CN116059366A - 一种搭载p16-siRNA的纳米药物的构建及其在治疗梗死后心室重塑中的应用 - Google Patents
一种搭载p16-siRNA的纳米药物的构建及其在治疗梗死后心室重塑中的应用 Download PDFInfo
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Abstract
本发明公开了一种搭载p16‑siRNA的纳米药物的构建及其在治疗梗死后心室重塑中的应用,属于医药技术领域。本发明人研究发现,携带p16‑siRNA的纳米药物经静脉注射后,可以靶向心肌梗死区成纤维细胞并敲低该细胞p16的表达,抑制心肌梗死区NLRP3‑炎症小体通路,可以显著降低心肌炎症、改善心脏功能、减少心肌梗死区及周围区纤维化,从而治疗心梗后心室重塑,为临床治疗心梗后心室重塑提供了一种新的治疗方案。
Description
技术领域
本发明属于医药技术领域,涉及一种搭载p16-siRNA的纳米药物的应用及其产品,具体涉及一种搭载p16-siRNA的纳米药物的构建及其在治疗梗死后心室重塑中的应用。
背景技术
心血管***疾病是导致死亡的第一要素,据《中国心血管病报告2017》公布的数据显示,我国农村、城市心血管疾病死亡占全部死因的比例分别高达45%和42%,即在每年的死亡人数中,其中将近一半的死因是心血管疾病所致。心肌梗死(myocardial infarction,MI)在心血管***疾病死亡原因中位居首位,其发生率和心室重构程度与衰老紧密相关,随年龄增长而逐渐增加。近一半的MI患者经历了不良的心室重塑,导致心肌细胞肥大、间质纤维化、左心室扩张和心力衰竭。作为MI的重要危险因素,炎性衰老是心室重构恶化的重要原因,显著增加了患者的死亡率。在规范化使用传统的血管紧张素转化酶抑制剂(ACEI)、β受体阻滞剂(β-blocker)、安体舒通等药物,仍无法控制梗死后心室重塑的进程,因此,亟待寻找控制梗死后心室重塑的关键药物治疗靶点和靶向性药物。
目前尚无关于以p16作为临床防治MI后心室重塑的新型关键靶点,以及搭载p16-siRNA 的纳米药物的应用和产品治疗MI后心室重塑的报道。
发明内容
本发明的目的是解决现有技术的不足,提供一种搭载p16-siRNA的纳米药物的应用及其产品。
本发明技术方案如下:
本发明的第一个目的是提供p16作为治疗靶点在制备或筛选治疗心肌梗死后的心室重塑的药物中的应用。
本发明的第二个目的是提供将p16作为靶点筛选用于下调α-SMA、POSTN、CollagenI、 IL-1β、IL-6、TNF-α、NLRP3、ASC、Caspase-1中的一种或多种蛋白的表达水平的制品中的应用。
本发明的第三个目的是提供一种治疗心肌梗死后的心室重塑的药物,所述药物包括抑制或沉默p16表达的核酸分子。所述抑制或沉默p16表达的核酸分子为主要活性成分。
进一步的,所述药物为纳米药物。
进一步的,所述沉默p16表达的核酸分子为p16-siRNA。
进一步的,所述p16-siRNA靶向p16编码区序列片段5′-TCTCAGAGGATCCCGGAAATTT-3′ (SEQ ID NO.4)
进一步的,所述p16-siRNA序列为:
正义链:
5′-TGCTGAGCGGAACGCAAATATCGCACGTTTTGGCCACTGACTGACGTGCGATATGCGTTCCGCTdTdT-3′(SEQ ID NO.1);
反义链:
5′-dTdTTGCTGAGCGGAACGCAAATATCGCACGTTTTGGCCACTGACTGACGTGCGATATGCGTTCCGCT-3′(SEQ ID NO.2)。
进一步的,所述阴性对照(NC)-siRNA序列为:
正义链:
5′-UUCUCCGAACGUGUCACGUTTdTdT-3′(SEQ ID NO.5);
反义链:
5′-dTdTACGUGACACGUUCGGAGAATT-3′(SEQ ID NO.6)。
进一步的,所述药物为搭载p16-siRNA的纳米颗粒。
进一步的,所述药物还包括递送***,所述递送***包括FH肽、脂质膜(Li)、中性粒细胞膜蛋白(NMP)、介孔二氧化硅纳米颗粒(MSN);
优选的,所述药物是由介孔二氧化硅纳米颗粒装载p16-siRNA,脂质膜包覆装载p16-siRNA 的介孔二氧化硅纳米颗粒,所述FH肽由脂质膜包裹后得到脂质膜包裹的FH肽,所述脂质膜包裹的FH肽、中性粒细胞膜蛋白(NMP)嵌入包覆装载p16-siRNA的介孔二氧化硅纳米颗粒的脂质膜,所述药物简称为FNLM-sip16(图12)。
进一步的,所述FH肽包括苯丙氨酸(F)组氨酸(H)。
进一步的,所述FH肽氨基酸序列为FHKHKSPALPPV(SEQ ID NO.3)。
在某一个特殊的实施例中,所述搭载p16-siRNA的纳米颗粒为搭载SEQ ID NO.1和SEQ ID NO.2所示的p16-siRNA,并包括由SEQ ID NO.3所示的FH肽、中性粒细胞膜蛋白(NMP)、脂质膜(Li)、介孔二氧化硅纳米颗粒(MSN)组成的递送***,该递送***简称FNLM,该搭载p16-siRNA的纳米颗粒FNLM-sip16。
经静脉注射所述FNLM-sip16,能够靶向MI区的成纤维细胞并敲低该细胞内p16的表达;同时,能够抑制MI区NLRP3-炎症小体信号通路,可以显著降低心肌炎症、改善心脏功能、减轻MI区及其周围区纤维化程度。
本发明的第四个目的是提供抑制或沉默p16表达的物质在制备治疗心肌梗死后的心室重塑的药物中的应用。
进一步的,所述沉默p16基因表达的物质为抑制或沉默p16表达的核酸分子或前述治疗心肌梗死后的心室重塑的药物。
本发明技术方案相对于现有技术有益效果在于:
本发明发现衰老标志分子p16在MI后的心室重塑进程中发挥重要作用,p16加剧MI后心室重塑区心脏成纤维细胞的炎性衰老及其衰老相关分泌表型,此作用与p16促进转录调控 NLRP3表达进而激活NLRP3炎症小体信号通路这一新的调控机制密切相关,因此,本发明确定p16作为临床防治MI后心室重塑的新型关键靶点。
针对目前临床上尚缺乏靶向MI后心脏内成纤维细胞的特异性抑制p16的小分子药物或生物活性药物的问题,本发明构建了搭载p16-siRNA的靶向心梗后成纤维细胞的纳米药物,即苯丙氨酸(F)组氨酸(H)肽-中性粒细胞膜蛋白(NMP)-脂质(Li)膜-介孔二氧化硅纳米颗粒(MSN) 核心-p16-siRNA,即FNLM-sip16。
我们通过科学实验证实:纳米药物p16-siRNA-FNLM经静脉注射,可以靶向心肌梗死区成纤维细胞并抑制该细胞p16的表达,从而下调心肌梗死区NLRP3-炎症小体信号通路,抑制衰老相关分泌表型的促炎症因子IL-1β、IL-6和TNF-α的表达,改善心脏功能、减轻MI区及其周围区纤维化程度,进而治疗MI后的心室重塑,改善心脏功能。同时,我们的研究表明,这种纳米药物在体内无明显的肝肾毒性,表明这种纳米药物的安全性高。
本发明为临床治疗MI后心室重塑提供了一种新的治疗方案。
附图说明
图1为WT及p16敲除(p16-KO)小鼠心梗4周后超声心动图的代表性图像(图1A)以及左心室射血分数(LVEF)和左心室缩短分数(LVFS)统计图(图1B);
图2为WT及p16-KO小鼠心梗4周后心脏组织Masson染色的代表性显微图像(图2A)和纤维化面积统计图(图2B);
图3为WT及p16-KO小鼠心梗4周后心肌梗死区Collagen I免疫组化染色代表性显微图像(图3A)及阳性面积占总面积比值统计图(图3B);
图4为WT及p16-KO小鼠心梗4周后心肌梗死区α-SMA免疫组化染色代表性显微图像(图 4A)及阳性面积占总面积比值统计图(图4B);
图5为WT及p16-KO小鼠心梗4周后心肌梗死区α-SMA和Collagen I蛋白表达水平的Western blot检测结果(图5A)及其统计图(图5B);
图6为WT及p16-KO小鼠心梗4周后血清BNP水平统计图;
图7为WT及p16-KO小鼠心梗4周后心肌梗死区IL-1β免疫组化染色代表性显微图像(图 7A)及阳性面积占总面积比值统计图(图7B);
图8为WT及p16-KO小鼠心梗4周后心肌梗死区IL-6免疫组化染色代表性显微图像(图 8A)及阳性面积占总面积比值统计图(图8B);
图9为WT及p16-KO小鼠心梗4周后心肌梗死区TNF-α免疫组化染色代表性显微图像(图 9A)及阳性面积占总面积比值统计图(图9B);
图10为WT及p16-KO小鼠心梗4周后梗死区pro-IL-1β、IL-1β和TNF-α的蛋白表达水平的Western blot检测结果(图10A)和统计图(图10B);
图11为WT及p16-KO小鼠心梗4周后梗死区NLRP3信号通路分子NLRP3和ASC的蛋白表达水平的Western blot检测结果(图11A)和统计图(图11B);
图12为FNLM-sip16构建模式图;
图13为FNLM-sip16透射电子显微镜的代表性图像;
图14为MSNs、MSNs-sip16和FNLM-sip16的Zeta电位统计图;
图15为MSNs、MSNs-sip16和FNLM-sip16的直径统计图;
图16为CY3荧光标记的FNLM-sip16与心脏成纤维细胞共孵育后的免疫荧光代表性显微图像;
图17为小鼠心脏梗死区和非梗死区由CY3标记的阴性对照(NC)组FNLM-siNC或FNLM-sip16的免疫荧光染色代表性显微图像;
图18为FNLM-siNC或FNLM-sip16处理后小鼠肝脏和肾脏的HE染色图像;
图19为FNLM-siNC或FNLM-sip16处理后小鼠血清谷丙转氨酶(ALT)水平统计图;
图20为FNLM-siNC或FNLM-sip16处理后小鼠血清谷草转氨酶(AST)水平统计图;
图21为FNLM-siNC或FNLM-sip16处理后小鼠血清肌酐水平统计图;
图22为FNLM-siNC或FNLM-sip16处理后小鼠血清尿素氮水平统计图;
图23为各处理组小鼠超声心动图的代表性图像(图23A)以及左心室射血分数(LVEF)统计图(图23B)和左心室缩短分数(LVFS)统计图(图23C);
图24为各处理组小鼠血清BNP水平统计图;
图25为心梗小鼠4周后心脏组织Masson染色的代表性显微图像(图25A)和纤维化面积统计图(图25B);
图26为心梗小鼠4周后心肌梗死区α-SMA、POSTN和Collagen I蛋白表达水平的Western blot检测结果(图26A)和统计图(图26B);
图27为心梗小鼠4周后心肌梗死区的α-SMA、POSTN和Collagen I免疫组化染色代表性显微图像(图27A)及阳性面积占总面积比值统计图(图27B);
图28为心梗小鼠4周后梗死区NLRP3信号通路分子NLRP3、ASC、Caspase-1、TNF-α、pro-IL-1β、IL-1β和IL-6的蛋白表达水平的Western blot检测结果(图28A)和统计图(图28B);
图29为心梗小鼠4周后心肌梗死区的TNF-α、IL-1β和IL-6免疫组化染色代表性显微图像 (图29A)及阳性面积占总面积比值统计图(图29B);
图30为心梗小鼠4周后心肌梗死区的NLRP3、ASC和Caspase-1免疫组化染色代表性显微图像(图30A)及阳性面积占总面积比值统计图(图30B);
图31为心梗小鼠4周后梗死区p16的蛋白表达水平的Western blot检测结果(图31A)和统计图(图31B);
图32为心梗小鼠4周后心肌梗死区的p16免疫组化染色代表性显微图像(图32A)及阳性面积占总面积比值统计图(图32B)。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。
除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。未单独注明来源的试剂或材料为本领域常规的试剂或材料,可按照本领域常识选取或按照常规方式处理或者按照厂商建议条件处理或使用,以及可通过常规渠道购买获得。文中所述的较佳实施方法与材料仅作示范之用。
实施例1关于p16作为心肌梗死后的心室重塑靶点的验证
(一)构建小鼠MI模型
使用12月龄野生型(WT)小鼠或p16敲除小鼠(p16-KO)进行MI模型构建。禁食4-6小时后,对小鼠进行称重并记录。根据体重,按照50mg/kg使用戊巴比妥钠进行腹腔注射麻醉。固定小鼠四肢,手术区域皮毛消毒处理后,使用备皮刀刮去小鼠颈胸部毛发。分离皮肤、肌肉等组织以使气管暴露,打开并连接动物呼吸机,观察到胸廓随机器频率规律起伏后固定。外科手术剪剪开小鼠胸前皮肤,约1厘米长斜切口,钝性分离胸前肌肉,暴露肋骨、肋间肌,找到第三肋间隙,钝头弯镊刺破肋间肌及胸膜,伸入自制拉钩固定胸骨,弯头镊钝性分离肋间肌,撑开肋骨,暴露心脏,探查左心耳,直视下用无菌缝合线结扎小鼠的心肌和血管,外科结确认牢固,张力适宜,结扎后局部心肌组织明显由红润变苍白。拔去气管插管针,见小鼠恢复正常呼吸心跳,置于保温电热毯上保持体温待其苏醒,进行编号。假手术组仅对小鼠进行开胸及进针、出针处理,并不结扎血管,其余步骤均与MI建模相同。
(二)动物模型分组
将实验动物分为4个组:
(1)12月龄WT+MI小鼠未结扎LAD的假手术组(WT+sham组,n=8);
(2)12月龄p16-KO+MI小鼠未结扎LAD的假手术组(p16-KO+sham组,n=8);
(3)建模成功后存活的WT+MI心肌梗死小鼠组(WT+MI组,n=8);
(4)建模成功后存活的p16-KO+MI心肌梗死小鼠组(p16-KO+MI组,n=8)。
(三)评估心梗4周后小鼠的心功能和梗死区纤维化及炎症水平
在心肌梗死造模4周后进行心脏超声心动图(心超)检测。使用高分辨率成像***检测小鼠心脏,于长轴和短轴方向测量左室收缩末期内径(LVESD),左室舒张末期内径(LVEDD),左室室壁厚度(LVPWT),室间隔厚度(IVST),并计算左室射血分数(LVEF)和左室缩短分数 (LVFS),评估小鼠心功能;ELISA技术检测外周血清的脑钠尿肽(BNP)水平。
进一步将小鼠以1%戊巴比妥钠麻醉,眼球取血后脱颈椎处死,打开胸腔,冲洗心脏干净后剪下,滤纸上去除周围组织。心脏标本取心尖部于4%的多聚甲醛中固定24h以上,梯度乙醇脱水,二甲苯透明石蜡包埋。石蜡切片厚度为5μm,37℃烘箱内烤片24h。通过Masson染色、免疫组织化学染色检测纤维化相关蛋白α-SMA和Collagen I,炎症蛋白IL-1β、IL-6和TNF-α。其余心脏组织以蛋白裂解液提取组织蛋白,通过免疫印迹检测纤维化相关蛋白炎症因子pro-IL-1β、IL-1β、TNF-α,NLRP3信号通路蛋白NLRP3、ASC。血液标本自然凝固,4℃3000g 离心25min后吸取留存血清检测其中BNP水平。
结果见图1-11:
图1为WT及p16敲除(p16-KO)小鼠心梗4周后超声心动图的代表性图像以及左心室射血分数(LVEF)和左心室缩短分数(LVFS)统计图。其中图1A为各组小鼠超声心动图,图1B为左心室射血分数(LVEF)统计图和左心室缩短分数(LVFS)统计图,由图1可以看出:与未造模小鼠相比,MI造模4周后小鼠左心室射血分数(LVEF)和左心室缩短分数(LVFS)均降低,小鼠心功能下降;与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周后左心室射血分数(LVEF)和左心室缩短分数(LVFS)均升高,小鼠心功能改善。这些结果说明:p16敲除可以改善小鼠心功能。
图2为WT及p16-KO小鼠心梗4周后心脏组织Masson染色的代表性显微图像和纤维化面积统计图。与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周后Masson染色阳性面积明显减小。结果显示:p16敲除明显降低了心梗小鼠心脏梗死区纤维化水平。
图3为WT及p16-KO小鼠心梗4周后心肌梗死区Collagen I免疫组化染色代表性显微图像及阳性面积占总面积比值统计图。
图4为WT及p16-KO小鼠心梗4周后心肌梗死区α-SMA免疫组化染色代表性显微图像及阳性面积占总面积比值统计图。
结果显示:与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周后Collagen I和α-SMA阳性面积明显减小。这一结果表明:p16敲除明显降低了心梗小鼠心脏梗死区Collagen I和α-SMA 的表达。
图5为WT及p16-KO小鼠心梗4周后心肌梗死区α-SMA和Collagen I蛋白表达水平的Western blot检测结果及其统计图。与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周后心脏梗死区α-SMA和Collagen I表达明显降低。结果显示:p16敲除明显降低了心梗小鼠心脏梗死区α-SMA和Collagen I的表达。同时结合前述实验结果,表明p16敲除明显减少心梗小鼠心脏梗死区纤维化蛋白表达,改善心梗小鼠心脏梗死区的纤维化。
图6为WT及p16-KO小鼠心梗4周后血清BNP水平统计图。结果显示:与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周后血清BNP水平明显降低。
图7为WT及p16-KO小鼠心梗4周后IL-1β免疫组化染色代表性显微图像及阳性面积占总面积比值统计图。
图8为WT及p16-KO小鼠心梗4周后IL-6免疫组化染色代表性显微图像及阳性面积占总面积比值统计图。
图9为WT及p16-KO小鼠心梗4周后TNF-α免疫组化染色代表性显微图像及阳性面积占总面积比值统计图。
与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周后IL-1β、IL-6和TNF-α阳性面积明显减小。结果显示:p16敲除明显降低了心梗小鼠心脏梗死区炎症因子IL-1β、IL-6和TNF-α的表达。
图10为WT及p16-KO小鼠心梗4周后梗死区pro-IL-1β、IL-1β和TNF-α的蛋白表达水平的Western blot检测结果和统计图。与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周心脏梗死区pro-IL-1β、IL-1β和TNF-α表达明显降低。结果显示:与WT组相比,p16敲除可明显减少心梗后梗死区的炎症因子,改善心梗后炎性衰老。
图11为WT及p16-KO小鼠心梗4周后梗死区NLRP3信号通路分子NLRP3和ASC的蛋白表达水平的Western blot检测结果和统计图。与WT+MI小鼠相比,p16-KO+MI小鼠MI造模4周心脏梗死区NLRP3和ASC表达明显降低。结果显示:与WT组相比,p16敲除可明显降低心梗小鼠NLRP3和ASC蛋白的表达。同时结合前述实验结果,表明p16敲除可以改善心梗小鼠心功能,缓解心梗小鼠心肌心和炎症,并减少NLRP3信号的激活。
实施例2一种搭载p16-siRNA的纳米药物的构建:
(一)构建搭载p16-siRNA的纳米药物
(1)培养人外周血中性粒细胞后,收集细胞质膜,对细胞质膜进行超声处理,获得中性粒细胞膜蛋白(NMP)。
(2)将3g十六烷基三甲基溴化铵(CTAB)和0.15mL三乙醇胺溶解在60mL蒸馏水中。在60℃下培养1小时后,添加16mL环己烷和4mL正硅酸乙酯(TEOS),连续搅拌24小时后离心,收集产物并用乙醇重悬数次后得到介孔纳米硅(MSNs)。为了去除残留的CTAB,用硝酸铵搅拌3小时,并重复两次。最终产物用蒸馏水稀释至浓度为20mg/mL,获得MSNs悬液。
(3)以SEQ ID NO.1、SEQ ID NO.2所示核苷酸序列,分别人工合成p16-siRNA片段,置于DEPC水,获得浓度为500nM的p16-siRNA悬液,将150μL p16-siRNA悬液与150μL步骤(2)制备的MSN悬液和700μL 3M氯化钙溶液在超声作用下混匀15分钟,获得装载 p16-siRNA的介孔二氧化硅纳米颗粒(MSNs-sip16)水悬浮液。
(4)为进行脂质涂层,将二硬脂酰磷脂酰乙醇胺(DMPC,厂家:阿拉丁,规格:D163596; 100mg)、磷脂聚乙二醇活性酯(DSPE-PEG-NHS,厂家:西安瑞禧生物科技有限公司,规格:R-0042;500mg)和阳离子脂质材料(2,3-二油酰基-丙基)-三甲胺DOTAP以76.2:3.8:20的摩尔比例溶解在氯仿中,并通过蒸发有机溶剂制备脂质膜(Li)。
(5)FH肽由中肽生化有限公司合成,其氨基酸序列为“FHKHKSPALPPV”。为了使FH肽在纳米颗粒上结合,采用脂质膜修饰包裹FH肽,脂质膜修饰包裹FH肽的其余操作与步骤(4)相同,区别在于DMPC+DSPE-PEG-NHS+DOTAP的体系内还添加FH肽,使 DSPE-PEG-NHS和FH肽以2:1的摩尔比混合,进行水合作用,并在室温下搅拌过夜,最终合成脂质膜包被的FH肽DSPE-PEG-FH。取部分DSPE-PEG-FH溶液与步骤(1)制备的中性粒细胞膜蛋白混合均匀,水浴超声5~10秒,然后室温静置6h,让中性粒细胞膜蛋白嵌入至 DSPE-PEG-FH,获得嵌入至DSPE-PEG-FH中的中性粒细胞膜蛋白。
(6)用步骤(3)制备的MSNs-sip16水悬浮液与步骤(4)脂质膜混合均匀,然后水浴超声5~10秒,再室温静置6h,然后将水合产物连续挤压通过1000、400、200纳米聚碳酸酯多孔膜,获得脂质膜包裹的装载p16-siRNA的介孔二氧化硅纳米颗粒体系。为进行中性粒细胞膜蛋白(NMP)***,添加与脂质膜包裹的装载p16-siRNA的介孔二氧化硅纳米颗粒体系相同体积的步骤5中嵌入DSPE-PEG-FH中的中性粒细胞膜蛋白混合,进行30分钟的超声处理。
(7)通过10000g离心5分钟,去除多余的脂质、蛋白质和肽,得到FH-NMP-LiMSNs-p16 siRNA,即FNLM-sip16(图12),并重新悬浮在PBS中使用。
上述产物观察结果见图12-15:
图12为FNLM-sip16构建模式图。所述介孔二氧化硅纳米颗粒装载p16-siRNA,脂质膜包覆装载p16-siRNA的介孔二氧化硅纳米颗粒,所述FH肽由脂质膜包裹后得到脂质膜包裹的 FH肽,所述脂质膜包裹的FH肽、中性粒细胞膜蛋白(NMP)嵌入包覆装载p16-siRNA的介孔二氧化硅纳米颗粒的脂质膜。
图13为FNLM-sip16透射电子显微镜的代表性图像,显示FNLM-sip16的结构形态。可以看出:成功的包装的FNLM-sip16存在脂质涂层和暗的单分散二氧化硅微球核。
图14为步骤(2)制备的MSNs、步骤(3)制备的MSNs-sip16和步骤(7)制备的FNLM-sip16的Zeta电位统计图,代表了胶体分散系稳定性。可以看出:其电位在±30V之内,较容易发生凝结或凝聚,从而释放siRNA。
图15为步骤(2)制备的MSNs、步骤(3)制备的MSNs-siR和步骤(7)制备的FNLM-siR的直径统计图,其直径采用动态光散射实验(Dynamic light scattering,DLS)测量,结果显示:脂质涂层的存在增加了纳米颗粒的尺寸,平均直径为175nm。
(二)FNLM-sip16与心脏成纤维细胞共孵育
为了制备CY3标记的FNLM-sip16,我们使用SilencerTMsiRNA标记试剂盒(Invitrogen, AM1632),按顺序添加无核酸酶水、10X标记缓冲液、dsRNA*和标记染料,在37℃下孵育1 小时。添加5M NaCl和预冷的100%乙醇并充分混合,在-20℃下孵育20-30分钟。在4℃下高速离心20分钟,去除上清液并用70%乙醇清洗沉淀颗粒。室温下高速离心5分钟,去除上清液,干燥沉淀颗粒,获得标记的RNA并悬浮于20μl的无核酸酶水中。
为了观察纳米粒的内体逃逸,将心脏成纤维细胞接种在共聚焦培养皿上,缺氧处理24小时后与FNLM-sip16共培养24小时。然后将细胞洗涤三次,并通过与DAPI一起培养进行细胞核染色并在荧光显微镜下观察。
结果见图16:
图16CY3荧光标记的FNLM-sip16与心脏成纤维细胞共孵育后的免疫荧光代表性显微图像,显示FNLM-sip16可以进入心脏成纤维细胞内。
实施例3纳米材料搭载p16-siRNA可在体内靶向小鼠MI区域:
(一)构建小鼠MI模型
使用12月龄野生型(WT)小鼠(购买自北京维通利华实验动物技术有限公司)进行MI模型构建。方法同实施实例1。
(二)小鼠纳米药物体内给药
在利用12月龄WT小鼠建立MI模型后,将动物分为三个组:
(1)12月龄WT小鼠未结扎左冠状动脉前降支(LAD)的假手术组(WT+sham组,n=6);建模成功后存活的WT+MI心肌梗死小鼠随机分为
(2)心肌梗死注射由FNLM装载阴性对照(NC)-siRNA的纳米药物组 (WT+MI+FNLM-siNC组,n=6),其中FNLM-siNC构建与FNLM-sip16构建方法完全一致,只是将p16-siRNA更换为SEQ ID NO.5和SEQ ID NO.6所示NC-siRNA;
(3)心肌梗死注射实施例2制备的搭载p16-siRNA的纳米药物组(WT+MI+FNLM-sip16 组,n=6)。
心肌梗死造模后隔天,WT+sham组尾静脉注射200μL PBS、WT+MI组尾静脉注射 200μLFNLM-sip16,持续1周。
注射后第1天或28天采集血样,以测定血清谷丙转氨酶(ALT)、谷草转氨酶(AST)评估肝功能,肌酐(Cr)和尿素氮(BUN)评估肾功能。然后处死小鼠,收集主要器官并用4%的PFA固定。然后将器官石蜡包埋并切片进行H&E染色。
结果见17-22:
图17为小鼠心脏梗死区和非梗死区WT+MI+FNLM-siNC组或WT+MI+FNLM-sip16组的免疫荧光染色代表性纤维图像,DAPI标记细胞核,CY3标记FNLM-siNC或FNLM-sip16。可以看出:心肌梗死后,FNLM-siNC和FNLM-sip16均向梗死区大量归巢,但在心脏的非梗死区鲜有归巢,说明它们可以靶向心肌梗死小鼠的心脏梗死区。
图18为FNLM-siNC或FNLM-sip16给药后小鼠肝脏和肾脏的HE染色图像。可以看出:FNLM-siNC及FNLM-sip16给药组的小鼠的肝脏和肾脏组织与对照组相比均无明显病理改变及炎症反应。
图19为FNLM-siNC或FNLM-sip16给药后小鼠血清谷丙转氨酶(ALT)水平统计图。
图20为FNLM-siNC或FNLM-sip16给药后小鼠血清谷草转氨酶(AST)水平统计图,显示小鼠肝脏功能。
可以看出:与对照组相比,FNLM-siNC及FNLM-sip16给药组的小鼠ALT、AST水平无明显改变,表明FNLM-siNC及FNLM-sip16在体内无明显肝脏毒性。
图21为FNLM-siNC或FNLM-sip16给药后小鼠血清肌酐(Cr)水平统计图。
图22为FNLM-siNC或FNLM-sip16给药后小鼠血清尿素氮(BUN)水平统计图,显示小鼠肾脏功能。
可以看出:与对照组相比,FNLM-siNC及FNLM-sip16处理组的小鼠血清尿素氮及肌酐水平交对照组无明显改变,表明FNLM-siNC及FNLM-sip16在体内无明显肾脏毒性。
实施例4
纳米材料搭载p16-siRNA进入MI区心脏成纤维细胞,改善心功能并减轻梗死区及其周围区的纤维化程度与炎症反应:
为了进一步证明我们构建的纳米材料进入MI区心脏成纤维细胞,可以改善心功能并减轻梗死区及其周围区的纤维化程度与炎症反应,我们使用12月龄的小鼠构建MI模型,分为12 月龄WT+MI小鼠未结扎左冠状动脉前降支(LAD)的假手术组(WT+sham组,n=6);FNLM-siNC 处理组(WT+MI+FNLM-siNC组,n=6)和FNLM-sip16处理组(WT+MI+FNLM-siNC组,n=6); FNLM-siNC处理组和FNLM-sip16处理组每周通过尾静脉注射1次FNLM-siNC或FNLM-sip16,WT+sham组注射PBS,持续4周,同实施例2。进行心脏超声心动图检测。使用高分辨率成像***检测小鼠心脏,并计算左室射血分数(LVEF)和左室缩短分数(LVFS),评估小鼠心功能;通过ELISA技术检测外周血清的脑钠尿肽(BNP)水平。进一步通过免疫组织化学染色检测小鼠心脏纤维化相关蛋白α-SMA、POSTN和Collagen I,炎症因子IL-1β、IL-6和TNF-α,NLRP3信号通路分子NLRP3、ASC和Caspase-1。其余心脏组织用于提取心脏蛋白,通过Western Blot检测纤维化相关蛋白α-SMA、POSTN和Collagen I,炎症因子IL-1β、IL-6 和TNF-α,以及NLRP3信号通路分子NLRP3、ASC和Caspase-1的蛋白表达水平。
结果见图23-32:
图23为各处理组小鼠的超声心动图、左心室射血分数(LVEF)及左心室缩短分数(LVFS)的统计图,其中图23A为超声心动图,图23B左心室射血分数(LVEF)统计图,图23C为左心室缩短分数(LVFS)统计图,由图23可以看出:与对照组相比,FNLM-sip16给药明显增高了小鼠心梗4周后的左心室射血分数(LVEF)和左心室缩短分数(LVFS),说明FNLM-sip16给药可以改善心梗小鼠的心功能。
图24为各处理组小鼠血清脑钠尿肽(BNP)水平统计图,可以看出:与对照组相比,FNLM-sip16给药组小鼠外周血BNP水平降低,提示FNLM-sip16给药可以改善心梗小鼠的心衰。
图25为心梗小鼠4周后心脏组织Masson染色的代表性纤维性图像和纤维化面积统计图,结果显示:与对照组相比,FNLM-sip16给药组梗死区及其周围区的纤维化面积明显减少。
图26为心梗小鼠4周后心脏梗死区α-SMA、POSTN和Collagen I蛋白表达水平的Western blot检测结果,结果发现:与FNLM-siNC处理组相比,FNLM-sip16给药后小鼠心肌梗死区的α-SMA、Collagen I和POSTN等促纤维化蛋白表达水平明显降低。
图27为通过免疫组织化学染色进一步检测心梗小鼠4周后心脏梗死区α-SMA、POSTN 和Collagen I,与FNLM-siNC组相比,FNLM-sip16给药后小鼠心肌梗死区的α-SMA、Collagen I和POSTN阳性面积显著降低。结果证明:与FNLM-siNC组相比,FNLM-sip16给药可以明显抑制心梗后梗死区的促纤维化蛋白表达,减轻心梗后纤维化程度。
图28为心梗小鼠4周后心脏梗死区NLRP3、ASC、Caspase-1、pro-IL-1β、IL-1β、IL-6和TNF-α蛋白表达水平的Western blot检测结果。结果显示:与对照组相比,FNLM-sip16给药后小鼠梗死区的NLRP3信号通路分子NLRP3、ASC和Caspase-1以及炎症因子IL-1β、IL-6和TNF-α的蛋白表达水平明显降低。
图29为免疫组织化学染色进一步检测心梗小鼠4周后心脏梗死区IL-1β、IL-6和TNF-α,结果显示:与FNLM-siNC组相比,FNLM-sip16处理后小鼠心肌梗死区的IL-1β、IL-6和TNF-α阳性面积明显降低。结果证明,FNLM-sip16给药可以明显减少心梗后梗死区的炎症因子,改善心梗后炎性衰老。
图30为免疫组织化学染色进一步检测心梗小鼠4周后心脏梗死区NLRP3、ASC和Caspase-1,结果显示:与FNLM-siNC组相比,FNLM-sip16给药后小鼠心肌梗死区的NLRP3、ASC和Caspase-1阳性面积明显减小。结果证明,FNLM-sip16给药可以明显减少心梗后梗死区NLRP3信号通路分子表达。
图31为心梗小鼠4周后心脏梗死区p16蛋白表达水平的Western blot检测结果,结果显示: FNLM-sip16给药可以明显抑制心梗区p16蛋白的表达,同时结合前述实验结果,表明抑制心梗区p16蛋白的表达能够治疗MI后的心室重塑,改善心脏功能。
图32为免疫组织化学染色进一步检测心梗小鼠4周后心脏梗死区p16,与FNLM-siNC组相比,FNLM-sip16给药后小鼠心肌梗死区的p16阳性细胞数目明显减少。以上结果证明 FNLM-sip16可以明显减少心梗区p16蛋白的表达,同时结合前述实验结果,表明抑制心梗区 p16蛋白的表达能够治疗MI后的心室重塑,改善心脏功能。
Claims (10)
1.p16作为治疗靶点在制备或筛选治疗心肌梗死后的心室重塑的药物中的应用。
2.将p16作为靶点筛选用于下调α-SMA、POSTN、Collagen I、IL-1β、IL-6、TNF-α、NLRP3、ASC、Caspase-1中的一种或多种蛋白的表达水平的制品中的应用。
3.一种治疗心肌梗死后的心室重塑的药物,其特征在于,所述药物包括抑制或沉默p16表达的核酸分子。
4.根据权利要求3所述的治疗心肌梗死后的心室重塑的药物,其特征在于,所述沉默p16表达的核酸分子为p16-siRNA。
5.根据权利要求4所述的治疗心肌梗死后的心室重塑的药物,其特征在于,所述p16-siRNA序列为:
正义链:
5′-TGCTGAGCGGAACGCAAATATCGCACGTTTTGGCCACTGACTGACGTGCGATATGCGTTCCGCTdTdT-3′(SEQ ID NO.1);
反义链:
5′-dTdTTGCTGAGCGGAACGCAAATATCGCACGTTTTGGCCACTGACTGACGTGCGATATGCGTTCCGCT-3′(SEQ ID NO.2)。
6.根据权利要求4所述的治疗心肌梗死后的心室重塑的药物,其特征在于,所述药物为搭载p16-siRNA的纳米颗粒。
7.根据权利要求6所述的治疗心肌梗死后的心室重塑的药物,其特征在于,所述药物还包括递送***,所述递送***包括FH肽、脂质膜、中性粒细胞膜蛋白、介孔二氧化硅纳米颗粒;
优选的,所述药物是由介孔二氧化硅纳米颗粒装载p16-siRNA,脂质膜包覆装载p16-siRNA的介孔二氧化硅纳米颗粒,所述FH肽由脂质膜包裹后得到脂质膜包裹的FH肽,所述脂质膜包裹的FH肽、中性粒细胞膜蛋白嵌入包覆装载p16-siRNA的介孔二氧化硅纳米颗粒的脂质膜。
8.根据权利要求7所述的治疗心肌梗死后的心室重塑的药物,其特征在于,所述FH肽氨基酸序列为FHKHKSPALPPV(SEQ ID NO.3)。
9.抑制或沉默p16表达的物质在制备治疗心肌梗死后的心室重塑的药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述沉默p16基因表达的物质为抑制或沉默p16表达的核酸分子或权利要求3所述治疗心肌梗死后的心室重塑的药物。
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