CN110172461B - 一种新型骨肉瘤肺转移模型的构建方法及其应用 - Google Patents
一种新型骨肉瘤肺转移模型的构建方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种新型骨肉瘤肺转移模型的构建方法及其应用。本发明针对REGγ设计了敲除效果显著的siRNA和shRNA的序列,并构建了包含上述序列的慢病毒载体,通过尾静脉注射细胞法来构建裸鼠骨肉瘤肺转移模型,结果证实包含本发明siRNA和shRNA的构建物能够显著降低REGγmRNA和蛋白表达量,且构建的骨肉瘤肺转移模型可明显观察细胞生长及转移情况。对于未来骨肉瘤靶向药物的探索提供了新的研究思路。
Description
技术领域
本发明涉及肿瘤分子生物学技术领域,具体地说,是一种新型骨肉瘤肺转移模型的构建方法及其应用。
背景技术
骨肉瘤是儿童和青少年中最常见原发性恶性骨肿瘤,发病率男性高于女性,骨肉瘤可发生在各个年龄段,尤其好发于青少年,约占青少年肿瘤的5%,每年全世界发病率为1-3/100万人。具有很强侵袭性,其恶性程度高,病人预后极差,尤其是早期发生肺转移后患者生存期急剧缩短。目前尚无有效治愈的治疗方法。骨肉瘤好发于生长快速的骨组织,疾病进展迅速,可于短时间内发生肺转移,治疗效果不佳,5年生存率低,不但对于骨肉瘤患者及家庭造成巨大痛苦,而且影响了我国医疗资源的配置。
蛋白酶体激活因子REGγ,也被称为PSME3(Proteasomeactivatorcomplexsubunit3)、PA28γ或11Sγ。属于蛋白酶体激活因子REG(又名11S)家族的成员之一,以非泛素化和非ATP依赖的方式降解蛋白质。近年来,越来越多的研究表明,REGγ在多种肿瘤中出现了异常表达并于肿瘤的发生发展密切相关。REGγ主要通过降解多种靶蛋白并调控相关的信号通路参与肿瘤的发生发展。针对REGγ与肿瘤的研究已成为国内外领域研究的热点,REGγ已经证实与多种肿瘤的增殖、迁移的恶性肿瘤学行为关系密切,但未有骨肉瘤的相关研究报道。
尽管目前术前新辅助化疗结合手术技术的提高,骨肉瘤患者的生存期及生活质量较以往得到了极大的改善,但对难治、复发及对化疗药物不敏感的骨肉瘤患者来说,其预后情况仍不乐观。近些年来,针对肿瘤细胞的基因靶向治疗已经成为国内外肿瘤领域研究的热点和前沿。同时,作为蛋白酶体系中重要的一员—REGγ对于多种肿瘤的发生发展过程的生物学作用前期已有大量研究报道。对于特定瘤肿,针对肿瘤细胞特异性靶点的靶向药物可以精准杀灭肿瘤细胞,通过研制针对骨肉瘤的新型靶向药物可极大的改善复发、难治性骨肉瘤患者的预后情况。而针对REGγ基因的肺转移小鼠模型的建立,对于未来骨肉瘤靶向药物的研制意义重大。本专利通过构建REGγ敲减细胞系,并进一步通过尾静脉注射骨肉瘤细胞法,构建一种新型在骨肉瘤中的表达及功能研究展开研究。
传统的骨肉瘤模型多是通过胫骨平台注射或皮下荷瘤,无法观察骨肉瘤的肺脏转移特性,尤其是其肺转移特点。
RNA干扰(RNA interference,RNAi)是一门基因阻断技术,为一种双链RNA(double-strandedRNA,dsRNA)分子在mRNA水平上阻断特异基因的表达或使其沉默的过程,即序列特异性的转录后基因沉默(Post-transcriptional gene silencing,PTGS)。采用RNAi技术研究基因在肿瘤发病过程中行使的功能是对肿瘤发病机制研究的重要补充。而且目前RNAi已成为肿瘤基因治疗的有效策略。利用RNAi技术可以抑制原癌基因、突变的抑癌基因、细胞周期相关基因、抗凋亡相关基因等表达来抑制肿瘤的发生和发展。
发明人针对现有技术的缺陷,设计了本专利一种新型骨肉瘤肺转移构建方法,这对于骨肉瘤肺转移模型的建立以及靶向药物的探索提供了新的研究思路,对于骨肉瘤的基础研究意义重大。
发明内容
本发明的第一个目的是针对现有技术中的不足,提供针对REGγ,具备药物和研发试剂开发前景的siRNA分子、相关产品及其用途。
本发明的第二个目的是针对现有技术的不足,提供一种新型骨肉瘤肺转移模型的构建方法。
为实现上述第一个目的,本发明采取的技术方案是:
第一方面,本发明提供了一种siRNA分子,所述的siRNA分子包含以下序列:
正义链:CAGAAGACTTGGTGGCAAA(SEQ ID NO:1),
反义链:TTTGCCACCAAGTCTTCTG(SEQ ID NO:2)。
第二方面,本发明提供了一种shRNA分子,所述的shRNA分子包含以下序列:
正义链:GGTTCTGAGAGTAAAATTATT(SEQ ID NO:3),
反义链:AATAATTTTACTCTCAGAACC(SEQ ID NO:4)。
第三方面,本发明提供了一种构建物,,所述的构建物包含以下shRNA分子序列:
正义链:GGTTCTGAGAGTAAAATTATT(SEQ ID NO:3),
反义链:AATAATTTTACTCTCAGAACC(SEQ ID NO:4)。
作为一个优选例,所述的构建物为慢病毒载体。
第四方面,本发明提供了一种慢病毒颗粒,所述的慢病毒颗粒包含如上所述的构建物、psPAX2和VsVg/PMD2G,所述慢病毒颗粒由以下体积配比的物质构成:psPAX2:VsVg/PMD2G:慢病毒载体=2:1:2,其中psPAX2与VsVg/PMD2G是慢病毒包装质粒。
第五方面,本发明提供了一种骨肉瘤稳转细胞系,所述的细胞系包含如上所述的构建物。
第六方面,本发明提供了如上所述的siRNA分子、shRNA分子、构建物、慢病毒颗粒、骨肉瘤稳转细胞系在非治疗目的的抑制骨肉瘤细胞增殖、迁移或侵袭中的应用。
第七方面,本发明提供了如上所述的siRNA分子、shRNA分子、构建物、骨肉瘤稳转细胞系、慢病毒颗粒在制备治疗骨肉瘤的药物中的应用。
第八方面,本发明提供了如上所述的siRNA分子、shRNA分子、构建物、骨肉瘤稳转细胞系、慢病毒颗粒在制备抑制骨肉瘤细胞增殖、迁移或侵袭的试剂中的应用。
作为一个优选例,所述的骨肉瘤细胞来自于骨肉瘤143B细胞。
为实现上述第二个目的,本发明采取的技术方案是:
一种新型骨肉瘤肺转移模型的构建方法,所述方法包括以下步骤:
取如上所述骨肉瘤稳转细胞系导入动物体内得到新型骨肉瘤肺转移模型。
作为一个优选例,所述构建方法还包括以下步骤:
动物活体成像系统检测骨肉瘤细胞肺脏分布、组织蜡块切片制作与免疫组化染色和肺转移组织HE染色。
作为一个优选例,所述骨肉瘤稳转细胞系导入动物体内是通过尾静脉注射细胞法将骨肉瘤稳转细胞系导入动物体内。
本发明优点在于:
1、针对REGγ设计了合适的siRNA和shRNA的序列,并构建了包含上述序列的慢病毒载体。实验表明,本发明的siRNA和shRNA的构建物能够显著降低REGγmRNA和蛋白表达量,敲除作用十分明显,显著优于其他siRNA和shRNA,因此能开发为小分子治疗药物,或研究骨肉瘤病理机制的研发制剂。
2、首次通过尾静脉注射法,注射REGγ敲除的骨肉瘤细胞,并构建对照组细胞观察比较;通过IVIS活体成像系统检测骨肉瘤细胞生长情况,能够动态观察到通过尾静脉接种的骨肉瘤细胞的生长及转移情况。
3、解剖肺组织,处理肿瘤组织,通过将肺组织固定、包埋制作转移灶蜡块,并制片行HE染色观察肿瘤组织生长情况,进而,将骨肉瘤组织剪碎处理,通过WB实验进一步从蛋白表达水平检测肿瘤细胞REGγ敲除情况,本发明的构建方法得到的模型可用于将来骨肉瘤药物REGγ靶点的检测。
附图说明
附图1是稳转细胞WB结果:143B shR中REGγ表达量明显减低。
附图2是骨肉瘤细胞尾静脉接种后第24h、24d IVIS成像。
附图3是骨肉瘤肺转移模型肺组织骨肉瘤转移情况。
附图4是骨肉瘤肺转移模型两组骨肉瘤转移瘤数量比较。
附图5是骨肉瘤转移模型组织HE染色情况。
附图6是骨肉瘤肺转移模型肿瘤组织REGγ表达情况。
具体实施方式
下面结合具体实施方式,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
以下实施例中所述的比例均为体积比。
实施例1
一、实验方法
1、骨肉瘤稳转细胞系的建立及荧光基因的转染
慢病毒表达载体名称:Lenti-EF1α-puro来源:美国贝勒医学院质粒库赠与。
骨肉瘤143B细胞转染REGγ的碱基序列:
shREGγ:“5’-CCGGGGTTCTGAGAGAGTAAAATTATTCTCGAGAATAATTTTACTCAGAACCTTTTTG-3'”,shNC为空载质粒,采用慢病毒法构建骨肉瘤细胞稳系。
1)首先根据REGγ基因的序列信息,构建相应shRNA到慢病毒表达载体上,即质粒构建;
2)再将psPAX2,VsVg/PMD2G(其中psPAX2和VsVg/PMD2G是慢病毒的包装质粒),及构建的慢病毒载体,按照体积比psPAX2:VsVg/PMD2G:慢病毒载体=2:1:2共转染293T细胞;
3)转染8h后更换培养基,并将培养皿移至病毒房培养,12h观察一次,若培养基变黄,及时补充少量新鲜培养基;
4)转染36-48h后,用注射器吸取上清至离心管中,5000rpm,离心10min,滤膜过滤后再用病毒浓缩柱进行浓缩;
5)用浓缩后的病毒去感染所需感染的细胞系,剩余病毒保存在-80℃备用;
6)感染36-48h后,(由于慢病毒载体上具有puromycin抗性)用含有puromycin的培养基进行筛选;
7)筛选几天后,通过细胞计数在96孔板中接种细胞,用正常培养基稀释细胞,混匀平均加至96孔板中,保证每个孔至多含有一个细胞;
8)每天观察细胞生长情况,若细胞不死亡并呈单个细胞团生长,待细胞生长到一定程度,消化传至12孔板中继续培养;
9)Westernblot方法检测以上单克隆细胞蛋白表达情况,进而判断基因敲低效率;
10)将shN143B和shR143B细胞接种于6孔板中,待细胞密度达到30-40%即可转染;按照Lipofectamine TM2000试剂盒操作流程进行转染,在100ul DMEM Free培养基中加入2μg/ml的PGL4-luc质粒,同时在100ul DMEM Free培养基中加入2ul转染试剂,混匀室温静置5min;
11)将上述2种稀释液混匀,室温静置30min后加入预先加入800ul Free培养基的细胞孔板中,分别补加各置于培养箱常规培养6-8h后,弃掉Free培养基,加用含血清DMEM培养基。
2、确定G418最佳筛选浓度
于常规DMEM培养基中培养shN143B和shR143B细胞,处理细胞并按每孔1×104个接种于12孔板中,置于37℃细胞培养箱中培养。G418按0、100、300、600、800、1000μg/ml设置6个浓度梯度。在细胞接种24h后,将G418按比例加入孔板中,每天观察细胞生长状况,在两周内细胞基本全部死亡的最低G418浓度,即为筛选质粒转染克隆细胞的最适宜浓度,实验重复一次进一步确定G418最适宜浓度。
3、单克隆细胞的筛选
转染36-48h后胰酶消化细胞,通过细胞计数在96孔板中接种细胞,并加入前述实验确定的最适浓度G418,保证每个孔至多含有一个细胞,随后每天观察细胞生长情况,更换培养基并维持上述浓度的G418,若细胞不死亡并呈单个细胞团生长,待细胞生长到一定程度,消化传至12孔板中继续培养。
4、荧光素酶基因的检测
将前述构建好的带有荧光标记基因的细胞铺12孔板,每个细胞分别按照103、5×103、104三个浓度铺板,并分别设置3个复孔,同时设置一组只加PBS空白对照组,24h后收样,lysis裂解液,放入-80℃冰箱内过夜,次日室温下摇床摇匀30min,将细胞样品12000r,10min离心,取150ul上清液,避光加入100ulluciferase底物,荧光分度仪观察细胞荧光值。
5、尾静脉注射细胞法构建裸鼠骨肉瘤肺转移模型
每只裸鼠尾静脉注射约5×105个骨肉瘤细胞。取对数生长期状态良好的shN143B和shR143B细胞,0.25%胰酶消化后制成细胞悬液,细胞数达到5×105个/ml,PBS洗两遍后加入100ul PBS重悬,重悬过程中注意避免吹打力度过大,影响细胞状态。将重悬的细胞悬液放置冰盒,分别于裸鼠尾部选取较粗的静脉,按照由远及近的方向用胰岛素注射器注射,注射前先要拿50℃左右的温水浸泡尾巴1min,每只裸鼠约注射细胞悬液100ul。
6、动物活体成像系统(IVIS)检测骨肉瘤细胞肺脏分布情况
利用尾静脉注射细胞后,通过IVIS系统检测裸鼠体内荧光表达情况。首先按150mg/kg体重的量腹腔注射荧光底物luciferin,异戊醛气体麻醉小鼠,5min后利用IVIS系统检测裸鼠体内荧光表达值。分组记录荧光值并拍照。
7、组织蜡块切片制作与免疫组化染色(1mmuno histochemical Staining,IHC)
收集的肺转移骨肉瘤组织石蜡包埋和切片石蜡制片。组织经固定、脱水、透明、浸蜡后包埋在石蜡里面进行切片。以下为脱水、固定、切片及组织染色详细步骤及注意事项:
1)固定及包埋:将组织放到包埋盒用多聚甲醛浸泡过夜,次日将组织脱水(50%、75%、85%、95%、100%,100%酒精各浸泡1小时),接着在二甲苯:乙醇=1:1;二甲苯1;二甲苯2中各浸泡30min(用镊子夹包埋盒时为避免毁坏组织,应夹在边框上),接着同石蜡包埋,石蜡提前放到水浴锅中融化(1号蜡1小时,2号可以过夜,3号1小时,组织放入蜡中),组织浸蜡后进行包埋,注意组织的朝向要竖直;
2)切片、脱蜡复水:从这一步开始,需要始终保持组织的湿润。将切片放在染缸中,依次放入“二甲苯2号(7min),二甲苯1号(7min),二甲苯:乙醇=1:1、100%、95%、85%、75%、50%乙醇各3min”(确保染缸内有足够的试剂来浸没组织,防止组织干燥);
3)修复:脱蜡复水后先放入盛有纯净水的载玻片染缸中清洗3min 2次,然后用100℃的乙二胺四乙酸(EDTA)修复20min(装有EDTA的烧杯用锡箔纸封口,提前放入100℃水浴中加热,放入组织片时有组织的一面靠玻璃放置,切片组织之间不可相互摩擦,以免损坏),室温放置恢复常温;
4)淬灭:恢复常温后用PBS洗3遍,每次3min(放在染缸里洗),然后用3%H2O2淬灭10min(H2O2重复利用,在染缸中淬灭过氧化酶的活性),随后用PBS洗3次,每次3min;
5)封闭:先将载玻片上的水甩干,然后用纸吸干组织周围的残留水分,再将100ul封闭液滴在组织上,放在湿盒中(不要吸干组织上的水,组织要始终保持湿润);
6)孵化一抗:同样要先吸干组织周围的水分,滴加一抗50ul(覆盖组织)左右,随后用封口膜覆盖以增强效果,封口膜覆盖时,尽量不要让组织上方有气泡,若有,则尽快用枪头轻轻赶走,敷好一抗的切片放在湿盒中,4℃过夜;
7)孵化二抗:次日先用PBS洗3次/3min,同样吸干组织周围水分后滴加二抗(100ul左右),湿盒中敷20min(常温);
8)显色:将二抗用吸水纸吸去,再用PBS洗3次/3min,再用辣根过氧化物酶(HRP)染色试剂敷10min,PBS洗3次/3min,随后在暗房中敷二氨基联苯胺(DAB),在显微镜下检测染色效果(先用30%的H2O2激活,体积比:DAB:H2O2=1000:1激活后只可用30min)。染色效果较好后终止染色,用自来水即可(染色太过用碳酸盐蘸洗一下即可);
9)苏木精染色:随后用苏木精染细胞核,在苏木精中静置5min,取出用自来水先清洗几次,随即用盐酸脱色,浸泡后立即取出,用自来水终止反应,浸泡1h以上;
10)脱水:接着用50%、75%、85%、95%、100%的乙醇;1:1的乙醇:二甲苯;二甲苯1号;二甲苯2号的顺序依次脱水3min;
11)封片:将树脂滴在组织周围,盖上盖玻片,斜立放置,让树脂自动散开,直至完全封住组织即可,静置晾干,拍片。
8、肺转移组织HE染色
取制好的组织切片于烘片机上62℃烘烤2h,行脱蜡复水步骤(依次放入100%二甲苯2号-7min,100%二甲苯1号-7min,二甲苯:乙醇=1:1,100%、95%、85%、75%、50%乙醇各3min),纯净水浸泡2遍,各3min,苏木精染色5min后,用水漂洗30min,脱水(依次放入50%、75%、85%、95%脱水),伊红染色2s后取出用水漂洗2遍,100%酒精各两遍每次3min,100%二甲苯2号各两遍,每遍7min,树胶封边,常温风干,拍照。
二、实验结果
1、带有荧光基因标记的骨肉瘤细胞株的构建
利用前期构建的REGγ敲减骨肉瘤细胞株shREGγ(shR)及对照组细胞株shNC(shN),其REGγ敲减情况可见图1。通过转染luc荧光质粒,并利用G418筛选单克隆细胞,构建成带有luc荧光基因的目的细胞系。通过荧光分度仪测量细胞荧光值,确定luc基因已转入细胞,可稳定发出荧光。
2、REGγ对骨肉瘤细胞的影响设计了多种shRNA,其中sh-REGγ-1、sh-REGγ-2、sh-REGγ-3的信息见表1。相比于对照,以上三种shRNA均能下调骨肉瘤细胞中REGγmRNA和蛋白表达水平,但sh-REGγ-1敲减水平最为明显,高达50%以上,显著高于sh-REGγ-2、sh-REGγ-3及其他的shRNA(p<0.05)。
表1.shRNA设计
3、裸鼠骨肉瘤肺转移模型的构建
利用构建好的shN143B luc和shR143B luc细胞通过尾静脉注射法,接种骨肉瘤细胞于4-6周左右裸鼠体内,3h后通过IVIS系统检测两组裸鼠均可见强度一致的荧光信号,相同条件下喂养小鼠24天,通过活体成像系统在6只小鼠的肺部均检测到骨肉瘤细胞的生物荧光信号,如图2所示,尾静脉注射shN143B luc细胞的裸鼠与注射shR143B luc细胞的裸鼠相比,其肿瘤细胞荧光信号强度及肺转移病灶明显强且范围广。
4、裸鼠骨肉瘤肺转移模型的检测
通过尾静脉注射骨肉瘤细胞构建肺转移模型裸鼠,正常饲养24d后,处死,解剖获得其肺组织,拍照。肺组织大体标本可见表面有数量不等、大小不一的肿瘤组织,呈白色半透明、质韧,对比可见shN143B细胞注射组较shR143B相比其肿瘤组织数量明显较多,肿瘤组织较大,如图3、4所示(p<0.05)。依次将每个肺组织内的肿瘤组织解剖取出,肿瘤组织经过固定、包埋后切片做HE染色结果如图5所示,可见肺组织中有类圆形肿瘤组织,肿瘤组织细胞致密类圆形。肿瘤组织经研磨、低温裂解后,收蛋白样行WB实验检测两组组织中REGγ的表达量,图6可见shN143B组REGγ表达量较shR143B明显较高。
本发明针对REGγ设计了合适的siRNA和shRNA的序列,并构建了包含该序列的慢病毒载体。实验表明,本发明的siRNA和shRNA的构建物能够显著降低REGγmRNA和蛋白表达量,敲除作用十分明显,显著优于其他siRNA和shRNA;另,本发明首次通过尾静脉注射法,注射REGγ敲除的骨肉瘤细胞,并构建对照组细胞观察比较;通过IVIS活体成像系统检测骨肉瘤细胞生长情况,能够动态观察到通过尾静脉接种的骨肉瘤细胞的生长及转移情况,本发明的构建方法得到的模型可用于将来骨肉瘤药物REGγ靶点的检测,为骨肉瘤患者提供了新的治疗方案,提高了患者的生存率。
以上所述仅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,在不脱离本发明原理的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海长征医院
<120> 一种新型骨肉瘤肺转移模型的构建方法及其应用
<130> /
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Claims (4)
1.shRNA分子、慢病毒载体、慢病毒颗粒以及骨肉瘤稳转细胞系中任一在非治疗目的的抑制骨肉瘤细胞增殖、迁移或侵袭中的应用,其特征在于,所述shRNA分子的序列为:
正义链:GGTTCTGAGAGTAAAATTATT,
反义链:AATAATTTTACTCTCAGAACC;
所述的慢病毒载体包含以下shRNA分子序列:
正义链:GGTTCTGAGAGTAAAATTATT,
反义链:AATAATTTTACTCTCAGAACC;
所述慢病毒颗粒由以下体积配比的物质构成:psPAX2:VsVg/PMD2G:慢病毒载体=2:1:2,
所述的骨肉瘤稳转细胞系包含慢病毒载体,所述的慢病毒载体包含以下shRNA分子序列:
正义链:GGTTCTGAGAGTAAAATTATT,
反义链:AATAATTTTACTCTCAGAACC。
2.一种新型骨肉瘤肺转移模型的构建方法,其特征在于,所述方法包括以下步骤:
将骨肉瘤稳转细胞系导入脊椎动物体内得到新型骨肉瘤肺转移模型,所述骨肉瘤稳转细胞系包含慢病毒载体,所述的慢病毒载体包含以下shRNA分子序列:正义链:GGTTCTGAGAGTAAAATTATT,反义链:AATAATTTTACTCTCAGAACC。
3.根据权利要求2所述构建方法,其特征在于,所述构建方法还包括以下步骤:
脊椎动物活体成像系统检测骨肉瘤细胞肺脏分布、组织蜡块切片制作与免疫组化染色和肺转移组织HE染色。
4.根据权利要求3所述构建方法,其特征在于,所述骨肉瘤稳转细胞系导入脊椎动物体内是通过尾静脉注射细胞法将骨肉瘤稳转细胞系导入脊椎动物体内。
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