CN111440800B - 靶向Galectin-3基因的miRNA-128-3p及其抗胰腺癌的用途 - Google Patents
靶向Galectin-3基因的miRNA-128-3p及其抗胰腺癌的用途 Download PDFInfo
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Abstract
本发明公开一种靶向Galectin‑3基因的miRNA‑128‑3p,所述miRNA‑128‑3p的序列:UCACAGUGAACCGGUCUCUUU。本发明还公开一种靶向Galectin‑3基因的miRNA‑128‑3p的药物载体,所述药物载体为HUCMSCs来源的外泌体。本发明另外还公开实验性验证靶向Galectin‑3基因的HUCMSCs外泌体携载miRNA‑128‑3p在抗胰腺癌中的用途,本发明的实施例中成功将miRNA‑128‑3p mimic转染至新生儿脐带组织来源HUCMSCs‑EXO,构建成工程化EXO;工程化EXO可靶向Galectin‑3抑制胰腺癌细胞的迁移、侵袭及增殖。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种靶向Galectin-3基因的HUCMSCs外泌体携载miRNA-128-3p及其抗胰腺癌的用途。
背景技术
胰腺癌(Pancreatic cancer)是临床上常见的消化系统恶性肿瘤。患者确诊后1年内死亡率约75%,5年生存率低于6%。在欧美国家,胰腺癌已是第4位常见的恶性肿瘤。在我国,胰腺癌的年发病率约511/10万,预计每年新增5万~6万例。多数胰腺癌患者早期无明显不适,发现时已处于晚期,错失了根治性手术切除的机会,只能以放化疗为主。但是化疗药物不能够特异性杀伤癌细胞,一些正常的细胞也可被杀伤,如骨髓、肠黏膜及毛囊。此外,一些癌细胞可以在化疗中存活下来并对化疗药物耐药,甚至有的化疗药物会刺激正常细胞突变成癌细胞,癌症干细胞不易受化疗药物的影响,经过一段时间的治疗后,癌症干细胞又会很快地引起癌症复发。因此寻找靶向性强、特异性好、副作用小的新型胰腺癌治疗方法显得尤为重要。
外泌体(Exosome,EXO)是直径为50~200nm的小囊泡,可由多种细胞分泌,具有脂质双分子膜结构,包裹少量胞质,胞质中含有母细胞中除了细胞器以外的各种分子成分,包括蛋白及核酸。EXO来源广泛,容易获取及保存,具有良好的生物相容性,因此,可以进行改造作为转运各种生物分子(如药物、蛋白和小分子RNA)的重要载体,与癌细胞相互作用,影响肿瘤发生、发展、侵袭及迁移等行为学表现。
间充质干细胞(mesenchymal stem cell,MSC)是源自早期中胚层和外胚层的多能干细胞,能够分化为成骨、成脂及成软骨细胞,具有显著的自我更新和多向分化能力。而脐带是连接母亲与新生儿之间的组织,其中有两条动脉及一条静脉,脐带羊膜和血管之间,由华通氏胶填充。从人脐带中可以分离得到脐带间充质干细胞(HUCMSCs),它具有良好的增殖和分化能力,与人骨髓间充质干细胞(human bone mesenchymal stem cell,HBMSC)相比,脐带间充质干细胞具有来源丰富、收集相对简便、增殖更新速度快等优势,是临床应用潜力较高的干细胞来源。
在肿瘤微环境中,炎性介质、低氧环境和其他肿瘤衍生因子诱导间充质干细胞的归巢能力,在肿瘤部位自发聚集,并通过与先天和后天免疫系统的相互作用逃避免疫反应。细胞外囊泡系统反映了亲代细胞的特征。来源于间充质干细胞的EXO具有自发定位肿瘤部位的能力。研究人员已经成功地将来源于间充质干细胞的EXO作为货物的载体。O'Brien等人发现miR-379在肿瘤细胞和组织中的表达降低,如乳腺癌、肝细胞癌和骨肉瘤。此外,据报道miR-379能够抑制肿瘤的增殖、迁移和侵袭。在乳腺癌的治疗中,通过慢病毒将miR-379转染间充质干细胞。与阴性对照组相比,获得的间充质干细胞EXO显示出高5倍的miR-379表达。将这些EXO通过静脉注射到患有乳腺癌的小鼠体内,经过一系列的对照研究后,间充质干细胞衍生的载有miR-379的EXO在治疗乳腺癌荷瘤小鼠后,肿瘤的生长明显受到抑制;该报道论证了HUCMSCs来源的EXO具有作为治疗载体的可行性。
半乳凝素(Gal)家族是一类能识别糖蛋白和糖脂的特异性低聚糖可溶性凝集素,Galectin-3(也称LGALS3)是Gal家族中的一员,其主要通过与相应配体相互作用发挥多种效应。Galectin-3广泛表达于正常组织和肿瘤组织中,参与多种生理和病理过程,包括调节细胞生长、抑制细胞调亡、介导细胞黏附、参与新生血管的形成和肿瘤的浸润与转移等。在之前的研究发现Galectin-3在胰腺癌组织中高表达,且高于癌旁组织;胰腺癌患者血清Galectin-3明显高于健康人及良性疾病胰腺病患者,其诊断胰腺癌的敏感性与CA19-9及CEA相仿,且与CEA及CA19-9均无相关性。根据Galectin-3的特异性表达情况,我们推断它可能参与到胰腺癌的发生发展过程中,可以作为胰腺癌靶向治疗的特异性靶点。有研究者发现胰腺癌细胞中过表达的Galectin-3通过结合并活化Ras信号诱导细胞增殖和侵袭。另外,Galectin-3可以调节胰腺癌细胞中MUC1和EGFR的细胞分布以及EGFR下游信号通路。这些结果证实了Galectin-3促进了胰腺癌的发生发展,并且近期已有研究证实,利用Galectin-3抑制剂,可以通过阻断
Galectin-3/EGFR/AKT/FOXO3信号通路发挥抗胰腺癌作用。
发明内容
发明目的:针对现有技术中存在的不足,本发明的目的是提供一种靶向Galectin-3基因的miRNA-128-3p、靶向Galectin-3基因的miRNA-128-3p的药物载体、靶向Galectin-3基因的miRNA-128-3p在制备抗胰腺癌药物方面的应用、实验性验证靶向Galectin-3基因的HUCMSCs外泌体携载miRNA-128-3p在抗胰腺癌的用途,
技术方案:为解决上述技术问题,本发明的实施例提供一种靶向Galectin-3基因的miRNA-128-3p,其特征在于,所述miRNA-128-3p的序列为:UCACAGUGAACCGGUCUCUUU。
本发明的实施例还提供一种靶向Galectin-3基因的miRNA-128-3p的药物载体,其特征在于,所述药物载体为HUCMSCs来源的外泌体。
靶向Galectin-3基因的miRNA-128-3p在制备抗胰腺癌药物方面的应用。
进一步的,所述药物以HUCMSCs来源的外泌体作为药物载体,装载miRNA-128-3p,靶向Galectin-3基因。
进一步的,所述药物通过以下过程制得:收集健康新生儿的新鲜脐带,通过组织贴壁法获得HUCMSCs;将miRNA-128-3p mimic转染至HUCMSCs,通过超速离心法提取HUCMSCs来源的外泌体。
本发明的实施例另外还提供实验性验证靶向Galectin-3基因的HUCMSCs外泌体携载miRNA-128-3p在抗胰腺癌中的用途,其特征在于,包括以下步骤:
(1)、利用Western Blot、qRT-PCR明确正常胰腺导管细胞及胰腺癌细胞系中Galectin-3的表达情况;
(2)、将miRNA-128-3p mimic(模拟物)转染至PANC-1细胞,用WesternBlot、qRT-PCR检测对PANC-1细胞中Galectin-3表达情况的影响;再利用细胞划痕实验、Transwell细胞侵袭实验及CCK-8细胞增殖检测验证miRNA-128-3p mimic对胰腺癌PANC-1细胞的迁移、侵袭及增殖等方面的影响;
(3)、收集了健康新生儿的新鲜脐带,通过组织贴壁法获得HUCMSCs;将miRNA-128-3p mimic转染至HUCMSCs,提取HUCMSCs来源的外泌体;用qRT-PCR检测HUCMSCs及HUCMSCs来源的外泌体中miRNA-128-3pmimic的含量;
(4)、使用工程化的外泌体刺激胰腺癌PANC-1细胞,用WesternBlot、qRT-PCR检测对PANC-1细胞中Galectin-3表达情况的影响;利用细胞划痕实验、Transwell细胞侵袭实验及CCK-8细胞增殖检测验证工程化的外泌体对胰腺癌PANC-1细胞的迁移、侵袭及增殖等方面的影响。
本发明的上述技术方案的有益效果如下:
(1)Galectin-3是胰腺癌的潜在治疗靶点,本发明中所提出miRNA-128-3p可通过靶向半乳凝素-3(Galectin-3)抑制胰腺癌细胞的迁移、侵袭及增殖。
(2)本发明的实施例中成功将miRNA-128-3p mimic转染至新生儿脐带组织来源HUCMSCs-EXO,构建成工程化EXO;工程化EXO可靶向Galectin-3抑制胰腺癌细胞的迁移、侵袭及增殖;证明了HUCMSCs-EXO携载miRNA-128-3p通过靶向Galectin-3在体外抑制胰腺癌PANC-1细胞的迁移、侵袭及增殖;从而,验证了靶向Galectin-3基因的HUCMSCs外泌体携载miRNA-128-3p在抗胰腺癌中的用途的可行性。
(3)本发明提出靶向Galectin-3基因的miRNA-128-3p在制备抗胰腺癌药物方面的应用,为治疗胰腺癌提供了新的药物可能。
附图说明
图1为实施例1中Galectin-3在良恶性标本之间的差异表达以及与生存期的关系图;其中,图1A为组织中Galectin-3的表达图;图1B生存曲线图。
图2为实施例1中正常胰腺导管上皮细胞及胰腺癌细胞系中Galectin-3的表达情况图;其中,图2A和图2B为Western Blot检测结果图;图2C为qRT-PCR检测结果图。
图3为实施例1中预测Galectin-3上游miRNA,选择合适的实验用细胞的情况图;其中,图3A为MiRNA数据库预测结果韦恩图;图3B为MiRNA-128-3p碱基序列图;图3C为细胞中miRNA-128-3p表达图;图3D为PANC-1细胞图。
图4为实施例2中转染miRNA-128-3p mimic至PANC-1细胞的情况图;其中,图4A为miRNA-128-3p mimic(红色荧光)转染至PANC-1细胞图;图4B转染后Western Blot检测PANC-1细胞中的Galectin-3表达图;
图4C为转染后WesternBlot检测Galectin-3表达的柱状图;图4D为qRT-PCR实验检测Galectin-3表达的柱状图。
图5为实施例2中miRNA-128-3p对PANC-1细胞迁移、侵袭及增殖的影响图;图5A和图5B为划痕实验的结果图;图5C和图5D为Transwell细胞侵袭实验的结果图;图5E为CCK-8细胞增殖-毒性实验的结果图。
图6为实施例3中组织贴壁法提取HUCMSCs的细胞图;其中,图6A和6B为组织贴壁法收集HUCMSCs的培养情况细胞图;图6C为显微镜下的观察呈梭形长条状图;图6D为HUCMSCs融合呈漩涡状。
图7为实施例3中鉴定HUCMSCs的结果图;图7A为成骨诱导分化的情况图;图7B为成脂诱导分化的情况图;图7C为流式细胞仪鉴定抗原表型的情况图;
图8为实施例3中验证HUCMSCs来源的EXO的情况图;图8A为透射电镜图;图8B为WesternBlot验证EXO蛋白marker的表达图;图8C为NTA分析图;图8D为PANC-1细胞(红色胞质、蓝色胞核)摄取EXO(绿色)的拍摄荧光图。
图9为实施例4中miRNA-128-3p mimic转染至HUCMSCs的情况图;其中,图9A为miRNA-128-3p mimic(红色荧光)转染至HUCMSCs的荧光图;图9B为转染后细胞中miRNA-128-3p水平的对比图;图9C为转染后EXO中miRNA-128-3p水平的对比图。
图10为实施例4中工程化EXO刺激PANC-1细胞后对Galectin-3表达的影响图;图10A和图10B为WesternBlot的检测结果图;图10C为qRT-PCR检测结果图。
图11为实施例4中工程化EXO对PANC-1细胞迁移、侵袭及增殖的影响图;其中,图11A和图11B为细胞划痕实验的实验结果图;图11C和图11D为Transwell细胞侵袭实验的实验结果图;图11E为CCK-8细胞增殖-毒性实验的实验结果图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合具体实施例进行详细描述。
一、实施例所采用的细胞株及来源:
人正常胰腺导管上皮细胞hTERT-HPNE、人胰腺癌细胞系BxPC-3,Capan-2,PANC-1、CFPAC-1购自中国科学院典型培养物保藏委员会细胞库,人胰腺癌细胞系PANC-1购自北纳生物细胞库。
收集南通大学附属医院妇产科健康新生儿的新鲜脐带,胎龄正常,正常生产,产妇无肝炎、梅毒、艾滋病等传染性疾病,并征得产妇及家属同意。
二、实施例中所采用的主要实验试剂及器材:
细胞培养过程所采用的试剂及器材:
(1)DMEM高糖培养基(Hyclone公司·美国)
(2)F12培养基(Hyclone公司·美国)
(3)人脐带间充质干细胞成骨诱导分化培养基试剂盒(广州赛业生物科技有限公司)
(4)人脐带间充质干细胞成脂诱导分化培养基试剂盒(广州赛业生物科技有限公司)
(5)细胞PBS(上海生工生物工程股份有限公司)
(6)胎牛血清(Gibco公司·美国)
(7)青霉素和链霉素(苏州新赛美生物科技有限公司)
(8)盐酸四环素溶液(上海生工生物工程股份有限公司)
(9)无血清细胞冻存液(苏州新赛美生物科技有限公司)
(10)Lipofectamine2000(Invitrogen公司·美国)
(11)CCK8试剂盒(上海生工生物工程股份有限公司)
(12)细胞培养皿、细胞培养瓶(Corning公司·美国)
(13)六孔、二十四孔、九十六孔细胞培养板(Corning公司·美国)
(14)2mL可立细胞冻存管(Corning公司·美国)
(15)15mL、50mL离心管(无锡耐思生物科技有限公司)
(16)200μL、0.5mL、1.5mL、2mL、5mL、10mLEP管(上海BBI生命科学有限公司)
超速离心提取EXO所采用的器材
(1)超速离心管(日立公司·日本)
(2)0.22μL无菌针头式过滤器滤头(Millipore公司·美国)
(3)20mL注射器(上海康德莱企业发展集团股份有限公司)
PCR所采用的器材
(1)Trizol(Ambion公司·美国)
(2)氯仿(国药集团化学试剂有限公司)
(3)异丙醇(国药集团化学试剂有限公司)
(4)DEPC水(上海生工生物工程股份有限公司)
(5)无水乙醇(国药集团化学试剂有限公司)
(6)逆转录试剂盒(Thermo Fisher Scientific公司·美国)
(7)PCR八联管(Roche公司·瑞士)
(8)200μL、0.5mL、1.5mL无酶EP管(上海生工生物工程股份有限公司)
(9)10μL、200μL、1mL无酶枪头(上海生工生物工程股份有限公司)
(10)QuantiNovaTM SYBR Green试剂盒(QIAGEN公司·德国)
2.1.2.4 Western Blot
(1)兔来源的TSG101、Calnexin单克隆抗体(Abcam公司·英国)
(2)兔来源的Galectin-3单克隆抗体(Cell Signaling Technologies公司·美国)
(3)鼠来源的CD81多克隆抗体(Cell Signaling Technologies公司·美国)
(4)鼠来源的GAPDH单克隆抗体(Proteintech公司·美国)
(5)辣根过氧化物酶标记的羊抗鼠或抗兔IgG(Pierce Biotechnology公司·美国)
(6)RIPA裂解液(Beyotime公司·美国)
(7)completeTM,EDTA-free Protease Inhibitor Cocktail(Sigma公司·德国)
(8)PhosSTOPTM(Sigma公司·德国)
(9)SDS-PAGE蛋白上样缓冲液(5×)(Beyotime公司·美国)
(10)异丙醇(国药集团化学试剂有限公司)
(11)甲醇(广东光华科技股份有限公司)
(12)Tris(国药集团化学试剂有限公司)
(13)甘氨酸(国药集团化学试剂有限公司)
(14)SDS(国药集团化学试剂有限公司)
(15)氯化钠(国药集团化学试剂有限公司)
(16)Tween-20(国药集团化学试剂有限公司)
(17)BSA(Sigma公司·德国)
(18)蛋白预染Marker(香港生物医学科技控股有限公司)
(19)30%丙烯酰胺溶液:丙烯酰胺29g、亚甲双丙1g,ddH2O定容至1L,完全溶解后经定性滤纸过滤,4℃避光保存。
(20)1.0M Tris-HCl(pH 6.8):Tris 12.11g,ddH2O溶解,逐滴加入浓盐酸直至pH调节至6.8,ddH2O定容至100mL,室温保存。
(21)1.5M Tris-HCl(pH 8.8):Tris 18.17g,ddH2O溶解,逐滴加入浓盐酸直至pH调节至8.8,ddH2O定容至100mL,室温保存。
(22)10%过硫酸铵:1gAPS溶于10mL ddH2O中并以每管0.5mL分装,-20℃密封保存。
(23)2×SDS匀浆缓冲液:6mL 10%SDS(pH7.2),1mL 1.0M Tris-HCl(pH6.8),0.2mLβ-巯基乙醇和2.8mLddH2O,充分混匀,4℃保存。
(24)TEMED
(25)10×电泳缓冲液:Tris 30.2g、SDS 10g以及甘氨酸188g,ddH2O定容至1L,充分溶解后于室温保存。
(26)1×电泳缓冲液:100mL 10×电泳缓冲液加ddH2O稀释定容至1L。
(27)1×转膜液:Tris 3.02g、甘氨酸18.8g、甲醇200mL,ddH2O定容至1L,混匀。
(28)TBST:Tris 2.42g、氯化钠8g、Tween-200.5mL,ddH2O定容到1L,混匀。
(29)5%脱脂牛奶:5g牛奶溶于100mLTBST中,充分混匀。
(30)PVDF膜(Roche公司·瑞士)
(31)Westernblot一抗稀释液(Beyotime公司·美国)
(32)Westernblot一抗洗脱液(Beyotime公司·美国)
(33)超敏ECL发光液(苏州新赛美生物科技有限公司)
细胞转染所采用的试剂及器材
(1)miRNA-128-3p mimic(广州市锐博生物科技有限公司)
UCACAGUGAACCGGUCUCUUU
(2)miRNA-128-3p mimic-NC(广州市锐博生物科技有限公司)
(3)miRNA-128-3p inhibitor(广州市锐博生物科技有限公司)
(5'->3'):UCCAGGAGCUCACAAUCUAGUG
(4)miRNA-128-3p inhibitor-NC(广州市锐博生物科技有限公司)
(5)红色荧光Cy3标记的miRNA-128-3p mimic(广州市锐博生物科技有限公司)
流式细胞仪鉴定抗原表型所使用的试剂
流式抗体(CD10-FITC-A、CD13-PE-Cy7-A、CD11b-PE-A、CD14-APC-A、
CD19-PE-A、CD34-PE-Cy7-A、CD45PerCP-Cy5-5-A、HLA-DR-FITC-A,Miltenyi
Biotec公司·德国)
细胞摄取EXO所使用的试剂
(1)DAPI(Cell Signaling Technologies公司·美国)
(2)PKH67绿色荧光细胞接头微型试剂盒(Sigma公司·德国)
(3)F-Actin骨架蛋白染色试剂盒(Abcam公司·英国)
实施例1胰腺癌细胞系中Galectin-3表达情况及上游miRNA预测
1、利用Western Blot、qRT-PCR明确正常胰腺导管细胞及胰腺癌细胞系中Galectin-3的表达情况。采用Western Blot、qRT-PCR操作方法,检测正常胰腺导管细胞及胰腺癌细胞系中Galectin-3的表达情况。
1.1利用GEPIA平台对Galectin-3与胰腺癌之间的生物信息学分析
在平台中发现,通过比较179例胰腺癌标本和171例正常标本中Galectin-3的表达,经统计学分析发现Galectin-3在胰腺癌中的表达明显高于正常人(图1A)。并且,在胰腺癌患者人群中,Galectin-3低表达组患者(89例)的生存时间较Galectin-3高表达组(89例)长(P=0.043),差异具有统计学意义(图1B)。
1.2 Galectin-3在正常胰腺细胞及胰腺癌细胞系的表达
选择正常胰腺细胞系(hTERT-HPNE),胰腺癌细胞系(BxPC-3,Capan-2,PANC-1,CFPAC-1)。WesternBlot结果显示:在上述5种细胞系中,胰腺癌细胞系的Galectin-3表达明显高于正常胰腺细胞系;而在胰腺癌细胞系中,以PANC-1细胞中Galectin-3表达最高(图2A)。同时,将Galectin-3的条带灰度值与相对应的内参对照GAPDH的条带灰度值相比,将所得的比值绘制柱状图(*指P<0.05,图2B)。qRT-PCR结果与Western Blot结果相一致(图2C)。
1.3预测Galectin-3的上游miRNA
为了研究Galectin-3基因的上游调控机制,我首先分别选择4个不同的miRNA数据库(mirDIP,miRSearch,miR-TarBase,TargetScan),通过韦恩图可以发现,4个数据库的交集有且仅有一个,即miRNA-128-3p(图3A),因此,预测miRNA-128-3p是调控Galectin-3的上游miRNA。从生物预测网站microRNA.org网站可以查询miRNA-128-3p碱基序列以及与Galectin-3之间的关系(图3B)。
1.4 miRNA-128-3p在正常胰腺细胞及胰腺癌细胞系中的表达
根据数据库结果,利用qRT-PCR方法检测正常胰腺细胞及胰腺癌细胞系中miRNA-128-3p的表达情况,qRT-PCR显示miRNA-128-3p表达趋势与细胞中Galectin-3呈负相关(图3C)。因为Galectin-3在PANC-1细胞中表达最高,而miRNA-128-3p表达最低,因此选择PANC-1细胞(图3D)来验证Galectin-3与胰腺癌靶向治疗之间的关系。
其中,Western Blot及qRT-PCR的实验方法如下:
(1)细胞蛋白提取:弃去细胞培养基,用PBS漂洗3次,按每个中皿加600μL细胞裂解液(97%强效裂解液,2%LCM,1%LPI),用细胞刮充分刮取细胞后,冰上裂解25min,移入2mL离心管中,4℃、12000g离心10min,吸取蛋白溶液上清,超微量紫外分光光度计测定蛋白浓度后加入蛋白溶液25%的SDS-PAGE蛋白上样缓冲液(5×),吹打均匀后沸水浴5min,-80℃长期保存。
(2)配置SDS-PAGE凝胶制备:将大小玻璃板用清洁剂冲洗干净后用双蒸水(ddH2O)冲洗三遍,晾干放入玻璃板架,固定后用ddH2O确认玻璃板密封,倒出ddH2O后在烘箱烘干玻璃板。根据所需蛋白分子量选择合适的分离胶浓度,按比例将适量ddH2O、30%丙烯酰胺溶液、1M Tris、10%SDS、10%过硫酸铵、TEMED混匀,加入玻璃板夹层,每块凝胶加分离胶约7mL,再加适量异丙醇封胶并排除气泡。约30min后分离胶凝固,倒出异丙醇,用ddH2O冲洗干净后用吸水纸吸去残余水分。按照6.8mLddH2O、1.7mL 30%丙烯酰胺溶液、1.25mL 1.5MTris、0.1mL 10%SDS、0.1mL 10%过硫酸铵、20μLTEMED的比例配置5%浓缩胶,加入玻璃板至满,插入上样梳,排除气泡。约15min后凝胶凝固后可使用,或浸没在电泳液中4℃冰箱保存。
(3)蛋白上样:将-80℃冻存的蛋白样品沸水浴5min,12000rpm离心3min,电泳液4℃预冷。用ddH2O冲洗凝胶玻璃板,装入电泳槽,内槽加入电泳液确认密封后,向外槽加入约300mL电泳液,缓慢拔出上样梳,开始加入蛋白样品及预染蛋白Marker。
(4)蛋白电泳:连接电泳仪,对准正负极,起始电压90V,待预染蛋白Marker的条带分散后,加电压至120V,玻璃板中的溴酚蓝移至底部时停止电泳,取出玻璃板,用ddH2O冲洗后放入转膜液。
(5)凝胶转膜:提前准备冰盒,玻璃皿,甲醇溶液、ddH2O、转膜液、PVDF膜、转膜夹、滤纸。将PVDF膜先放入甲醇溶液极化,再放入ddH2O中。膜液中玻璃板的薄板翘起,切除凝胶中的浓缩胶以及溴酚蓝以下部分,将转膜夹按照“黑色板、海绵、滤纸、凝胶、PVDF膜、滤纸、海绵、透明板”的顺序放置,排除气泡,夹紧,按照转膜夹黑色板对应转膜槽黑色面的顺序放置。接上电泳仪,冰上转膜,设置转膜电流300mA,根据蛋白分子量大小设置合适的转膜时间。
(6)封闭PVDF膜:转膜结束后,取出PVDF膜,可见膜上预染蛋白Marker的条带,将PVDF膜用TBST漂洗后正面朝上放入5%的脱脂牛奶-TBST溶液,室温摇床封闭2h。
(7)孵育一抗:将封闭好的PVDF膜用ddH2O冲洗,将一抗根据说明书推荐比例用TBST稀释,4℃孵育PVDF膜正面约12~16h,将PVDF膜用TBST在摇床上摇晃清洗3次,每次10min。
(8)孵育二抗:根据一抗来源,选择合适的二抗,按照1:2500用TBST稀释,常温孵育PVDF膜1h。将PVDF膜用TBST在摇床上摇晃清洗3次,每次10min。
(9)显影:超敏ECL发光液A液和B液按照1:1混合在避光EP管。PVDF膜正面朝上,将显影液加在PVDF膜上,开始显影。
qRT-PCR
(1)细胞RNA提取:培养皿中细胞融合80~90%时,弃去培养基,用PBS漂洗3次,加入1mLTrizol冰上裂解细胞10min,将裂解后的液体移入2mL无酶EP管,加入0.2mL氯仿,振荡混匀,置于冰上。在4℃低温离心机12000rpm离心15min,可见EP管中上层RNA水相,中间层,下层红色的酚-氯仿相。将上层移入2mL无酶EP管中,加入等体积异丙醇,振荡混匀后静置20min,4℃低温离心机12000rpm离心15min,可见管底RNA沉淀。弃去上清,加入1mL75%乙醇溶解有机溶剂,振荡EP管,4℃低温离心机7500rpm离心11min,弃上清,重复一次,收集RNA沉淀。室温干燥后,将RNA沉淀用20μL DEPC水溶解,取2μL用超微量分光光度计测量浓度和纯度。RNA长期保存需放置-80℃冻存。
(2)PCR引物处理:外购引物干粉-20℃冻存,使用前取出,冰上放置5min后4℃低温离心机12000rpm离心1min。按照引物说明书向干粉中加入对应剂量的DEPC水配制成储存浓度为100μM的引物原液,振荡混匀,-20℃冻存。
(3)逆转录:购置Thermo Fisher Scientific逆转录试剂盒,冰上融化,根据RNA浓度,取出2μg RNA的体积。配置20μL的逆转录总体系:Oligo(dT)primer 1μL,5×ReactionBuffer 1μL,dNTPMix 2μL,Ribolock RNase Inhibitor 1μL,RevertAid ReverseTranscriptase 1μL,加入取出的RNA体积,最后加DEPC水加量至20μL。在PCR仪中逆转录,反应条件:42℃×60min,然后72℃×5min,4℃∞。向逆转录产物(cDNA)中加入5倍ddH2O稀释至100μL,长期保存需放置-20℃冻存。
(4)qRT-PCR:将cDNA、引物(10μM)、SYBR Green冰上避光融化,20μl qRT-PCR反应体系包括:2μL cDNA,0.5μL上游引物,0.5μL下游引物,7μL DEPC水,10μL SYBR Green,离心去除气泡。预热Roche LightCycler480 PCR仪预热30min,设置合适的参数,扩增DNA:95℃预变性5min,95℃变性15s、60℃退火30s、72℃延伸30s(共循环45次),95℃×1min,42℃×1min,50℃×1min,50℃×30s。反应结束,统计mRNA表达水平。
实施例2 miRNA-128-3p mimic对PANC-1细胞的作用
采用转染细胞的方法将miRNA-128-3p mimic转染至PANC-1细胞,采用WesternBlot、qRT-PCR方法检测对PANC-1细胞中Galectin-3表达情况的影响;再利用细胞划痕实验的方法、Transwell细胞侵袭实验的方法及CCK-8细胞增殖检测验证miRNA-128-3p mimic对胰腺癌PANC-1细胞的迁移、侵袭及增殖等方面的影响。
2.1将miRNA-128-3p mimic转染至PANC-1细胞
从上海锐博生物公司购置miRNA-128-3p mimic、miRNA-128-3p mimic-NC对照、用于抑制miRNA-128-3p的miRNA-128-3p inhibitor、miRNA-128-3p inhibitor-NC对照,其中miRNA-128-3p mimic带有红色荧光,利用转染试剂Lipofectamine2000分别将上述四种物质转染至PANC-1细胞,48小时后使用倒置荧光显微镜拍摄PANC-1细胞中的红色荧光(图4A),PANC-1细胞中成功显示红色荧光,提示转染成功。
其中,转染细胞方法如下:
转染细胞:
(1)细胞种板:将细胞铺在在六孔板中,在5%CO2的37℃恒温细胞培养箱培养,待细胞融合至50%~60%水平,弃去培养基,用PBS漂洗2次,换新鲜培养基,准备转染。
(2)制备转染试剂:将5μLLipofectamine2000和250μL基础培养基混合,室温放置5min。将外购的miRNA-128-3p mimic、miRNA-128-3p mimic-NC、miRNA-128-3p inhibitor、miRNA-128-3p inhibitor-NC冰上放置5min后4℃低温离心机12000rpm离心1min。按照引物说明书向干粉中加入对应剂量的DEPC水稀释,按照说明书分别吸取5μL miRNA-128-3pmimic、miRNA-128-3p mimic-NC、红色荧光cy3标记的miRNA-128-3p mimic、10μLmiRNA-128-3p inhibitor、miRNA-128-3p inhibitor-NC加入250μL基础培养基中,室温放置5min,每份加入一份上述Lipofectamine2000溶液制成转染混合液,室温静置30min,六孔板每孔加一份转染混合液,5%CO2的37℃恒温细胞培养箱转染48小时。
(3)观察细胞荧光:将转染红色荧光cy3标记的miRNA-128-3p mimic的细胞培养皿用倒置荧光显微镜观察,用绿色荧光激发红色荧光cy3,观察细胞转染情况。
2.2转染后PANC-1细胞的Galectin-3表达水平
将转染后的四组细胞进行Western Blot实验(实验方法与实施例1中方法相同)检测Galectin-3表达水平变化;进行qRT-PCR(实验方法与实施例1中方法相同)检测miRNA-128-3p表达水平变化。Western Blot实验结果显示:转染miRNA-128-3p mimic组的Galectin-3表达较对照组降低,转染miRNA-128-3p inhibitor组的Galectin-3表达较对照组升高(图4B),将Galectin-3的条带灰度值与相对应的内参对照GAPDH的条带灰度值相比,将所得的比值绘制柱状图(*指P<0.05,图4C)。qRT-PCR实验结果与Western Blot实验结果一致(图4D)。
2.3转染后PANC-1细胞的行为学改变
将转染后的四组细胞分别进行细胞划痕实验,Transwell细胞侵袭实验,CCK-8细胞增殖-毒性检测。划痕实验结果显示,转染miRNA-128-3p mimic后,由于Galectin-3的表达被抑制,PANC-1细胞迁移能力明显低于对照组;转染miRNA-128-3p inhibitor后,PANC-1细胞迁移能力高于对照组(图5A、5B)。
Transwell细胞侵袭实验及CCK-8细胞增殖-毒性检测。提示转染后的PANC-1细胞的侵袭能力(图5C、5D)及增殖能力(图5E)改变与划痕实验所示的迁移能力改变有一致的趋势。
其中,细胞划痕实验,Transwell细胞侵袭实验,CCK-8细胞增殖-毒性检测的实验方法如下:
细胞划痕实验
上述转染成功的细胞继续在六孔板中培养,细胞融合至90%上下,弃去培养基,用PBS漂洗2遍,弃去上清。用记号笔在培养皿底画平行线,利用直尺,使用10μL移液枪头与平行线垂直划细胞划痕。PBS漂洗2遍去除细胞碎片,加基础培养基。每隔12h拍摄同一位点的划痕图片并比较。
Transwell细胞侵袭实验:
(1)细胞计数:将转染后的细胞用胰酶消化,消化结束吹打均匀离心后用PBS漂洗2遍,用3mL基础培养基重悬细胞,利用细胞计数板法进行细胞计数。
(2)准备小室:取出-80℃保存的BD matrigel在4℃过夜液化,冰上用5倍量基础培养基稀释BD matrigel,混匀后向每个小室加入100μL,5%CO2的37℃恒温细胞培养箱过夜使BD matrigel凝固。
(3)细胞铺板:在24孔板中加入600μL完全培养基,放入事先准备的transwell小室,小室中按照每孔2×104个细胞加入细胞悬液,再加入适量基础培养基将每孔细胞悬液量均加至200μL。放入5%CO2的37℃恒温细胞培养箱培养。
(4)收集结果:培养48小时后将小室取出,吸去其中的培养基,用4%多聚甲醛溶液固定30min,用PBS漂洗小室2次,再用0.1%结晶紫染料浸泡小室30min,用PBS漂洗小室2次,用棉签擦去未迁移细胞。显微镜下观察小室上细胞数量并统计。
CCK-8细胞增殖检测:
(1)细胞铺板:准备96孔板,周围一圈每孔加100μL PBS,将转染结束的4种细胞用完全培养基重悬,细胞计数后按照每孔105个细胞加入孔中,加入适量完全培养基将每孔总量配制成加至200μL,每种样品设置4个复孔,每组样品分别设置贴壁0h,24h、48h共3个时间点。
(2)准备CCK-8试剂:根据实验孔的数量,按照每孔100μL CCK-8溶液(CCK-8试剂与RPMI-1640基础培养基1:9的比例)配置。
(3)数据测量:细胞铺板约6~8h后细胞贴壁,弃去培养基,每孔加入100μL CCK-8溶液,5%CO2的37℃恒温细胞培养箱反应1h后使用酶标仪测定450nm波长处OD值,每隔12h重复测量一次OD值,绘制曲线图统计数据。
实施例3收集HUCMSCs并提取EXO
收集了健康新生儿的新鲜脐带,通过组织贴壁法获得HUCMSCs;将miRNA-128-3pmimic转染至HUCMSCs,提取HUCMSCs来源的外泌体;采用qRT-PCR的方法检测HUCMSCs及HUCMSCs来源的外泌体中miRNA-128-3p mimic的含量。
3.1收集HUCMSCs并观察形态学
收集健康婴儿的新鲜脐带,长度为10~12cm,胎龄正常,产妇无肝炎、梅毒、艾滋病等传染性疾病,获取产妇及家属同意。利用组织贴壁法收集HUCMSCs,培养约(图6A,6B),显微镜下观察细胞呈梭形长条状,核浆较大(图6C);收集细胞传代培养,细胞呈螺旋状(图6D)
其中,细胞培养方法如下:
(1)收集HUCMSCs:选择胎龄正常,无肝炎、梅毒、艾滋病等传染性疾病的产妇,获取产妇及家属同意,采集脐带前核对保存脐带产妇的信息(姓名、年龄、既往史等)。先将无菌脐带采集瓶放置4℃预冷,采集过程严格无菌操作。
在新生儿娩出后,剪断脐带,用无菌纱布擦除脐带的胎脂、羊水等污物;用无菌钳持脐带,先用75%酒精充分冲洗,然后用500mL生理盐水冲洗干净,放入脐带采集瓶冷藏。在无菌烧杯中将脐带血管内的血液洗净,剪为2~3cm长的小段,放入玻璃培养皿中,以5mm左右的间距接种于加有用培养液孵育过的6孔或者12孔培养板中,放入培养箱倒置2~3个小时,加入适量培养液,放入培养箱内培养,每日观察。
(2)完全培养基的配制:DMEM高糖完全培养基含10%的胎牛血清10%的青-链霉素;F12完全培养基含20%的胎牛血清及20%的青-链霉素。上述血清每100mL完全培养基加入20μL盐酸四环素溶液,血清使用前需复温。基础培养基、完全培养基及青-链霉素保存在4℃冰箱;胎牛血清及盐酸四环素保存在-20℃冰箱。
(3)细胞复苏:将-80℃冻存的细胞种放入37℃恒温水浴锅中迅速融化;用1000μL移液枪将融化的细胞种转移至5mL离心管,配平后1000rpm离心4min;弃去上层冻存液,加入1mL完全培养基,吹打均匀后将细胞悬液移入加有完全培养基的培养皿中,轻摇培养皿使细胞分布均匀。放入5%CO2的37℃培养箱中培养。
(5)细胞培养:细胞复苏约24小时后,细胞贴壁,观察细胞形态。每24小时用PBS漂洗后弃去陈旧培养基,换新的培养基。
(6)细胞传代:当培养皿中细胞融合80%~90%的时候,弃去陈旧培养基,用PBS漂洗两遍,加入适量胰酶消化细胞,显微镜下观察细胞,细胞变圆皱缩后,加入3倍量的完全培养基终止消化。用移液枪反复吹打细胞,使细胞悬浮后移入离心管中,1000rpm离心5分钟。弃去上清,加入2mLPBS吹打细胞,1000rpm离心5分钟,弃去上清,加入完全培养基,吹打均匀后将细胞悬液移入加有完全培养基的培养皿中,轻摇培养皿使细胞分布均匀。放入5%CO2的37℃培养箱中培养。
(7)细胞保种:当培养皿中细胞融合80%~90%的时候,弃去陈旧培养基,用PBS漂洗两遍,加入适量胰酶消化细胞,显微镜下观察细胞,细胞变圆皱缩后,加入3倍量的完全培养基终止消化。用移液枪反复吹打细胞,使细胞悬浮后移入离心管中,1000rpm离心5分钟。弃去上清,加入2mL PBS吹打细胞,1000rpm离心5分钟,弃去上清,加入1mL无血清细胞冻存液,吹打均匀后移入2mL Coring细胞冻存管中,-80℃长期保存。
3.2鉴定HUCMSCs
利用成骨诱导分化液对HUCMSCs进行诱导分化,大约18天后,用茜素红染色,可见胞浆内被染成红色的钙质,HUCMSCs被成功诱导后向成骨细胞方向分化(图7A),使用成脂诱导分化液对HUCMSCs进行诱导分化,大约20天后,用油红O染液染色,可见细胞内被染成红色的脂滴,证明HUCMSCs被向成脂细胞方向分化(图7B)。利用流式细胞仪鉴定HUCMSCs抗原表型,证明HUCMSCs阳性表达CD10、CD13,阴性表达CD11b、CD14、CD19、CD34、CD45、HLA-DR(图7C)。根据实验结果,可以证明从脐带提取出的细胞是HUCMSCs。
其中,MSC成脂诱导分化、MSC成骨诱导分化、流式细胞仪鉴定MSC抗原表型的具体 实验方法如下:
MSC成脂诱导分化
(1)配置成脂分化诱导液:将诱导分化试剂盒的试剂置于2~8℃环境中融化,根据试剂盒说明书配置诱导分化A液(含A液基础培养基、人脐带间质干细胞专用胎牛血清、地塞米松、胰岛素、IBMX、罗格列酮、双抗和谷氨酰胺)及B液(B液基础培养基、人脐带间质干细胞专用胎牛血清、胰岛素、双抗和谷氨酰胺)。
(2)细胞种板:将间质干细胞细胞铺在在六孔板中,在5%CO2的37℃恒温细胞培养箱培养。
(3)诱导分化:根据诱导分化试剂盒说明书,细胞在六孔板融合60%上下时,小心地将间质干细胞完全培养基吸走,向六孔板中加入2mL人脐带间质干细胞成脂诱导分化培养基A液。诱导3天后,吸走A液,加入2mL人脐带间质干细胞成脂诱导分化培养基B液。24h后再换A液进行诱导。交替诱导5次,用B液维持培养,每2天用B液换液,观察细胞中质地变大变圆后可以进行染色。
(4)细胞染色:将油红O贮存液与蒸馏水按照3:2比例混匀后用中性滤纸过滤,配制成油红O染料工作液。将待染色细胞弃去培养上清,加入2mL4%多聚甲醛固定30min。弃去多余固定液,用PBS漂洗2次。每孔中加入1mL油红O染色工作液染色30min,弃去油红O染液,用PBS漂洗2次,显微镜下观察染色效果。
MSC成骨诱导分化
(1)配置成骨分化诱导液:将诱导分化试剂盒的试剂置于2~8℃环境中融化,根据试剂盒说明书配置诱导分化液(含基础培养基、抗坏血酸、β-甘油磷酸钠、人脐带间质干细胞专用胎牛血清、双抗和谷氨酰胺、地塞米松)。
(2)细胞种板:将间质干细胞铺在在六孔板中,在5%CO2的37℃恒温细胞培养箱培养。
(3)诱导分化:根据诱导分化试剂盒说明书,细胞在六孔板中融合60%上下时,弃去培养基吸走,加入2mL人脐带间质干细胞成骨诱导分化完全培养基。每隔3天换液(使用前需预热至37℃),细胞培养3周后,用茜素红进行染色。
(4)细胞染色:将待染色细胞弃去培养上清,加入2mL4%多聚甲醛固定30min。弃去多余固定液,用PBS漂洗2次。每孔中加入1mL茜素红染液染色30min,弃去茜素红染液,用PBS漂洗2次,显微镜下观察染色效果。
流式细胞仪鉴定MSC抗原表型
(1)收集细胞悬液:当培养皿中MSC融合80%~90%的时候,弃去陈旧培养基,用PBS漂洗两遍,加入适量胰酶消化细胞,显微镜下观察细胞,细胞变圆皱缩后,加入3倍量的完全培养基终止消化。用移液枪反复吹打细胞,使细胞悬浮后移入离心管中,1000rpm离心5分钟。弃去上清,加入2mL PBS吹打细胞,1000rpm离心5分钟,弃去上清,重复用PBS洗涤一次,弃去上清,加入2mLPBS重悬细胞。
(2)抗体孵育:将细胞悬液分别与适量抗鼠CD10-FITC-A、CD13-PE-Cy7-A、CD11b-PE-A、CD14-APC-A、CD19-PE-A、CD34-PE-Cy7-A、CD45PerCP-Cy5-5-A、HLA-DR-FITC-A抗体,4℃避光孵育30min,1000rpm离心5min,弃去上清,加入2mLPBS重悬细胞,用流式细胞仪检测CD10、CD13、CD11b、CD14、CD19、CD34、CD45、HLA-DR表达情况。
3.3提取HUCMSCs来源的EXO并鉴定
培养皿中HUCMSCs融合至90%水平后,用DMEM基培处理后收集上清,提取EXO。用100μL预冷过的PBS重悬获得待测样本。使用透射电子显微镜(TEM)拍摄待测样本,可见圆形类似杯托状微粒图片,直径约为120nm上下(图8A);Western Blot结果显示重悬后的样品里EXO特异性蛋白TSG101、CD81阳性表达,而内质网蛋白钙连蛋白Calnexin阴性表达(图8B);对样本进行浓度粒径检测(NTA)提示其中粒子直径在100nm至160nm之间(图8C)。证实所提取样本为EXO。
其中,提取HUCMSCs来源的EXO及鉴定方法如下:
提取EXO
(1)收集细胞EXO上清:细胞培养皿中细胞融合度90%上下,弃去培养基,用PBS漂洗两次,加入去血清培养基,在5%CO2的37℃恒温细胞培养箱饥饿培养细胞48小时,收集细胞上清4℃备用,长期保存需放置-80℃冻存。
(2)去除细胞杂质:将细胞上清在4℃下300g离心10min,收集上清至无菌EP管,再4℃下3000g离心30min,收集上清至无菌EP管。再4℃下16500g离心60min,收集上清至无菌EP管。用无菌一次性针头式滤器(0.22um)过滤上清。
(3)超速离心:清洗消毒5mL超速离心管,预冷超速离心转子,将过滤后的细胞上清装入超离管中,配平后放入超速离心机,4℃超速离心150000g×2h,去除上清,加入4mL预冷PBS重悬沉淀,4℃超速离心150000g×2h,去除上清,用100μL PBS重悬EXO,可在4℃冰箱保存一周。
纳米颗粒跟踪分析技术(Nanosight Tracking Analysis,NTA)
将EXO溶于1mLPBS中,振荡混匀使EXO均匀分布。将EXO悬液注入纳米颗粒跟踪分析仪,计算出纳米颗粒的流体力学半径和浓度。
透射电子显微镜(Transmission Electron Microscope,TEM)
取5μLEXO悬液,悬浮于已经覆膜的铜网,室温静置,待铜网干透后,放入投射电镜,观察EXO形态并照相。
3.4 EXO被PANC-1细胞摄取
将所获得的EXO用绿色荧光染料PKH67染色后与PANC-1细胞共培养6小时左右,多聚甲醛固定细胞后分别用蓝色DAPI及红色鬼笔环肽将细胞核和细胞微丝骨架染成蓝色及红色,拍摄荧光图片(图8D),可见PANC-1细胞可以主动摄取EXO。
其中,PANC-1细胞摄取EXO的方法如下:
PANC-1细胞摄取EXO:
(1)EXO染色:将100μLEXO-PBS悬液与500μL Diluent C混匀配制成A液,再将4μLpkh67染料与500μLDiluent C配制成B液。将A液及B液混合,4min后加入1mL 5%BSA终止染色,增加连接效率,封闭非特异性。超速离心提取EXO,去除多余染料,用500μLPBS重悬后。
(2)细胞摄取EXO:将细胞铺在在六孔板中,在5%CO2的37℃恒温细胞培养箱培养,待细胞融合至50%~60%水平,弃去培养基,用PBS漂洗2次,换新鲜培养基,加入染色后EXO悬液,5%CO2的37℃恒温细胞培养箱共孵育至少5h。
(3)细胞染色:细胞染色后弃去培养上清,PBS漂洗两遍,每孔加入1mL4%多聚甲醛固定细胞30min,弃去多余固定液,PBS漂洗两遍;加入2mL DAPI室温染色30min,弃去多余染液,PBS漂洗两遍;根据F-Actin骨架蛋白染色试剂盒说明书配置染色工作液,每孔加入2mL工作液,室温染色30min,弃去多余染液,PBS漂洗两遍。在倒置荧光显微镜观察,用紫外光激发蓝色DAPI荧光,用绿色荧光激发激发F-Actin骨架蛋白红色染料,用蓝色光激发绿色pkh67染料,将不同的荧光照片用ImageJ软件合并。
实施例4制备工程化的EXO并验证对PANC-1细胞的作用
使用工程化的外泌体刺激胰腺癌PANC-1细胞,采用Western Blot、qRT-PCR的方法检测对PANC-1细胞中Galectin-3表达情况的影响;利用细胞划痕实验的方法、Transwell细胞侵袭实验的方法及CCK-8细胞增殖检测的方法验证工程化的外泌体对胰腺癌PANC-1细胞的迁移、侵袭及增殖等方面的影响。
4.1将治疗性物质转染至HUCMSCs
将miRNA-128-3p mimic、miRNA-128-3p mimic-NC对照、用于抑制miRNA-128-3p的miRNA-128-3p inhibitor以及miRNA-128-3p inhibitor-NC对照利用转染试剂Lipofectamine2000转染至HUCMSCs(转染细胞的方法与实施例2中的方法相同),48小时后使用倒置荧光显微镜拍摄,HUCMSCs中显示红色荧光(图9A),提示转染成功。qRT-PCR结果显示转染miRNA-128-3p mimic的HUCMSCs中miRNA-128-3p表达水平较对照组升高;转染miRNA-128-3p inhibitor的HUCMSCs中miRNA-128-3p表达水平较对照组降低(*指P<0.05,图9B)。
4.2收集工程化EXO
从转染后的HUCMSCs中提取EXO(提取EXO的方法与实施例3中相同);qRT-PCR(PCR方法与实施例1中相同)结果显示转染miRNA-128-3p mimic的HUCMSCs-EXO中miRNA-128-3p表达水平较对照组升高,转染miRNA-128-3p inhibitor的HUCMSCs-EXO中miRNA-128-3p表达水平较对照组降低(*指P<0.05,图9C)。
4.3 EXO刺激PANC-1细胞后Galectin-3表达水平的变化
在HUCMSCs中分别转染miRNA-128-3p mimic、miRNA-128-3p mimic-NC对照、用于抑制miRNA-128-3p的miRNA-128-3p inhibitor以及miRNA-128-3p inhibitor-NC对照,控制HUCMSCs细胞数、Lipofectamine2000量,转染治疗性物质的量基本一致,收集获得的EXO。每两天将等量EXO分组与PANC-1细胞共培养,重复3次,收集细胞。将EXO刺激后的细胞进行Western Blot实验(Western Blot实验的实验方法与实施例1中相同)检测Galectin-3表达水平变化;进行qRT-PCR检测miRNA-128-3p表达水平变化。
Western Blot实验结果显示:转染miRNA-128-3p mimic-EXO组的Galectin-3表达较对照组降低,转染miRNA-128-3p inhibitor-EXO组的Galectin-3表达较对照组升高(图10A、10B)。qRT-PCR实验结果与Western Blot实验结果一致(图10C)。
4.4工程化EXO刺激PANC-1细胞后细胞的行为学改变
将转染后细胞分别进行细胞划痕实验,Transwell细胞侵袭实验,CCK-8细胞增殖-毒性检测(细胞划痕实验,Transwell细胞侵袭实验,CCK-8细胞增殖实验的实验方法与实施例2中的相同)。划痕实验结果显示,加有miRNA-128-3p mimic-EXO后,由于Galectin-3的表达被抑制,PANC-1细胞迁移能力明显低于对照组;加有miRNA-128-3p inhibitor-EXO的PANC-1细胞迁移能力高于对照组。(图11A、11B)。Transwell细胞侵袭实验及CCK-8细胞增殖-毒性检测。提示加有miRNA-128-3p mimic-EXO后,PANC-1细胞的侵袭能力(图11C、11D)及增殖能力(图11E)改变与划痕实验所示的迁移能力改变有一致的趋势。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 南通大学附属医院
<120> 靶向 Galectin-3 基因的 miRNA-128-3p 及其抗胰腺癌的用途
<140> 2020103102642
<141> 2020-04-20
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> miRNA-128-3p(miRNA-128-3p)
<400> 1
ucacagugaa ccggucucuu u 21
Claims (1)
1.靶向 Galectin-3 基因的miRNA-128-3p在制备抗胰腺癌药物方面的应用,其特征在于,所述药物以 HUCMSCs来源的外泌体作为药物载体,装载miRNA-128-3p,靶向Galectin-3基因,所述miRNA-128-3p的序列为UCACAGUGAACCGGUCUCUUU。
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