CN115025223B - 一种用于激活休眠肿瘤细胞的setd4蛋白抑制剂的投递蛋白 - Google Patents
一种用于激活休眠肿瘤细胞的setd4蛋白抑制剂的投递蛋白 Download PDFInfo
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Abstract
本发明公开了一种用于激活休眠肿瘤细胞的SETD4蛋白抑制剂的投递蛋白,所述SETD4蛋白抑制剂包括DEK蛋白,所述DEK蛋白具有SEQ ID NO.25所示保守区域。本发明提供一种SETD4蛋白抑制剂,特别是DEK蛋白在激活休眠肿瘤细胞、清除肿瘤细胞及治愈肿瘤中的应用,癌症患者的不同时期,定向在静息肿瘤细胞中转入DEK基因或投递DEK蛋白,可以激活静息肿瘤细胞,使其失去对肿瘤治疗的抵抗性,体外实验验证临床乳腺癌病人静息细胞清除率为100%。结合标准的放化疗治疗从而彻底清除休眠肿瘤细胞,进而实现人类各种癌症的治愈治疗,杜绝肿瘤的复发。
Description
(一)技术领域
本发明涉及一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用,特别是DEK蛋白联合临床的肿瘤切除、放化疗、靶向及免疫治疗,激活并清除休眠肿瘤细胞,从而实现肿瘤治愈,以及在临床上治愈各种癌症的应用。
(二)背景技术
癌症目前依然是人类健康的主要疾病之一。目前癌症的治疗方法主要为外科手术、放疗、化疗、靶向治疗及免疫治疗为主。尽管这些治疗能够清除并杀死大部分的肿瘤细胞,然而却无法清除肿瘤内一小类群的休眠肿瘤细胞。肿瘤内的休眠肿瘤细胞数量虽少,但是具有极强的抵抗各种临床治疗的特征,它们通常具有极强的体内形成肿瘤和体外形成肿瘤球的能力。激活的休眠肿瘤细胞能够促进肿瘤的恶化和转移,是恶性肿瘤形成的主要因素。休眠肿瘤细胞能够在临床的放化疗、靶向及免疫治疗中存活,在治疗后能够快速激活并产生大量的肿瘤细胞,从而诱发肿瘤病人术后的肿瘤复发和转移。因此如何杀死并清除休眠肿瘤细胞是肿瘤临床治愈上无法突破的瓶颈问题。
研究报道,含有SET结构域的家族蛋白(SETD)具有组蛋白精氨酸甲基化转移酶的活性,在调控染色质结构及在细胞增殖过程中调控基因的转录表达上起重要的作用。我们经十余年的研究,建立了独有的卤虫胚胎干细胞休眠及激活的研究模型。使用该模型,我们筛选获得了细胞休眠调控的SETD4蛋白,阐明了SETD4通过催化组蛋白H4K20me3促进异染色质的形成,从而表观遗传调控卤虫休眠胚胎形成过程的分子机制(文献1)。在此基础上,我们发现SETD4以同样的机制表观遗传调控乳腺肿瘤细胞的休眠,揭示了由SETD4调控干细胞休眠的进化保守机制,同时发现SETD4休眠肿瘤细胞存在于各种不同类型临床肿瘤病人中如肝癌,肺癌,胰腺癌,卵巢癌,子宫癌等,与早期患者相比晚期肿瘤患者的SETD4休眠肿瘤细胞数量显著增加(文献2)。因此使用SETD4蛋白能够标记这类休眠的肿瘤细胞,为清除休眠肿瘤细胞提供了重要的靶点。这是首次在各种肿瘤中分子标记了休眠肿瘤细胞的研究报道,也是首次发现通过组蛋白修饰 (H4K20me3)的肿瘤细胞休眠的表观遗传机制的研究报道。
以前的研究表明,DEK作为细胞核因子蛋白能够与染色质相结合并参与调控细胞增殖、分化、凋亡、衰老,DNA损伤修复及干细胞特征维持。此外作为癌蛋白在各种肿瘤细胞中高量表达并与肿瘤的复发及转移相关。DEK蛋白能够被肿瘤细胞合成分泌并被另外的细胞所摄入,因此是具有细胞内及细胞间活性的蛋白。此外,DEK蛋白能够以自由及包含在外泌体中的形式从细胞中释放并进入到其目标细胞中。我们通过卤虫动物模型的研究发现了DEK,在卤虫休眠胚胎激活的过程中高量表达,并发现DEK通过降低SETD4蛋白的表达,降低H4K20me3从而在休眠细胞的激活起到了重要的作用(文献3)。本发明中,我们发现DEK蛋白在激活的休眠的肿瘤干细胞及肿瘤细胞中高量表达,抑制DEK蛋白的表达能够显著抑制休眠肿瘤细胞的激活,添加外源DEK蛋白能够促进休眠肿瘤细胞的激活。并发现激活后的休眠肿瘤细胞失去抵抗放化疗的能力,使用放化疗和DEK蛋白同时处理能够清除休眠肿瘤细胞,从而实现了肿瘤的完全治愈。
文献
1.Li Dai,Sen Ye,Hua-Wei Li,Dian-Fu Chen,Hong-Liang Wang,Sheng-NanJia,Cheng Lin, Jin-Shu Yang,Fan Yang,Hiromichi Nagasawa and Wei-Jun Yang*.SETD4 regulates cell quiescence and catalyzes the trimethylation of H4K20during diapause formation of Artemia.Molecular and Cellular Biology 37(7).pii,e00453-16(2017).
2.Sen Ye,Yan-Fu Ding,Wen-Huan Jia,Xiao-Li Liu,Jing-Yi Feng,Qian Zhu,Sun-Li Cai, Yao-Shun Yang,Qian-Yun Lu,Xue-Ting Huang,Jin-Shu Yang,Sheng-NanJia,Guo-Ping Ding, Yue-Hong Wang,Jiao-Jiao Zhou,Yi-Ding Chen and Wei-JunYang*.SET domain-containing protein 4 epigenetically controls breast cancerstem cell quiescence.Cancer Research 79(18),4729–4743(2019).
3.Wen-Huan Jia,An-Qi Li,Jing-Yi Feng,Yan-Fu Ding,Sen Ye,Jin-Shu Yangand Wei-Jun Yang*.DEK terminates diapause by activation of quiescent cells inthe crustacean Artemia.Biochemical Journal 476(12),1753–1769(2019).
(三)发明内容
本发明目的是提供一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用,特别是外源DEK蛋白能够进入并激活休眠的肿瘤细胞,使其失去抵抗放化疗的能力。临床手术、放化疗、靶向及免疫治疗和DEK蛋白同时处理能够清除休眠肿瘤细胞,从而实现了肿瘤的完全治愈。
本发明采用的技术方案是:
第一方面,本发明提供一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞药物中的应用。
本发明所涉及的SETD4蛋白抑制剂包括DEK蛋白,所述DEK蛋白具有SEQ ID NO.25所示保守序列。
进一步,所述DEK蛋白具有SEQ ID NO.2所示NLS结构域的氨基酸序列95%以上相似性。
进一步,所述DEK蛋白具有SEQ ID NO.3所示SAP结构域的氨基酸序列95%以上相似性。
进一步,所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域的氨基酸序列95%以上相似性。
进一步,所述DEK蛋白具有SEQ ID NO.4所示pseudo-SAP结构域或SEQ ID NO.3所示SAP 结构域中的一种或两种,且同时具有SEQ ID NO.2所示NLS结构域。
进一步,所述DEK蛋白具有SEQ ID NO.1或SEQ ID NO.22所示氨基酸序列95%以上相似性。
更进一步,所述DEK蛋白选自人源DEK蛋白,具有SEQ ID NO.1所示氨基酸序列,编码基因序列为SEQ ID NO.5所示。
更进一步,所述DEK蛋白选自鼠源DEK蛋白,具有SEQ ID NO.22所示氨基酸序列,编码基因序列为SEQ ID NO.21所示。
本发明所述休眠肿瘤细胞或肿瘤包括头颈部肿瘤,胸部肿瘤,消化系统肿瘤,泌尿生殖系统肿瘤,骨及软组织肿瘤,淋巴及血液系统肿瘤,其它肿瘤;其中头颈部肿瘤包括脑癌、眼癌、耳部肿瘤、颌骨肿瘤、颈部肿瘤、鼻腔癌、鼻窦癌、鼻咽癌、牙龈癌、舌癌、软硬腭癌、颌骨癌、口底癌、口咽癌、唇癌、上颌窦癌、颜面部皮肤黏膜的癌症、喉癌、涎腺肿瘤、甲状腺癌、脑膜瘤、室管膜瘤、垂体瘤、上皮神经母细胞瘤、神经外胚层肿瘤和副神经节肿瘤;胸部肿瘤包括肺癌、食管癌、乳腺癌、纵膈肿瘤和胸腺癌;消化系统肿瘤包括胃癌、大肠癌、肝癌、胰腺癌、胆管癌、小肠癌;泌尿生殖系统肿瘤包括肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、宫颈癌、子宫癌、卵巢癌、输卵管癌和阴道癌;骨及软组织肿瘤包括尤文氏肉瘤、脂肪肿瘤、卡波济肉瘤、平滑肌肿瘤、横纹肌肿瘤、血管肿瘤、滑膜肉瘤、纤维肉瘤和骨癌;淋巴及血液系统肿瘤包括恶性淋巴瘤、多发性骨髓瘤、白血病;其它肿瘤包括心脏肿瘤、间皮肿瘤、纤维母细胞肿瘤、滋养细胞肿瘤和黑色素瘤。
第二方面,本发明提供一种用于激活休眠肿瘤细胞的SETD4蛋白抑制剂投递蛋白,所述投递蛋白包括投递DEK蛋白,所述投递DEK蛋白是含有DEK蛋白的医学可接受的载体,所述载体包括外泌体、脂质体或纳米材料。在第二方面所述的DEK蛋白、休眠肿瘤细胞或肿瘤的定义及适用范围与第一方面限定相同。
进一步,所述外泌体是指一种由细胞分泌的大小为30-150nm的具有双层膜结构的囊泡,当所述载体为外泌体时,所述投递DEK蛋白是由肿瘤细胞系培养液中分离获得含有DEK蛋白的外泌体,或将DEK蛋白编码基因接入各种基因表达载体,在各种细胞系(推荐肿瘤细胞)中过量表达DEK蛋白并产生含有DEK蛋白的外泌体。
进一步,所述将DEK蛋白编码基因接入基因表达载体获得的含DEK蛋白的外泌体按如下方法之一制备:(1)构建过表达DEK蛋白的质粒并将其转染到各种细胞系中制备外泌体:将DEK 基因插入到pEGFP-C1质粒的EcoRⅠ和XbaⅠ位点上,筛选获得重组质粒pEGFP-C1-DEK;利用脂质体助转剂lipo8000(碧云天,货号:C0533)将上述重组质粒转入细胞系中,过量表达DEK 蛋白后收集细胞培养液,从培养液中分离纯化含有DEK蛋白的外泌体溶液A;(2)构建过表达 DEK蛋白的慢病毒并将其感染各种细胞系构建表达DEK蛋白的细胞株制备外泌体:将DEK基因分别插入到pLent-N-GFP的慢病毒表达载体的EcoRⅠ和XbaⅠ位点上,筛选获得重组慢病毒表达载体pLent-N-GFP-DEK;将重组慢病毒表达载体pLent-N-GFP-DEK和慢病毒包装质粒混合物共同转染293T细胞,转染72小时后收集细胞培养上清即为病毒液,浓缩纯化,获得过表达DEK 蛋白的慢病毒;用过表达DEK蛋白的慢病毒感染细胞系并构建过表达DEK蛋白的细胞株;收集过表达DEK蛋白的细胞株的细胞培养液,从培养液中分离纯化含有DEK蛋白的外泌体溶液B。所述含有DEK蛋白的外泌体溶液A或含有DEK蛋白的外泌体溶液B实际均为含有DEK蛋白的外泌体溶液,为了区分不同方法获得的外泌体而命名,字母本身没有含义。
进一步,从肿瘤细胞系培养液中分离外泌体的方法有多种,都可以使用,本发明推荐由肿瘤细胞系培养液中分离获得外泌体采用外泌体抽提试剂法制备:从肿瘤细胞系培养液中加入外泌体分离试剂(品牌:Thermo Fisher,货号:4478359),上下颠倒3次混匀后4℃孵育过夜,第二天,上述混合液10000rpm 4℃离心60分钟,去掉上清,用PBS重悬离心管底部的沉淀,即为含DEK 蛋白的粗外泌体。
进一步,方法(1)和(2)细胞系均优选4T1、EMT6、MCF7。
进一步,方法(1)和(2)从培养液中分离纯化含有DEK蛋白的外泌体溶液的方法均为:利用外泌体抽提试剂从细胞培养液中分离获得外泌体,通过流式分选仪分选得到GFP阳性和粒径范围在50-200nm的含有DEK蛋白的外泌体。
进一步,方法(2)所述慢病毒包装质粒混合物源自慢病毒包装使用试剂盒(Lentiviral Packaging Kit,购自上海翊圣生物,货号:41102ES10),由质量比5:3:2的pMDL,VSVG和 pRSV-Rev组成。
进一步,所述脂质体是指由磷脂双分子层人工膜在水溶液中形成的微囊,所述载体为脂质体时,含DEK蛋白的脂质体采用薄膜水化法制备:取二棕榈酰磷脂酰胆碱(DPPC)、胆固醇、二硬脂酰磷脂酰乙酰胺-甲氧基聚乙二醇(2000DSPE-mPEG2000)溶于氯仿中,减压旋转蒸发成均匀的薄膜,真空过夜彻底挥发掉残留的氯仿,加入含DEK蛋白的PBS溶液,冰浴25KHz超声20min 使脂质体膜脱落,在振荡器上震荡5min充分水化,形成浊液,然后将浊液转移到EP管中,用探头超声在功率135W下超声30min,得到透明均一的蓝色脂质体悬液;用10kD的超滤管以12000g 的转速4℃超滤,每五分钟取出吹打一次并补充PBS洗去游离蛋白,收集截留液,即为含DEK 蛋白的脂质体悬液;所述DEK蛋白的PBS溶液浓度优选2mg/ml;所述二棕榈酰磷脂酰胆碱与胆固醇质量比为1:0.1,所述二棕榈酰磷脂酰胆碱与二硬脂酰磷脂酰乙酰胺-甲氧基聚乙二醇质量比为1:0.1,所述氯仿体积用量以二棕榈酰磷脂酰胆碱质量计为0.1mL/mg;所述二棕榈酰磷脂酰胆碱与DEK蛋白的PBS溶液中DEK蛋白质量比为1:0.2。
进一步,所述纳米材料是指可作为传导或输送药物的载体,由天然或人工合成的高分子构成的大小在10-1000nm之间的固态胶体颗粒,所述载体为纳米材料时,含DEK蛋白的纳米材料按采用改良溶剂挥发法制备:将DEK蛋白的PBS溶液,转入聚乳酸-羟基乙酸共聚物(PLGA)的二氯甲烷溶液中,25KHz超声1分钟形成初乳,将初乳转入体积浓度1%聚乙烯醇(PVA)水溶液中,再次25KHz超声5分钟形成复乳,搅拌4h,待有机溶剂挥发后,18000r/min离心收集沉淀,将沉淀-55℃冷冻干燥24h,获得含DEK蛋白的PLGA纳米粒;优选的,所述DEK蛋白与聚乳酸-羟基乙酸共聚物质量比为1:0.1,所述DEK蛋白的PBS溶液浓度为25mg/L;所述PLGA 的二氯甲烷溶液浓度为20mg/mL,所述PVA水溶液体积用量以DEK蛋白的PBS溶液中DEK蛋白质量计为1mL/mg。
第三方面,本发明提供一种SETD4蛋白抑制剂在制备激活休眠肿瘤细胞试剂中的应用,所述试剂包括DEK蛋白。所述激活是将SETD4蛋白抑制剂投递到休眠肿瘤细胞中,达到激活休眠肿瘤细胞的目的。所述试剂能够用于肿瘤的临床治疗和基础科学研究。
本发明所述激活休眠肿瘤细胞试剂还包括SETD4蛋白抑制剂及清除肿瘤细胞的药物。所述 SETD4蛋白抑制剂包括DEK蛋白;所述清除肿瘤细胞的药物推荐紫杉醇或5-氟尿嘧啶。本发明所述清除肿瘤细胞的方法是将SETD4蛋白抑制剂与放化疗等常规方法结合达到清除休眠肿瘤细胞的目的,具体是指在进行放化疗等常规治疗的同时,将SETD4蛋白抑制剂(特别是DEK蛋白,优选通过外泌体,脂质体及纳米材料)投递到肿瘤细胞中,达到激活并清除休眠肿瘤细胞的目的,以实现无转移,无复发的临床肿瘤治愈。在第三方面所述的DEK蛋白、休眠肿瘤细胞或肿瘤的定义及适用范围与第一方面限定相同。所述SETD4蛋白抑制剂投递到休眠肿瘤细胞采用第二方面所述投递蛋白。
第四方面,本发明提供一种SETD4蛋白抑制剂在制备抗肿瘤药物中的应用,所述抗肿瘤药物包括所述的SETD4蛋白抑制剂及清除肿瘤细胞的药物,所述SETD4蛋白抑制剂包括DEK蛋白;所述清除肿瘤细胞的药物推荐紫杉醇或5-氟尿嘧啶。所述的应用是在进行临床手术、放化疗、靶向或免疫治疗的同时将SETD4蛋白抑制剂(特别是DEK蛋白以外泌体,脂质体及纳米载体的形式),通过腹腔、静脉注射或瘤体直接注射投递到肿瘤中,同时与清除肿瘤细胞的药物组合使用达到清除肿瘤的目的。在第四方面所述的DEK蛋白、休眠肿瘤细胞或肿瘤的定义及适用范围与第一方面限定相同。所述SETD4蛋白抑制剂投递到休眠肿瘤细胞采用第二方面所述投递蛋白。
第五方面,本发明提供一种应用SETD4蛋白抑制剂治疗肿瘤的方法,所述方法为:在各种时期的肿瘤病人中,通过静脉注射、腹腔注射或瘤体内注射SETD4蛋白抑制剂,将SETD4蛋白抑制剂投递到肿瘤中,从而激活休眠肿瘤细胞,并在临床手术、放化疗、靶向或免疫治疗的作用下杀死并彻底清除休眠的肿瘤细胞,实现无转移,无复发的临床肿瘤治愈;所述SETD4蛋白抑制剂 (特别是DEK蛋白)进入细胞核,结合在SETD4基因的启动子上,关闭SETD4的表达,下调异染色质的形成,开放大部分基因的表达,激活休眠的肿瘤细胞,再通过放化疗等常规方法,杀死激活后的休眠肿瘤细胞;所述SETD4蛋白抑制剂包括DEK蛋白,所述DEK蛋白以投递DEK蛋白的形式注射,所述投递DEK蛋白是指含有DEK蛋白的外泌体、脂质体或纳米材料,所述投递 DEK蛋白同第二方面所述。在第五方面所述的DEK蛋白、休眠肿瘤细胞或肿瘤的定义及适用范围与第一方面限定相同。
本发明通过放化疗在人及小鼠乳腺癌中筛选获得了能够抵抗放化疗治疗的休眠肿瘤细胞,同时发现了一个可以激活休眠肿瘤细胞的功能蛋白及其基因,该基因的DNA全长为1128bp(SEQ ID NO.5),翻译编码一个由375个氨基酸组成的蛋白质(SEQ ID NO.1),即DEK蛋白。本发明外源DEK蛋白(包括肿瘤细胞分泌的含有DEK蛋白的外泌体)能够进入并激活休眠的肿瘤细胞,使其失去抵抗放化疗的能力,激活后的休眠肿瘤细胞,再通过放化疗等常规方法清除休眠肿瘤细胞。本发明在荷瘤小鼠中通过放化疗和含DEK蛋白外泌体同时处理能够激活并清除体内肿瘤中的肿瘤休眠细胞,从而实现了荷瘤小鼠的完全治愈,明确了使用放化疗等常规方法和DEK或含 DEK蛋白的外泌体同时处理能够清除休眠肿瘤细胞(图7),本实验中观察到小鼠在治疗后肿瘤未见复发、未见转移。
本发明DEK蛋白激活休眠肿瘤细胞的机理为:在体内将DEK蛋白投递到肿瘤组织中,DEK 蛋白能够进入到休眠的肿瘤细胞中,结合在SETD4,MYC及TP53等基因的启动子上,下调SETD4 和p53蛋白的表达,上调MYC蛋白的表达。通过降低H4K20me3水平,降低异染色质结构水平并上升常染色质结构的水平,进而上调细胞增殖分裂相关的胞内信号转导、基因表达、细胞周期和细胞代谢、细胞的转录翻译、细胞呼吸、细胞代谢、DNA修复等途径,下调细胞滞育、染色体沉默、基因沉默、低氧代谢、p53信号通路、上皮间充质转化等途径,从而激活休眠的肿瘤细胞,在放化疗的条件下杀死并清除休眠的肿瘤细胞,以实现无转移,无复发的临床肿瘤治愈。
与现有的肿瘤临床治疗的方法相比,本发明有益效果主要体现在:由于在肿瘤中存在的休眠肿瘤细胞具有抵抗包括放疗,化疗,靶向及免疫治疗等各种临床肿瘤治疗的能力,是肿瘤恶化、转移及预后肿瘤复发的主要原因,本发明提供一种SETD4蛋白抑制剂,特别是DEK蛋白在激活休眠肿瘤细胞、清除肿瘤细胞及治愈肿瘤中的应用。
本发明将外源DEK蛋白投递到休眠肿瘤细胞,外源DEK蛋白能够与染色质相结合,降低异染色质的同时升高常染色质的结构,从而引起一系列的基因转录及表达,进而诱导肿瘤细胞从休眠状态转换到激活状态(分子机制见图1),使其失去对各种临床治疗抵抗能力,同时结合放化疗等目前已有的治疗方法达到清除体内休眠肿瘤细胞,在临床上实现无复发癌症治愈目的。本发明在癌症患者的不同时期,定向在静息肿瘤细胞或组织中转入DEK基因或投递DEK蛋白,可以激活静息肿瘤细胞,使其失去对肿瘤治疗的抵抗性,结合标准的放化疗治疗从而彻底清除休眠肿瘤细胞。本发明SETD4蛋白抑制剂对于4T1休眠细胞、EMT6休眠细胞和MCF7休眠细胞清除率为100%,体外实验验证临床乳腺癌病人静息细胞清除率为100%。本发明方法可用于很早期的肿瘤治疗,当肿瘤的尺寸小到无法用手术进行治疗时,可以通过DEK外泌体和化疗的联合使用,激活并彻底清除休眠肿瘤细胞,杜绝肿瘤的复发,进而实现人类各种癌症的治愈治疗。本发明方法还可用于原位的肿瘤已经被手术切除,但是全身还存在转移肿瘤细胞的病人,通过DEK外泌体和化疗的联合使用,激活并清除转移到其它地方的休眠肿瘤细胞。
本发明是首例关于SETD4蛋白抑制剂激活休眠肿瘤细胞的研究和报道,也是首次使用DEK 蛋白与标准临床治疗联合处理治愈癌症,并且无复发与转移。本发明在各种人类癌治愈治疗的临床上有着重大的应用价值。
(四)附图说明
图1为含外源DEK蛋白的外泌体激活休眠肿瘤细胞,结合放化疗将其杀死的模式图。
图2为4T1、EMT6和MCF7细胞在贴壁培养时的光镜照片。标尺,50μm。
图3为4T1、EMT6和MCF7肿瘤的光镜照片。标尺,4mm。
图4为从4T1、EMT6和MCF7肿瘤中消化下来的细胞光镜照片。标尺,50μm。
图5为4T1、EMT6和MCF7肿瘤中抵抗放化疗的细胞的光镜照片。标尺,50μm。
图6为4T1、EMT6和MCF7肿瘤中抵抗放化疗的肿瘤细胞的SETD4、H3S10ph和PCNA的免疫印迹结果,内参分别为GAPDH和H3。
图7为4T1、EMT6和MCF7肿瘤中休眠肿瘤细胞的ALDH1、CD44和CD24的免疫荧光结果。标尺,50μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。
图8为4T1、EMT6和MCF7细胞来源的粗外泌体的粒径分布曲线。
图9为4T1、EMT6和MCF7细胞来源的粗外泌体的透射电镜照片。标尺,100nm。箭头指示外泌体。
图10为4T1、EMT6和MCF7细胞来源的粗外泌体的DEK、CD9、CD81和CD63的免疫印迹结果。
图11为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图,内参分别为H3和GAPDH。
图12为4T1、EMT6和MCF7中激活的休眠肿瘤细胞的ALDH1、CD44和CD24的免疫荧光结果。标尺,50μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。
图13为4T1、EMT6和MCF7中激活的休眠肿瘤细胞的单颗细胞体外成球的光镜照片和统计结果。标尺,2mm。红色箭头指示肿瘤球。
图14为4T1、EMT6和MCF7中100颗、10颗或者1颗激活的休眠肿瘤细胞在体内原位成瘤的光镜照片和统计结果,设置5个平行对照。标尺,1cm。
图15为4T1、EMT6和MCF7休眠肿瘤细胞实体瘤切片中的ALDH1、Ki67、SETD4和DEK的免疫荧光结果。a为检测ALDH1、Ki67和SETD4的免疫荧光结果。b为检测ALDH1、Ki67 和DEK的免疫荧光结果。标尺,10μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。箭头指示ALDH1阳性且Ki67阴性的肿瘤细胞。
图16为4T1、EMT6和MCF7激活肿瘤细胞实体瘤切片中的ALDH1、Ki67、SETD4和DEK的免疫荧光结果。a为检测ALDH1、Ki67和SETD4的免疫荧光结果。b为检测ALDH1、Ki67 和DEK的免疫荧光结果。标尺,10μm。DAPI表示细胞核的染色。Merge表示四个荧光通道的叠加图。箭头指示ALDH1阳性且Ki67阳性的肿瘤细胞。
图17为4T1、EMT6和MCF7休眠肿瘤细胞激活前后的SETD4和DEK的免疫印迹结果(a)及其定量统计图(b、c),内参分别为GAPDH和H3。
图18为pFastBacTM HT A质粒的图谱。
图19为人源DEK蛋白(hDEK-GFP)、鼠源DEK蛋白(mDEK-GFP)、人源结构域NLS突变DEK蛋白(hDEKΔNLS-GFP)和鼠源结构域NLS突变DEK蛋白(mDEKΔNLS-GFP)的考马斯亮蓝染色。箭头指示蛋白的位置。M表示蛋白分子量标准。
图20为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEKΔNLS-GFP蛋白或DEK-GFP蛋白后的DEK-GFP、DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图,内参分别为H3和GAPDH。
图21为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEKΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的GFP和cCasp3的免疫荧光结果及其统计图,标尺,50μm。DAPI表示细胞核的染色。
图22为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEKΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的台盼蓝染色结果及其死亡率统计图。标尺,50μm。
图23为pLent-U6-RFP-Puro质粒的图谱。
图24为在激活的4T1、EMT6和MCF7休眠肿瘤细胞中进行DEK干涉后的免疫印迹和体外成球试验。a为DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图,内参分别为H3和GAPDH。b为体外成球试验的光镜照片和成球率的统计图,标尺,400μm。
图25为在激活的4T1、EMT6和MCF7休眠肿瘤细胞中干涉DEK一周后加入PBS、 DEKΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的DEK-GFP、DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图,内参分别为H3和GAPDH。
图26为在激活的4T1、EMT6和MCF7休眠肿瘤细胞中干涉DEK一周后加入PBS、 DEKΔNLS-GFP蛋白或DEK-GFP蛋白后的体外成球试验的光镜照片和成球率的统计图,标尺,400 μm。
图27为在4T1(a)、EMT6(b)和MCF7(c)休眠肿瘤细胞中加入PBS、DEKΔNLS-GFP蛋白或DEK-GFP蛋白后细胞的透射电镜照片及细胞核中异染色质的相对水平统计图。标尺,1μm。
图28为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEKΔNLS-GFP蛋白或DEK-GFP蛋白后的H4K20me1、H4K20me2、H4K20me3、HP1-α、H3K9ac、H3K9me3和H3K27me3的免疫印迹结果及其定量统计图,内参分别为H4和H3。
图29为结合位点分析法(ChIP-Seq)分析在激活后的MCF7休眠肿瘤细胞中DEK结合位点的峰在染色体上的分布情况。
图30为ChIP-Seq分析在激活后的MCF7休眠肿瘤细胞中DEK结合位点的峰在基因区域中的分布情况。
图31为ChIP-Seq分析在激活后的MCF7休眠肿瘤细胞中DEK结合位点的峰所在基因的基因本体富集分析。
图32为ChIP-Seq分析在激活后的MCF7休眠肿瘤细胞中DEK在SETD4、TP53和MYC基因区域上结合信号的可视化结果。
图33为ATAC-Seq分析激活前后的MCF7休眠肿瘤细胞中开放基因的整体信号情况。a为 reads相对于TSS区域的分布曲线,b为reads相对于TSS区域的热图。
图34为ATAC-Seq分析在激活前后的MCF7休眠肿瘤细胞中开放程度上升的基因进行基因本体富集分析的结果。
图35为ATAC-Seq分析激活前后的MCF7休眠肿瘤细胞在SETD4、TP53和MYC基因区域上ATAC-Seq信号的可视化结果。
图36为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞基因差异的结果。a为差异基因的聚类热图,b为差异基因的火山图。padj表示校正后的p值。
图37为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞中上调基因的基因本体富集分析。
图38为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞中下调基因的基因本体富集分析。
图39为RNA-Seq分析激活前后的MCF7休眠肿瘤细胞中差异基因的GSEA分析。a为在激活后的细胞中上调的基因集,b为在激活后的细胞中下调的基因集。
图40为MYC靶向基因集和p53靶向基因集的GSEA分析结果。a为GSEA图,b为基因集中显著差异基因的表达量热图。
图41为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、DEKΔNLS-GFP蛋白或DEK-GFP蛋白后的p53、p21、PUMA和MYC的免疫印迹结果及其定量统计图,内参为β-actin。
图42为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的cCasp3的免疫荧光结果及其统计结果,标尺,50μm。
图43为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的台盼蓝染色结果及其死亡率统计图。标尺,50μm。
图44为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的H4K20me1、H4K20me2、H4K20me3、HP1-α、H3K9ac、H3K9me3和H3K27me3的免疫印迹结果及其定量统计图,内参分别为H4和H3。
图45为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS和粗外泌体后的p53、p21、PUMA和MYC的免疫印迹结果及其定量统计图,内参为β-actin。
图46为pEGFP-C1质粒的图谱。
图47为pLent-N-GFP质粒的图谱。
图48为4T1、EMT6和MCF7细胞来源的含有DEK-GFP或者DEKΔNLS-GFP蛋白的外泌体的粒径分布曲线。
图49为4T1、EMT6和MCF7细胞来源的含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体的透射电镜照片。标尺,100nm。箭头指示外泌体。
图50为4T1、EMT6和MCF7细胞来源的含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体的DEK-GFP、DEK、CD9、CD81和CD63的免疫印迹结果。
图51为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体后的DEK-GFP、DEK、H3S10ph、PCNA和SETD4的免疫印迹结果及其定量统计图,内参分别为H3和GAPDH。
图52为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体后的cCasp3的免疫荧光结果及其统计结果,标尺,50μm。
图53为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体后的台盼蓝染色结果及其死亡率统计图。标尺,50μm。
图54为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体后的H4K20me1、H4K20me2、H4K20me3、HP1-α、H3K9ac、H3K9me3和H3K27me3的免疫印迹结果及其定量统计图,内参分别为H4和H3。
图55为在4T1、EMT6和MCF7休眠肿瘤细胞中加入PBS、含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体后的p53、p21、PUMA和MYC的免疫印迹结果及其定量统计图,内参为β-actin。
图56为在小鼠中腹腔注射含外源DEK-GFP蛋白的分选外泌体后小鼠血浆中含有GFP阳性外泌体的比例检测的流式分析结果和统计图。
图57为在小鼠中腹腔注射含有DEK-GFP或者DEKΔNLS-GFP蛋白的分选外泌体后的肿瘤切片中GFP的免疫荧光结果。标尺,50μm。DAPI表示细胞核染色。
图58为在小鼠中腹腔注射含有DEK-GFP蛋白的分选外泌体24小时后的肿瘤切片中SETD4 的免疫荧光结果及其比例统计。标尺,50μm。DAPI表示细胞核染色。
图59为4T1荷瘤小鼠在放疗组、放疗且注射含有DEKΔNLS-GFP蛋白的分选外泌体组和放疗且注射含有DEK-GFP蛋白的外泌体组中肿瘤切片的SETD4免疫荧光结果及其比例统计。标尺, 50μm。DAPI表示细胞核染色。
图60为EMT6荷瘤小鼠在放化疗组、放化疗且注射含有DEKΔNLS-GFP蛋白的分选外泌体组和放化疗且注射含有DEK-GFP蛋白的分选外泌体组中肿瘤切片的SETD4免疫荧光结果及其比例统计。标尺,50μm。DAPI表示细胞核染色。
图61为4T1荷瘤小鼠的放疗及分选外泌体注射方案和瘤径曲线。
图62为4T1荷瘤小鼠在接受放疗和分选外泌体的联合治疗后的肺转移灶情况。a为肺组织切片的苏木素伊红染色结果(标尺,1mm),b为肺转移灶数目的统计图;图中1、2、3、4表示放大区域。
图63为4T1荷瘤小鼠在接受放疗和分选外泌体的联合治疗后的生存曲线。
图64为EMT6荷瘤小鼠的放化疗及分选外泌体注射方案和瘤径曲线。
图65为MCF7荷瘤小鼠的化疗及分选外泌体注射方案和瘤径曲线。
图66为临床病人的乳腺癌切片中SETD4细胞比例与TNM分期的对应关系。a为临床病人的样本信息,b为临床分期Ⅰ、Ⅱ和Ⅲ的病人乳腺癌切片中的SETD4细胞比例。
图67为从临床乳腺癌病人样品中获得休眠肿瘤细胞,并且利用MCF7含DEK-GFP蛋白的分选外泌体结合化疗将其杀死的数据。a为从临床乳腺癌病人样品中获得抵抗放化疗的休眠肿瘤细胞,标尺,50μm。b为在休眠肿瘤细胞中加入MCF7含DEK-GFP蛋白的外泌体使其激活,DAPI表示细胞核染色,标尺,50μm。c为在休眠肿瘤细胞中加入MCF7含DEK-GFP蛋白的外泌体将其杀死,标尺,50μm。
图68为在各种人肿瘤细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞的数据。a为在肺癌 H226细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞,标尺,50μm。b为在胃癌MKN45细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞,标尺,50μm。c为在前列腺癌PC-3细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞,标尺,50μm。d为在宫颈癌HeLa细胞中通过DEK 结合化疗激活并清除休眠肿瘤细胞,标尺,50μm。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
本发明实施例中百分浓度除特别说明外均为体积浓度。
本发明所用磷酸缓冲液(PBS),配方:终浓度137mM氯化钠、终浓度2.7mM氯化钾、终浓度10mM的十二水合磷酸氢二钠和终浓度1.76mM磷酸二氢钾,溶剂为水,pH调至7.4。
本发明实施例中免疫印迹试验所用一抗及相应二抗:
表1、蛋白对应的一抗和二抗
实施例1、小鼠移植瘤的获取
1、细胞系
MCF7细胞系,人乳腺癌细胞,分子表征为luminal A型,购自中国科学院典型培养物保藏委员会细胞库,目录号为TCHu74,细胞培养基是EMEM培养基(Eagle's minimumEssential Medium, Corning,目录号:10-009-cv)中添加10%的胎牛血清和1%的青霉素-链霉素。
4T1细胞系,典型的三阴性乳腺癌细胞,特征类似于临床Ⅳ期的乳腺癌,购自美国菌种保藏中心(ATCC),目录号为CRL-2539,细胞培养基是DMEM培养基(Dulbecco’sModification of Eagle's Medium,Corning,目录号:R10-013-cv)中添加10%的胎牛血清和1%的青霉素-链霉素。
EMT6细胞系,小鼠乳腺癌细胞,购自美国菌种保藏中心(ATCC),目录号为CRL-2755,细胞培养基是Waymouth’s培养基(Gibco,目录号:31220072)中添加体积浓度15%的胎牛血清和1%的青霉素-链霉素。
从液氮罐中取出第五代4T1、EMT6和MCF7细胞的冻存管各1支,37℃水浴3分钟后,1000 rpm 25℃离心5分钟,去掉上清,分别加入1mL上述的培养基重悬,重悬液分别接种到含9mL 培养基的直径10cm的细胞培养皿中。将细胞培养皿培养在37℃,5%CO2的湿润环境中,贴壁细胞的光镜照片见图2。
2、小鼠移植瘤的获取
MCF7细胞选取6-8周龄的Nod/Scid雌鼠(购自上海斯莱克实验动物有限责任公司)作为接种对象。1000万MCF7细胞中加入1mL含2500U/mL胰蛋白酶和0.02%乙二胺四乙酸(EDTA)的磷酸缓冲液(PBS),25℃静置孵育40秒,加入0.5mL胎牛血清中和,将壁上的细胞吹打下来, 1000rpm 25℃离心5分钟,去上清。取底部的0.5g细胞沉淀用1mL体积比1:1的EMEM培养基(Corning,目录号:10-009-cv)与基质胶(Corning BioCoat,目录号:354234)的混合液重悬,获得1mL 含1000万MCF7细胞的悬液。采用1mL的微量注射器将MCF7细胞悬液原位注射到 Nod/Scid雌鼠腋下的乳腺脂肪垫中,每只Nod/Scid鼠接种100万颗MCF7细胞。
4T1细胞选取6-8周龄的BALB/c雌鼠(北京维通利华实验动物技术有限公司)作为接种对象。 1000万4T1细胞中加入1mL含2500U/mL胰蛋白酶和0.02%EDTA的PBS,25℃静置孵育40秒,加入0.5mL胎牛血清中和,将壁上的细胞吹打下来,1000rpm 25℃离心5分钟,去上清。取底部的0.5g细胞沉淀用1mL的DMEM培养基重悬,获得1mL含1000万4T1细胞的悬液。采用1mL 的微量注射器将4T1细胞悬液原位注射到BALB/c雌鼠腋下的乳腺脂肪垫中,每只BALB/c雌鼠接种100万颗4T1细胞。
EMT6细胞选取6-8周龄的BALB/c雌鼠(北京维通利华实验动物技术有限公司)作为接种对象。400万EMT6细胞中加入0.5mL含2500U/mL胰蛋白酶和0.02%EDTA的PBS,25℃静置孵育40秒,加入0.25mL胎牛血清中和,将壁上的细胞吹打下来,1000rpm 25℃离心5分钟,去上清。取底部的0.2g细胞沉淀,用2mL的Waymouth’s培养基重悬,获得2mL含400万EMT6 细胞的悬液。采用1mL的微量注射器将EMT6细胞悬液原位注射到BALB/c雌鼠腋下的乳腺脂肪垫中,每只BALB/c雌鼠接种20万颗EMT6细胞。
所有的小鼠都饲养在无菌的环境中,水和鼠粮持续供应,垫料及时更换,饲养至肿瘤体积达到500mm3左右时,安乐死小鼠,用于下一步的获取实体瘤。小鼠实验均通过动物实验的伦理审查,实验者在进行实验时遵守实验动物的福利伦理原则。
实施例2、抵抗放化疗的休眠肿瘤细胞的获取和鉴定
1、实体瘤中获取抵抗放化疗的休眠肿瘤细胞
将实施例1中三种荷瘤小鼠安乐死,手术取出实体瘤,光镜照片见图3。先用剪刀将瘤子物理切割成小块,再进一步剪成碎末,将1.5g瘤子碎末放入30mL消化液(DMEM培养基中含有 1250U/mL的胰蛋白酶,0.01%EDTA,2000U/mL胶原酶Ⅳ)中,37℃水浴20分钟,每隔5分钟手动摇匀。消化后的细胞液通过40μm的尼龙膜过滤,取滤液,即为从实体瘤中消化下来的细胞,光镜照片见图4。
上述实体瘤中消化下来的细胞按80万颗/孔的密度接种在超低吸附的六孔盘(品牌:Corning,货号:3471)中。每孔加入3mL含有10%血清替代物(Thermo FisherScientific Cat#10828028),终浓度100nM紫杉醇和终浓度1mM的5-氟尿嘧啶的DMEM/F12培养基(Corning,目录号: 10-092-cv),在37℃培养1个月,每隔3天换一次培养液,前两周中每周照射30Gy X射线一次,总共照射2次(两次间隔7天),每次照射10分钟。一个月后,使用死细胞去除试剂盒(Miltenyi Biotec,目录号:130-090-101)筛选出抵抗放化疗的肿瘤细胞,即休眠肿瘤细胞(DCCs),分别获得休眠肿瘤细胞4T1-DCCs、EMT6-DCCs、MCF7-DCCs,光镜照片见图5。
2、休眠肿瘤细胞的特征鉴定
(1)免疫印迹实验检测PCNA、H3S10ph和SETD4
分别将步骤1中得到3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)从培养基中分离出来,在每种细胞各400万颗中加入0.2mL细胞裂解液(50mM Tris-HCl(pH 7.4),150mM NaCl,1%乙基苯基聚乙二醇(NP-40),0.1%十二烷基硫酸钠(SDS)),冰上放置10分钟,12000 rpm 4℃离心10分钟,将上清转移到新的离心管中,加入0.05mL的蛋白上样缓冲液(250mM三羟甲基氨基甲烷盐酸盐,pH 6.8;0.1g/ml十二烷基硫酸钠;0.005g/ml溴酚蓝;50%甘油和0.05g/ml β-巯基乙醇,溶剂为水),沸水浴10分钟,12000rpm 25℃离心10分钟,取上清,即为蛋白样品,转至新的离心管中用于后续的上样。取3μL蛋白样品进行聚丙烯酰胺凝胶电泳,将凝胶中的蛋白转移到PVDF膜(聚偏二氟乙烯膜)上(转膜液:25mM三羟甲基氨基甲烷,pH 8.3,192mM甘氨酸和10%甲醇,溶剂为水)。TBS缓冲液(三羟甲基氨基甲烷12.1g和氯化钠17.5g溶到1500mL 蒸馏水中,滴加盐酸将pH调至7.4,蒸馏水定容至2000mL)洗1遍,5分钟;封闭液(品牌: Roche,货号:11921681001)封闭1小时。分别将Mouse monoclonalanti-PCNA(abcam,目录号:ab29), Rabbit monoclonal anti-H3S10ph(Cell signalingtechnology,目录号:53348)和Mouse monoclonal anti-SETD4Santa Cruz,目录号:sc-134221)的抗体各2μg加入到2mL的封闭液中配置成一抗稀释液,再将PVDF膜完全浸泡在所述稀释液中,4℃孵育过夜。第二天,PVDF膜用含0.1%吐温的 TBS洗4遍,每遍7分钟。将16.7μL的表1对应二抗加入到50mL的封闭液中配置成二抗稀释液,将PVDF膜完全浸泡在二抗稀释液中,25℃孵育40分钟。PVDF膜用含0.1%吐温的TBS洗 4遍,每遍7分钟。在PVDF膜中加入辣根过氧化氢酶底物Clarity Max Western ECL Substrate (BIO-RAD,货号:1705062),浸没处理,用显影仪检测。免疫印迹实验发现,3种休眠肿瘤细胞 (4T1-DCCs、EMT6-DCCs、MCF7-DCCs)均表达极低水平的细胞分裂指标蛋白PCNA和 H3S10ph,但是高量表达细胞静息蛋白SETD4,免疫印迹图见图6。
(2)免疫荧光检测ALDH1、CD44和CD24
分别将步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)用4%的多聚甲醛固定。固定后的细胞悬液分别滴在粘附性的载玻片上。待细胞稳定附着后,用PBS洗 1遍,5分钟。将载玻片分别浸入含0.25%Triton-100的PBS中25℃孵育10分钟,PBS洗1遍, 5分钟。再将载玻片分别浸入含1.5%驴血清的PBS(封闭液)中25℃孵育1小时。将Rabbit monoclonal anti-ALDH1A1(abcam,目录号:ab52492)、Rat monoclonal anti-CD44,FITC (eBiosciences,目录号:11-0441-82)和Alexa 647anti-CD24(BioLegend,目录号:311109) 抗体各2μg加入到200μL封闭液(品牌:Roche,货号:11921681001)中配置成一抗稀释液,将一抗稀释液滴加到载玻片中的细胞样品上,浸没即可,4℃孵育过夜。第二天,PBS洗3遍,每次5 分钟。将1μg Alexa Fluor 594荧光标记驴抗兔(Thermo Fisher scientific,货号R37119)的抗体加入到200μL的封闭液(品牌:Roche,货号:11921681001)中配置成二抗稀释液,将二抗稀释液滴加到ALDH1的载玻片中的细胞样品上,浸没即可,25℃孵育2小时。将200μL细胞核染色DAPI (碧云天,目录号:C1005)滴加到载玻片中的细胞样品上,浸没即可,25℃孵育10分钟。用50%甘油封片。样品用荧光显微镜进行观察,发现上述细胞均表达高水平的ALDH1和CD44及低水平的CD24(图7),表明分离获得的休眠肿瘤细胞为肿瘤干细胞。
实施例3、肿瘤细胞培养液来源粗外泌体的制备和鉴定
1、肿瘤细胞培养液的获取
500万颗的MCF7细胞系接种到90mm3的细胞培养皿中,培养基是10mL添加1%的青霉素- 链霉素的EMEM培养基(同实施例1)。37℃培养24小时后收集细胞的培养液,1000rpm4℃离心10分钟,取上清,12000rpm 4℃离心20分钟,取上清,即为MCF7细胞培养液。
500万颗的4T1细胞系接种到90mm3的细胞培养皿中,培养基是10mL添加1%的青霉素-链霉素的DMEM培养基(同实施例1)。37℃培养24小时后收集细胞的培养液,1000rpm 4℃离心 10分钟,取上清,12000rpm 4℃离心20分钟,取上清,即为4T1细胞培养液。
500万颗的EMT6细胞系接种到90mm3的细胞培养皿中,培养基是10mL添加1%的青霉素- 链霉素的Waymouth’s培养基(同实施例1)。37℃培养24小时后收集细胞的培养液,1000rpm 4℃离心10分钟,取上清,12000rpm 4℃离心20分钟,取上清,即为EMT6细胞培养液。
2、粗外泌体的制备
在20mL步骤1中获得的MCF7、4T1和MCF7细胞培养液中分别加入1mL的外泌体分离试剂(品牌:Thermo Fisher,货号:4478359),上下颠倒3次混匀后4℃孵育过夜。第二天,上述混合液10000rpm 4℃离心60分钟,去掉上清,用200μL的PBS重悬离心管底部的沉淀(沉淀即为粗外泌体),利用BCA蛋白质定量检测试剂盒(品牌:生工,货号:C503021)测定粗外泌体悬液中的蛋白总量,用PBS配制蛋白浓度为20μg/mL的粗外泌体溶液,用于后续的使用。
3、粗外泌体的鉴定
(1)粗外泌体粒径分布
使用粒径检测仪器(型号:ZetaView,厂家:Particle Metrix)对步骤2中获得的20μg/mL粗外泌体溶液进行检测,发现粗外泌体的粒径范围均为50-300nm,主体粒径大小均在150nm附近(图8)。
(2)粗外泌体形态特征
将2μL的步骤2中获得的20μg/mL粗外泌体溶液滴加到铜网上并覆盖铜网表面(直径 3.05mm,孔径90μm,厚度18μm,200目),25℃孵育10秒钟,滤纸吸除多余的粗外泌体。将2μL 含2%醋酸双氧铀的水溶液滴加到铜网上,25℃孵育10秒钟,滤纸吸除多余的醋酸双氧铀。将铜网放在40℃中烘干5分钟后,放入透射电子显微镜(型号:JEM-1200EX,厂家:NEC)中,电压调节到120kV,观察到双层膜结构的茶托状囊泡,符合外泌体的形态特征(图9)。
(3)免疫印迹检测粗外泌体中DEK、CD9、CD81和CD63
使用Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP)、Rabbitmonoclonal anti-CD9 (Abcam,目录号:ab92726)、Rabbit monoclonal anti-CD81(Abcam,目录号:ab109201)和Mouse monoclonal anti-CD63(Abcam,目录号:ab59479)的抗体,对步骤2中获得的20μg/mL粗外泌体溶液开展了DEK和外泌体通用的指标分子CD9、CD81和CD63的免疫印迹分析(方法同实施例2步骤2),发现粗外泌体表达高水平的DEK蛋白和CD9、CD81和CD63蛋白(图10)。以上的结果逐一验证了粗外泌体的大小、形态和分子特征,并且说明粗外泌体中含有DEK蛋白。
实施例4、肿瘤细胞培养液来源粗外泌体激活休眠肿瘤细胞
1、粗外泌体激活休眠肿瘤细胞
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度50ng/mL;37℃培养20小时后收集细胞,使用Rabbit polyclonal anti-DEK(Proteintech,目录号: 16448-1-AP)、Mouse monoclonal anti-PCNA(abcam,目录号:ab29),Rabbit monoclonal anti-H3S10ph (Cell signaling technology,目录号:53348)和Mouse monoclonal anti-SETD4(Santa Cruz,目录号:sc-134221)的抗体,以H3和GAPDH为内参,开展了免疫印迹分析(方法同实施例2步骤2),发现DEK,PCNA和H3S10ph的水平上升,SETD4的水平下降(图11)。研究结果表明外源添加粗外泌体能够激活休眠肿瘤细胞。将上述收集的细胞记为激活的休眠肿瘤细胞(A-DCCs),分别获得激活的休眠肿瘤细胞4T1 A-DCCs、EMT6 A-DCCs、MCF7 A-DCCs。
2、免疫荧光检测激活的休眠肿瘤细胞ALDH1、CD44和CD24
将上述获得的激活的休眠肿瘤细胞开展ALDH1、CD44和CD24的免疫荧光分析(同实施例 2步骤2),同样发现表达高水平的ALDH1和CD44及低水平的CD24(图12)。
实施例5、激活后的休眠肿瘤细胞具有体外成球和体内成瘤的能力
1、体外肿瘤球形成的能力检测
将实施例4中获取的激活的休眠肿瘤细胞(4T1 A-DCCs、EMT6 A-DCCs、MCF7 A-DCCs) 通过流式细胞分选仪,按1颗/孔的密度接种到超低吸附96孔板(品牌:Corning,货号:3469a)中。每个孔中加入200μL含10%血清替代物的DMEM/F12培养基,37℃连续培养三周,发现该细胞表现出极高的体外肿瘤球形成能力(图13)。
2、小鼠体内成瘤的能力检测
分别将100颗、10颗和1颗实施例4中获取的激活的休眠肿瘤细胞(4T1 A-DCCs、EMT6 A-DCCs、MCF7 A-DCCs)稀释在DMEM培养基和基质胶(Corning BioCoat,目录号:354234) 以体积比1:1的混合液100μL中,即为不同细胞含量的细胞液。将100μL的各细胞液原位注射到6-8周龄的Nod/Scid雌鼠腋下的乳腺脂肪垫中(EMT6和4T1各个细胞含量分别注射5只,MCF7 中100颗注射5只,10颗注射4只,1颗注射2只),将小鼠饲养在无菌的环境中,水和鼠粮持续供应,垫料及时更换。饲养时间在半年以内,瘤子的直径到达伦理上限时安乐死小鼠,手术取出肿瘤部位进行拍照(图14),发现该细胞表现出极强的小鼠体内成瘤的能力。
实施例6、实体瘤内休眠及激活的休眠肿瘤细胞的鉴定
1、休眠肿瘤细胞
将实施例2方法获得的100万颗4T1-DCCs和100万颗EMT6-DCCs细胞分别原位接种至8 周BALB/c雌鼠的脂肪垫中,将100万颗MCF7-DCCs细胞原位接种至8周Nod/Scid雌鼠脂肪垫中。待瘤子体积达到500mm3左右时,分别安乐死小鼠,手术取出4T1-DCCs、EMT6-DCCs和MCF7-DCCs肿瘤。将上述肿瘤分别放入4%多聚甲醛中4℃过夜,4℃浸泡到质量浓度30%蔗糖水溶液中脱水48小时,浸泡后的肿瘤捞出,放到10x10x5mm的敞开长方体型塑料材质的模具中,往其中加满OCT包埋剂(SAKURA,目录号:4583),将模具放在干冰上静置5分钟,将包埋块取出,-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。肿瘤切片用PBS洗1遍,5分钟。放入含0.25%Triton-100的PBS中,25℃孵育10分钟。放入含1.5%驴血清的PBS中25℃孵育1 小时。按实施例2步骤2方法分别开展Mouse monoclonal anti-ALDH1A1(BDPharmingen,目录号: 611195)抗体、Rat monoclonal anti-Ki67(eBioscience,目录号:14-5698-82)抗体和Rabbit polyclonal anti-SETD4(Sigma-Aldrich,目录号:HPA024073)抗体以及Mouse monoclonal anti-ALDH1A1(BD Pharmingen,目录号:611195)抗体、Ratmonoclonal anti-Ki67(eBioscience,目录号:14-5698-82)抗体和Rabbit polyclonalanti-DEK(Proteintech,目录号:16448-1-AP)抗体的免疫荧光分析,Alexa Fluor 488荧光标记驴抗鼠免疫球蛋白(Thermo Fisher scientific,货号A21202)对应ALDH1,AlexaFluor 594荧光标记羊抗大鼠免疫球蛋白(Thermo Fisher scientific,货号A11007)对应Ki67,Alexa Fluor 647荧光标记驴抗兔免疫球蛋白(Thermo Fisher scientific,货号A31573)对应SETD4和DEK样品,用荧光显微镜进行观察。结果发现ALDH1阳性且Ki67阴性的肿瘤细胞中,SETD4高表达且DEK低表达(图15),说明DEK在这一类静息的肿瘤细胞中低表达。
2、激活肿瘤细胞
将步骤1中4T1-DCCs、EMT6-DCCs、MCF7-DCCs分别替换为实施例4方法获得的4T1A-DCCs、EMT6 A-DCCs、MCF7 A-DCCs,其他操作相同。
ALDH1阳性且Ki67阳性的肿瘤细胞中,SETD4低表达且DEK高表达(图16),说明DEK在这一类激活的肿瘤细胞中高表达。
实施例7、激活后的休眠肿瘤细胞DEK蛋白显著上升,SETD4蛋白显著下降
采用实施例2步骤2的方法,使用Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP)抗体和Mouse monoclonal anti-SETD4(Santa Cruz,目录号:sc-134221)抗体,开展了休眠肿瘤细胞(实施例2步骤1获取)和激活的休眠肿瘤细胞(实施例4获取)的免疫印迹分析(同实施例2),发现与休眠肿瘤细胞相比,激活的休眠肿瘤细胞高量表达DEK蛋白,但是SETD4 表达显著下降(图17)。研究结果表明DEK蛋白对休眠肿瘤细胞的激活有着重要的作用。
实施例8、外源DEK蛋白及结构域NLS突变DEK蛋白的制备
1、制备表达外源DEK蛋白及结构域突变DEK蛋白的杆状病毒
先用引物F3(CCGGAATTCATGGTGAGCA)和引物R3(CGCTCTAGATCAAGAAATTAG) 对SEQID NO.6和SEQ ID NO.8进行PCR扩增,得到含有酶切位点的人源hEGFP-DEK序列和人源hEGFP-DEK△NLS序列。再对上述的序列和pFastBacTMHT A质粒同时进行EcoRⅠ和XbaⅠ的双酶切处理和连接处理。
同样地,用引物F4(AGAATTCATGGTGAGCAAGGGCGA)和引物R4 (GCTCTAGATCAAGAAATTAGCTCTTTTACAGTTGT)对SEQ ID NO.10和SEQ ID NO.12进行PCR扩增,得到含有酶切位点的鼠源hEGFP-DEK序列和鼠源hEGFP-DEK△NLS序列。再对上述的序列和pFastBacTMHT A质粒同时进行EcoRⅠ和XbaⅠ的双酶切处理和连接处理。
分别将人源hEGFP-DEK基因(核苷酸序列:SEQ ID NO.6,氨基酸序列:SEQ IDNO.7)、人源hEGFP-DEK△NLS(核定位序列NLS缺失)基因(核苷酸序列:SEQ ID NO.8、氨基酸序列: SEQ ID NO.9)、鼠源mEGFP-DEK基因(核苷酸序列:SEQ ID NO.10,氨基酸序列:SEQ IDNO.11)、鼠源mEGFP-DEK△NLS(核定位序列NLS缺失)基因(核苷酸序列:SEQ ID NO.12,氨基酸序列: SEQ ID NO.13)连入pFastBacTMHT A质粒(购自Thermo Fisher,货号:10712024)的EcoRⅠ和 XbaⅠ的酶切位点(图18)中。
将连入上述基因后的pFastBacTMHT A质粒转化大肠杆菌DH10Bac感受态细胞(购自Gibco, 货号:10361012),42℃水浴45秒。在转化后的大肠杆菌中加入1mL LB培养基(胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,溶剂为水,pH=7.4),37℃220rpm摇4小时。1000rpm离心 5分钟,去掉上清,加入100μL LB培养基重悬。100μL重悬液涂布至含有终浓度30μL/mL卡那霉素、终浓度15μL/mL庆大霉素、终浓度12μL/mL四环霉素、40μL(20mg/ml)X-gal(5-溴-4- 氯-3-吲哚-β-D-吡喃半乳糖苷,溶剂为二甲基甲酰胺),4μL(200mg/ml)IPTG(异丙基-β-D-硫代半乳糖苷,溶剂为超蒸水)的LB固体培养基(LB培养基中加入15g/L琼脂)中,37℃避光培养 48小时,挑选其中的白色单菌落接种到2mL的LB培养基中,37℃220rpm摇过夜。第二天用试剂盒(购自Invitrogen,货号:K210002)进行质粒抽提,得到重组杆状病毒质粒(Bacmid)。将重组Bacmid利用Cellfectin转染试剂(Gibco,货号:10362100)转染至Sf9细胞(ATCC来源,货号: CRL-1711)中。转染后的细胞接种在Sf-900培养基(Gibco,货号:10902179)中,27℃培养72小时,收集细胞培养液,10000xg离心20分钟,取上清为病毒液,分别获得携带人源hDEK-GFP 基因、鼠源mDEK-GFP基因、人源hDEK△NLS-GFP基因、鼠源mDEK△NLS-GFP基因的重组杆状病毒。
2、外源DEK蛋白及结构域突变DEK蛋白的表达和纯化
Sf9细胞按照50万/mL的密度接种到100ml的Sf-900培养基(Gibco,货号:10902179)中,27℃培养72小时,加入10mL滴度为108pfu/mL的上述重组杆状病毒(携带人源DEK-GFP、鼠源 DEK-GFP或人源DEKΔNLS-GFP、鼠源DEKΔNLS-GFP基因,共4个基因序列),27℃培养5天,1200 rpm离心10分钟,去掉上清,细胞沉淀用裂解液(50mM磷酸二氢钠和300mM氯化钠溶于双蒸水中,pH=8.0)重悬。重悬后的溶液进行超声处理(39瓦特,10秒超声,50秒停顿,超声时长 3分钟)。超声后的溶液12000rpm 4℃离心20分钟,取上清即为蛋白溶液。利用His标签蛋白纯化试剂盒(碧云天,货号:P2226)对上述的蛋白溶液进行纯化,纯化后的蛋白溶于PBS中,制备成20μg/mL的蛋白溶液,即外源DEK蛋白的PBS溶液(mDEK-GFP、hDEK-GFP)、NLS突变DEK蛋白的PBS溶液(hDEKΔNLS-GFP、mDEKΔNLS-GFP)。考马斯亮蓝染色发现蛋白的纯度较高,蛋白大小正确(图19)。
实施例9、外源DEK蛋白的添加激活休眠肿瘤细胞
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL 的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL,使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL。其中4T1-DCCs和EMT6-DCCs中各自分别加入mDEK-GFP和mDEKΔNLS-GFP, MCF7-DCCs中分别加入hDEK-GFP和hDEKΔNLS-GFP,37℃培养20小时后收集细胞。取部分细胞,使用Rabbit polyclonal anti-DEK(Proteintech,目录号:16448-1-AP),Mouse monoclonalanti-PCNA(abcam,目录号:ab29),Rabbit monoclonal anti-H3S10ph(Cell signalingtechnology,目录号:53348)和Mouse monoclonal anti-SETD4(Santa Cruz,目录号:sc-134221)的抗体,开展了免疫印迹分析(方法同实施例2步骤2,二抗见表1),发现DEK,PCNA和H3S10ph的水平上升,SETD4的水平下降(图20)。研究结果表明外源添加DEK蛋白能够激活休眠肿瘤细胞,将其记为外源DEK蛋白激活的休眠肿瘤细胞,分别获得外源DEK蛋白激活的4T1A-DCCs、EMT6 A-DCCs、MCF7 A-DCCs。
实施例10、结合化疗,外源DEK蛋白添加能够清除休眠肿瘤细胞
1、DEK蛋白结合化疗对休眠肿瘤细胞cCasp3信号的影响
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL 的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL,使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL,其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEKΔNLS-GFP, MCF7-DCCs中分别添加hDEK-GFP和hDEKΔNLS-GFP。37℃培养20小时后收集细胞,使用切割的Rabbitmonoclonal anti-cleaved Caspase-3(abcam,货号:ab32042)抗体开展免疫荧光实验(具体操作同实施例2步骤2,二抗更替为Alexa Fluor 594荧光标记驴抗兔,Thermo Fisher,货号: R37119)。样品用荧光显微镜进行观察,发现cCasp3的信号大量出现,细胞出现大量的凋亡(图 21)。
2、DEK蛋白结合化疗处理不同时间对休眠肿瘤细胞的影响
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL 的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL,使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL,其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEKΔNLS-GFP, MCF7-DCCs中分别添加hDEK-GFP和hDEKΔNLS-GFP。37℃培养0小时、5小时、20小时、25 小时、30小时后收集细胞样品,台盼蓝染色检测死亡的细胞,发现原本抵抗放化疗的休眠肿瘤细胞逐渐全部死亡(图22)。
以上的结果表明结合化疗,外源DEK蛋白添加能够清除休眠肿瘤细胞。
实施例11、在激活的休眠肿瘤细胞中干涉DEK使细胞重新进入静息状态
1、制备表达靶向DEK的小发卡RNA(shRNA)慢病毒
(1)序列
靶向人源DEK的干涉序列1:GGATAGTTCAGATGATGAAC,SEQ ID NO.14,记为shDEK #H1;
靶向人源DEK的干涉序列2:GTGATGAAGATGAAAAGAAA,SEQ ID NO.15,记为shDEK #H2;
靶向鼠源DEK的干涉序列1:GTGAAGAAATTACTGGCTGAT,SEQ ID NO.16,记为shDEK #M1;
靶向鼠源DEK的干涉序列2:CGAACTCGTGAAGAGGATCTT,SEQ ID NO.17,记为shDEK #M2;
杂乱的干涉序列:ATGTTAACAGCTGTACTGGTG,SEQ ID NO.18,记为shCTRL。
(2)重组慢病毒表达载体
将5段靶向DEK的shRNA序列分别插入到pLent-U6-RFP-Puro慢病毒表达载体(维真生物,货号:LT88024,图23)的BamHⅠ和MluⅠ位点上,分别获得含人源DEK1(SEQ ID NO.14)重组慢病毒表达载体,含人源DEK2(SEQ ID NO.15)重组慢病毒表达载体,含鼠源DEK1(SEQID NO.16)重组慢病毒表达载体,含鼠源DEK2(SEQ ID NO.17)重组慢病毒表达载体和含杂乱序列(SEQ ID NO.18)重组慢病毒表达载体。
(3)靶向DEK的shRNA慢病毒
将上述含人源DEK1(SEQ ID NO.14)重组慢病毒表达载体和慢病毒包装质粒混合物(pMDL, VSVG,pRSV-Rev,质量比例为5:3:2,购自上海翊圣生物,货号:41102ES10)共同转染293T 细胞(ATCC,货号:ACS-4500),具体为:取一个洁净无菌离心管,加入750μL不含抗生素和血清的DMEM培养液,分别加入4.5μg慢病毒包装质粒混合物和1.5μg含人源DEK1(SEQID NO.14)重组慢病毒表达载体,并用枪轻轻吹打混匀,再加入24μl脂质体转染试剂Lipo8000 (Beyotime,目录号:C0533),用枪轻轻吹打混匀,获得转染试剂和质粒的混合物。将转染试剂和质粒的混合物750μL均匀滴加到293T细胞中,37℃培养72小时。转染72小时后收集细胞培养上清,即为病毒液。取6个SW28离心管,每个SW28离心管中加入约32ml的病毒液。取6 支10ml的移液管,分别吸取4ml质量浓度20%的蔗糖水溶液,将移液管一直插入到每支SW28 离心管的底部,缓慢将蔗糖水溶液打出4ml。用PBS调整各管的重量,使对应的SW28离心管之间的重量相差不超过0.1g。按次序将所有6个SW28离心管放入Beckman SW28超速离心转头中, 4℃,25000rpm离心2小时。小心将SW28管子从转头中取出。倒掉上清,将SW28离心管倒扣在纸巾上放置10分钟使剩余的上清流干。吸掉剩余的液滴。每管中加入1ml不含钙和镁的PBS 重悬管底的沉淀。将SW28离心管插入到50ml锥底离心管中,盖上盖子,4℃溶解2小时,每隔 20分钟轻轻震荡。4℃,500rpm离心1分钟,使溶液集中于管底。用200μl移液器轻柔吹打使沉淀重悬,避免产生泡沫。将所有管中的液体集中到一个新的离心管中,即为浓缩纯化后的病毒液 6ml,记为慢病毒shDEK#H1(即人源DEK干涉序列1)。使用试剂盒(GeneCopoeia,货号:LT005) 进行病毒滴度的测定,病毒滴度为108pfu/mL。
将含人源DEK1(SEQ ID NO.14)重组慢病毒表达载体分别替换为含人源DEK2(SEQID NO.15)重组慢病毒表达载体,含鼠源DEK1(SEQ ID NO.16)重组慢病毒表达载体,含鼠源DEK2 (SEQ ID NO.17)重组慢病毒表达载体和含杂乱序列(SEQ ID NO.18)重组慢病毒表达载体,其他操作相同。最终分别获得靶向DEK的shRNA慢病毒shDEK#H1,shDEK#H2(人源DEK干涉序列2,6ml,病毒滴度为108pfu/mL),shDEK#M1(鼠源DEK干涉序列2,6ml,病毒滴度为108pfu/mL),shDEK#M2(鼠源DEK干涉序列2,6ml,病毒滴度为108pfu/mL),shCTRL(干涉对照6ml,病毒滴度为108pfu/mL)。
2、在激活的休眠肿瘤细胞中干涉DEK使细胞重新进入静息状态
(1)干涉DEK使休眠肿瘤细胞重新进入静息状态
分别将实施例4获取的3种激活的休眠肿瘤细胞(4T1A-DCCs、EMT6 A-DCCs、MCF7A-DCCs)以10万颗细胞/孔的密度接种到低贴6孔板中,培养基为3mL的DMEM/Ham's F-12,37℃培养2小时后,在每个孔中加入终浓度6μg/mL的聚凝胺(polybrene);在4T1A-DCCs和EMT6A-DCCs培养孔中各自分别加入10μL 108pfu/mL的步骤1制备的慢病毒shDEK#M1、shDEK#M2、shCTR,同时不添加慢病毒作为未处理。在MCF7A-DCCs培养孔中分别加入10μL108pfu/mL的步骤1制备的慢病毒shDEK#H1、shDEK#H2、shCTRL,同时不添加慢病毒作为未处理。37℃培养24小时后更换成新的培养基,继续培养72小时,收集细胞(记为干涉DEK后的细胞),开展Rabbit polyclonal anti-DEK、Rabbit monoclonal anti-H3S10ph、Mousemonoclonal anti-PCNA和Mouse monoclonal anti-SETD4抗体的免疫印迹实验(方法同实施例2步骤2,二抗见表1),发现Ki67,PCNA和H3S10ph的水平下降,SETD4的水平上升(图24中a)。
(2)干涉DEK后的休眠肿瘤细胞成瘤能力受到抑制
将上述步骤(1)干涉DEK后的细胞1万颗接种到3mL含10%血清替代物的DMEM/Ham's F-12 培养基中,37℃培养一周,发现形成肿瘤球的能力受到抑制(图24中b)。
以上结果表明在激活的休眠肿瘤细胞中干涉DEK使细胞重新进入静息状态。
3、DEK干涉导致的休眠肿瘤细胞中添加外源DEK可重新激活休眠肿瘤细胞
(1)分别将步骤2中慢病毒shDEK#M1和shCTRL干涉DEK后的4T1A-DCCs细胞按4000颗细胞/孔的密度接种到5mL含10%血清替代物的DMEM/Ham's F-12培养基中。shDEK#M1干涉DEK后的4T1A-DCCs分别添加12.5μL的PBS、12.5μL 20μg/mL实施例8步骤2中制备的mDEK-GFP的PBS溶液和12.5μL 20μg/mL实施例8步骤2中制备的mDEKΔNLS-GFP的PBS溶液,使mDEK-GFP和mDEKΔNLS-GFP在培养基中的终浓度均为50ng/mL;shCTRL干涉DEK后的 4T1A-DCCs细胞不添加PBS或蛋白。37℃培养20小时后收集细胞(记为鼠源干涉序列 1+mDEK-GFP、鼠源干涉序列1+mDEK△NLS-GFP、鼠源干涉序列1+PBS、shCTRL),开展DEK-GFP、 DEK、H3S10ph、PCNA和SETD4抗体的免疫印迹实验(方法同实施例2步骤2,一抗和二抗见表1)。同样条件下,对步骤2中慢病毒shDEK#M2和shCTRL干涉DEK后的EMT6A-DCCs细胞(mDEK-GFP和mDEKΔNLS-GFP),shDEK#H2和shCTRL干涉DEK后的MCF7A-DCCs细胞 (hDEK-GFP和hDEKΔNLS-GFP)进行免疫印迹实验。
发现PCNA和H3S10ph的水平回升,SETD4的水平回降(图25)。
(2)将上述步骤(1)重新加入DEK蛋白的shDEK干涉细胞(即鼠源干涉序列1+mDEK-GFP 的4T1A-DCCs和EMT6A-DCCs细胞、人源干涉序列1+mDEK-GFP的MCF7A-DCCs细胞)1万颗接种到3mL含10%血清替代物的DMEM/Ham's F-12培养基中,37℃培养一周,体外成球试验的光镜照片和成球率的统计图发现形成肿瘤球的能力得到恢复(图26)。
以上结果表明DEK干涉导致的休眠肿瘤细胞中添加外源DEK可重新激活休眠肿瘤细胞。
实施例12、外源DEK蛋白激活休眠肿瘤细胞的调控机制
1、外源DEK蛋白引起异染色质的下降,常染色质的上升
(1)将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL 的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL,使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL,其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEKΔNLS-GFP, MCF7-DCCs中分别添加hDEK-GFP和hDEKΔNLS-GFP。37℃培养20小时后收集细胞。将细胞加入2.5%的戊二醛水溶液中4℃固定过夜。第二天,倒掉固定液,用PBS漂洗样品三次,每次15 分钟。用1%的锇酸水溶液25℃孵育样品1-2h。取出锇酸废液,用PBS漂洗样品三次,每次15 分钟。用梯度浓度(30%,50%,70%,80%,90%和95%)的乙醇水溶液25℃孵育样品各15分钟。100%乙醇25℃孵育样品20分钟。纯丙酮25℃孵育样品20分钟。Spurr包埋剂(二氧化乙烯环己烯、聚丙烯乙二醇的二缩水甘油醚、壬基琥珀酸酐和二甲基乙醇胺四种物质聚合而成,购自SPI-CHEM公司)与纯丙酮的混合液(V/V=1/1)25℃孵育样品1小时。Spurr包埋剂与丙酮的混合液(V/V=3/1)25℃孵育样品3小时。纯Spurr包埋剂25℃孵育样品过夜。第二天,将样品捞出,放在10x10x5mm的敞开长方体型塑料材质的包埋模具(SAKURA,货号:4566)里,模具中倒满新的Spurr包埋剂,放到70℃烘箱中静置过夜。第二天,得到包埋好的样品。样品在LEICA EM UC7型超薄切片机中切片,获得70nm的切片。在切片中滴加柠檬酸铅溶液(21.33g的硝酸铅和1.76g的柠檬酸钠加入到30mL的双蒸水中,用力振荡30分钟,至溶液呈乳白色浑浊状后,加入1mol/L的氢氧化钠水溶液8mL,使溶液变得清亮透明,再加双蒸水定容到50mL),浸没即可,25℃染色10分钟,滤纸吸除多余的染色液。在切片中滴加醋酸双氧铀50%乙醇饱和溶液(2g 醋酸双氧铀加到100mL的50%乙醇中,充分搅拌10分钟,静置1天后取上清液使用),浸没即可, 25℃染色10分钟,滤纸吸除多余的染色液。用透射电镜观察完成染色的切片。发现在休眠肿瘤细胞中添加了PBS或外源NLS突变DEK蛋白后,细胞核内异染色质水平没有显著性的改变,而在休眠肿瘤细胞中添加了外源DEK蛋白后,细胞核内异染色质水平显著下降(图27)。
(2)将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL 的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS突变DEK蛋白的PBS溶液25μL,使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL,其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEKΔNLS-GFP, MCF7-DCCs中分别添加hDEK-GFP和hDEKΔNLS-GFP。37℃培养20小时后收集细胞。使用Mouse monoclonalanti-H4K20me1(Santa Cruz,目录号:sc-134221)、Rabbit polyclonal anti-H4K20me2(abcam,目录号:ab9052)、Rabbit polyclonal anti-H4K20me3(abcam,目录号:ab9053)、Rabbit polyclonal anti-HP1α(Cell Signaling Technology,目录号:2616)、Rabbitpolyclonal anti-H3K9ac (abcam,目录号:ab10812)、Rabbit monoclonal anti-H3K9me3(abcam,目录号:ab176916)和 Mouse monoclonal anti-H3K27me3(abcam,目录号:ab6002),在上述细胞中开展免疫印迹分析(方法同实施例2步骤2,H4K20me1和H3K27me3对应偶联辣根过氧化酶驴抗鼠二抗,H4K20me2、 H4K20me3、H3K9ac、HP1α和H3K9me3对应偶联辣根过氧化酶驴抗兔二抗)。发现与在休眠肿瘤细胞中添加了PBS或外源NLS突变DEK蛋白相比,在休眠肿瘤细胞中添加了外源DEK蛋白后,组成型异染色质的分子指标H4K20me3和异染色质形成关键蛋白HP1-α的水平显著下降,常染色质的分子指标H3K9ac的水平显著上升,兼性异染色质的分子指标H3K9me3和H3K27me3 的水平没有显著性的变化(图28)。
2、DEK蛋白在染色质上结合位点的基因检测
(1)染色质免疫沉淀试验的高通量测序(ChIP-seq):收集实施例9中的外源DEK蛋白激活的MCF7 A-DCCs细胞,使用试剂盒EZ ChIP Kit(Millipore,目录号:17-371)和DEK(Proteintech,目录号:16448-1-AP)抗体开展DEK蛋白的染色质免疫沉淀试验(ChIP-Seq)。1g细胞中加入 1mL 4%多聚甲醛(溶于PBS)25℃孵育10分钟。加入1mL 1M甘氨酸(溶于PBS)25℃孵育10 分钟。1000rpm离心5分钟,去上清,在细胞沉淀中加入1.5mL裂解液(50mMTris,pH8.1,1% SDS和0.1μmol/L蛋白酶抑制剂混合物(Sigma-Aldrich,货号:P8340)),吹打均匀,冰上放置 10分钟。悬液进行超声(39瓦特,10秒超声,50秒停顿,总超声时长2分钟)。4℃12000rpm 离心10分钟,取上清,加入60μL的ChIP Blocked Protein G Agarose(Sigma-Aldrich,货号: 16-201D)和3μg Rabbit polyclonal anti-DEK抗体4℃孵育过夜。3000rpm离心2分钟,去掉上清,取沉淀。TE缓冲液(10mM Tris-HCI和1mM EDTA,pH=8.0)洗3遍,每遍5分钟。洗涤后的沉淀中加入200μL洗脱液(10μL 20%SDS水溶液;20μL 1M碳酸氢钠水溶液和170μL双蒸水) 25℃孵育15分钟,3000rpm离心2分钟,将上清转移至新的离心管中。在上清中加入8μL 5M氯化钠水溶液,65℃水浴孵育5小时。加入1μL RNase A 37℃水浴孵育30分钟。加入4μL 0.5M EDTA 水溶液;8μL 1M Tris-HCl和1μL Proteinase K(Sigma-Aldrich,货号:20-298),45℃水浴孵育 1小时。利用DNA纯化试剂盒(Solarbio,货号:D1300)提取DNA。将获得的DNA构建文库 (包括末端修复、加A、加接头、长度筛选、PCR扩增)并进行高通量测序(该操作委托北京诺禾致源科技股份有限公司完成)。
(2)测序结果的分析:DEK结合位点的峰分布在所有23条染色体上(图29),DEK结合位点的峰主要集中在启动子区域,占64.35%(其中内含子区域占10.5%,基因间区域占11.81%)(图 30)。对DEK结合位点的峰对应的基因进行基因本体富集分析(GO富集分析)发现,DEK结合的基因主要与胞内信号转导、个体发育和蛋白结合相关(图31)。对DEK的结合信号进行可视化分析发现,DEK结合在SETD4、TP53和MYC基因的启动子区域上(图32)。
3、外源DEK蛋白引起染色质的开放水平上升
(1)易接近转座酶核染色质区域的高通量测序(ATAC-seq):收集实施例2步骤1中获得的MCF7-DCCs休眠肿瘤细胞和实施例9中获得的外源DEK蛋白激活的MCF7 A-DCCs细胞,开展易接近转座酶核染色质区域的高通量测序(ATAC-Seq)。提取上述两种细胞样品的细胞核,加入转座酶mix,mix中包含转座酶和两种等摩尔的接头Adapter 1和Adapter 2,37℃下孵育30分钟。将产物进行引物扩增、片段长度选择和纯化,获得文库后上机测序,该操作委托北京诺禾致源科技股份有限公司完成。
(2)测序结果的分析:激活后的休眠肿瘤细胞中的整体信号强度显著高于休眠的肿瘤细胞,表明休眠肿瘤细胞在激活后开放的基因区域大幅增加(图33)。将在激活的休眠肿瘤细胞中信号上调的基因进行基因本体富集分析(GO富集分析),发现上调的基因主要与胞内信号转导、基因表达、细胞周期、代谢过程、生物合成过程、催化活性和激酶活性等生物过程相关(图34)。对休眠肿瘤细胞和外源DEK蛋白激活的休眠肿瘤细胞的ATAC-seq信号进行UCSC基因浏览器可视化分析发现,激活的休眠肿瘤细胞中的SETD4和TP53基因的ATAC信号显著低于休眠肿瘤细胞,激活的休眠肿瘤细胞中的MYC基因的ATAC信号显著高于休眠肿瘤细胞(图35),说明休眠肿瘤细胞在外源DEK蛋白激活之后,SETD4和TP53基因的开放程度下降,MYC基因的开放程度上升。
4、外源DEK蛋白引起基因表达的变化
(1)转录组的高通量测序(RNA-seq):收集实施例2步骤1中获得的MCF7-DCCs休眠肿瘤细胞和实施例9中获得的外源DEK蛋白激活的MCF7 A-DCCs细胞,开展转录组的高通量测序 (RNA-seq)。采用 Stranded RNA-Seq Kit(Clontech,货号:634836),在上述的细胞 1g样品中加入1mL TRIzol试剂(Thermo Fisher),抽提总RNA,通过Oligo(dT)磁珠富集带polyA 尾的mRNA。使用Illumina的/> UltraTM RNA Library Prep Kit进行转录组文库的构建,上机测序。使用HISAT2软件将测序片段的序列比对到参考基因组UCSC人类参考基因组hg38 上,根据基因比对在参考基因组上的位置信息,统计每个基因从起始到终止范围内覆盖的信号数量,进行基因表达水平的定量分析。进一步对基因表达水平的数据进行统计学分析,筛选不同样本间表达水平显著差异的基因。
(2)测序结果的分析:发现在激活后的休眠肿瘤细胞中,有2119个上调的基因和3338个下调的基因(图36)。对得到的差异基因进行基因本体分析(GO分析),发现在激活后的休眠肿瘤细胞中,上调的基因主要与细胞激活过程相关(GO期转换成G1期、细胞增殖、细胞转录、细胞呼吸和代谢过程)(图37),下调的基因主要与细胞静息相关(细胞周期的负调控、甲基化依赖的染色质沉默、p53通路、翻译终止、细胞死亡和蛋白泛素化)(图38)。在基因的表达水平上开展基因集富集分析(GSEA分析),发现在激活后的休眠肿瘤细胞中,表达水平上调的基因集主要包括DNA复制、G2M检查点、有丝分裂纺锤体、氧化磷酸化、三羧酸循环、E2F靶向基因、MYC 靶向基因和脂肪酸代谢(图39),表达水平下调的基因集主要包括低氧、炎症反应、补体、凝血、上皮间充质转化、p53信号通路、TNFA信号通路、JAK-STAT3信号通路和Kras信号通路。MYC 信号通路中有25个基因显著上调,p53信号通路中有30个基因显著下调(图40)。
5、外源DEK蛋白添加引起P53水平的下调和MYC水平的上调
将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL的PBS、实施例8步骤2中制备的20μg/mL的外源DEK蛋白的PBS溶液25μL及20μg/mL的NLS 突变DEK蛋白的PBS溶液25μL,使得培养基中外源DEK蛋白和NLS突变DEK蛋白终浓度均为50ng/mL,其中4T1-DCCs和EMT6-DCCs中各自分别添加mDEK-GFP和mDEKΔNLS-GFP, MCF7-DCCs中分别添加hDEK-GFP和hDEKΔNLS-GFP。37℃培养20小时后收集细胞。使用Mouse monoclonalanti-p53(Santa Cruz,目录号:sc-126)、Rabbit monoclonal anti-p21(Cell SignalingTechnology,目录号:2947)、Rabbit polyclonal anti-PUMA(abcam,目录号:ab9643)和Mouse monoclonal anti-c-Myc(Santa Cruz,目录号:sc-40)的抗体,在上述细胞中开展免疫印迹分析(方法同实施例2步骤2,二抗见表1)。发现与休眠肿瘤细胞和休眠肿瘤细胞中添加DEK△NLS-GFP 蛋白20小时相比,在休眠肿瘤细胞中添加DEK-GFP蛋白20小时的细胞中,p53、p21和PUMA 的水平显著下降,MYC的水平显著上升(图41)。
实施例13、结合化疗,肿瘤细胞培养液来源粗外泌体添加能够清除休眠肿瘤细胞
1、粗外泌体结合化疗检测细胞cCasp3信号
将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μLPBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度50ng/mL,37℃培养20小时后收集细胞,使用Rabbit monoclonal anti-cleavedCaspase-3(abcam, 货号:ab32042)抗体开展免疫荧光实验(具体操作同实施例2步骤2,二抗更替为Alexa Fluor 594 荧光标记驴抗兔,Thermo Fisher,货号:R37119)。样品用荧光显微镜进行观察,发现cCasp3 的信号大量出现,细胞出现大量的凋亡(图42)。
2、粗外泌体结合化疗处理时间对休眠肿瘤细胞活力影响
将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs)各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μLPBS (作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度50 ng/mL,37℃培养0小时、5小时、20小时、25小时、30小时后收集细胞样品,台盼蓝染色检测死亡的细胞,发现原本抵抗放化疗的休眠肿瘤细胞逐渐全部死亡(图43)。
以上的结果表明结合化疗,肿瘤细胞培养液来源粗外泌体添加能够清除休眠肿瘤细胞。
实施例14、肿瘤细胞培养液来源粗外泌体激活休眠肿瘤细胞的调控机制
1、粗外泌体的添加引起异染色质水平的下调和常染色质水平的上调
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体的PBS溶液使其在培养基中的终浓度50ng/mL,37℃培养20小时后收集细胞。使用Mouse monoclonal anti-H4K20me1(Santa Cruz,目录号:sc-134221)、Rabbit polyclonal anti-H4K20me2(abcam,目录号:ab9052)、Rabbit polyclonal anti-H4K20me3(abcam,目录号:ab9053)、Rabbitpolyclonal anti-HP1α(Cell Signaling Technology,目录号:2616)、Rabbit polyclonalanti-H3K9ac(abcam,目录号:ab10812)、Rabbit monoclonal anti-H3K9me3(abcam,目录号:ab176916)和Mouse monoclonal anti-H3K27me3(abcam,目录号:ab6002)的抗体,在上述细胞中开展免疫印迹分析(方法同实施例2步骤2,二抗见表1)。发现与在休眠肿瘤细胞中添加PBS相比,在休眠肿瘤细胞中添加了粗外泌体后,组成型异染色质的分子指标H4K20me3和异染色质形成关键蛋白HP1-α的水平显著下降,常染色质的分子指标 H3K9ac的水平显著上升,兼性异染色质的分子指标H3K9me3和H3K27me3的水平没有显著性的变化(图44)。
2、粗外泌体的添加引起P53信号通路的下调和MYC信号通路的上调
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物的DMEM/F12培养基中,每种细胞分别添加25μL PBS(作为对照)和25μL实施例3步骤2制备的20μg/mL粗外泌体PBS溶液使其在培养基中的终浓度50ng/mL,37℃培养20小时后收集细胞。使用Mouse monoclonal anti-p53(Santa Cruz,目录号:sc-126)、Rabbit monoclonal anti-p21(Cell SignalingTechnology,目录号:2947)、Rabbit polyclonal anti-PUMA(abcam,目录号:ab9643)和Mouse monoclonal anti-c-Myc(Santa Cruz,目录号:sc-40)的抗体,在上述细胞中开展免疫印迹分析(方法同实施例2步骤2,二抗见表1)。发现与在休眠肿瘤细胞中添加PBS相比,在休眠肿瘤细胞中添加了粗外泌体后,p53、p21和PUMA 的水平显著下降,MYC的水平显著上升(图45)。
实施例15、含有外源DEK蛋白及结构域NLS突变DEK蛋白的分选外泌体的制备
1、构建过表达外源DEK蛋白及结构域NLS突变DEK蛋白的质粒
先用引物F1(CCGGAATTCTATGTCCGCCT)和引物R1(CGCTCTAGATCAAGAAATTAG) 对SEQID NO.5和SEQ ID NO.19进行PCR扩增,得到涵盖酶切位点的人源DEK基因序列。再对上述的序列和pEGFP-C1质粒同时进行EcoRⅠ和XbaⅠ的双酶切处理和连接处理。
同样地,用引物F2(AGAATTCTATGTCGGCGGCGGCGG)和引物R2 (GCTCTAGATCAAGAAATTAGCTCTTTTACAGTTGT)对SEQ ID NO.21和SEQ ID NO.23进行PCR扩增,得到涵盖酶切位点的鼠源DEK基因序列。再对上述的序列和pEGFP-C1质粒同时进行EcoRⅠ和XbaⅠ的双酶切处理和连接处理。
将人源hDEK基因(核苷酸序列:SEQ ID NO.5,氨基酸序列:SEQ ID NO.1)、人源hDEK △NLS(核定位序列NLS缺失)(核苷酸序列:SEQ ID NO.19,氨基酸序列:SEQ ID NO.20)、鼠源mDEK基因(核苷酸序列:SEQ ID NO.21,氨基酸序列:SEQ ID NO.22)、鼠源mDEK△NLS (核定位序列NLS缺失)基因(核苷酸序列:SEQ ID NO.23,氨基酸序列:SEQ ID NO.24)分别插入到pEGFP-C1质粒(优宝生物,货号:VT1118)(图46)的EcoRⅠ和XbaⅠ位点上。分别获得重组质粒pEGFP-C1-hDEK,pEGFP-C1-mDEK,pEGFP-C1-hDEK△NLS和 pEGFP-C1-mDEK△NLS,即为过表达外源DEK蛋白及结构域NLS突变DEK蛋白的质粒。
2、构建过表达外源DEK蛋白及结构域NLS突变DEK蛋白的慢病毒
先用引物F1(CCGGAATTCTATGTCCGCCT)和引物R1(CGCTCTAGATCAAGAAATTAG) 对SEQID NO.5和SEQ ID NO.19进行PCR扩增,得到涵盖酶切位点的人源DEK基因序列。再对上述的序列和pLent-N-GFP质粒同时进行EcoRⅠ和XbaⅠ的双酶切处理和连接处理。
同样地,用引物F2(AGAATTCTATGTCGGCGGCGGCGG)和引物R2 (GCTCTAGATCAAGAAATTAGCTCTTTTACAGTTGT)对SEQ ID NO.21和SEQ ID NO.23进行 PCR扩增,得到涵盖酶切位点的鼠源DEK基因序列。再对上述的序列和pLent-N-GFP质粒同时进行EcoRⅠ和XbaⅠ的双酶切处理和连接处理。
将步骤1的hDEK基因、hDEK△NLS基因、mDEK基因、mDEK△NLS基因分别插入到pLent-N-GFP 的慢病毒表达载体(维真生物,货号:LT88008)(图47)的EcoRⅠ和XbaⅠ位点上。分别获得重组慢病毒表达载体pLent-N-GFP-hDEK,pLent-N-GFP-mDEK,pLent-N-GFP-hDEK△NLS和pLent-N-GFP-mDEK△NLS。采用实施例11方法,将上述重组慢病毒表达载体和慢病毒包装质粒混合物(pMDL,VSVG,pRSV-Rev,质量比例为5:3:2)共同转染293T细胞,转染72小时后收集细胞培养上清即为病毒液,浓缩纯化后进行病毒滴度的测定,获得过表达外源DEK蛋白及结构域NLS突变DEK蛋白的慢病毒,即慢病毒hDEK,mDEK,hDEK△NLS和mDEK△NLS。
3、分选外泌体的获取与鉴定
(1)分选外泌体的制备
1)过表达质粒转染方法:
①分别将实施例2获得的4T1-DCCs、EMT6-DCCs和MCF7-DCCs细胞按照每个10cm细胞培养盘约300万细胞进行接种,加入10mL含10%血清和1%抗生素的DMEM培养液,37℃培养过夜。第二天,倒去原来的培养基,加入新的含10%血清和1%抗生素的DMEM培养液。
②取一个洁净无菌离心管,加入750μL不含抗生素和血清的DMEM培养液,分别加入步骤 1制备的15μg重组质粒(pEGFP-C1-hDEK,pEGFP-C1-mDEK,pEGFP-C1-hDEK△NLS或pEGFP-C1-mDEK△NLS),并用枪轻轻吹打混匀,再加入24μl脂质体转染试剂Lipo8000(Beyotime,目录号:C0533),用枪轻轻吹打混匀,获得转染试剂和质粒的混合物。
③将转染试剂和质粒的混合物750μL均匀滴加到上述①的细胞培养盘(4T1-DCCs、EMT6-DCCs细胞分别加入pEGFP-C1-mDEK和pEGFP-C1-mDEK△NLS;MCF7-DCCs细胞加入 pEGFP-C1-hDEK和pEGFP-C1-hDEK△NLS)中,37℃培养72小时。收集细胞培养液,利用外泌体抽提试剂(Invitrogen,目录号:4478359)从细胞培养液中分离获得外泌体(同实施例3步骤2中的方法)。将外泌体通过流式分选仪(Beckman moflo Astrios EQ),分选得到GFP阳性和粒径范围在 50-150nm的来自不同肿瘤细胞的含有外源DEK蛋白的分选外泌体及结构域NLS突变DEK蛋白的分选外泌体,分别为MCF7含hDEK-GFP蛋白的外泌体、MCF7含hDEK△NLS-GFP蛋白的外泌体、4T1含mDEK-GFP蛋白的外泌体、4T1含mDEK△NLS-GFP蛋白的外泌体、EMT6含mDEK-GFP蛋白的外泌体、EMT6-含mDEK△NLS-GFP蛋白的外泌体。
2)过表达慢病毒转染方法:分别将实施例2获得的4T1-DCCs、EMT6-DCCs和MCF7-DCCs 细胞按照每个10cm细胞培养盘约300万细胞进行接种,加入10mL含10%血清和1%抗生素的 DMEM培养液,37℃培养过夜。第二天,在每个孔中加入终浓度6μg/mL的聚凝胺(polybrene) 和10μL的步骤2制备的108pfu/mL慢病毒(4T1-DCCs、EMT6-DCCs细胞分别加入mDEK和 mDEK△NLS;MCF7-DCCs细胞加入hDEK和hDEK△NLS)。37℃培养24小时后更换成新的培养基,继续培养72小时,收集细胞培养液,利用外泌体抽提试剂(Invitrogen,目录号:4478359)从细胞培养液中分离获得外泌体(同实施例3步骤2中的方法)。将外泌体通过流式分选仪(Beckman moflo Astrios EQ),分选得到GFP阳性和粒径范围在50-150nm的不同来源的含有外源DEK蛋白的分选外泌体和结构域NLS突变DEK蛋白的分选外泌体,分别为MCF7含hDEK-GFP蛋白的分选外泌体、MCF7含hDEK△NLS-GFP蛋白的分选外泌体、4T1含mDEK-GFP蛋白的分选外泌体溶液、4T1 含mDEK△NLS-GFP蛋白的分选外泌体、EMT6含mDEK-GFP蛋白的分选外泌体、EMT6-含mDEK △NLS-GFP蛋白的分选外泌体。
利用BCA蛋白质定量检测试剂盒(品牌:生工,货号:C503021)测定分选外泌体中的蛋白总量,将分选外泌体以200μg/mL的浓度溶解到PBS中,制成分选外泌体PBS溶液,用于后续的使用。
(2)分选外泌体的鉴定:使用粒径检测仪器(型号:ZetaView,厂家:ParticleMetrix)对步骤(1) 过表达慢病毒转染方法中获得的200μg/mL分选外泌体PBS溶液进行检测,发现分选外泌体的粒径范围均为30-150nm,主体粒径大小均在70nm附近(图48)。对步骤(1)中获得的200μg/mL 分选外泌体PBS溶液进行透射电子显微镜的检测(方法同实施例3步骤3),观察到双层膜结构的茶托状囊泡,符合外泌体的形态特征(图49)。使用Rabbitpolyclonal anti-DEK(Proteintech,目录号:16448-1-AP)、Rabbit monoclonal anti-CD9(Abcam,目录号:ab92726)、Rabbit monoclonal anti-CD81(Abcam,目录号:ab109201)和Mouse monoclonal anti-CD63(Abcam,目录号:ab59479) 的抗体,对步骤(1)中获得的200μg/mL分选外泌体溶液开展了DEK、CD9、CD81和CD63的免疫印迹分析(方法同实施例2步骤2,一抗、二抗见表1),发现分选外泌体表达高水平的CD9、 CD81和CD63蛋白(图50)。以上的结果逐一验证了分选外泌体的大小、形态和分子特征。
实施例16、含有外源DEK蛋白的分选外泌体添加激活休眠肿瘤细胞
分别将实施例2步骤1中得到的3种休眠肿瘤细胞(4T1-DCCs、EMT6-DCCs、MCF7-DCCs) 各100万颗接种至10mL含有10%血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中。4T1-DCCs细胞分别添加2.5μL PBS、2.5μL200μg/mL实施例15步骤3 中过表达慢病毒转染方法制备的4T1含mDEK-GFP蛋白的分选外泌体PBS溶液、2.5μL200μg/mL 实施例15步骤3中过表达慢病毒转染方法制备的4T1含mDEK△NLS-GFP蛋白的分选外泌体PBS 溶液,使4T1含mDEK-GFP蛋白的分选外泌体和4T1含mDEK△NLS-GFP蛋白的分选外泌体在培养基中的终浓度均为50ng/mL。同样条件下,EMT6-DCCs添加PBS、EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液、EMT6-含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液;MCF7-DCCs细胞添加PBS、MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液、MCF7含hDEK△NLS-GFP蛋白的分选外泌体PBS溶液。37℃培养20小时后收集细胞,使用Rabbit polyclonal anti-DEK、Rabbitmonoclonal anti-H3S10ph、Mouse monoclonal anti-PCNA和Mouse monoclonal anti-SETD4的抗体,开展了免疫印迹分析(方法同实施例2步骤2,二抗见表1),发现DEK,PCNA和H3S10ph的水平上升,SETD4的水平下降(图51)。研究结果表明含有外源DEK蛋白的分选外泌体添加能够激活休眠肿瘤细胞。
实施例17、结合化疗,含有外源DEK蛋白的分选外泌体添加能够清除休眠肿瘤细胞
1、分选外泌体结合化疗对休眠肿瘤细胞cCasp3信号的影响
同实施例16方法,37℃培养20小时后收集细胞,使用切割的半胱氨酸蛋白酶(cCasp3,abcam, 货号:ab32042)抗体开展免疫荧光实验(具体操作同实施例2步骤2,二抗更替为Alexa Fluor 594 荧光标记驴抗兔,Thermo Fisher,货号:R37119)。样品用荧光显微镜进行观察,发现cCasp3的信号大量出现,细胞出现大量的凋亡(图52)。
2、分选外泌体结合化疗处理时间对休眠肿瘤细胞的影响
同实施例16方法,37℃培养20小时改为在37℃培养0小时、5小时、20小时、25小时、30 小时后收集细胞样品,台盼蓝染色检测死亡的细胞,发现原本抵抗放化疗的休眠肿瘤细胞逐渐全部死亡(图53)。
以上的结果表明结合化疗,含有外源DEK蛋白的分选外泌体添加能够清除休眠肿瘤细胞。
实施例18、含有外源DEK蛋白的分选外泌体激活休眠肿瘤细胞的调控机制
1、分选外泌体的添加引起异染色质水平的下调和常染色质水平的上调
同实施例16方法,37℃培养20小时后收集细胞。使用H4K20me1、H4K20me2、H4K20me3、 HP1-α、H3K9ac、H3K9me3和H3K27me3的抗体,在上述细胞中开展免疫印迹分析(方法同实施例2步骤2,一抗、二抗见表1)。发现与在休眠肿瘤细胞中添加PBS和含外源结构域突变DEK 蛋白的分选外泌体相比,在休眠肿瘤细胞中添加了含外源DEK蛋白的分选外泌体后,组成型异染色质的分子指标H4K20me3和异染色质形成关键蛋白HP1-α的水平显著下降,常染色质的分子指标H3K9ac的水平显著上升,兼性异染色质的分子指标H3K9me3和H3K27me3的水平没有显著性的变化(图54)。
2、分选外泌体的添加引起P53信号通路的下调和MYC信号通路的上调
同实施例16方法,37℃培养20小时后收集细胞。使用Mouse monoclonal anti-p53(Santa Cruz,目录号:sc-126)、Rabbit monoclonal anti-p21(Cell SignalingTechnology,目录号:2947)、Rabbit polyclonal anti-PUMA(abcam,目录号:ab9643)和Mouse monoclonal anti-c-Myc(Santa Cruz,目录号:sc-40)的抗体,在上述细胞中开展免疫印迹分析(方法同实施例2步骤2,二抗见表1)。发现与在休眠肿瘤细胞中添加PBS和含外源结构域突变DEK蛋白的分选外泌体相比,在休眠肿瘤细胞中添加了含外源DEK蛋白的分选外泌体后,p53、p21和PUMA的水平显著下降,MYC 的水平显著上升(图55)。
实施例19、在荷瘤小鼠的血液和肿瘤组织中检测注射的含有外源DEK蛋白的分选外泌体
1、分选外泌体腹腔注射到荷瘤小鼠体内后在血液中滞留
将100万颗4T1和100万颗EMT6细胞分别原位接种至8周BALB/c雌鼠的脂肪垫中,将100 万颗MCF7细胞原位接种至8周Nod/Scid雌鼠脂肪垫中,待瘤子体积达到500mm3左右时,分为 4T1组、EMT6组和MCF7组,每组9只小鼠。4T1组6只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL的4T1含mDEK-GFP蛋白的分选外泌体PBS溶液,3只荷瘤小鼠腹腔不注射物质作为未处理对照。EMT6 组6只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL的EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液,3只荷瘤小鼠腹腔不注射物质作为未处理对照。MCF7组6只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL的MCF7含 mDEK-GFP蛋白的分选外泌体PBS溶液,3只荷瘤小鼠腹腔不注射物质作为未处理对照。分别在注射24小时后和7天后安乐死小鼠,收集小鼠的血浆,利用外泌体抽提试剂盒(Invitrogen,货号: 4484450)分离血浆内的外泌体,在100μL外泌体中加入10μL的质量浓度4%的9μm硫酸乳胶珠子(溶于超蒸水中),25℃孵育10分钟。1000rpm 25℃离心5分钟,去掉上清,用100μL PBS 重悬。将重悬液放到流式分析仪中检测GFP+的珠子比例。发现在荷瘤小鼠的血液中检测到注射的含有外源DEK蛋白的分选外泌体(图56),说明分选外泌体腹腔注射到荷瘤小鼠体内后能在血液中滞留。
2、分选外泌体腹腔注射到荷瘤小鼠体内后进入肿瘤组织
将100万颗4T1和100万颗EMT6细胞分别原位接种至8周BALB/c雌鼠的脂肪垫中,将100 万颗MCF7细胞原位接种至8周Nod/Scid雌鼠脂肪垫中,待瘤子体积达到500mm3左右时,分为 4T1组、EMT6组和MCF7组,每组6只小鼠。4T1组3只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK-GFP蛋白的分选外泌体PBS溶液,3只小鼠按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液。EMT6组3只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液,3只小鼠按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液。MCF7组3只荷瘤小鼠腹腔按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含mDEK-GFP蛋白的分选外泌体PBS溶液,3只小鼠按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液。
在注射24小时后安乐死小鼠,手术取出4T1、EMT6和MCF7肿瘤,将上述肿瘤放入4%多聚甲醛中4℃过夜,浸泡到质量浓度30%蔗糖水溶液中脱水48小时,浸泡后的肿瘤捞出,放到 10x10x5mm的敞开长方体型塑料材质的模具中,往其中加满OCT包埋剂(SAKURA,目录号: 4583),将模具放在干冰上静置5分钟,将包埋块取出,-80℃保存。用冰冻切片机将包埋块切成 10μm的肿瘤切片。对肿瘤切片开展Rabbit polyclonal anti-SETD4(Sigma-Aldrich,目录号: HPA024073)抗体的免疫荧光实验(方法同实施例2)。发现在肿瘤组织中有大量的GFP信号(图 57),表明含有外源蛋白DEK或结构域突变DEK蛋白的分选外泌体注射到荷瘤小鼠体内后进入肿瘤组织。
实施例20、注射含有外源DEK蛋白的分选外泌体激活荷瘤小鼠体内休眠肿瘤细胞
将100万颗4T1和100万颗EMT6细胞分别原位接种至8周BALB/c雌鼠的脂肪垫中,将100 万颗MCF7细胞原位接种至8周Nod/Scid雌鼠脂肪垫中,待瘤子体积达到500mm3左右时,分为4T1组、EMT6组、MCF7组,每组6只小鼠。4T1组3只荷瘤小鼠腹腔不注射物质作为未处理对照,3只荷瘤小鼠腹腔按20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK-GFP蛋白的分选外泌体PBS溶液。EMT6组3只荷瘤小鼠腹腔不注射物质作为未处理对照,3只荷瘤小鼠腹腔按20g小鼠体重注射总蛋白量为 20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK-GFP 蛋白的分选外泌体PBS溶液。MCF7组3只荷瘤小鼠腹腔不注射物质作为未处理对照,3只荷瘤小鼠腹腔按20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL MCF7含mDEK-GFP蛋白的分选外泌体PBS溶液。在注射24小时后安乐死小鼠,手术取出4T1、EMT6和MCF7肿瘤,将上述肿瘤放入4%多聚甲醛中4℃过夜,浸泡到质量浓度30%蔗糖水溶液中脱水48小时,浸泡后的肿瘤捞出,放到10x10x5mm的敞开长方体型塑料材质的模具中,往其中加满OCT包埋剂(SAKURA,目录号:4583),将模具放在干冰上静置5 分钟,将包埋块取出,-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。对肿瘤切片开展Rabbit polyclonal anti-SETD4(Sigma-Aldrich,目录号:HPA024073)抗体的免疫荧光实验(方法同实施例2)。发现相比于未处理组,腹腔注射含外源DEK蛋白的分选外泌体后,SETD4细胞占总细胞的比例显著降低(图58),表明注射含外源DEK蛋白的分选外泌体能够100%激活荷瘤小鼠体内休眠肿瘤细胞。
实施例21、结合放化疗,注射含有外源DEK蛋白的分选外泌体能够清除荷瘤小鼠体内休眠肿瘤细胞
1、外泌体结合放疗对休眠肿瘤细胞的影响
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种100万颗4T1细胞,分为放疗+PBS组、放疗 +4T1含mDEK-GFP蛋白的分选外泌体组、放疗+4T1含mDEK△NLS-GFP蛋白的分选外泌体组,每组3只。在接种后的第6天和第9天,放疗+PBS组每只荷瘤小鼠腹腔注射PBS,PBS注射量为100μL;放疗+4T1含mDEK-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK-GFP蛋白的分选外泌体PBS溶液;放疗+4T1含mDEK△NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20Gy X射线辐照。在接种后第21天,安乐死实验小鼠,手术取出瘤子,将肿瘤放入4%多聚甲醛中4℃过夜,浸泡到质量浓度30%蔗糖水溶液中脱水48小时,浸泡后的肿瘤捞出,放到10x10x5mm的敞开长方体型塑料材质的模具中,往其中加满OCT包埋剂(SAKURA,目录号:4583),将模具放在干冰上静置5分钟,将包埋块取出,-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。在肿瘤切片中开展Rabbit polyclonal anti-SETD4 (Sigma-Aldrich,目录号:HPA024073)抗体的免疫荧光分析(方法同实施例2),发现相比于放疗 +PBS组和放疗+4T1含mDEK△NLS-GFP蛋白的外泌体组,放疗+4T1含mDEK-GFP蛋白的外泌体组中,只发现极少量SETD4阳性细胞的存在(图59)。结果表明结合放疗,注射含有外源DEK 蛋白的分选外泌体能够100%清除4T1荷瘤小鼠体内休眠肿瘤细胞。
2、外泌体结合放化疗对休眠肿瘤细胞的影响
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种20万颗EMT6细胞,分为放化疗+PBS组、放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组、放化疗+EMT6-含mDEK△NLS-GFP蛋白的分选外泌体组,每组3只。在接种后的第6天、第9天和第12天,放化疗+PBS组每只荷瘤小鼠腹腔注射PBS,PBS注射量为100μL;放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组按每20g 小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200 μg/mL EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液;放化疗+EMT6-含mDEK△NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6-含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20Gy X射线辐照,在接种后的第6天、第12天和第18天分别腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇。在接种后第21天,安乐死实验小鼠,手术取出瘤子,将肿瘤放入4%多聚甲醛中4℃过夜,浸泡到质量浓度30%蔗糖水溶液中脱水48小时,浸泡后的肿瘤捞出,放到10x10x5mm的敞开长方体型塑料材质的模具中,往其中加满OCT包埋剂(SAKURA,目录号:4583),将模具放在干冰上静置5分钟,将包埋块取出,-80℃保存。用冰冻切片机将包埋块切成10μm的肿瘤切片。在肿瘤切片中开展SETD4抗体(Sigma-Aldrich,目录号:HPA024073)的免疫荧光分析(方法同实施例2),发现相比于放化疗+PBS组和放化疗+EMT6-含mDEK△NLS-GFP蛋白的外泌体组,放化疗+EMT6含mDEK-GFP蛋白的外泌体组中,只发现极少量SETD4阳性细胞的存在(图60)。结果表明结合放化疗,注射含有外源DEK蛋白的分选外泌体能够100%清除EMT6荷瘤小鼠体内休眠肿瘤细胞。
实施例22、结合放化疗,注射含有外源DEK蛋白的分选外泌体能够治愈乳腺癌
1、放疗结合分选外泌体治愈4T1移植瘤小鼠
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种100万颗4T1细胞,分为未处理组、放疗+PBS 组、放疗+4T1含mDEK-GFP蛋白的分选外泌体组、放疗+4T1含mDEK△NLS-GFP蛋白的分选外泌体组,每组11只。在接种后的第6天和第9天,未处理组不注射物质;放疗+PBS组每只荷瘤小鼠腹腔注射PBS,PBS注射量为100μL;放疗+4T1含mDEK-GFP蛋白的分选外泌体组按每20g 小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200 μg/mL 4T1含mDEK-GFP蛋白的分选外泌体PBS溶液;放疗+4T1含mDEK△NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL 4T1含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20Gy X射线辐照。在接种细胞后每周测量肿瘤的长度和宽度,利用公式(长度×宽度×宽度/2)计算出肿瘤体积,绘制肿瘤的生长曲线,发现无处理组在接种细胞4周后瘤体直径达到伦理上限,单纯放疗组和放疗且注射含外源结构域突变DEK蛋白的分选外泌体组在接种细胞8周后流体直径达到约400mm3,而在放疗且注射含外源DEK蛋白的分选外泌体组中,没有发现肿瘤(图61)。
在接种细胞后的第8周,对所有实验小鼠安乐死后手术取出肺组织的样品,固定包埋后冷冻切片。切片在PBS中洗1遍,5分钟;苏木素染色液染色10分钟;自来水中冲洗掉多余的染色液,1分钟;蒸馏水洗1遍,2分钟;伊红染色液染色2分钟;用50%的甘油封片。染色后的片子在显微镜下观察,发现相比于放疗+PBS组、放疗+4T1含mDEK△NLS-GFP蛋白的分选外泌体组,在放疗+4T1含mDEK-GFP蛋白的分选外泌体组中找不到肿瘤的转移灶(图62)。
对所有的实验鼠进行生存曲线分析,发现相比于未处理组的35天、放疗+PBS组的56天和放疗+4T1含mDEK△NLS-GFP蛋白的外泌体组的57天,在放疗+4T1含mDEK-GFP蛋白的外泌体组中,小鼠的中位生存期延长到了100天(图63)。结果表明使用含外源DEK蛋白的分选外泌体和放疗能够完全治愈4T1肿瘤,消除肿瘤的复发和转移,改善小鼠的生存,治疗后1年内小鼠的肿瘤未见复发且未见转移。
2、放化疗结合分选外泌体治愈EMT6移植瘤小鼠
在6-8周BALB/c雌鼠的腋下乳腺脂肪垫中接种20万颗EMT6细胞,分为未处理组、放化疗 +PBS组、放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组、放化疗+EMT6-含mDEK△NLS-GFP蛋白的分选外泌体组,每组7只。在接种后的第6天、第9天和第12天,未处理组不注射物质;放化疗+PBS组每只荷瘤小鼠腹腔注射100μL的PBS;放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3中过表达慢病毒转染方法制备的200μg/mL EMT6含mDEK-GFP蛋白的分选外泌体PBS溶液;放化疗+EMT6-含mDEK△NLS-GFP蛋白的分选外泌体组按每20g小鼠体重注射总蛋白量为20μg的量注射实施例15步骤3 中过表达慢病毒转染方法制备的200μg/mL EMT6-含mDEK△NLS-GFP蛋白的分选外泌体PBS溶液。在接种后的第7天、第10天和第13天分别接受一次腋下范围内的3分钟45秒的20GyX射线辐照,在接种后的第6天、第12天和第18天分别腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇。在接种细胞后每周测量肿瘤的长度和宽度,利用公式(长度×宽度×宽度/2)计算出肿瘤体积,绘制肿瘤的生长曲线,发现未处理组在接种细胞3周后瘤体直径达到伦理上限,放化疗+PBS 组和放化疗+EMT6-含mDEK△NLS-GFP蛋白的外泌体组在接种细胞6周后瘤体直径达到伦理上限,而在放化疗+EMT6含mDEK-GFP蛋白的分选外泌体组中,肿瘤的生长受到显著抑制(图64)。结果表明使用含外源DEK蛋白的分选外泌体和放化疗能够完全治愈EMT6肿瘤,抑制肿瘤的复发。
3、化疗结合分选外泌体治愈MCF7移植瘤小鼠
在6-8周Nod/Scid雌鼠的腋下乳腺脂肪垫中接种100万颗MCF7细胞,分为未处理组、化疗 +PBS组、化疗+MCF7含hDEK-GFP蛋白的分选外泌体组、化疗+MCF7含hDEK△NLS-GFP蛋白的分选外泌体组,每组5只。在接种后的第3-8周,未处理组不注射物质;化疗+PBS组每只小鼠每周腹腔注射1次PBS和1次剂量为3mg/kg小鼠体重的紫杉醇,PBS注射量为100μL;化疗+MCF7 含hDEK-GFP蛋白的分选外泌体组每周腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇和1次剂量为1μg/g小鼠体重的实施例15步骤3中过表达慢病毒转染方法制备的200μg/mLMCF7含 hDEK-GFP蛋白的分选外泌体PBS溶液;化疗+MCF7含hDEK△NLS-GFP蛋白的分选外泌体组每周腹腔注射1次剂量为3mg/kg小鼠体重的紫杉醇和1次剂量为1μg/g小鼠体重的实施例15步骤 3中过表达慢病毒转染方法制备的200μg/mL MCF7含hDEK△NLS-GFP蛋白的分选外泌体PBS溶液。在接种细胞后每周测量肿瘤的长度和宽度,利用公式(长度×宽度×宽度/2)计算出肿瘤体积,绘制肿瘤的生长曲线,发现未处理组在接种细胞5周后瘤体直径达到伦理上限,化疗+PBS组和化疗+MCF7含hDEK△NLS-GFP蛋白的外泌体组在接种细胞12周后瘤体直径到伦理上限,而在化疗+MCF7含hDEK-GFP蛋白的外泌体组中,没有发现肿瘤(图65)。结果表明使用含外源DEK 蛋白的分选外泌体和化疗能够完全治愈MCF7肿瘤,消除肿瘤的复发。
实施例23、休眠肿瘤细胞的数量与临床乳腺癌的恶化程度密切相关
我们收集了8例Ⅰ期、12例Ⅱ期和3例Ⅲ期的乳腺癌病人石蜡包埋样品,切片后进行SETD4 的免疫荧光实验(方法同实施例2),发现Ⅲ期样品中的SETD4细胞比例远远高于Ⅰ期和Ⅱ期样品(图66)。结果表明静息肿瘤细胞的数量越多,临床乳腺癌的肿瘤恶化程度越高。
实施例24、临床乳腺癌样品来源休眠肿瘤细胞的获取,激活和杀死
1、病人样品中获取抵抗放化疗的肿瘤细胞
利用肿瘤解离试剂盒(美天旎,货号:130-095-929)处理2个病人的临床乳腺癌样品,得到消化下来的肿瘤细胞,并将其按80万颗/孔的密度接种在超低吸附的六孔盘(品牌:Corning,货号:3471) 中。每孔加入3mL含有10%血清替代物(Thermo Fisher ScientificCat#10828028),终浓度100nM 紫杉醇和终浓度1mM的5-氟尿嘧啶的DMEM/F12培养基(Corning,目录号:10-092-cv),在37℃培养1个月,每隔3天换一次培养液,前两周中每周照射30Gy X射线一次,总共照射2次(两次间隔7天),每次照射10分钟。一个月后,使用死细胞去除试剂盒(Miltenyi Biotec,目录号: 130-090-101)筛选出抵抗放化疗的肿瘤细胞,即为休眠肿瘤细胞(图67中a)。
2、DEK蛋白结合化疗对休眠肿瘤细胞SETD4和Ki67信号的影响
分别将上述2个病人来源的休眠肿瘤细胞各20万颗接种至10mL含有10%血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中。在上述细胞中分别添加25μL PBS、25μL 200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液、25μL 200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的 MCF7含hDEK△NLS-GFP蛋白的分选外泌体PBS溶液,使MCF7含hDEK-GFP蛋白的分选外泌体和MCF7含hDEK△NLS-GFP蛋白的分选外泌体在培养基中的终浓度均为50ng/mL。37℃培养20 小时后收集细胞,使用Rabbit polyclonal anti-DEK(Proteintech,货号:16448-1-AP)、Rabbit polyclonal anti-SETD4(Sigma-Aldrich,货号:HPA024073)和Rabbitmonoclonal anti-Ki67(abcam, 货号:ab16667)抗体开展免疫荧光实验(具体操作同实施例2步骤2,使用的二抗均是Alexa Fluor 594荧光标记驴抗兔,Thermo Fisher,货号:R37119)。样品用荧光显微镜进行观察,发现相较于添加PBS的对照组和添加MCF7含hDEK△NLS-GFP蛋白的分选外泌体PBS溶液的对照组,在休眠肿瘤细胞中添加MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液后,细胞中SETD4的水平下降,Ki67的水平上升(图67中b)。研究结果表明含有外源DEK蛋白的分选外泌体添加能够100%激活临床乳腺癌样本来源的休眠肿瘤细胞。
3、DEK蛋白结合化疗对休眠肿瘤细胞死亡的影响
分别将上述2个病人来源的休眠肿瘤细胞各20万颗接种至10mL含有10%血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中。在上述细胞中分别添加25μL PBS、25μL 200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的MCF7含hDEK-GFP蛋白的分选外泌体PBS溶液、25μL 200μg/mL实施例15步骤3中过表达慢病毒转染方法制备的 MCF7含hDEK△NLS-GFP蛋白的分选外泌体PBS溶液,使MCF7含hDEK-GFP蛋白的分选外泌体和MCF7含hDEK△NLS-GFP蛋白的分选外泌体在培养基中的终浓度均为50ng/mL。37℃培养30 小时后收集细胞样品,台盼蓝染色检测死亡的细胞,发现原本抵抗放化疗的休眠肿瘤细胞在添加 MCF7含hDEK-GFP蛋白的分选外泌体后全部死亡(图67中c)。研究结果表明添加含有外源DEK 蛋白的分选外泌体结合化疗能够100%杀死临床乳腺癌样本来源的休眠肿瘤细胞。
实施例25、在各种人肿瘤细胞中通过DEK结合化疗激活并清除休眠肿瘤细胞
1.肺癌细胞系H226来源粗外泌体结合化疗100%激活并清除H226休眠肿瘤细胞
H226细胞系(来源见表2),细胞培养基是RPMI-1640培养基(Gibco,目录号:31800022) 中添加10%的胎牛血清和1%的青霉素-链霉素。
1000万颗H226细胞系接种到20mL的培养基中。37℃培养24小时后收集细胞的培养液,1000 rpm 4℃离心10分钟,取上清,12000rpm 4℃离心20分钟,取上清,即为H226的细胞培养液。在细胞培养液中加入1mL的外泌体分离试剂(品牌:Thermo Fisher,货号:4478359),上下颠倒 3次混匀后4℃孵育过夜。第二天,上述混合液10000rpm 4℃离心60分钟,去掉上清,用200μL 的PBS重悬离心管底部的沉淀(沉淀即为粗外泌体),利用BCA蛋白质定量检测试剂盒(品牌:生工,货号:C503021)测定粗外泌体悬液中的蛋白总量,用PBS配制蛋白浓度为20μg/mL的粗外泌体溶液,用于后续的使用。
100万颗H226细胞中加入0.1mL含2500U/mL胰蛋白酶和0.02%乙二胺四乙酸(EDTA) 的磷酸缓冲液(PBS),25℃静置孵育40秒,加入0.2mL胎牛血清中和,将壁上的细胞吹打下来, 1000rpm 25℃离心5分钟,去上清,沉淀用1mL的细胞培养基重悬。将上述的重悬液按80万颗 /孔的密度接种在超低吸附的六孔盘(品牌:Corning,货号:3471)中。每孔加入3mL含有10%血清替代物(Thermo Fisher Scientific Cat#10828028),终浓度100nM紫杉醇和终浓度1mM的5- 氟尿嘧啶的DMEM/F12培养基(Corning,目录号:10-092-cv),在37℃培养1个月,每隔3天换一次培养液,前两周中每周照射30Gy X射线一次,总共照射2次(两次间隔7天),每次照射10 分钟。一个月后,使用死细胞去除试剂盒(Miltenyi Biotec,目录号:130-090-101)筛选出抵抗放化疗的休眠肿瘤细胞。
将1000颗上述的休眠肿瘤细胞接种至2mL含有10%血清替代物、终浓度100nM紫杉醇和终浓度1mM 5-氟尿嘧啶的DMEM/F12培养基中,并加入5μL上述制备的20μg/mL粗外泌体溶液使其在培养基中的终浓度为50ng/mL。37℃培养30小时后,发现细胞的死亡率为100%,H226 来源粗外泌体结合化疗100%激活并清除H226休眠肿瘤细胞。
2.胃癌细胞系MKN45来源粗外泌体结合化疗100%激活并清除MKN45休眠肿瘤细胞
同实施例25步骤1方法(细胞系更替为MKN45),发现MKN45来源粗外泌体结合化疗100%激活并清除MKN45休眠肿瘤细胞。
3.前列腺癌细胞系PC-3来源粗外泌体结合化疗100%激活并清除PC-3休眠肿瘤细胞
同实施例25步骤1方法(细胞系更替为PC-3,培养基更替为:F-12培养基(Gibco,目录号:21700075)中添加10%的胎牛血清和1%的青霉素-链霉素),发现PC-3来源粗外泌体结合化疗 100%激活并清除PC-3休眠肿瘤细胞。
4.宫颈癌细胞系HeLa来源粗外泌体结合化疗100%激活并清除HeLa休眠肿瘤细胞
同实施例25步骤1方法(细胞系更替为HeLa,培养基更替为:MEM培养基(Gibco,目录号:41500034)中添加10%的胎牛血清和1%的青霉素-链霉素),发现HeLa来源粗外泌体结合化疗 100%激活并清除HeLa休眠肿瘤细胞。
研究结果表明使用DEK激活并清除休眠肿瘤细胞可以适用于各种不同类型的人癌症的治疗。
表2、DEK蛋白对于肿瘤细胞激活并清除效果
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序列表
<110> 浙江大学
<120> 一种用于激活休眠肿瘤细胞的SETD4蛋白抑制剂的投递蛋白
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 375
<212> PRT
<213> 人源DEK蛋白(Humanized)
<400> 1
Met Ser Ala Ser Ala Pro Ala Ala Glu Gly Glu Gly Thr Pro Thr Gln
1 5 10 15
Pro Ala Ser Glu Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser
20 25 30
Glu Glu Glu Glu Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Lys
35 40 45
Glu Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu
50 55 60
Arg Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Ile
65 70 75 80
Ala Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe
85 90 95
Phe Leu Ser Lys Lys Lys Thr Asp Glu Leu Arg Asn Leu His Lys Leu
100 105 110
Leu Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly
115 120 125
Gln Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Val Gln Tyr Lys Lys
130 135 140
Lys Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile
145 150 155 160
Cys Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val
165 170 175
Lys Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro
180 185 190
Leu Pro Lys Ser Lys Lys Thr Cys Ser Lys Gly Ser Lys Lys Glu Arg
195 200 205
Asn Ser Ser Gly Met Ala Arg Lys Ala Lys Arg Thr Lys Cys Pro Glu
210 215 220
Ile Leu Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys
225 230 235 240
Glu Glu Ser Ser Asp Asp Glu Asp Lys Glu Ser Glu Glu Glu Pro Pro
245 250 255
Lys Lys Thr Ala Lys Arg Glu Lys Pro Lys Gln Lys Ala Thr Ser Lys
260 265 270
Ser Lys Lys Ser Val Lys Ser Ala Asn Val Lys Lys Ala Asp Ser Ser
275 280 285
Thr Thr Lys Lys Asn Gln Asn Ser Ser Lys Lys Glu Ser Glu Ser Glu
290 295 300
Asp Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro
305 310 315 320
Thr Asp Glu Glu Leu Lys Glu Thr Ile Lys Lys Leu Leu Ala Ser Ala
325 330 335
Asn Leu Glu Glu Val Thr Met Lys Gln Ile Cys Lys Lys Val Tyr Glu
340 345 350
Asn Tyr Pro Thr Tyr Asp Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr
355 360 365
Thr Val Lys Glu Leu Ile Ser
370 375
<210> 2
<211> 17
<212> PRT
<213> NLS结构域(Humanized)
<400> 2
Lys Lys Glu Arg Asn Ser Ser Gly Met Ala Arg Lys Ala Lys Arg Thr
1 5 10 15
Lys
<210> 3
<211> 35
<212> PRT
<213> SAP结构域(Humanized)
<400> 3
Leu Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile Cys Glu Val Leu
1 5 10 15
Asp Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val Lys Arg Ile Leu
20 25 30
Asn Phe Leu
35
<210> 4
<211> 39
<212> PRT
<213> pseudo-SAP结构域(Humanized)
<400> 4
Ile Glu Arg Ile His Phe Phe Leu Ser Lys Lys Lys Thr Asp Glu Leu
1 5 10 15
Arg Asn Leu His Lys Leu Leu Tyr Asn Arg Pro Gly Thr Val Ser Ser
20 25 30
Leu Lys Lys Asn Val Gly Gln
35
<210> 5
<211> 1128
<212> DNA
<213> DEK基因(Humanized)
<400> 5
atgtccgcct cggcccctgc tgcggagggg gagggaaccc ccacccagcc cgcgtccgag 60
aaagaacccg aaatgcccgg tcccagagag gagagcgagg aggaagagga cgaggacgac 120
gaggaggagg aggaggagga aaaagaaaag agtctcatcg tggaaggcaa gagggaaaag 180
aaaaaagtag agaggttgac aatgcaagtc tcttccttac agagagagcc atttacaatt 240
gcacaaggaa aggggcagaa actttgtgaa attgagagga tacatttttt tctaagtaag 300
aagaaaaccg atgaacttag aaatctacac aaactgcttt acaacaggcc aggcactgtg 360
tcctcattaa agaagaatgt gggtcagttc agtggctttc catttgaaaa aggaagtgtc 420
caatataaaa agaaggaaga aatgttgaaa aaatttagaa atgccatgtt aaagagcatc 480
tgtgaggttc ttgatttgga gagatcaggt gtaaatagtg aactagtgaa gaggatcttg 540
aatttcttaa tgcatccaaa gccttctggc aaaccattgc cgaaatctaa aaaaacttgt 600
agcaaaggca gtaaaaagga acggaacagt tctggaatgg caaggaaggc taagcgaacc 660
aaatgtcctg aaattctgtc agatgaatct agtagtgatg aagatgaaaa gaaaaacaag 720
gaagagtctt cagatgatga agataaagaa agtgaagagg agccaccaaa aaagacagcc 780
aaaagagaaa aacctaaaca gaaagctact tctaaaagta aaaaatctgt gaaaagtgcc 840
aatgttaaga aagcagatag cagcaccacc aagaagaatc aaaacagttc caaaaaagaa 900
agtgagtctg aggatagttc agatgatgaa cctttaatta aaaagttgaa gaaaccccct 960
acagatgaag agttaaagga aacaataaag aaattactgg ccagtgctaa cttggaagaa 1020
gtcacaatga aacagatttg caaaaaggtc tatgaaaatt atcctactta tgatttaact 1080
gaaagaaaag atttcataaa aacaactgta aaagagctaa tttcttga 1128
<210> 6
<211> 1881
<212> DNA
<213> EGFP-DEK基因(Humanized)
<400> 6
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720
ggactcagat ctcgagctca agcttcgaat tctatgtccg cctcggcccc tgctgcggag 780
ggggagggaa cccccaccca gcccgcgtcc gagaaagaac ccgaaatgcc cggtcccaga 840
gaggagagcg aggaggaaga ggacgaggac gacgaggagg aggaggagga ggaaaaagaa 900
aagagtctca tcgtggaagg caagagggaa aagaaaaaag tagagaggtt gacaatgcaa 960
gtctcttcct tacagagaga gccatttaca attgcacaag gaaaggggca gaaactttgt 1020
gaaattgaga ggatacattt ttttctaagt aagaagaaaa ccgatgaact tagaaatcta 1080
cacaaactgc tttacaacag gccaggcact gtgtcctcat taaagaagaa tgtgggtcag 1140
ttcagtggct ttccatttga aaaaggaagt gtccaatata aaaagaagga agaaatgttg 1200
aaaaaattta gaaatgccat gttaaagagc atctgtgagg ttcttgattt ggagagatca 1260
ggtgtaaata gtgaactagt gaagaggatc ttgaatttct taatgcatcc aaagccttct 1320
ggcaaaccat tgccgaaatc taaaaaaact tgtagcaaag gcagtaaaaa ggaacggaac 1380
agttctggaa tggcaaggaa ggctaagcga accaaatgtc ctgaaattct gtcagatgaa 1440
tctagtagtg atgaagatga aaagaaaaac aaggaagagt cttcagatga tgaagataaa 1500
gaaagtgaag aggagccacc aaaaaagaca gccaaaagag aaaaacctaa acagaaagct 1560
acttctaaaa gtaaaaaatc tgtgaaaagt gccaatgtta agaaagcaga tagcagcacc 1620
accaagaaga atcaaaacag ttccaaaaaa gaaagtgagt ctgaggatag ttcagatgat 1680
gaacctttaa ttaaaaagtt gaagaaaccc cctacagatg aagagttaaa ggaaacaata 1740
aagaaattac tggccagtgc taacttggaa gaagtcacaa tgaaacagat ttgcaaaaag 1800
gtctatgaaa attatcctac ttatgattta actgaaagaa aagatttcat aaaaacaact 1860
gtaaaagagc taatttcttg a 1881
<210> 7
<211> 626
<212> PRT
<213> EGFP-DEK(Humanized)
<400> 7
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser
225 230 235 240
Gly Leu Arg Ser Arg Ala Gln Ala Ser Asn Ser Met Ser Ala Ser Ala
245 250 255
Pro Ala Ala Glu Gly Glu Gly Thr Pro Thr Gln Pro Ala Ser Glu Lys
260 265 270
Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser Glu Glu Glu Glu Asp
275 280 285
Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Lys Glu Lys Ser Leu Ile
290 295 300
Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu Arg Leu Thr Met Gln
305 310 315 320
Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Ile Ala Gln Gly Lys Gly
325 330 335
Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe Phe Leu Ser Lys Lys
340 345 350
Lys Thr Asp Glu Leu Arg Asn Leu His Lys Leu Leu Tyr Asn Arg Pro
355 360 365
Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly Gln Phe Ser Gly Phe
370 375 380
Pro Phe Glu Lys Gly Ser Val Gln Tyr Lys Lys Lys Glu Glu Met Leu
385 390 395 400
Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile Cys Glu Val Leu Asp
405 410 415
Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val Lys Arg Ile Leu Asn
420 425 430
Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro Leu Pro Lys Ser Lys
435 440 445
Lys Thr Cys Ser Lys Gly Ser Lys Lys Glu Arg Asn Ser Ser Gly Met
450 455 460
Ala Arg Lys Ala Lys Arg Thr Lys Cys Pro Glu Ile Leu Ser Asp Glu
465 470 475 480
Ser Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys Glu Glu Ser Ser Asp
485 490 495
Asp Glu Asp Lys Glu Ser Glu Glu Glu Pro Pro Lys Lys Thr Ala Lys
500 505 510
Arg Glu Lys Pro Lys Gln Lys Ala Thr Ser Lys Ser Lys Lys Ser Val
515 520 525
Lys Ser Ala Asn Val Lys Lys Ala Asp Ser Ser Thr Thr Lys Lys Asn
530 535 540
Gln Asn Ser Ser Lys Lys Glu Ser Glu Ser Glu Asp Ser Ser Asp Asp
545 550 555 560
Glu Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro Thr Asp Glu Glu Leu
565 570 575
Lys Glu Thr Ile Lys Lys Leu Leu Ala Ser Ala Asn Leu Glu Glu Val
580 585 590
Thr Met Lys Gln Ile Cys Lys Lys Val Tyr Glu Asn Tyr Pro Thr Tyr
595 600 605
Asp Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr Thr Val Lys Glu Leu
610 615 620
Ile Ser
625
<210> 8
<211> 1830
<212> DNA
<213> EGFP-DEKΔNLS基因(Humanized)
<400> 8
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720
ggactcagat ctcgagctca agcttcgaat tctatgtccg cctcggcccc tgctgcggag 780
ggggagggaa cccccaccca gcccgcgtcc gagaaagaac ccgaaatgcc cggtcccaga 840
gaggagagcg aggaggaaga ggacgaggac gacgaggagg aggaggagga ggaaaaagaa 900
aagagtctca tcgtggaagg caagagggaa aagaaaaaag tagagaggtt gacaatgcaa 960
gtctcttcct tacagagaga gccatttaca attgcacaag gaaaggggca gaaactttgt 1020
gaaattgaga ggatacattt ttttctaagt aagaagaaaa ccgatgaact tagaaatcta 1080
cacaaactgc tttacaacag gccaggcact gtgtcctcat taaagaagaa tgtgggtcag 1140
ttcagtggct ttccatttga aaaaggaagt gtccaatata aaaagaagga agaaatgttg 1200
aaaaaattta gaaatgccat gttaaagagc atctgtgagg ttcttgattt ggagagatca 1260
ggtgtaaata gtgaactagt gaagaggatc ttgaatttct taatgcatcc aaagccttct 1320
ggcaaaccat tgccgaaatc taaaaaaact tgtagcaaag gcagttgtcc tgaaattctg 1380
tcagatgaat ctagtagtga tgaagatgaa aagaaaaaca aggaagagtc ttcagatgat 1440
gaagataaag aaagtgaaga ggagccacca aaaaagacag ccaaaagaga aaaacctaaa 1500
cagaaagcta cttctaaaag taaaaaatct gtgaaaagtg ccaatgttaa gaaagcagat 1560
agcagcacca ccaagaagaa tcaaaacagt tccaaaaaag aaagtgagtc tgaggatagt 1620
tcagatgatg aacctttaat taaaaagttg aagaaacccc ctacagatga agagttaaag 1680
gaaacaataa agaaattact ggccagtgct aacttggaag aagtcacaat gaaacagatt 1740
tgcaaaaagg tctatgaaaa ttatcctact tatgatttaa ctgaaagaaa agatttcata 1800
aaaacaactg taaaagagct aatttcttga 1830
<210> 9
<211> 609
<212> PRT
<213> EGFP-DEKΔNLS基因(Humanized)
<400> 9
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser
225 230 235 240
Gly Leu Arg Ser Arg Ala Gln Ala Ser Asn Ser Met Ser Ala Ser Ala
245 250 255
Pro Ala Ala Glu Gly Glu Gly Thr Pro Thr Gln Pro Ala Ser Glu Lys
260 265 270
Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser Glu Glu Glu Glu Asp
275 280 285
Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Lys Glu Lys Ser Leu Ile
290 295 300
Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu Arg Leu Thr Met Gln
305 310 315 320
Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Ile Ala Gln Gly Lys Gly
325 330 335
Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe Phe Leu Ser Lys Lys
340 345 350
Lys Thr Asp Glu Leu Arg Asn Leu His Lys Leu Leu Tyr Asn Arg Pro
355 360 365
Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly Gln Phe Ser Gly Phe
370 375 380
Pro Phe Glu Lys Gly Ser Val Gln Tyr Lys Lys Lys Glu Glu Met Leu
385 390 395 400
Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile Cys Glu Val Leu Asp
405 410 415
Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val Lys Arg Ile Leu Asn
420 425 430
Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro Leu Pro Lys Ser Lys
435 440 445
Lys Thr Cys Ser Lys Gly Ser Cys Pro Glu Ile Leu Ser Asp Glu Ser
450 455 460
Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys Glu Glu Ser Ser Asp Asp
465 470 475 480
Glu Asp Lys Glu Ser Glu Glu Glu Pro Pro Lys Lys Thr Ala Lys Arg
485 490 495
Glu Lys Pro Lys Gln Lys Ala Thr Ser Lys Ser Lys Lys Ser Val Lys
500 505 510
Ser Ala Asn Val Lys Lys Ala Asp Ser Ser Thr Thr Lys Lys Asn Gln
515 520 525
Asn Ser Ser Lys Lys Glu Ser Glu Ser Glu Asp Ser Ser Asp Asp Glu
530 535 540
Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro Thr Asp Glu Glu Leu Lys
545 550 555 560
Glu Thr Ile Lys Lys Leu Leu Ala Ser Ala Asn Leu Glu Glu Val Thr
565 570 575
Met Lys Gln Ile Cys Lys Lys Val Tyr Glu Asn Tyr Pro Thr Tyr Asp
580 585 590
Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr Thr Val Lys Glu Leu Ile
595 600 605
Ser
<210> 10
<211> 1896
<212> DNA
<213> EGFP-DEK基因(Murine)
<400> 10
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720
ggactcagat ctcgagctca agcttcgaat tctatgtcgg cggcggcggc ccccgctgcg 780
gagggagagg acgcccccgt gccgccctca tccgagaagg aacccgagat gccgggtccc 840
agggaagaga gtgaggagga ggaggaggat gacgaagacg atgatgaaga ggacgaggag 900
gaagaaaaag aaaagagtct tatcgtggaa ggcaagagag agaagaagaa agtagagaga 960
ctgacgatgc aagtgtcttc cttacagaga gagccattta cagtgacaca agggaagggt 1020
cagaaacttt gtgaaattga aaggatacat ttctttctga gtaagaaaaa accagatgaa 1080
cttagaaatc tacacaaact gctttacaac aggccgggca cagtgtcctc gttgaagaag 1140
aacgtgggtc agttcagtgg ctttccattc gaaaaaggca gtacccagta taaaaagaag 1200
gaagaaatgt tgaaaaagtt tcgaaatgcc atgttaaaga gcatctgtga ggttcttgat 1260
ttagagaggt caggcgtgaa cagcgaactc gtgaagagga tcttgaactt cttaatgcat 1320
ccaaagcctt ctggcaaacc attaccaaaa tccaaaaaat cttccagcaa aggtagtaaa 1380
aaggaacgga acagttctgg aacaacaagg aagtcaaagc aaactaaatg ccctgaaatt 1440
ctgtcagatg agtctagtag tgatgaagat gagaagaaaa ataaggaaga gtcttcggaa 1500
gatgaagaga aagaaagtga agaggagcaa ccaccaaaaa agacatctaa aaaagaaaaa 1560
gcaaaacaga aagctactgc taaaagtaaa aaatctgtga agagtgctaa tgttaagaag 1620
gcagacagca gtaccaccaa gaagaatcaa aaaagttcca aaaaagagtc tgaatctgaa 1680
gacagttctg atgatgaacc cttaattaaa aaattgaaaa agccacctac agatgaagag 1740
ctaaaggaaa cagtgaagaa attactggct gatgctaact tggaagaagt cacaatgaag 1800
cagatttgca aagaggtata tgaaaattat cctgcttatg atttgactga gaggaaagat 1860
ttcattaaaa caactgtaaa agagctaatt tcttga 1896
<210> 11
<211> 631
<212> PRT
<213> EGFP-DEK基因(Murine)
<400> 11
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser
225 230 235 240
Gly Leu Arg Ser Arg Ala Gln Ala Ser Asn Ser Met Ser Ala Ala Ala
245 250 255
Ala Pro Ala Ala Glu Gly Glu Asp Ala Pro Val Pro Pro Ser Ser Glu
260 265 270
Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser Glu Glu Glu Glu
275 280 285
Glu Asp Asp Glu Asp Asp Asp Glu Glu Asp Glu Glu Glu Glu Lys Glu
290 295 300
Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu Arg
305 310 315 320
Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Val Thr
325 330 335
Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe Phe
340 345 350
Leu Ser Lys Lys Lys Pro Asp Glu Leu Arg Asn Leu His Lys Leu Leu
355 360 365
Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly Gln
370 375 380
Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Thr Gln Tyr Lys Lys Lys
385 390 395 400
Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile Cys
405 410 415
Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val Lys
420 425 430
Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro Leu
435 440 445
Pro Lys Ser Lys Lys Ser Ser Ser Lys Gly Ser Lys Lys Glu Arg Asn
450 455 460
Ser Ser Gly Thr Thr Arg Lys Ser Lys Gln Thr Lys Cys Pro Glu Ile
465 470 475 480
Leu Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys Glu
485 490 495
Glu Ser Ser Glu Asp Glu Glu Lys Glu Ser Glu Glu Glu Gln Pro Pro
500 505 510
Lys Lys Thr Ser Lys Lys Glu Lys Ala Lys Gln Lys Ala Thr Ala Lys
515 520 525
Ser Lys Lys Ser Val Lys Ser Ala Asn Val Lys Lys Ala Asp Ser Ser
530 535 540
Thr Thr Lys Lys Asn Gln Lys Ser Ser Lys Lys Glu Ser Glu Ser Glu
545 550 555 560
Asp Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro
565 570 575
Thr Asp Glu Glu Leu Lys Glu Thr Val Lys Lys Leu Leu Ala Asp Ala
580 585 590
Asn Leu Glu Glu Val Thr Met Lys Gln Ile Cys Lys Glu Val Tyr Glu
595 600 605
Asn Tyr Pro Ala Tyr Asp Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr
610 615 620
Thr Val Lys Glu Leu Ile Ser
625 630
<210> 12
<211> 1845
<212> DNA
<213> EGFP-DEKΔNLS基因(Murine)
<400> 12
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 720
ggactcagat ctcgagctca agcttcgaat tctatgtcgg cggcggcggc ccccgctgcg 780
gagggagagg acgcccccgt gccgccctca tccgagaagg aacccgagat gccgggtccc 840
agggaagaga gtgaggagga ggaggaggat gacgaagacg atgatgaaga ggacgaggag 900
gaagaaaaag aaaagagtct tatcgtggaa ggcaagagag agaagaagaa agtagagaga 960
ctgacgatgc aagtgtcttc cttacagaga gagccattta cagtgacaca agggaagggt 1020
cagaaacttt gtgaaattga aaggatacat ttctttctga gtaagaaaaa accagatgaa 1080
cttagaaatc tacacaaact gctttacaac aggccgggca cagtgtcctc gttgaagaag 1140
aacgtgggtc agttcagtgg ctttccattc gaaaaaggca gtacccagta taaaaagaag 1200
gaagaaatgt tgaaaaagtt tcgaaatgcc atgttaaaga gcatctgtga ggttcttgat 1260
ttagagaggt caggcgtgaa cagcgaactc gtgaagagga tcttgaactt cttaatgcat 1320
ccaaagcctt ctggcaaacc attaccaaaa tccaaaaaat cttccagcaa aggtagttgc 1380
cctgaaattc tgtcagatga gtctagtagt gatgaagatg agaagaaaaa taaggaagag 1440
tcttcggaag atgaagagaa agaaagtgaa gaggagcaac caccaaaaaa gacatctaaa 1500
aaagaaaaag caaaacagaa agctactgct aaaagtaaaa aatctgtgaa gagtgctaat 1560
gttaagaagg cagacagcag taccaccaag aagaatcaaa aaagttccaa aaaagagtct 1620
gaatctgaag acagttctga tgatgaaccc ttaattaaaa aattgaaaaa gccacctaca 1680
gatgaagagc taaaggaaac agtgaagaaa ttactggctg atgctaactt ggaagaagtc 1740
acaatgaagc agatttgcaa agaggtatat gaaaattatc ctgcttatga tttgactgag 1800
aggaaagatt tcattaaaac aactgtaaaa gagctaattt cttga 1845
<210> 13
<211> 614
<212> PRT
<213> EGFP-DEKΔNLS(Murine)
<400> 13
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser
225 230 235 240
Gly Leu Arg Ser Arg Ala Gln Ala Ser Asn Ser Met Ser Ala Ala Ala
245 250 255
Ala Pro Ala Ala Glu Gly Glu Asp Ala Pro Val Pro Pro Ser Ser Glu
260 265 270
Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser Glu Glu Glu Glu
275 280 285
Glu Asp Asp Glu Asp Asp Asp Glu Glu Asp Glu Glu Glu Glu Lys Glu
290 295 300
Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu Arg
305 310 315 320
Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Val Thr
325 330 335
Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe Phe
340 345 350
Leu Ser Lys Lys Lys Pro Asp Glu Leu Arg Asn Leu His Lys Leu Leu
355 360 365
Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly Gln
370 375 380
Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Thr Gln Tyr Lys Lys Lys
385 390 395 400
Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile Cys
405 410 415
Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val Lys
420 425 430
Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro Leu
435 440 445
Pro Lys Ser Lys Lys Ser Ser Ser Lys Gly Ser Cys Pro Glu Ile Leu
450 455 460
Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys Glu Glu
465 470 475 480
Ser Ser Glu Asp Glu Glu Lys Glu Ser Glu Glu Glu Gln Pro Pro Lys
485 490 495
Lys Thr Ser Lys Lys Glu Lys Ala Lys Gln Lys Ala Thr Ala Lys Ser
500 505 510
Lys Lys Ser Val Lys Ser Ala Asn Val Lys Lys Ala Asp Ser Ser Thr
515 520 525
Thr Lys Lys Asn Gln Lys Ser Ser Lys Lys Glu Ser Glu Ser Glu Asp
530 535 540
Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro Thr
545 550 555 560
Asp Glu Glu Leu Lys Glu Thr Val Lys Lys Leu Leu Ala Asp Ala Asn
565 570 575
Leu Glu Glu Val Thr Met Lys Gln Ile Cys Lys Glu Val Tyr Glu Asn
580 585 590
Tyr Pro Ala Tyr Asp Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr Thr
595 600 605
Val Lys Glu Leu Ile Ser
610
<210> 14
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 14
ggatagttca gatgatgaac 20
<210> 15
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 15
gtgatgaaga tgaaaagaaa 20
<210> 16
<211> 21
<212> DNA
<213> 未知(Unknown)
<400> 16
gtgaagaaat tactggctga t 21
<210> 17
<211> 21
<212> DNA
<213> 未知(Unknown)
<400> 17
cgaactcgtg aagaggatct t 21
<210> 18
<211> 21
<212> DNA
<213> 未知(Unknown)
<400> 18
atgttaacag ctgtactggt g 21
<210> 19
<211> 1077
<212> DNA
<213> DEKΔNLS基因(Humanized)
<400> 19
atgtccgcct cggcccctgc tgcggagggg gagggaaccc ccacccagcc cgcgtccgag 60
aaagaacccg aaatgcccgg tcccagagag gagagcgagg aggaagagga cgaggacgac 120
gaggaggagg aggaggagga aaaagaaaag agtctcatcg tggaaggcaa gagggaaaag 180
aaaaaagtag agaggttgac aatgcaagtc tcttccttac agagagagcc atttacaatt 240
gcacaaggaa aggggcagaa actttgtgaa attgagagga tacatttttt tctaagtaag 300
aagaaaaccg atgaacttag aaatctacac aaactgcttt acaacaggcc aggcactgtg 360
tcctcattaa agaagaatgt gggtcagttc agtggctttc catttgaaaa aggaagtgtc 420
caatataaaa agaaggaaga aatgttgaaa aaatttagaa atgccatgtt aaagagcatc 480
tgtgaggttc ttgatttgga gagatcaggt gtaaatagtg aactagtgaa gaggatcttg 540
aatttcttaa tgcatccaaa gccttctggc aaaccattgc cgaaatctaa aaaaacttgt 600
agcaaaggca gttgtcctga aattctgtca gatgaatcta gtagtgatga agatgaaaag 660
aaaaacaagg aagagtcttc agatgatgaa gataaagaaa gtgaagagga gccaccaaaa 720
aagacagcca aaagagaaaa acctaaacag aaagctactt ctaaaagtaa aaaatctgtg 780
aaaagtgcca atgttaagaa agcagatagc agcaccacca agaagaatca aaacagttcc 840
aaaaaagaaa gtgagtctga ggatagttca gatgatgaac ctttaattaa aaagttgaag 900
aaacccccta cagatgaaga gttaaaggaa acaataaaga aattactggc cagtgctaac 960
ttggaagaag tcacaatgaa acagatttgc aaaaaggtct atgaaaatta tcctacttat 1020
gatttaactg aaagaaaaga tttcataaaa acaactgtaa aagagctaat ttcttga 1077
<210> 20
<211> 358
<212> PRT
<213> DEKΔNLS(Humanized)
<400> 20
Met Ser Ala Ser Ala Pro Ala Ala Glu Gly Glu Gly Thr Pro Thr Gln
1 5 10 15
Pro Ala Ser Glu Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser
20 25 30
Glu Glu Glu Glu Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Lys
35 40 45
Glu Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu
50 55 60
Arg Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Ile
65 70 75 80
Ala Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe
85 90 95
Phe Leu Ser Lys Lys Lys Thr Asp Glu Leu Arg Asn Leu His Lys Leu
100 105 110
Leu Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly
115 120 125
Gln Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Val Gln Tyr Lys Lys
130 135 140
Lys Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile
145 150 155 160
Cys Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val
165 170 175
Lys Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro
180 185 190
Leu Pro Lys Ser Lys Lys Thr Cys Ser Lys Gly Ser Cys Pro Glu Ile
195 200 205
Leu Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys Glu
210 215 220
Glu Ser Ser Asp Asp Glu Asp Lys Glu Ser Glu Glu Glu Pro Pro Lys
225 230 235 240
Lys Thr Ala Lys Arg Glu Lys Pro Lys Gln Lys Ala Thr Ser Lys Ser
245 250 255
Lys Lys Ser Val Lys Ser Ala Asn Val Lys Lys Ala Asp Ser Ser Thr
260 265 270
Thr Lys Lys Asn Gln Asn Ser Ser Lys Lys Glu Ser Glu Ser Glu Asp
275 280 285
Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro Thr
290 295 300
Asp Glu Glu Leu Lys Glu Thr Ile Lys Lys Leu Leu Ala Ser Ala Asn
305 310 315 320
Leu Glu Glu Val Thr Met Lys Gln Ile Cys Lys Lys Val Tyr Glu Asn
325 330 335
Tyr Pro Thr Tyr Asp Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr Thr
340 345 350
Val Lys Glu Leu Ile Ser
355
<210> 21
<211> 1143
<212> DNA
<213> DEK基因(Murine)
<400> 21
atgtcggcgg cggcggcccc cgctgcggag ggagaggacg cccccgtgcc gccctcatcc 60
gagaaggaac ccgagatgcc gggtcccagg gaagagagtg aggaggagga ggaggatgac 120
gaagacgatg atgaagagga cgaggaggaa gaaaaagaaa agagtcttat cgtggaaggc 180
aagagagaga agaagaaagt agagagactg acgatgcaag tgtcttcctt acagagagag 240
ccatttacag tgacacaagg gaagggtcag aaactttgtg aaattgaaag gatacatttc 300
tttctgagta agaaaaaacc agatgaactt agaaatctac acaaactgct ttacaacagg 360
ccgggcacag tgtcctcgtt gaagaagaac gtgggtcagt tcagtggctt tccattcgaa 420
aaaggcagta cccagtataa aaagaaggaa gaaatgttga aaaagtttcg aaatgccatg 480
ttaaagagca tctgtgaggt tcttgattta gagaggtcag gcgtgaacag cgaactcgtg 540
aagaggatct tgaacttctt aatgcatcca aagccttctg gcaaaccatt accaaaatcc 600
aaaaaatctt ccagcaaagg tagtaaaaag gaacggaaca gttctggaac aacaaggaag 660
tcaaagcaaa ctaaatgccc tgaaattctg tcagatgagt ctagtagtga tgaagatgag 720
aagaaaaata aggaagagtc ttcggaagat gaagagaaag aaagtgaaga ggagcaacca 780
ccaaaaaaga catctaaaaa agaaaaagca aaacagaaag ctactgctaa aagtaaaaaa 840
tctgtgaaga gtgctaatgt taagaaggca gacagcagta ccaccaagaa gaatcaaaaa 900
agttccaaaa aagagtctga atctgaagac agttctgatg atgaaccctt aattaaaaaa 960
ttgaaaaagc cacctacaga tgaagagcta aaggaaacag tgaagaaatt actggctgat 1020
gctaacttgg aagaagtcac aatgaagcag atttgcaaag aggtatatga aaattatcct 1080
gcttatgatt tgactgagag gaaagatttc attaaaacaa ctgtaaaaga gctaatttct 1140
tga 1143
<210> 22
<211> 380
<212> PRT
<213> DEK(Murine)
<400> 22
Met Ser Ala Ala Ala Ala Pro Ala Ala Glu Gly Glu Asp Ala Pro Val
1 5 10 15
Pro Pro Ser Ser Glu Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu
20 25 30
Ser Glu Glu Glu Glu Glu Asp Asp Glu Asp Asp Asp Glu Glu Asp Glu
35 40 45
Glu Glu Glu Lys Glu Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys
50 55 60
Lys Lys Val Glu Arg Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu
65 70 75 80
Pro Phe Thr Val Thr Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu
85 90 95
Arg Ile His Phe Phe Leu Ser Lys Lys Lys Pro Asp Glu Leu Arg Asn
100 105 110
Leu His Lys Leu Leu Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys
115 120 125
Lys Asn Val Gly Gln Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Thr
130 135 140
Gln Tyr Lys Lys Lys Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met
145 150 155 160
Leu Lys Ser Ile Cys Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn
165 170 175
Ser Glu Leu Val Lys Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro
180 185 190
Ser Gly Lys Pro Leu Pro Lys Ser Lys Lys Ser Ser Ser Lys Gly Ser
195 200 205
Lys Lys Glu Arg Asn Ser Ser Gly Thr Thr Arg Lys Ser Lys Gln Thr
210 215 220
Lys Cys Pro Glu Ile Leu Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu
225 230 235 240
Lys Lys Asn Lys Glu Glu Ser Ser Glu Asp Glu Glu Lys Glu Ser Glu
245 250 255
Glu Glu Gln Pro Pro Lys Lys Thr Ser Lys Lys Glu Lys Ala Lys Gln
260 265 270
Lys Ala Thr Ala Lys Ser Lys Lys Ser Val Lys Ser Ala Asn Val Lys
275 280 285
Lys Ala Asp Ser Ser Thr Thr Lys Lys Asn Gln Lys Ser Ser Lys Lys
290 295 300
Glu Ser Glu Ser Glu Asp Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys
305 310 315 320
Leu Lys Lys Pro Pro Thr Asp Glu Glu Leu Lys Glu Thr Val Lys Lys
325 330 335
Leu Leu Ala Asp Ala Asn Leu Glu Glu Val Thr Met Lys Gln Ile Cys
340 345 350
Lys Glu Val Tyr Glu Asn Tyr Pro Ala Tyr Asp Leu Thr Glu Arg Lys
355 360 365
Asp Phe Ile Lys Thr Thr Val Lys Glu Leu Ile Ser
370 375 380
<210> 23
<211> 1092
<212> DNA
<213> DEKΔNLS(Murine)
<400> 23
atgtcggcgg cggcggcccc cgctgcggag ggagaggacg cccccgtgcc gccctcatcc 60
gagaaggaac ccgagatgcc gggtcccagg gaagagagtg aggaggagga ggaggatgac 120
gaagacgatg atgaagagga cgaggaggaa gaaaaagaaa agagtcttat cgtggaaggc 180
aagagagaga agaagaaagt agagagactg acgatgcaag tgtcttcctt acagagagag 240
ccatttacag tgacacaagg gaagggtcag aaactttgtg aaattgaaag gatacatttc 300
tttctgagta agaaaaaacc agatgaactt agaaatctac acaaactgct ttacaacagg 360
ccgggcacag tgtcctcgtt gaagaagaac gtgggtcagt tcagtggctt tccattcgaa 420
aaaggcagta cccagtataa aaagaaggaa gaaatgttga aaaagtttcg aaatgccatg 480
ttaaagagca tctgtgaggt tcttgattta gagaggtcag gcgtgaacag cgaactcgtg 540
aagaggatct tgaacttctt aatgcatcca aagccttctg gcaaaccatt accaaaatcc 600
aaaaaatctt ccagcaaagg tagttgccct gaaattctgt cagatgagtc tagtagtgat 660
gaagatgaga agaaaaataa ggaagagtct tcggaagatg aagagaaaga aagtgaagag 720
gagcaaccac caaaaaagac atctaaaaaa gaaaaagcaa aacagaaagc tactgctaaa 780
agtaaaaaat ctgtgaagag tgctaatgtt aagaaggcag acagcagtac caccaagaag 840
aatcaaaaaa gttccaaaaa agagtctgaa tctgaagaca gttctgatga tgaaccctta 900
attaaaaaat tgaaaaagcc acctacagat gaagagctaa aggaaacagt gaagaaatta 960
ctggctgatg ctaacttgga agaagtcaca atgaagcaga tttgcaaaga ggtatatgaa 1020
aattatcctg cttatgattt gactgagagg aaagatttca ttaaaacaac tgtaaaagag 1080
ctaatttctt ga 1092
<210> 24
<211> 363
<212> PRT
<213> DEKΔNLS(Murine)
<400> 24
Met Ser Ala Ala Ala Ala Pro Ala Ala Glu Gly Glu Asp Ala Pro Val
1 5 10 15
Pro Pro Ser Ser Glu Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu
20 25 30
Ser Glu Glu Glu Glu Glu Asp Asp Glu Asp Asp Asp Glu Glu Asp Glu
35 40 45
Glu Glu Glu Lys Glu Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys
50 55 60
Lys Lys Val Glu Arg Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu
65 70 75 80
Pro Phe Thr Val Thr Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu
85 90 95
Arg Ile His Phe Phe Leu Ser Lys Lys Lys Pro Asp Glu Leu Arg Asn
100 105 110
Leu His Lys Leu Leu Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys
115 120 125
Lys Asn Val Gly Gln Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Thr
130 135 140
Gln Tyr Lys Lys Lys Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met
145 150 155 160
Leu Lys Ser Ile Cys Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn
165 170 175
Ser Glu Leu Val Lys Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro
180 185 190
Ser Gly Lys Pro Leu Pro Lys Ser Lys Lys Ser Ser Ser Lys Gly Ser
195 200 205
Cys Pro Glu Ile Leu Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu Lys
210 215 220
Lys Asn Lys Glu Glu Ser Ser Glu Asp Glu Glu Lys Glu Ser Glu Glu
225 230 235 240
Glu Gln Pro Pro Lys Lys Thr Ser Lys Lys Glu Lys Ala Lys Gln Lys
245 250 255
Ala Thr Ala Lys Ser Lys Lys Ser Val Lys Ser Ala Asn Val Lys Lys
260 265 270
Ala Asp Ser Ser Thr Thr Lys Lys Asn Gln Lys Ser Ser Lys Lys Glu
275 280 285
Ser Glu Ser Glu Asp Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys Leu
290 295 300
Lys Lys Pro Pro Thr Asp Glu Glu Leu Lys Glu Thr Val Lys Lys Leu
305 310 315 320
Leu Ala Asp Ala Asn Leu Glu Glu Val Thr Met Lys Gln Ile Cys Lys
325 330 335
Glu Val Tyr Glu Asn Tyr Pro Ala Tyr Asp Leu Thr Glu Arg Lys Asp
340 345 350
Phe Ile Lys Thr Thr Val Lys Glu Leu Ile Ser
355 360
<210> 25
<211> 375
<212> PRT
<213> 未知(Unknown)
<400> 25
Met Ser Ala Xaa Ala Pro Ala Ala Glu Gly Glu Xaa Xaa Pro Xaa Xaa
1 5 10 15
Pro Xaa Ser Glu Lys Glu Pro Glu Met Pro Gly Pro Arg Glu Glu Ser
20 25 30
Glu Glu Glu Glu Xaa Xaa Asp Asp Glu Glu Xaa Glu Glu Glu Glu Lys
35 40 45
Glu Lys Ser Leu Ile Val Glu Gly Lys Arg Glu Lys Lys Lys Val Glu
50 55 60
Arg Leu Thr Met Gln Val Ser Ser Leu Gln Arg Glu Pro Phe Thr Xaa
65 70 75 80
Xaa Gln Gly Lys Gly Gln Lys Leu Cys Glu Ile Glu Arg Ile His Phe
85 90 95
Phe Leu Ser Lys Lys Lys Xaa Asp Glu Leu Arg Asn Leu His Lys Leu
100 105 110
Leu Tyr Asn Arg Pro Gly Thr Val Ser Ser Leu Lys Lys Asn Val Gly
115 120 125
Gln Phe Ser Gly Phe Pro Phe Glu Lys Gly Ser Xaa Gln Tyr Lys Lys
130 135 140
Lys Glu Glu Met Leu Lys Lys Phe Arg Asn Ala Met Leu Lys Ser Ile
145 150 155 160
Cys Glu Val Leu Asp Leu Glu Arg Ser Gly Val Asn Ser Glu Leu Val
165 170 175
Lys Arg Ile Leu Asn Phe Leu Met His Pro Lys Pro Ser Gly Lys Pro
180 185 190
Leu Pro Lys Ser Lys Lys Xaa Xaa Ser Lys Gly Ser Lys Lys Glu Arg
195 200 205
Asn Ser Ser Gly Xaa Xaa Arg Lys Xaa Lys Xaa Thr Lys Cys Pro Glu
210 215 220
Ile Leu Ser Asp Glu Ser Ser Ser Asp Glu Asp Glu Lys Lys Asn Lys
225 230 235 240
Glu Glu Ser Ser Xaa Asp Glu Xaa Lys Glu Ser Glu Glu Glu Pro Pro
245 250 255
Lys Lys Thr Xaa Lys Xaa Glu Lys Xaa Lys Gln Lys Ala Thr Xaa Lys
260 265 270
Ser Lys Lys Ser Val Lys Ser Ala Asn Val Lys Lys Ala Asp Ser Ser
275 280 285
Thr Thr Lys Lys Asn Gln Xaa Ser Ser Lys Lys Glu Ser Glu Ser Glu
290 295 300
Asp Ser Ser Asp Asp Glu Pro Leu Ile Lys Lys Leu Lys Lys Pro Pro
305 310 315 320
Thr Asp Glu Glu Leu Lys Glu Thr Xaa Lys Lys Leu Leu Ala Xaa Ala
325 330 335
Asn Leu Glu Glu Val Thr Met Lys Gln Ile Cys Lys Xaa Val Tyr Glu
340 345 350
Asn Tyr Pro Xaa Tyr Asp Leu Thr Glu Arg Lys Asp Phe Ile Lys Thr
355 360 365
Thr Val Lys Glu Leu Ile Ser
370 375
Claims (2)
1.一种用于激活休眠肿瘤细胞的SETD4蛋白抑制剂的投递蛋白,其特征在于所述投递蛋白包括投递DEK蛋白,所述投递DEK蛋白为含有DEK蛋白的外泌体;所述DEK蛋白具有SEQID NO.22所示氨基酸序列。
2.如权利要求1所述的投递蛋白,其特征在于,所述含有DEK蛋白的外泌体按如下方法制备:构建过表达DEK蛋白的慢病毒并将其感染细胞系构建表达DEK蛋白的细胞株制备外泌体:将DEK基因分别插入到pLent-N-GFP的慢病毒表达载体的EcoRⅠ和XbaⅠ位点上,筛选获得重组慢病毒表达载体pLent-N-GFP-DEK;将上述重组慢病毒表达载体和慢病毒包装质粒混合物共同转染293T细胞,转染72小时后收集细胞培养上清即为病毒液,浓缩纯化,获得过表达DEK蛋白的慢病毒;用过表达DEK蛋白的慢病毒感染细胞系并构建过表达DEK蛋白的细胞株;收集过表达DEK蛋白的细胞株中的细胞培养液,从培养液中分离纯化含有DEK蛋白的外泌体溶液B;所述慢病毒包装质粒混合物为质量比5:3:2的pMDL、VSVG和pRSV-Rev;所述细胞系包括乳腺癌、肺癌、胃癌、前列腺癌或宫颈癌。
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