CN116656619A - 用于胰腺癌治疗的工程化外泌体 - Google Patents
用于胰腺癌治疗的工程化外泌体 Download PDFInfo
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- CN116656619A CN116656619A CN202310684413.5A CN202310684413A CN116656619A CN 116656619 A CN116656619 A CN 116656619A CN 202310684413 A CN202310684413 A CN 202310684413A CN 116656619 A CN116656619 A CN 116656619A
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明属于医学领域,具体涉及一种用于胰腺癌治疗的工程化外泌体。所述工程化外泌体含有BRCA1/2 siRNA。本发明通过BRCA1/2 siRNA实现针对BRCA1、BRCA2靶向干预,从而将非同源重组修复缺陷的胰腺癌转变为同源重组修复缺陷的胰腺癌。本发明建立的胰腺癌细胞靶向药物递送系统,能够实现针对胰腺癌细胞的组织特异性干预,避免对正常细胞的影响,给胰腺癌患者带来了福音。
Description
技术领域
本发明属于医学领域,具体涉及一种用于胰腺癌治疗的工程化外泌体。
背景技术
胰腺癌(pancreatic cancer)是一种恶性程度高,早期筛查、诊断以及治疗都有难度的消化道恶性肿瘤。根据GLOBOCAN 2012统计,胰腺癌在全球每年可导致超过331000人死亡;其中中国的新发病例占全球的19.4%,死亡病例占全球的19.27%。胰腺癌起病隐匿,治疗效果较差,手术和放化疗效果不佳,患者5年生存率小于8%,中位生存期小于20个月。大部分患者发现时肿瘤分期较晚,失去进行手术的机会;化疗是中、晚期胰腺癌的首选治疗方法。奥沙利铂是治疗胰腺癌的常用化疗药物之一,其作为铂类化合物,能与DNA形成交联,造成DNA损伤,从而杀死肿瘤细胞。DNA双链断裂(DNAdouble-strandbreak,DSB)是细胞中最致命的DNA损伤形式,如果无法及时正确修复DSB可能会导致细胞基因组不稳定从而造成细胞死亡,铂类等化疗药物则是通过造成DNA损伤从而发挥抗肿瘤作用。
同源重组修复是利用细胞姐妹染色单体上同源的DNA序列作为模板修复断裂DNA的方式,需要BRCA1、BRCA2等蛋白的协作,同源重组修复准确率高,有利于保证DNA完整性,维持细胞正常功能。然而,肿瘤细胞中的DNA同源重组损伤修复机制,会修复化疗造成的DNA断裂损伤,导致胰腺癌耐药。因此,减少胰腺癌细胞中BRCA1、BRCA2的表达,从而抑制胰腺癌细胞的同源重组损伤修复机制,能够增强胰腺癌细胞的化疗敏感性。
siRNA是一种双链分子,通常为19-21个碱基对,能够通过切断同源mRNA来调控特定基因的表达。通过向胰腺癌细胞中输送靶向BRCA1、BRCA2的siRNA,能够减少胰腺癌细胞中BRCA1、BRCA2的表达,从而抑制胰腺癌细胞的同源重组损伤修复机制。尽管siRNA技术在肿瘤治疗具有很高的潜力,但迄今为止,临床应用仍然受到限制。一方面siRNA都是分子量大的或带负电的分子,它们不能通过被动扩散的方式穿过细胞膜的脂质双分子层。另一方面,裸露的siRNA在人体内环境中很脆弱,容易受到酶促降解和清除,使siRNA很难到达肿瘤部位发挥功能。因此,迫切需要一种能够将siRNA安全、高效地递送至胰腺癌以实现其作用的载体。
外泌体(Exosome)是一种直径约为40-160nm的细胞外囊泡。可以选择性分装蛋白质、脂质和核酸等释放到细胞外,释放到细胞外的外泌体,可以被驻留在微环境中的细胞捕获,也可以随生物体液被携带到远距组织器官,携带和传递重要的信号分子,构成一种全新的细胞之间的信息传递系统。为了最大限度地提高递送效率,克服siRNA传递过程的障碍,从而提高BRCA1/2 siRNA造成胰腺癌HR缺陷的有效性,基于外泌体的siRNA递送系统具有显著的优势。工程化的设计使其表面修饰特定分子来获得胰腺癌特异性靶向性,其具有以下优势:(1)良好的生物相容性和低毒性;(2)使siRNA免受体内环境破坏,延长siRNA在血液中的循环时间;(3)可以使siRNA特异性的靶向到胰腺癌,以减少全身的副作用。
虽然通过外泌体输送BRCA1/2 siRNA可以实现对于BRCA1、BRCA2的靶向抑制,但是如果外泌体不能特异性识别胰腺癌细胞的话,那么这种外泌体也能够抑制正常细胞中的BRCA1、BRCA2,导致正常细胞的基因组不稳定,有可能促进正常细胞发生癌变,因此输送BRCA1/2 siRNA的外泌体需要具有肿瘤组织特异性。尿激酶型纤溶酶原激活物(PlasminogenActivator,Urokinase,PLAU)在胰腺癌中特异性地高表达,其与尿激酶型纤溶酶原激活物受体(urokinase plasminogen activatorreceptor,uPAR)特异性结合组成尿激酶型纤溶酶原激活物系统,该系统可以通过细胞内外信号转导、趋化因子活化、蛋白水解等途径介导胰腺癌细胞增殖、迁移、黏附等功能,与胰腺癌多种生理、病理过程相关,在胰腺癌的发生发展中发挥重要作用。
因此,亟需发明一种能特异性识别胰腺癌细胞的外泌体。
发明内容
在本发明中,申请人利用胰腺癌高表达该系统的特性,通过PLAU特异性结合的uPAR中的结合结构域ATF获得胰腺癌的靶向性。CD63是一种在外泌体包膜上表达的一种四跨膜蛋白,该蛋白可作为外泌体区别于其他细胞外囊泡的标志物。利用在外泌体包膜上表达重组融合CD63-ATF蛋白,可以制作靶向胰腺癌的特异性外泌体。
293T细胞是一种有极低的免疫原性以及旁分泌性的细胞系。其分泌的纳米级外泌体,可以通过靶向递送BRCA1/2 siRNA发挥治疗作用。本发明通过在293T细胞表达CD63-ATF重组蛋白以获得胰腺癌靶向性的纳米级外泌体,该外泌体可以通过向胰腺癌细胞靶向递送BRCA1/2 siRNA,从而发挥只针对胰腺癌细胞的治疗作用,避免对正常细胞的毒副作用。
通常存在同源重组修复缺陷的肿瘤细胞,对放化疗和PARP抑制剂的治疗更敏感。本发明建立了一种人为诱导胰腺癌细胞发生同源重组修复缺陷的方法,从而提升胰腺癌细胞对放化疗的敏感性;而且这种技术只针对胰腺癌细胞,具有胰腺癌组织特异性,从而避免对正常组织可能带来的毒副作用。
具体的,本发明的技术方案如下:
本发明第一个方面公开了一种用于胰腺癌治疗的工程化外泌体,所述工程化外泌体含有BRCA1/2 siRNA。
优选的,所述工程化外泌体包膜上表达重组融合CD63-ATF蛋白。
本发明第二个方面公开了一种用于胰腺癌治疗的工程化外泌体的制备方法,包括:
S1:提取表达重组蛋白CD63-ATF的靶向性外泌体;
S2:将BRCA1/2 siRNA装载入S1中的靶向性外泌体得到用于胰腺癌治疗的工程化外泌体。
优选的,在S2中,所述BRCA1基因siRNA的核苷酸序列如SEQ ID NO:5-9中至少一种;所述BRCA2基因siRNA的核苷酸序列如SEQ ID NO:10-14中至少一种。
优选的,所述S1包括:
S11:制备CD63-ATF融合蛋白;
S12:构建表达载体pLentai-PuroR-CMV-CD63-ATF-EGFR;
S13:提取重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR;
S14:转染重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR到细胞中,提取表达重组蛋白CD63-ATF的靶向性外泌体。
优选的,在S11中,根据CD63-ATF融合蛋白的基因序列设计PCR引物的核苷酸序列如SEQ ID NO:1-4所示。
优选的,在S12中,将CD63-ATF融合蛋白与pLentai-PuroR-CMV-EGFR载体连接后转化大肠杆菌得到表达载体pLentai-PuroR-CMV-CD63-ATF-EGFR。
优选的,在S14中,所述细胞为293T细胞。
本发明第三个方面公开了一种治疗胰腺癌的药物,所述药物包括上述的工程化外泌体或上述的方法制备得到的工程化外泌体。
本发明第四个方面公开了上述的工程化外泌体或上述的方法制备得到的工程化外泌体在制备抗胰腺癌药物中的应用。
与现有的技术相比,本发明具有以下优点:
非同源重组修复缺陷的胰腺癌,占胰腺癌的绝大多数。目前尚无将这些非同源重组修复缺陷的胰腺癌转变为同源重组修复缺陷的胰腺癌的技术。本发明通过BRCA1/2siRNA实现针对BRCA1、BRCA2靶向干预,从而将非同源重组修复缺陷的胰腺癌转变为同源重组修复缺陷的胰腺癌。
本发明建立的胰腺癌细胞靶向药物递送系统,能够实现针对胰腺癌细胞的组织特异性干预,避免对正常细胞的影响,给胰腺癌患者带来了福音。
附图说明
图1为实施例中的酶切验证结果示意图。其中,*NC=未酶切对照,1kb=1kbDNAMarker,E=XmaI-BamHI酶切。
图2为实施例中的测序结果示意图:经过比对,序列插入成功,测序通过,在测序结果上找到引物序列。
图3为实施例中流式细胞仪检测空载体(左)与重组质粒(右)EGFP荧光强度。
图4为实施例中对两组EGFR荧光强度进行统计学分析示意图,**P<0.01。
图5为实施例中验证293T细胞中CD63和ATF的表达示意图。
图6为实施例中电镜下观察到的工程化外泌体示意图。
图7为实施例中通过NTA法检测外泌体的粒径的示意图;图中,左,普通外泌体;右,工程化外泌体。
图8为实施例中工程化外泌体(Transfected)对比普通外泌体(Mock)CD63及ATF的表达示意图。
图9为实施例中工程化外泌体孵育细胞的荧光示意图。其中,正常细胞(左)和胰腺癌细胞(右)红色荧光强弱。
图10为实施例中工程化外泌体孵育细胞的荧光示意图。其中,正常细胞(左)和胰腺癌细胞(右)红色荧光强弱统计学分析,**P<0.01。
图11为实施例中工程化外泌体介导胰腺癌细胞BRCA1/2表达下调的示意图。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细描述,但并不因此将本发明限制在所述的实施例范围之中。
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明所用试剂和原料均市售可得。
实施例1
本实施例公开了一种用于胰腺癌治疗的工程化外泌体,具体包括以下步骤:
1.方法:
1.1CD63-ATF融合基因的构建
1.1.1引物的设计与合成
根据融合蛋白CD63-ATF的基因序列(CD63-ATF融合基因序列如SEQ ID NO:21所示),自行设计PCR引物如下
P1:5’-CCACCGCCATGGTGGCGCCCGGGA-3’(CCCGGG为XmaI识别位点)(SEQ ID NO:1);
P2:5’-AGTGGCTACGAGGTGATGT-3’(SEQ ID NO:2);
P3:5’-AGTTCTCAAATGGAAGACCAT-3’(SEQ ID NO:3);
P4:5’-GACTGCGCAGCCGCTGCAGGATCCGC-3’(GGATCC为BamHI识别位点)(SEQ ID NO:4)。
1.1.2CD63、uPAR(ATF)基因片段的扩增
以293T细胞cDNA为模板,以CD63基因的P1、P2为引物,扩增CD63基因,ATF指uPAR氨基末端片段,反应体系如下所示:
PCR反应条件为:98℃预变性5min,98℃变性30s,61℃退火1min,72℃延伸2min,35cycle,72℃延伸10min,4℃保存。
以293T cDNA为模板,以uPAR(ATF)序列的P3、P4为引物,扩增ATF序列,反应体系如下:
PCR反应条件为:98℃预变性5min,98℃变性30s,61℃退火1min,72℃延伸2min,35cycle,72℃延伸10min,4℃保存。
1.1.3PCR产物的纯化
将扩增后的PCR产物经过1%琼脂糖凝胶电泳分析后,切下目的条带,经过DNA凝胶回收试剂盒纯化,获得纯化后的PCR产物。
1.1.4CD63-ATF融合基因的扩增
使用重叠、延伸、拼接PCR(SOE-PCR)的方法,以CD63、ATF的PCR回收产物为模板,以P1、P4为引物,扩增融合基因CD63-ATF,反应体系如下:
PCR反应条件为:98℃预变性8min,98℃变性30s,64℃退火1min,72℃延伸2min,42cycle,72℃延伸10min,4℃保存。
扩增后的PCR产物经过1%琼脂糖凝胶电泳分析后,切下目的条带,经过DNA凝胶回收纯化试剂盒纯化。
1.2表达载体pLentai-PuroR-CMV-CD63-ATF-EGFR的构建
1.2.1pLentai-PuroR-CMV-EGFRVector酶切步骤
用BamHI和XmaI酶切载体pLentai-PuroR-CMV-EGFR,配置反应体系:
混匀后在37度水浴锅中孵育2小时;1.5%琼脂糖凝胶电泳,可见7700bp和848bp两个条带,取7700bp载体条带回收。(10XNEbufferr3.1来自New Englandbiolabs货号B6003S)1.2.2T4连接步骤
建立T4连接体系
混匀后,16摄氏度连接过夜。(10X T4连接Buffer来自Thermo Scientific货号B69)
1.2.3连接产物的转化
(1)-80℃冰箱中取出感受态TOP10大肠杆菌细胞,放置于超净工作台冰盒上,当其融化为冰水混合物时,加入5μl连接产物,轻轻吹打使其混匀,冰浴30min;
(2)42℃水浴90s,冰浴4min;
(3)加入800μl培养基,37℃振荡培养4h;
(4)吸取300μl培养产物涂布在Amp抗性(10μg/ml)的固体培养平板,37℃培养箱培养过夜。
1.3重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR的提取
使用质粒小提试剂盒(中国甄选(Labselect)有限公司)进行质粒提取。
(1)取1至5ml菌液,12000rpm,离心1分钟,吸弃上清,随后加入200μl溶液P1,充分混合后加入200μl溶液P2,温和上下翻转6~8次至溶液变得清亮粘稠,立刻加入250μl溶液P3,温和翻转6到8次。随后12000rpm,离心10分钟。
(2)将上清转移至吸附柱,12000rpm,离心1分钟;弃去废液,加入700μl Washingbuffer,12000rpm,离心1分钟,重复两次。将吸附柱放入回收管,12000rpm离心2分钟,弃废液后室温静置至吸附柱内剩余液体晾干。将吸附柱转移至新的EP管,滴加洗脱液20μl,室温下静置5分钟,12000rpm,离心2分钟。收集溶液,Thermo NanoDrop-2000检测质粒浓度后置于-20℃保存。
1.4293T细胞转染重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR
使用Thermo Fisher Scientific公司lipofectamine试剂盒进行细胞转染,步骤如下:
(1)使用处于对数生长期的293T细胞进行实验。细胞消化、计数后按每孔2×105的密度接种于6孔板,培养过夜,当细胞贴壁后进行转染。
(2)配制溶液1:MEM培养基(250μl)+lipo3000(3.75μl)。配制溶液2:MEM培养基(250μl)+质粒(5μg)+P3000。溶液1和2分别在室温下静置15分钟,随后混合,并孵育5分钟。
(3)细胞用PBS润洗后加入(2)中的混合液,培养6小时换成DMEM完全培养基。
1.5通过Western blot方法鉴定CD63-ATF融合蛋白在293T细胞中的表达。
(1)蛋白裂解:首先配制裂解液:200μl RIPA裂解液加入4μl蛋白酶抑制剂,4μl磷酸酶抑制剂和2μl PMSF,冰上混合,现配现用。向细胞沉淀或外泌体沉淀中加入200μl裂解液,然后在冰上孵育20分钟后,12000×g,4℃下离心5分钟,收集上清。使用BCA定量法测定蛋白质的浓度。
(2)蛋白变性:向上清中加入SDS-PAGE蛋白上样缓冲液(5×),随后100℃水浴10分钟,使蛋白充分变性。
(3)电泳:制备8%SDS-PAGE凝胶,上样后于80V恒压电泳30分钟,然后在120V恒压下电泳,直至溴酚蓝距凝胶下缘0.5cm时终止电泳。
(4)转膜,取出SDS-PAGE凝胶,按电源阴极、海绵、滤纸、SDS-PAGE凝胶、PVDF膜、滤纸、海绵、电源阳极的顺序固定后,放置于转膜槽,在100V恒压下转膜90分钟。
(5)封闭,取出PVDF膜,放置在用TBST配制的含5%脱脂奶粉的封闭液中,放置于室温下封闭1小时。
(6)孵育一抗:取出PVDF膜,用封闭液按1:1000的比例稀释一抗,将膜置于一抗溶液中,放在摇床上轻摇,4℃孵育过夜。
(7)孵育二抗:取出PVDF膜,用TBST洗涤3次后,室温下孵育二抗,1小时后用TBST洗涤3次。
(8)显色:配制ECL发光液:增强液:稳定液=1:1。将ECL发光液滴加于PVDF膜的蛋白结合面。用滤纸吸去多余的发光液后,用X光胶片压片,随后将胶片依次放入显影液、定影液后,用水冲洗,拍照并统计结果。
1.6表达重组蛋白CD63-ATF的靶向性外泌体的提取
吸弃293T细胞旧培养液,用PBS润洗3遍,为确保收集到的培养上清中无外界外泌体混杂因素的影响,将培养基换成10%去除外泌体的FBS(超速离心去除外泌体)配制的DMEM培养基,常规培养36小时后收集培养液。取50ml培养液在300×g,4℃下离心10分钟,弃去沉淀。随后2000×g,4℃下离心10分钟,弃去沉淀。随后10000×g,4℃下离心30分钟,弃去沉淀。随后在100000×g,4℃下离心70分钟,此时的沉淀为外泌体。吸弃上清,随后加入200μl PBS重悬,然后100000×g,4℃下离心70分钟,吸弃上清,加入50μlPBS重悬。置于-80℃冰箱保存。
1.7BRCA1/2 siRNA序列及靶向位点
设计并合成靶向BRCA1/2位点的siRNA
1.8通过电穿孔方式将BRCA1/2 siRNA装载入靶向性外泌体中构建工程化外泌体
将纯化的靶向性外泌体和BRCA1/2混合在电穿孔缓冲液中,使用Bio-Rad genepulser Xcell电穿孔系统在电穿孔比色杯中以350V的电压进行电穿孔,随后将混合物在37℃下孵育30min以恢复外泌体包膜。
1.9通过透射电子显微镜观察外泌体
将10μl外泌体悬液滴加至Formvar-碳膜包被的载样铜网,静置1分钟后风干。使用PBS润洗Formvar膜面3次,保持铜网另一面干燥。使用1%戊二醛室温下固定20分钟。使用ddH2O润洗两次后,用2%乙酸铀酰染色15分钟。随后使用甲基纤维素-UA润洗10分钟后风干。在透射电子显微镜下观察并拍照。
1.10外泌体的粒径鉴定
用ddH2O稀释外泌体悬液,外泌体颗粒浓度控制在1x107/ml至1x109/ml之间,使用ZetaView PMX110仪器在405nm激光下测定样本中例子数量和大小,采用纳米颗粒示踪分析(Nanoparticle TrackingAnalysis,NTA)软件分析外泌体大小。
1.11外泌体示踪与靶向性鉴定
(1)外泌体染色
采用BCA定量法测定外泌体含量,取500μg外泌体,与4μg/ml的Dil红色染料等体积混合,4℃避光孵育2小时。随后用1ml PBS将外泌体重悬后,100000×g,4℃下离心70分钟,吸弃上清并用50μl PBS重悬,此为红色荧光标记的外泌体(Dil-Exosome)。将其放置在-80℃冰箱中避光保存。
(2)外泌体与细胞孵育
采用不同种细胞系共同孵育带Dil荧光标记的外泌体,在相同时间内,观察胰腺癌对比其他种细胞系对工程化外泌体的摄取情况。以下操作均避光进行。将5x105个细胞接种于放置有细胞爬片的6孔板,12小时后将50μg Dil-Exosome加入培养液,24小时后吸弃培养液,随后用PBS润洗3次并加入1ml 4%多聚甲醛常温下固定细胞15分钟。将固定液吸出后,加入1ml 0.1%Triton X-100,孵育细胞5分钟,随后用PBS洗涤3次。最后用预混DAPI的抗荧光淬灭剂的封片液封片,并避光15分钟。在荧光显微镜下观察外泌体吞噬情况。运用流式细胞仪检测外泌体吞噬情况。
1.12通过实时荧光定量PCR实验检测胰腺癌细胞BRCA1/2表达水平
实验所需引物由上海生工生物工程股份有限公司合成。本部分涉及的引物序列见下表:
使用Roche公司的SYBR Green PCR Master Mix进行实验,体系如下:
将上述反应体系置于LightCycler 96实时荧光定量PCR仪。cDNA的PCR扩增采用两步法进行。
条件为:步骤1,预变性:95℃,30秒,设置1个循环;步骤2,PCR反应:95℃,5秒,60℃,30秒,设置40个循环。每个样品设置3个复孔。以GAPDH作为内参,随后检测每个样本的Ct值(荧光强度达到所设阈值时所需要的循环数),目的基因的相对表达水平用2-△△CT法计算:
△CT(实验组)=CT(目的基因,实验组)-CT(内参基因,实验组)
△CT(对照组)=CT(目的基因,对照组)-CT(内参基因,对照组)
△△CT=△CT(实验组)-△CT(对照组)
2.结果
2.1重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR的鉴定
将重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR用XmaI和BamHI双酶切,结果见图1,可见两条大约为8000和1300bp大小条带,分别为pLentai-PuroR-CMV-EGFRvector线性片段和CD63-ATF融合基因片段,成功构建了重组表达质粒pLentai-PuroR-CMV-CD63-ATF-EGFR。将重组质粒送检测序,测序到P1,P4引物证明重组CD63-ATF整合成功,如图1和图2所示。
2.2重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR转染至293T细胞
将重组质粒转染至293T细胞,293T细胞表达EGFP而发出绿色荧光,使用流式细胞仪检测荧光强度,如图3和图4所示。表明工程质粒可以在细胞内表达整合重组的CD63-ATF基因。
2.3融合基因CD63-ATF的验证
转染了空载体的293T细胞内低表达CD63和ATF,而转染了工程质粒的293T细胞内高表达CD63和ATF,表明重组质粒能成功在293T细胞中表达整合的CD63和ATF,如图5所示。
2.4工程化外泌体的提取、制备和鉴定
收集转染了工程质粒的293T细胞培养液,通过超速离心法提取培养液中的外泌体,通过电穿孔的方式将BRCA1/2 siRNA递送到外泌体中,构建为为工程化外泌体,如图6所示,在电子显微镜下观察外泌体的结构,该结构与一般外泌体没有明显差异,表明改造的工程化外泌体没有改变其结构性质。
如图7所示,通过NTA检测工程化外泌体的粒径,左图为转染了空载体的293T细胞培养液提取的外泌体,右图为转染了工程质粒的293T细胞培养液提取的工程化外泌体。其粒径大小没有明显差异,表明改造的工程化外泌体没有改变外泌体的结构性质。图8显示了工程化外泌体(Transfected)对比普通外泌体(Mock)CD63及ATF的表达。
2.5工程化外泌体靶向性的测定
为了验证工程化外泌体的靶向性,使用红色染料Dil标记工程化外泌体,并用于孵育正常胰腺上皮细胞(HPDE)和胰腺癌细胞(PANC1),通过流式细胞仪测定红色荧光,发现胰腺癌细胞中的红色荧光较多,如图9和图10所示,表明工程化外泌体倾向于富集在肿瘤细胞中而非正常组织细胞,表明该工程化外泌体有对胰腺癌的靶向性。
2.6工程化外泌体造成胰腺癌细胞内BRCA1/2表达下调
通过实时荧光定量PCR方法,检测转染普通外泌体(control)和工程化外泌体(CD63-ATF)的胰腺癌细胞中BRCA1/2的表达。如图11所示,工程化外泌体介导胰腺癌细胞BRCA1/2表达下调。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种用于胰腺癌治疗的工程化外泌体,其特征在于,所述工程化外泌体含有BRCA1/2siRNA。
2.根据权利要求1所述的工程化外泌体,其特征在于,所述工程化外泌体包膜上表达重组融合CD63-ATF蛋白。
3.一种用于胰腺癌治疗的工程化外泌体的制备方法,其特征在于,包括:
S1:提取表达重组蛋白CD63-ATF的靶向性外泌体;
S2:将BRCA1/2 siRNA装载入S1中的靶向性外泌体得到用于胰腺癌治疗的工程化外泌体。
4.根据权利要求3所述的方法,其特征在于,在S2中,所述BRCA1基因siRNA的核苷酸序列如SEQ ID NO:5-9中至少一种;所述BRCA2基因siRNA的核苷酸序列如SEQ ID NO:10-14中至少一种。
5.根据权利要求3所述的方法,其特征在于,所述S1包括:
S11:制备CD63-ATF融合蛋白;
S12:构建表达载体pLentai-PuroR-CMV-CD63-ATF-EGFR;
S13:提取重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR;
S14:转染重组质粒pLentai-PuroR-CMV-CD63-ATF-EGFR到细胞中,提取表达重组蛋白CD63-ATF的靶向性外泌体。
6.根据权利要求5所述的方法,其特征在于,在S11中,根据CD63-ATF融合蛋白的基因序列设计PCR引物的核苷酸序列如SEQ ID NO:1-4所示。
7.根据权利要求5所述的方法,其特征在于,在S12中,将CD63-ATF融合蛋白与pLentai-PuroR-CMV-EGFR载体连接后转化大肠杆菌得到表达载体pLentai-PuroR-CMV-CD63-ATF-EGFR。
8.根据权利要求5所述的方法,其特征在于,在S14中,所述细胞为293T细胞。
9.一种治疗胰腺癌的药物,其特征在于,所述药物包括权利要求1-2所述的工程化外泌体或权利要求3-8所述的方法制备得到的工程化外泌体。
10.根据权利要求1-2所述的工程化外泌体或权利要求3-8所述的方法制备得到的工程化外泌体在制备抗胰腺癌药物中的应用。
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