CN105214087A - Pcsk9单克隆抗体在制备治疗炎症免疫性疾病药物中的应用 - Google Patents
Pcsk9单克隆抗体在制备治疗炎症免疫性疾病药物中的应用 Download PDFInfo
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Abstract
本发明属于医药生物技术领域，具体涉及PCSK9单克隆抗体在制备治疗炎症免疫性疾病药物中的应用。本发明以银屑病作为炎症免疫性疾病研究的试验床，发现PCSK9在治疗炎症免疫性疾病中发挥重要作用。本发明为炎症免疫性疾病的治疗提供了一种新的治疗方法，可进一步制备外用和系统应用的PCSK9单克隆抗体，开发治疗炎症免疫性疾病(如银屑病)的新药。该药副作用小，成本低，疗效显著。
Description
一、技术领域
本发明属于医药生物技术领域，具体涉及PCSK9单克隆抗体在制备治疗炎症免疫性疾病药物中的应用。
二、背景技术
炎症免疫性疾病发病率高，全世界至少有数亿患者，其中包括多种难治性疾病，如红斑狼疮、银屑病、湿疹、类风湿性关节炎、皮肌炎、硬皮病、克隆病等等，由于可以累及多器官，导致心、肝肾、血管、肺、关节和脑等损伤，死亡率仅次于恶性肿瘤。该类疾病由于病因和发病机制相当复杂，目前仍然无法根治。常用治疗药物主要是糖皮质激素和免疫抑制剂，但仅能控制疾病的发展。由于这些药物不良反应大，限制了长期应用。近年来，针对疾病发病机制研究的生物制剂由于治疗靶位准确，副反应小而成为今后药物研发的主要方向，但目前此类药物疗效显著者很少，因价格昂贵又限制了其广泛应用。银屑病由于皮损治疗效果易于观察，成为研究生物制剂治疗炎症免疫性疾病的实验床。
PCSK9是前蛋白转换酶家族中的一员，前蛋白转换酶在肝脏内作为一种不活跃酶原分泌。PCSK9基因cDNA的大小为3617bp，编码692个氨基酸组成的PCSK9蛋白,见序列表。PCSK9前体在内质网内经过分子内自动催化分离其N-末端前肽，分离后的N-末端前区与催化区相连，允许成熟的PCSK9蛋白离开内质网并进入分泌途径。PCSK9分泌至细胞外后，在细胞表面的第一表皮生长因子样区域与LDL受体结合，PCSK9-LDL受体复合体可进入溶酶体降解，从而导致细胞表面LDL受体下降，即PCSK9水平与LDL受体成负相关关系。而多项研究显示，PCSK9基因的突变功能丧失可使不同人种的LDL-C水平及冠心病发生率明显下降。
鉴于沉默PCSK9对降低LDL-C及冠心病发生率的显著作用，已有多项治疗方案正在研发阻断PCSK9的药物。目前研究最多且较先进的PCSK9抑制剂为单克隆抗体，两种不同的单克隆抗体已分别获得FDA及欧盟委员会批准上市，用于治疗某些使用目前治疗选择不能使其低密度脂蛋白胆固醇得到控制的高胆固醇血症患者。
此外，尚有多项研究发现，PCSK9在肿瘤、肝硬化等多种增生、炎症性疾病的发病中起到一定作用。
三、发明内容
本发明需要解决的问题是:PCSK9在治疗炎症免疫性疾病中的作用和机制研究，以及PCSK9单克隆抗体在制备治疗炎症免疫性疾病药物中的应用。本发明以银屑病作为炎症免疫性疾病研究的试验床，发现PCSK9在治疗炎症免疫性疾病中发挥重要作用。本发明首先建立PCSK9敲除转基因小鼠模型，在此基础上建立IMQ诱导的银屑病样炎症模型，证实PCSK9抑制后对银屑病样炎症皮损具有非常明显的治疗作用。然后通过培养人角质形成细胞研究抑制PCSK9明显减轻炎症的机制，发现敲除PCSK9能够通过NFkb途径明显抑制机体的炎症和免疫反应；同时敲除PCSK9基因也能明显抑制皮肤角质形成细胞的异常增生，促进细胞凋亡。目前尚未见研究报道和发明专利涉及PCSK9基因的类似作用。
本发明的有益效果是：本发明为炎症免疫性疾病的治疗提供了一种新的治疗方法，可进一步制备外用和系统应用的PCSK9单克隆抗体，开发治疗炎症免疫性疾病(如银屑病)的新药。该药副作用小，成本低，疗效显著。
四、附图说明,,
图1连续涂药5天后，C57BL/6小鼠的背部涂药区域出现明显红斑、鳞屑及皮损增厚，而PCSK9基因敲除小鼠的背部涂药区域仅出现轻微的红斑、鳞屑及皮损增厚。
图2每天对小鼠背部涂药区域皮损进行评分，C57BL/6小鼠的红斑、鳞屑、皮损增厚及总分(红斑+鳞屑+皮损增厚)均明显高于PCSK9基因敲除小鼠的评分(P<0.05)。
图3涂药5天后，取小鼠背部皮肤组织(处理区及非处理区)，进行HE染色。C57BL/6小鼠的IMQ处理区皮肤可见表皮增厚，皮突延长，棘层增厚，角化过度伴角化不全，Kogoj脓疡及Munro脓疡等典型银屑病病理变化，而PCSK9基因敲除小鼠的IMQ处理区皮肤可见轻度表皮增厚及角化过度，未见皮突延长，棘层增厚，角化过度伴角化不全，Kogoj脓疡及Munro脓疡等典型银屑病病理变化；两组小鼠的未处理区皮肤均表现为正常皮肤组织结构。
图4免疫荧光法检测示：C57BL/6小鼠的IMQ处理区皮肤PCSK9表达较未处理区明显上调，而PCSK9基因敲除小鼠的IMQ处理区皮肤及未处理区皮肤均未见PCSK9表达。
图5C57BL/6小鼠及PCSK9基因敲除小鼠的IMQ处理区皮肤NF-kB的表达较之各自的未处理区均明显上调，但PCSK9基因敲除小鼠的IMQ处理区皮肤的NF-kB的表达较之C57BL/6小鼠明显降低。
图6si-PCSK9转染后的人原代角质形成细胞的存活数较之si-Con转染后细胞明显降低(P<0.05)
图7si-PCSK9转染后的人原代角质形成细胞凋亡明显增加(P<0.05)，而增殖(S+G2/M期细胞比例)则明显降低(P<0.05)。
五、具体实施方式
1、PCSK9敲除小鼠对IMQ诱导银屑病样炎症反应明显减轻
主要试剂：5％咪喹莫特乳膏(IMQ)，Pcsk9一抗(abcam，ab31762)，NF-kB一抗(abcam，ab16502)
实验动物：C57BL/6(B6)小鼠，雄性，7只；C57BL/6-PCSK9-/-小鼠，雌雄各5只
实验方法：
(1).将两组不同基因型小鼠背部脱毛处理后，每天涂抹62.5mg的5％咪喹莫特乳膏，连续5天。
(2).涂药前及开始涂药后每天做评分(红斑，鳞屑，皮损增厚及总分)并拍照存档(评分为两名研究者分别打分后取平均值)。
(3).实验最后一天，处死所有小鼠，并取背部皮肤组织(处理区及非处理区)。
(4).HE染色观察各组皮损形态，免疫荧光法测PCSK9及NF-kB在4组小鼠皮损中的表达和分布。
实验结果：
(1)连续涂药5天后，7只C57BL/6小鼠的背部涂药区域均出现明显红斑、鳞屑及皮损增厚，符合银屑病样皮损外观，而10只PCSK9基因敲除小鼠的背部涂药区域仅出现轻微的红斑、鳞屑及皮损增厚(见图1)。
(2)每天对小鼠背部涂药区域皮损进行评分，C57BL/6小鼠的红斑、鳞屑、皮损增厚及总分(红斑+鳞屑+皮损增厚)均明显高于PCSK9基因敲除小鼠的评分(P<0.05)。
(3)涂药5天后，取小鼠背部皮肤组织(处理区及非处理区)，进行HE染色并观察病理变化，C57BL/6小鼠的IMQ处理区皮肤可见表皮增厚，皮突延长，棘层增厚，角化过度伴角化不全，Kogoj脓疡及Munro脓疡等典型银屑病病理变化，而PCSK9基因敲除小鼠的IMQ处理区皮肤可见轻度表皮增厚及角化过度，未见皮突延长，棘层增厚，角化过度伴角化不全，Kogoj脓疡及Munro脓疡等典型银屑病病理变化；两组小鼠的未处理区皮肤均表现为正常皮肤组织结构(见图3)。
(4)对小鼠背部皮肤组织(处理区及非处理区)进行免疫荧光法检测PCSK9及NF-kB表达情况：1)C57BL/6小鼠的IMQ处理区皮肤PCSK9表达较未处理区明显上调，而PCSK9基因敲除小鼠的IMQ处理区皮肤及未处理区皮肤均未见PCSK9表达(见图4)；2)C57BL/6小鼠及PCSK9基因敲除小鼠的IMQ处理区皮肤NF-kB的表达较之各自的未处理区均明显上调，但PCSK9基因敲除小鼠的IMQ处理区皮肤的NF-kB的表达较之C57BL/6小鼠明显降低(见图5)。
2、si-PCSK9转染可增强人角质形成细胞的凋亡并抑制其增殖
实验材料及试剂：
(1)人原代角质形成细胞(LifelineCellTechnology,FC-0064)；
(2)DermaLife角质形成细胞培养液(LifelineCellTechnology,LL-0007),si-PCSK9(SantaCruz,sc-45482),Lipofectamine3000转染试剂(ThermalFisher，L-3000001)，AnnexinV(BD)，PI(BD)
实验方法：
(1)培养人原代角质形成细胞，种植于6孔板，当细胞密度为60-70％时，进行si-RNA(si-Con&si-PCSK9)转染，转染后24h/48h/72h分别用MTT法测每孔细胞活性，两组细胞每个时间点各计数3个孔，取平均值，绘制细胞活性曲线；
(2)培养人原代角质形成细胞，种植于6孔板，当细胞密度为60-70％时，进行si-RNA(si-Con&si-PCSK9)转染，转染后24h/48h/72h分别取细胞进行AnnexinV及PI染色监测细胞凋亡及细胞周期，两组细胞每个时间点各计数3个孔，取平均值。
实验结果：
(1)si-PCSK9转染后的人原代角质形成细胞的存活数较之si-Con转染后细胞明显降低(P<0.05，见图6)；
(2)si-PCSK9转染后的人原代角质形成细胞凋亡较之si-Con转染后细胞凋亡明显增加(P<0.05)，而增殖(S+G2/M期细胞比例)则明显降低(P<0.05)(见图7)。
Claims (1)
1.PCSK9单克隆抗体在制备治疗炎症免疫性疾病药物中的应用，所述PCSK9属前蛋白转换酶家族。
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| US15/965,907 US11214625B2 (en) | 2015-10-29 | 2018-04-28 | Application of PCSK9 inhibitors in the preparation of drugs for the treatment of inflammatory immune diseases |
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