分子可被用于预防和治疗朊蛋白疾病。
本发明目的采用下述方案来实现：
一对对PrP106-126聚集有抑制作用的手性氨基酸修饰的磷脂分子,手性氨基酸修饰的磷脂分子具有如下分子结构：
将手性氨基酸修饰的磷脂分子加工成单层手性磷脂囊泡，与PrP106-126单体和寡聚体共同孵育，然后使用全功能微孔板检测仪观察PrP106-126的聚集和纤维化情况；具体操作过程如下
(1)将手性氨基酸修饰的磷脂分子与未修饰的磷脂分子在烧瓶中混合，溶于三氯甲烷和甲醇的混合溶液，超声至完全溶解后用旋转蒸发仪除去三氯甲烷和甲醇，在烧瓶底部形成一层透明薄膜，之后向烧瓶中加入缓冲液使干燥的脂质膜再水化，得到多层囊泡悬浮液，将多层囊泡悬浮液通过小型挤出机挤出，得到单层手性磷脂囊泡；
(2)将PrP106-126多肽粉末溶解在1,1,1,3,3,3-六氟-2-丙醇中，震荡后用氮气吹干溶液，之后将PrP106-126重新溶解在水中，并用缓冲液稀释至所需浓度，得到PrP106-126单体，单体在37℃孵育24h-48h成为寡聚体；
(3)将单层手性磷脂囊泡与PrP106-126单体和寡聚体共同孵育，并加入荧光分子硫黄素T，加注于96孔板中，通过全功能微孔板检测仪观察PrP106-126单体的聚集和纤维化情况。
步骤1)中手性氨基酸修饰的磷脂分子与未修饰的磷脂分子的质量比为1：1三氯甲烷和甲醇的体积比为2：1，缓冲液为浓度为150nM的NaCl缓冲溶液中，其中所述溶剂为去离子水，水化温度为70℃并不断搅拌，单层手性磷脂囊泡浓度为1mg/mL，挤出机的滤膜尺寸为100nm，并循环挤出20-30次。
步骤2)中PrP106-126在1,1,1,3,3,3-六氟-2-丙醇中中的浓度为1mg/mL，震荡时间为1-2小时，震荡速率为500r/min，氮气吹扫，PrP106-126在水中的浓度为2.3mM，缓冲液为NaCl缓冲溶液，缓冲液浓度为150mM，所述溶剂为去离子水。
步骤3)中单层手性磷脂囊泡的浓度为1mg/mL，PrP106-126的浓度为100μM，二者按照体积比1：1混合，荧光分子硫黄素T的浓度为50μM，96孔板为黑色底部透光，两者共同在酶标仪中的孵育温度为37℃。
通过对全功能微孔板检测仪底部读取模式观察荧光强度的变化，判断PrP106-126的聚集和纤维化，读取数据间隔为10分钟，每次检测之前先振板2秒；全功能微孔板检测仪激发和发射波长分别为445nm和485nm。本发明的有益效果为：
1、本发明发现手性磷脂分子对PrP106-126单体和寡聚体的聚集和纤维化过程具有显著的抑制效果。
2、制备的手性磷脂分子，具有出色的生物相容性和低毒性，可用作药物包覆材料进行药物运输。
附图说明
附图1.L-/D-Asp-DPPE的合成过程。
附图2.ThT监测的PrP106-126单体纤维化动力学曲线。
附图3.ThT监测的PrP106-126单体纤维化动力学曲线。
附图4.ThT监测的PrP106-126单体纤维化动力学曲线。
附图5.ThT监测的PrP106-126寡聚体纤维化动力学曲线。
附图6.ThT监测的PrP106-126寡聚体纤维化动力学曲线。
附图7.ThT监测的PrP106-126寡聚体纤维化动力学曲线。
附图8.手性磷脂单分子可抑制PrP106-126引起的细胞毒性。
附图9.手性磷脂单分子可抑制PrP106-126引起的细胞毒性。
附图10.PrP106-126与L--Asp-DPPE引起细胞内钙离子的荧光图。
附图11.PrP106-126与D-Asp-DPPE引起细胞内钙离子的荧光图。
附图12.L-/D-Asp-DPPE在金片表面的吸附能力。
附图13.PrP单体在L-/D-Asp-DPPE表面的吸附能力。
附图14.PrP寡聚体在L-/D-Asp-DPPE表面的吸附能力。
附图15.PrP单体在L-Asp-DPPE表面的响应值。
附图16.PrP单体在D-Asp-DPPE表面的响应值。
附图17.单体亲和力拟合曲线。
附图18.PrP寡聚体在L-Asp-DPPE表面的响应值。
附图19.PrP寡聚体在D-Asp-DPPE表面的响应值。
附图20.寡聚体亲和力拟合曲线。
具体实施方式
为使本发明的内容、技术方案和优点更加清楚明白，以下结合具体实施例和附图进一步阐述本发明，这些实施例仅用于说明本发明，而本发明不仅限于以下实施例。
实施例1
手性磷脂分子的制备
以手性氨基酸修饰的磷脂分子L-Asp-DPPE为例，在干燥的250mL圆底烧瓶中依次加入0.4mmol Fmoc-L-天冬氨酸-4-叔丁酯(Fmoc-Asp(OtBu)-OH)(164mg)，0.4mmol二棕榈酰基磷脂酰乙醇胺(DPPE)(276mg)，1mmol 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·I)(192mg)，1mmol 1-羟基苯并三唑(HOBT)(136mg)和1mmol 4-二甲氨基吡啶(DMAP)(122mg)，然后加入100mL氯仿。将此反应加热至50℃并搅拌24h反应结束后，待冷却至室温，然后通过旋转蒸发仪除去溶剂，并用硅胶柱纯化反应后的产物。将收集的粗产物溶于20％哌啶和氯仿的混合物中，在室温下搅拌1小时以脱除Fmoc保护基团，然后通过旋转蒸发仪除去溶剂。将三氟乙酸，三异丙基硅烷和蒸馏水加入到残余物中并在相同条件下搅拌以脱除OtBu保护基团，最后用氮气流除去溶剂。之后，将粗产物再次通过硅胶柱纯化，得到手性氨基酸修饰的磷脂分子L-Asp-DPPE。手性氨基酸修饰的磷脂分子D-Asp-DPPE以同样的方法得到。附图1显示了L-Asp-DPPE和D-Asp-DPPE的合成过程。
实施例2
手性磷脂囊泡的制备
以L手性磷脂为例，在干燥的烧瓶里加入同等质量的手性磷脂分子L-Asp-DPPE和二棕榈酰磷脂酰胆碱(DPPC),加入三氯甲烷和甲醇使其溶解，并旋转蒸发去掉溶剂，形成一层薄膜，向烧瓶里加入缓冲液，加热到70℃，并且搅拌一个小时得到囊泡悬浮液。然后，通过小型挤出机进行21次循环挤出得到直径为100nm的手性囊泡，存于-4℃且保质期为一周。
实施例3
PrP106-126单体和寡聚体溶液的制备
将PrP106-126多肽粉末溶解在1,1,1,3,3,3-六氟-2-丙醇中，浓度为1mg/mL。在室温下震荡2小时后，用轻柔的氮气吹干溶液以除去1,1,1,3,3,3-六氟-2-丙醇。之后将PrP106-126重新溶解在蒸馏水中，初始浓度为5.3mM，储存在-20℃备用。使用前，将多肽PrP106-126用氯化钠缓冲液(150mM)稀释至所需浓度。
实施例4
通过全功能微孔板检测仪观察手性磷脂囊泡对PrP106-126单体和寡聚体的聚集和纤维化过程的影响。PrP106-126寡聚体是由PrP106-126单体在37℃下孵育48h获得。将L-/D-Asp-DPPE囊泡(0.1mg/mL、0.3mg/mL、0.5mg/mL)分别与PrP106-126单体和寡聚体(100μM)在37℃下共同孵育，并使用ThT(50μM)标记PrP106-126实时观察荧光强度的变化。附图2-附图7显示了ThT监测的PrP106-126单体和寡聚体的纤维化动力学过程，即L-/D-Asp-DPPE能够显著抑制PrP106-126单体和寡聚体的纤维化
实施例5
通过细胞凋亡实验评估手性磷脂分子对PrP106-126的抑制效果，按实施例1所述方法，将手性磷脂分子和PrP106-126与细胞共同孵育72h,用流式细胞仪检测。附图8和附图9显示L-/D-Asp-DPPE可抑制PrP106-126引起的细胞毒性。
实施例6
通过使用FRET技术测定细胞内钙离子的浓度来评估手性磷脂分子的抑制效果，首先将细胞转染Cyto-Ca2+FRET探针，然后利用FRET显微镜观察荧光强度的变化情况附10和附图11显示了L-/D-Asp-DPPE和PrP106-126加入细胞后，细胞内钙离子浓度的变化情况。
实施例7
通过石英晶体微天平和表面等离子体共振亲和力测定的方法，以评价这对手性磷脂分子与PrP106-126单体和寡聚体的亲和力大小。首先将传感器裸金片在1mM正十八烷基硫醇的乙醇溶液中浸泡12小时形成疏水单层，然后在25℃条件下分别吸附1mg/mL的手型磷脂囊泡得到自组装磷脂单层膜(附图12)，再依次吸附PrP106-126单体和寡聚体。附图13和附图14显示L-/D-Asp-DPPE表面对PrP106-126吸附能力，附图15-附图20显示了手性磷脂表面对PrP106-126的亲和力。
综上所述，本发明发现手性磷脂分子对于与如朊蛋白疾病相关的蛋白PrP106-126单体和寡聚体的聚集和纤维化过程显示出强烈的抑制效果，因此可被用于治疗和预防朊蛋白疾病。鉴于磷脂分子优异的生物相容性和低毒性，可将该对手性磷脂分子加工成脂质体，作为药物包覆材料进行载体运输。
本发明未尽事宜为公知技术。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
Claims (6)
1.一对对PrP106-126聚集有抑制作用的手性氨基酸修饰的磷脂分子,其特征在于：手性氨基酸修饰的磷脂分子具有如下分子结构：
2.一种如权利要求1所述的一对对PrP106-126聚集有抑制效果的手性氨基酸修饰的磷脂分子的应用，其特征在于：将手性氨基酸修饰的磷脂分子加工成单层手性磷脂囊泡，与PrP106-126单体和单体的聚集体在全功能酶标仪共同孵育，然后使用全功能微孔板检测仪观察PrP106-126的聚集和纤维化情况；
具体操作过程如下
(1)将手性氨基酸修饰的磷脂分子与未修饰的磷脂分子在烧瓶中混合，溶于三氯甲烷和甲醇的混合溶液，超声至完全溶解后用旋转蒸发仪除去三氯甲烷和甲醇，在烧瓶底部形成一层透明薄膜，之后向烧瓶中加入缓冲液使干燥的脂质膜再水化，得到多层囊泡悬浮液，将多层囊泡悬浮液通过小型挤出机挤出，得到单层手性磷脂囊泡；
(2)将PrP106-126多肽粉末溶解在1,1,1,3,3,3-六氟-2-丙醇中，震荡后用氮气吹干溶液，之后将PrP106-126重新溶解在水中，并用缓冲液稀释至所需浓度，得到PrP106-126单体，单体在37℃孵育24h-48h成为寡聚体；
(3)将单层手性磷脂囊泡与PrP106-126单体和寡聚体共同孵育，并加入荧光分子硫黄素T，加注于96孔板中，通过全功能微孔板检测仪观察PrP106-126单体的聚集和纤维化情况。
3.根据权利要求2所述的一对对PrP106-126聚集有抑制效果的手性氨基酸修饰的磷脂分子的应用，其特征在于：所述步骤1)中手性氨基酸修饰的磷脂分子与未修饰的磷脂分子的质量比为1：1三氯甲烷和甲醇的体积比为2：1，缓冲液为浓度为150nM的NaCl缓冲溶液中，其中所述溶剂为去离子水，水化温度为70℃并不断搅拌，单层手性磷脂囊泡浓度为1mg/mL，挤出机的滤膜尺寸为100nm，并循环挤出20-30次。
4.根据权利要求2所述的一对对PrP106-126聚集有抑制效果的手性氨基酸修饰的磷脂分子的应用，其特征在于：所述步骤2)PrP106-126在1,1,1,3,3,3-六氟-2-丙醇中中的浓度为1mg/mL，震荡时间为1-2小时，震荡速率为500r/min，氮气吹扫，PrP106-126在水中的浓度为2.3mM，缓冲液为NaCl缓冲溶液，缓冲液浓度为150mM，所述溶剂为去离子水。
5.根据权利要求2所述的一对对PrP106-126聚集有抑制效果的手性氨基酸修饰的磷脂分子的应用，其特征在于：所述步骤3)中单层手性磷脂囊泡的浓度为1mg/mL，PrP106-126的浓度为100μM，二者按照体积比1：1混合，荧光分子硫黄素T的浓度为50μM，96孔板为黑色底部透光，两者共同在酶标仪中的孵育温度为37℃。
6.根据权利要求2所述的一对对PrP106-126聚集有抑制效果的手性氨基酸修饰的磷脂分子的应用，其特征在于：通过对全功能微孔板检测仪底部读取模式观察荧光强度的变化，判断PrP106-126的聚集和纤维化，读取数据间隔为10分钟，每次检测之前先振板2秒；全功能微孔板检测仪激发和发射波长分别为445nm和485nm。
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| EP0169812A1 (de) * | 1984-07-25 | 1986-01-29 | Ciba-Geigy Ag | Phosphatidylverbindungen, Verfahren zu ihrer Herstellung und ihre Verwendung |
| CN111808131A (zh) * | 2019-04-11 | 2020-10-23 | 中国科学院大连化学物理研究所 | 一对有效抑制Aβ（1-40）聚集和纤维化的手性磷脂分子及其制备方法和应用 |
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