CN116474103B - Pdcd2l作为靶点在制备治疗炎症药物中的应用 - Google Patents
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Abstract
本发明属于生物技术领域,具体涉及PDCD2L作为靶点在制备治疗炎症药物中的应用。本发明研究发现,内皮细胞炎症是炎症干预的关键环节,且PDCD2L在内皮细胞炎症中发挥着重要作用,是干预内皮细胞炎症的关键靶点。
Description
技术领域
本发明属于生物技术领域,具体涉及PDCD2L作为靶点在制备治疗炎症药物中的应用。
背景技术
随着老龄化进程的不断加快和生活方式的改变,炎症性疾病已成为影响全球人群健康的重要威胁之一。炎症是一种由有害刺激和条件(如感染和组织损伤等)所触发的适应性反应,是多种生理和病理过程的基础。在一般情况下,炎症作为人体的自动防御反应是有益的,但如果机体调节不当,就会造成机体组织严重的损伤,包括许多传染性疾病和自身免疫性疾病、恶性肿瘤、心血管疾病、糖尿病等慢性非传染性重大疾病。
而内皮细胞在全身、局部性炎症扮演着重要角色,可以释放炎症因子、招募巨噬细胞、炎症细胞粘附穿梭进入组织,能在肺炎、血管炎、血管增生、动脉粥样硬化、炎症因子风暴、自身免疫性疾病等疾病、症状中起到早期介导作用,是干预系统性炎症的关键点。
尽管许多类型炎症的病理学已经得到了很好的研究,但目前对于炎症靶点的研究仍需深入,也缺乏针对内皮细胞炎症有效的治疗靶点和药物。因此研究炎症靶点,深入揭示炎症的生物学、分子基础和调控机制对于阐明其致病机制、发展有效的干预手段具有重要意义。
故基于此,提出本发明技术方案。
发明内容
为了解决现有技术存在的问题,本发明提供了一种PDCD2L作为靶点在制备治疗炎症药物中的应用。
为便于理解本发明,对相关原理进行解释说明:
Programmed cell death protein 2-like(PDCD2L)与细胞增殖、凋亡和小鼠胚胎发育相关,并能延迟S期细胞周期。PDCD2L穿梭于细胞核和细胞质之间,并与CRM1直接相互作用,而CRM1是蛋白质和核糖核蛋白颗粒(Ribonucleoprotein Particle,RNP)从细胞核输出的主要转运受体,PDCD2L在mRNA转运过程中具有潜在的功能。本发明也首次证明了PDCD2L与内皮细胞炎症存在重要关联,能够显著影响内皮细胞炎症因子的表达,进而调控炎症进程,在开发针对内皮细胞的炎症药物方面具有应用前景。
优选地,所述炎症为内皮细胞炎症。
优选地,所述药物包含针对PDCD2L的siRNA生物制剂。
优选地,所述PDCD2L的siRNA序列包括两组;其中:
第一组(siPDCD2L_1)的正向序列为SEQ ID NO.1:
5’-GGAATTTGGAACAATTCTATT-3’;
第一组的反向序列为SEQ ID NO.2:
5’-TAGAATTGTTCCAAATTCCTT-3’;
第二组(siPDCD2L_2)的正向序列为SEQ ID NO.3:
5’-GTGTTGCAGATGAGGATGATT-3’;
第二组的反向序列为SEQ ID NO.4:
5’-AATCATCCTCATCTGCAACAC-3’。
优选地,所述药物包含小分子生物抑制剂,所述小分子生物抑制剂为穿心莲内酯类化合物。
优选地,所述药物为口服制剂。
优选地,所述药物为注射制剂。
优选地,所述药物为喷雾制剂。
本发明的有益效果为:
本发明研究发现,内皮细胞炎症是炎症干预的关键环节,且PDCD2L在内皮细胞炎症中发挥着重要作用,是干预内皮细胞炎症的关键靶点,同时小分子穿心莲内酯作为PDCD2L抑制剂能够抑制炎症,安全性高,给药方式简单,具有较好的市场应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是过表达PDCD2L的内皮细胞过表达炎症因子mRNA结果图(***p<0.001;N=3);其中:
图1中的A是细胞中PDCD2L的mRNA表达量增加结果图;
图1中的B是炎症因子ICAM1的mRNA表达量增加结果图;
图1中的C是炎症因子IL-6的mRNA表达量增加结果图;
图1中的D是炎症因子IL-1β的mRNA表达量增加结果图;
图1中的E是炎症因子IL-8的mRNA表达量增加结果图;
图1中的F是单核细胞粘附增加的结果图。
图2是敲低PDCD2L的表达后内皮细胞炎症因子mRNA表达量降低结果图(*p<0.05;**p<0.01;***p<0.001;****p<0.0001;N=3);其中:
图2中的A是第一条沉默PDCD2L的样本和正常样本比较,PDCD2L和炎症因子的mRNA降低结果图;
图2中的B是第一条沉默PDCD2L的样本和正常样本比较,ICAM-1的mRNA表达量降低结果图;
图2中的C是第一条沉默PDCD2L的样本和正常样本比较,IL-6的mRNA表达量降低结果图;
图2中的D是第二条沉默PDCD2L的样本和正常样本比较,PDCD2L和炎症因子的mRNA降低结果图;
图2中的E是第二条沉默PDCD2L的样本和正常样本比较,ICAM-1的mRNA表达量降低结果图;
图2中的F是第一条沉默PDCD2L的样本和正常样本比较,IL-6的mRNA表达量降低结果图。
图3是穿心莲内酯影响PDCD2L的热稳定性结果图;其中:
图3中的A是相同温度下,穿心莲内酯浓度升高对PDCD2L含量的影响图;
图3中的B是不同温度下,相同穿心莲浓度对PDCD2L的稳定性的影响图。
图4是为穿心莲内酯降低内皮细胞的炎症结果图(*与对照组比较,p<0.05;**与对照组相比,p<0.01;#与模型组相比,p<0.05;##与模型组相比p<0.01,N=3;误差条代表平均值±标准差),其中:
图4中的A是穿心莲内酯对EA.hy926细胞活力的影响;
图4中的B是用ELISA法检测不同浓度穿心莲内酯处理LPS诱导的EA.hy926细胞培养基中ICAM1的含量;
图4中的C是通过qPCR检测不同浓度穿心莲内酯处理的LPS诱导的EA.hy926细胞后,细胞内ICAM1的相对比率;
图4中的D是通过qPCR检测不同浓度穿心莲内酯处理的LPS诱导的EA.hy926细胞后,细胞内IL-6的相对比率;
图4中的E是通过qPCR检测不同浓度穿心莲内酯处理的LPS诱导的EA.hy926细胞后,细胞内IL-1β的相对比率;
图4中的F是用15μM 穿心莲内酯处理LPS诱导EA.hy926细胞及过表达PDCD2L的EA.hy926细胞,通过WB检测细胞中VCAM1、ICAM1及PDCD2L的表达;
图4中的G是对图4中的F中PDCD2L的WB定量分析结果;
图4中的H是对图4中的F中ICAM1的WB定量分析结果;
图4中的I是对图4中的F中VCAM1的WB定量分析结果。
图5是穿心莲内酯有效降低了LPS诱导的PDCD2L过表达细胞中的ROS水平结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
实施例1 过表达PDCD2L影响LPS诱导的内皮细胞炎症增加
具体方法如下:
(1)PDCD2L过表达质粒的构建及慢病毒包装:PDCD2L的过表达质粒由金斯瑞合成将合成的质粒转化至stbl3菌中,按照质粒大提试剂盒进行质粒提取,随后根据慢病毒包装试剂盒说明书(百瑞极)进行病毒包装。
(2)构建过表达PDCD2L的EA.hy926细胞:病毒转染前一天,将细胞以1×105个/孔铺到24孔板中,使细胞在慢病毒转染时的数量为2×105,加入适量病毒悬液。继续培养24小时,用新鲜培养基替换含有病毒的培养基。病毒感染48小时后加入含有puro的培养基(终浓度为2μg/mL)进行筛选。
(3)WB验证EA.hy926细胞中PDCD2L过表达:将EA.hy926和过表达PDCD2L的EA.hy926细胞用PBS清洗干净后,吸干残余液体,加入适量RIPA(中)裂解液及蛋白酶抑制剂,冰上充分裂解细胞后,12000 rpm,4℃离心10 min,取上清。通过BCA试剂盒检测细胞裂解液的蛋白浓度,取50μg蛋白上样跑胶,转膜,5%脱脂奶粉室温封闭60 min,4℃摇床孵育PDCD2L和GAPDH抗体过夜,PBS清洗3遍后,室温孵育兔抗30 min,PBS清洗3遍后显影。
(4)PCR验证EA.hy926细胞中PDCD2L过表达和炎症因子检测:将EA.hy926和过表达PDCD2L的EA.hy926细胞计数后,取相等细胞量铺入6孔板中,待细胞贴壁后加入LPS(终浓度为1 μg/mL),刺激24 h后收集细胞。加入200 μL氯仿,混匀后,12000 rpm离心5 min,收集上清约400 μL至新的1.5 mL离心管中。然后加入500 μL异丙醇,混匀后静置5 min,4℃、12000rpm离心10 min,弃去上清得到沉淀,最后加入1mL 75%乙醇,4℃、12000 rpm离心10 min,清洗2次,将乙醇挥干后加入10 μL水,混匀,测定浓度后,取2.5 μg的量,加入2 μL 5X逆转录酶,加水补至10 μL,并进行逆转录。引物如表1 所示。
表1 siPDCD2L及IL6、PDCD2L、GAPDH序列
注:物种均为人。
结果如图1所示:过表达后,细胞中PDCD2L的mRNA表达量明显增加(如图1中A所示),相关的炎症因子ICAM1(如图1中B所示),IL-6(如图1中C所示),IL-1β(如图1中D所示),IL-8(如图1中E所示),SOD2(如图1中F所示),mRNA表达均明显升高,具有显著的统计学差异,与此同时,本专利发现过表达PDCD2L细胞,因为炎症粘附因子的增加,也增加了炎症细胞的粘附(如图1中G所示)。
实施例2 siRNA降低PDCD2L表达后抑制LPS诱导的内皮细胞炎症
具体方法如下:
(1)细胞实验操作:将500 mL的1640基础培养基、50 mL血清、5 mL双抗混合均匀后,作为EA.hy926细胞的培养基。将EA.hy926细胞消化后计数,并铺到六孔板中,按照lipo8000(碧云天生物科技)说明书进行操作,加入合成的siRNA(见序列表),4~6h后换液,继续培养24h后加入LPS刺激细胞,LPS终浓度为1μg/mL,刺激24h后收集细胞及细胞培养基。
(2)qPCR检测PDCD2L在EA.hy926细胞上的表达水平:为防止细胞被吹起,贴侧壁缓慢加入PBS清洗细胞3次,再加入1 mL Trizol,吹打细胞后收集到1.5 mL离心管中。加入200μL氯仿,混匀后,12000 rpm离心5 min,收集上清约400 μL至新的1.5 mL离心管中。然后加入500 μL异丙醇,混匀后静置5 min,4℃、12000 rpm离心10 min,弃去上清得到沉淀,最后加入1mL 75%乙醇,4℃、12000 rpm离心10 min,清洗2次,将乙醇挥干后加入10 μL水,混匀,测定浓度后,取2.5 μg的量,加入2 μL 5X逆转录酶,加水补至10 μL,并进行逆转录。
将以上步骤获得的cDNA为模版,使用实时荧光定量核酸扩增检测系统(qPCR)扩增PDCD2L和GAPDH,并分析其相对表达量(引物均由擎科生物合成)。具体的,如图2中A所示,qPCR结果表明沉默PDCD2L的样本和正常样本比较,显著降低了PDCD2L和炎症因子的mRNA;如图2中B所示,ICAM1 mRNA表达含量降低;如图2中C所示,IL-6的mRNA表达含量降低;如图2中D所示,qPCR结果表明第二条沉默PDCD2L的样本和正常样本比较,显著降低了PDCD2L和炎症因子的mRNA;如图2中E所示,ICAM1 mRNA表达含量降低(第二条siRNA);如图2中F所示,IL-6的mRNA表达含量降低(第二条siRNA)。*p<0.05,**p<0.01,***p<0.001。
实施例3 穿心莲内酯(Andro)抑制PDCD2L降低LPS诱导的内皮细胞炎症及活性氧(ROS)水平
具体方法如下:
(1)PDCD2L重组蛋白的表达:将PDCD2L的PET28载体转化至感受态细胞中(具体步骤参照擎科生物的DH5α感受态细胞说明书)。转化成功后,在培养基中加入卡那霉素,挑取单克隆抗体进行扩增,扩增后加入IPTG进行诱导。然后离心获得菌体,超声破碎后,4℃、4000 rpm离心30 min获得上清,过His标签亲和层析柱(具体操作参考碧云天说明书),使用含20 mM咪唑的PBS进行清洗,200 mM咪唑的PBS进行蛋白的洗脱。将收集的蛋白进行超滤(3500 rpm,4℃),超滤后过凝胶过滤色谱。取适宜浓度洗脱蛋白,加入5× loadingbuffer,电泳后用考马斯亮蓝染胶确定蛋白纯度,保存纯度较高的PDCD2L蛋白。
(2)穿心莲内酯与EA.hy926细胞裂解液中PDCD2L的细胞热转移实验(CETSA)实验:对于剂量依赖的CETSA实验,将40μL的EA.hy926细胞裂解液(5 mg/mL)与1μL不同浓度穿心莲内酯或DMSO在37℃共孵育1.5 h,使得穿心莲内酯的终浓度为0、15.6、31.3、62.5、125、250、500、1000、2000 μM,然后在65℃下加热4 min,随后20000 g、4℃离心20分钟,取上清20μL进行WB。对于温度依赖的CETSA,将40μL的EA.hy926细胞裂解液(5 mg/mL)与1μL穿心莲内酯或DMSO在37℃共孵育1.5h,使得穿心莲内酯的终浓度为400 μM,分别在50℃、60℃、70℃下加热4 min,随后20000 g、4℃离心20分钟,取上清20 μL进行WB。
结果如图3中A所示,随着穿心莲内酯浓度的升高,上清中PDCD2L蛋白增多,随着温度的升高,穿心莲内酯与对照组比较,上清中PDCD2L的含量增多,说明穿心莲内酯可能和PDCD2L具有结合作用,增强了其稳定性。如图3中B所示,进行同一温度下(65℃),不同穿心莲浓度对稳定性的影响,PDCD2L蛋白稳定性与穿心莲内酯浓度呈现相关性。
(3)穿心莲内酯降低LPS诱导的细胞炎症因子的水平:对EA.hy926细胞加入LPS及不同浓度穿心莲内酯进行刺激,LPS终浓度为1 μg/mL,刺激24h后收集细胞及细胞培养基,按照上述方法检测炎症因子的表达水平。随后,用15μM的穿心莲内酯浓度刺激LPS诱导的内皮细胞炎症,采用WB的方法检测了细胞中ICAM1、VCAM1、PDCD2L的表达。
结果如图4中所示,图4中的A为穿心莲内酯对EA.hy926细胞活力的影响,结果显示IC50值为24.5 μM。然后采用ELISA法检测不同浓度穿心莲内酯处理LPS诱导的EA.hy926细胞培养基中ICAM1的含量,结果如图4中的B所示,随着穿心莲内酯浓度的升高,ICAM1的含量下降。之后采用qPCR的方法检测了细胞中ICAM1、IL-6和IL-1β的含量,结果如图4中的C、D、E所示,穿心莲内酯降低了由LPS诱导的内皮细胞的炎症水平。通过WB的方法检测到,穿心莲内酯可以减轻LPS诱导的内皮细胞中VCAM1和ICAM1的表达,如图4中的F所示,其定量结果如图4中的G、H、I所示。
(4)穿心莲内酯降低LPS诱导的PDCD2L过表达细胞中的ROS水平:用LPS诱导EA.hy926细胞及过表达PDCD2LEA.hy926细胞后,同时15 μM的穿心莲内酯进行干预,刺激24h后采用DCFH-DA探针检测细胞中ROS的表达水平。
结果如图5所示,穿心莲内酯有效降低了LPS诱导的PDCD2L过表达细胞中的ROS水平。
综上所述,本发明得到如下结论:
1、本发明构建了PDCD2L过表达载体,包被了该载体的慢病毒感染EA.hy926细胞,结果显示过表达PDCD2L能够促进EA.hy926细胞炎症因子的表达以及对巨噬细胞的粘附。
2、本发明设计了两条针对PDCD2L的siRNA(见序列表),结果显示siRNA能够有效降低mRNA的表达,并降低了EA.hy926细胞对LPS刺激的炎症反应,发挥了抗内皮细胞炎症的作用。
3、本专利同时采用高通量药物筛选方法,基于PDCD2L靶点对其抑制剂进行了筛选,发现穿心莲内酯能够结合PDCD2L,降低炎症因子的表达。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (4)
1.PDCD2L的抑制剂在制备治疗炎症药物中的应用,其特征在于:
所述炎症为内皮细胞炎症;
所述药物包含针对PDCD2L的siRNA类型生物抑制剂;
所述PDCD2L的siRNA序列包括两组;其中:
第一组的正向序列为SEQ ID NO.1:
5’-GGAATTTGGAACAATTCTATT-3’;
第一组的反向序列为SEQ ID NO.2:
5’-TAGAATTGTTCCAAATTCCTT-3’;
第二组的正向序列为SEQ ID NO.3:
5’-GTGTTGCAGATGAGGATGATT-3’;
第二组的反向序列为SEQ ID NO.4:
5’-AATCATCCTCATCTGCAACAC-3’。
2.根据权利要求1所述的应用,其特征在于,所述药物为口服制剂。
3.根据权利要求1所述的应用,其特征在于,所述药物为注射制剂。
4.根据权利要求1所述的应用,其特征在于,所述药物为喷雾制剂。
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