CN111961688A - 具有胆管转移特性的肝癌模型构建方法及相应细胞 - Google Patents
具有胆管转移特性的肝癌模型构建方法及相应细胞 Download PDFInfo
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Abstract
本发明提供了一种具有胆管转移特性的肝癌模型构建方法,其包括如下步骤:S1:对大鼠成体肝干细胞进行扩增、培养并保存;S2:采用基因转染技术将含有Bmi1基因的慢病毒载体转入大鼠成体肝干细胞,获得稳定过表达Bmi1的大鼠成体肝干细胞;S3:利用稳定过表达Bmi1的大鼠成体肝干细胞,采用选择连续培养方法进行培养,使其在体外进一步分化,筛选出致瘤能力强及稳定的肝癌细胞;S4:采用筛选的肝癌细胞,通过动物体内原位肝脏移植瘤模型,可在肝脏形成肝癌且伴胆管癌栓,具有胆管转移特性。本发明同时提供了相应的具有胆管转移特性的肝癌模型及相应细胞。
Description
技术领域
本发明涉及一种具有胆管转移特性的肝癌模型构建方法及相应细胞。
背景技术
肝细胞癌(HCC)是最常见的原发性肝癌,是全球第三大癌症相关死亡原因。干细胞是一种特殊的细胞群,具有广泛的自我更新、分化和修复的能力。对于HCC或肝内胆管细胞癌,已经提出致癌过程以“卵圆细胞”开始,其具有双重分化能力并且可以转化为肝细胞或胆管细胞。这一过程被假设为正常组织中的干/祖细胞是癌症的来源。在肝脏中,认为涉及干/祖细胞作为癌症发展来源的致癌作用是可能的。基于功能获得和功能丧失分析结果,原发性肝癌可能源自从C57BL/6小鼠胎肝中分离的肝干细胞。众所周知,HCC主要发生在成年期,但尚不清楚HCC是否也可能源于成体肝祖细胞(HPCs)的恶性转化。
致癌基因B细胞特异性莫洛尼鼠白血病病毒整合位点1(Bmi1)是多梳抑制复合物1的重要辅助因子,其涉及多种细胞过程,包括调节细胞周期,细胞凋亡和通过赋予自我更新维持干细胞能力。Bmi1最初被确定与癌蛋白c-Myc协同诱导B细胞或T细胞白血病。自从这一发现以来,已经在几种人类癌症中检测到Bmi1的异常过表达,包括前列腺癌,结肠直肠癌,HCC,非小细胞肺癌,乳腺癌和胶质母细胞瘤。据报道在HCC中经常观察到Bmi1表达增加,且其有助于肿瘤发生能力。Bmi1敲低不仅降低了HCC细胞系的增殖和侵袭,而且还显着增加了化学敏感性。研究报道发现Bmi1在具有合并胆管癌栓的HCC中高度表达,胆管癌栓是HCC的特殊类型,具有相对更丰富的干细胞标志物表达。最近的研究表明,Bmi1 在调节正常和癌症干细胞(CSCs)的自我更新中起作用。例如,Bmi1对于正常造血干细胞以及白血病干细胞和祖细胞的自我更新是必需的。 Bmi1还被证明可以调节来自其他肿瘤类型(如前列腺癌和胰腺癌)的CSCs 的自我更新和增殖,Bmi1的敲低显着降低了HCC细胞系中的侧群细胞比率。这些侧群细胞被认为具有类似CSC的特性,以上这些发现表明Bmi1 在维持干性中的重要作用及其对干细胞恶性转化的可能贡献。然而,Bmi1 在驱动HPCs癌变的确切作用仍不清楚。
肝癌合并胆管癌栓占肝癌的7%-11%,其研究资料相对较少,目前没有具有胆管转移特性的肝癌模型构建方法。有效的动物体内模拟该过程有助于探索这一特殊疾病的发生、发展过程。因此,如何提供一种具有胆管转移特性的肝癌模型构建方法成为了业界需要解决的问题。
发明内容
针对现有技术的缺点,本发明的目的是提供一种具有胆管转移特性的肝癌模型构建方法以及相应的细胞。
为了实现上述目的,一方面,本发明提供了一种具有胆管转移特性的肝癌模型构建方法,其包括如下步骤:
S1:对大鼠成体肝干细胞进行扩增、培养并保存;
S2:采用基因转染技术将含有Bmi1基因的慢病毒载体转入大鼠成体肝干细胞,获得稳定过表达Bmi1的大鼠成体肝干细胞;
S3:利用稳定过表达Bmi1的大鼠成体肝干细胞,采用选择连续培养方法进行培养,使其在体外进一步分化,筛选出致瘤能力强及稳定的肝癌细胞;
S4:采用筛选的肝癌细胞,通过动物体内原位肝脏移植瘤模型,可在肝脏形成肝癌且伴胆管癌栓,具有胆管转移特性。
本发明的方法可以构建具有胆管转移特性的肝癌细胞。
根据本发明另一具体实施方式,步骤S2中,通过基因测序确认 Bmi1基因是否已插入慢病毒载体。
根据本发明另一具体实施方式,步骤S2中,通过PCT和Western blot检测细胞过表达效果,筛选出稳定过表达Bmi1的大鼠肝干细胞 (WB-F344-Bmi1)。
根据本发明另一具体实施方式,步骤S3包括多次重复的选择连续培养方法,每一周期所述选择连续培养方法内容包括:
a:反复更换细胞培养液,持续时间为4周;
b:细胞培养液采用连续选择改良的Richter培养液;
c:每周期培养后需观察细胞形态并记录;
d:每周期培养后将细胞分为三组,一组为用于验证致瘤性的评估组,一组为用于冷冻存样的冻存组,一组为用于下一培养周期的继续培养组。
根据本发明另一具体实施方式,通过观察形态,筛选出形态学上稳定的肝癌细胞。
根据本发明另一具体实施方式,通过裸鼠皮下成瘤,来评估细胞的致瘤性,筛选出致瘤能力强的肝癌细胞。
根据本发明另一具体实施方式,致瘤性包括成瘤率和肿瘤大小。
根据本发明另一具体实施方式,步骤S3中,通过免疫组织化学方法确定裸鼠皮下肿瘤类型为肝癌。
另一方面,本发明提供了一种采用上述方法制备的细胞,其具有胆管转移特性。
与现有技术相比,本发明具备如下有益效果:
本发明优选Bmi1基因,转染大鼠成体肝干细胞,获得稳定过表达 Bmi1的成体肝干细胞。利用连续选择传代方法,通过对每一周期的细胞形态学、皮下致瘤性和成瘤大小、肿瘤分子表型的变化进行评估,优选出恶性转化的细胞;再利用裸鼠原位肝癌模型形成肝癌并发生胆管转移。
下面结合附图对本发明作进一步的详细说明。
附图说明
图1是实施例1中,PCR与Western blot结果显示:转染Bmi1 的大鼠肝干细胞(WB-F344-Bmi1)稳定过表达Bmi1;
图2是实施例1中,光学显微镜下,选择连续培养法后,对比正常组细胞,WB-F344-Bmi1细胞形态、生长方式发生明显改变;且 WB-F344-Bmi1细胞可裸鼠皮下形成肿瘤,随着接种时间延长增殖生长,组织类型为低分化肝癌,表达AFP-,ALB+,CK19-,Bmi1+;
图3是实施例1中,原位肝脏移植瘤模型,WB-F344-Bmi1组观察裸鼠外形见皮肤发黄,MR提示胆囊明显增大,肝内胆管扩张;解剖动物发现:WB-F344-Bmi1组肝脏淤胆改变,肝外胆管存在癌栓;
图4是实施例1中,光学显微镜下,A图示WB-F344-Bmi1组肝脏内形成肿瘤,侵犯肝内胆管;B图示肝内胆管扩张、增生;
图5是实施例1中,光学显微镜下,A图为HE染色显示肝内胆管存在癌栓,且癌细胞浸润包绕胆管;B图为通过CK19标记胆管上皮细胞。
具体实施方式
实施例1
本实施例提供了一种具有胆管转移特性的肝癌模型构建方法及相应细胞。
本实施例的具体实验方案如下:
S1.实施Fisher 344系大鼠成体肝干细胞WB-F344的扩增及培养
(1)WB-F344的复苏:常规消毒超净工作台,在无菌15mL离心管中加入10mL DMEM培养基,从液氮罐中取出冻存的大鼠成体肝干细胞 WB-F344一管,立即放置37℃的水浴箱中,并振荡,观察细胞悬液的解冻情况,待至约2/3解冻后把冻存管经75%酒精喷洒消毒后放入超净台,用吸管把细胞悬液转移至含有10mL培养基的离心管中,并吹打匀,进行 1000rpm室温离心5分钟,弃去上清液,加入含有10%血清的5mL DMEM 培养液,吹打均匀后转移至25cm2培养瓶中,放入37℃,5%CO2细胞培养箱中。
(2)细胞换液及传代:当大鼠成体肝干细胞WB-F344扩增到 70%-80%覆盖瓶底时进行传代。首先弃去瓶中旧液,用0.1M PBS清洗1 遍,弃去PBS,加入2mL 0.25%胰蛋白酶/0.04%EDTA消化液,消化2-3 分钟,加入2mL含有10%血清的DMEM培养液中和液,用吸管吹打至所有细胞脱壁,把细胞悬液转移至15mL离心管中,进行1000rpm室温离心5 分钟,弃去上清液,加入5mL 10%血清的DMEM培养液,吹打均匀后等分至4个25cm2培养瓶中(即1:4传代),每瓶培养基补充至5mL,然后培养瓶放入37℃,5%CO2的细胞培养箱。
(3)大鼠成体肝干细胞WB-F344冻存:当ESC扩增到70%-80%覆盖瓶底时进行冻存。首先弃去瓶中旧液,用0.1M PBS清洗1遍,弃去 PBS,加入2mL 0.25%胰蛋白酶/0.04%EDTA消化液,消化2-3分钟,加入2mL 10%血清的DMEM培养液中和液,用吸管吹打至所有细胞脱壁,把细胞悬液转移至15mL离心管中,进行1000rpm室温离心5分钟,弃去上清液,加入细胞冻存液重悬细胞,调整细胞浓度为1.0-1.2×107/mL。将细胞悬液转移至冻存管,然后放置于含有异丙醇的梯度冻存盒,-80℃冻存。再转移至液氮罐保存。
大鼠成体肝干细胞WB-F344在传代后6-8小时后,逐渐在培养瓶中贴壁生长,细胞呈圆形,细胞核大,有一个或几个核仁,胞质胞浆少。形成边缘清楚的细胞克隆,克隆呈“巢状”或“岛状”,其中细胞排列紧密,与周围存在明显边界。每3-4天即可1:4传代一次。
S2.稳定过表达稳定细胞WB-F344-Bmi1的构建
(1)慢病毒的包装及质粒的构建由广州永诺生物科技有限公司协作完成。慢病毒表达载体,本次实验采用即pCDH-CMV-Puro载体。本次实验选择Rat Bmi1序列:NC_005116.4(85360439..85370283)进行扩增采用扩增引物序列:
Bmi1 Forward 5′-AGCAGCAATGACTGTGATGC-3′
Reverse 5′-CAGTCTCAGGTATCAACCAG-3′
Bmi1-Rat表达载体测序结果表明大鼠Bmi1基因已成功插入到载体中,测序序列正确。Blast结果比对显示已经测出来的部分序列与Pubmed 上序列100%一致。利用293T细胞包装慢病毒,滴度检测结果为5× 108TU/mL。
(2)感染方法
按实验需要将细胞铺板。细胞数以第2天密度约30%为宜。37℃培养过夜。感染前,从冰箱取出并在37℃水浴中快速融化病毒,用新鲜完全培养基稀释成所需浓度。吸去细胞原有培养基,将稀释好的病毒液加入细胞中。加入Polybrene(终浓度5ug/mL),轻轻摇匀。37℃培养过夜。感染后第二天,吸去含病毒的培养液,换上新鲜的完全培养液,继续37℃培养。换上含适当浓度的Puromycin的新鲜完全培养液,筛选稳定转染的细胞株。10天至12天后可获得稳定表达目的蛋白的细胞。利用PCR和 Western blot检测细胞过表达Bmi1效果。稳定构建过表达Bmi1的大鼠肝干细胞为WB-F344-Bmi1;WB-F344-NC作为阴性对照组,按照步骤1方法培养及冻存。
S3.稳定过表达细胞WB-F344-Bmi1的选择连续培养法
(1)稳定过表达细胞WB-F344-Bmi1及其对照组WB-F344-NC扩增到 80%-90%覆盖瓶底时开始进行选择连续培养。首先弃去瓶中旧液,用 0.1M PBS清洗2遍,弃去PBS,加入5mL连续选择改良的Richter培养液, 随后放入37℃,5%CO2的细胞培养箱中继续培养。依据培养瓶中旧液的情况决定是否换液,通常间隔48小时-60小时全量换液1次。注意重复以上步骤时:PBS清洗需要轻柔,避免细胞成片脱落,反复给予细胞换液持续4周,此过程中始终不给予0.25%胰蛋白酶/0.04%EDTA消化,保持细胞紧密生长状态。
(2)选择连续培养持续4周结束后,显微镜下观察细胞形态学改变及细胞边界是否清晰,细胞是否形成团块,并拍照记录。首先弃去瓶中旧液,用0.1M PBS清洗1遍,弃去PBS,加入2mL 0.25%胰蛋白酶 /0.04%EDTA消化液,消化3-4分钟,加入3mL连续选择改良的Richter 培养液中和,用吸管吹打至所有细胞脱壁,把细胞悬液转移至15mL离心管中,进行1000rpm室温离心5分钟,弃去上清液,加入5mL连续选择改良的Richter培养液,吹打均匀后等分至4个25cm2培养瓶(即1:4传代),每瓶培养基补充至5mL,然后培养瓶放入37℃,5%CO2的细胞培养箱中。2瓶细胞用于裸鼠的皮下成瘤实验评估其致瘤性,1瓶细胞冻存,1 瓶细胞继续进行选择连续培养。
(3)选择连续培养4周时间为1周期,本次实验持续培养细胞长达10 周期。每一周期的操作如(1),(2)和(4)步骤。
(4)裸鼠动物皮下移植瘤
每一周期选择连续培养后的细胞,需要进行裸鼠皮下注射评价致瘤能力。消化、中和、重悬细胞,细胞计数,离心后弃去上清液,加入0.1M PBS,吹打均匀,调整细胞浓度为5.0×106/mL。给予裸鼠背部两侧皮下注射200ul。每一周期皮下成瘤的裸鼠为3只。观察4周后处死动物,评价皮下肿瘤的成瘤率和肿瘤大小。联合HE染色及免疫组织化学方法检测CK19,AFP,ALB标记的表达以评估肿瘤类型,并评估肿瘤表达的标记是否已稳定。通过每周期的裸鼠皮下成瘤评估及免疫组织化学方法确定肿瘤类型,发现稳定过表达细胞WB-F344-Bmi1的致瘤性逐步增加,第1周期选择连续培养后的细胞,形态学、肿瘤组织标记逐步稳定,表现为低分化肝癌。而WB-F344-NC对照组从第一周期到第10周期始终无法在裸鼠皮下形成肿瘤。
S4.裸鼠原位肝脏移植瘤模型制作
本实验采取第1周期后的WB-F344-Bmi1过表达稳定细胞作为实验细胞。WB-F344-NC作为阴性对照组。
(1)选取BALB/c系雄性裸鼠,4周龄,体重12-15g。10%水合氯醛麻醉动物,70ul/10g。腹腔注射麻醉成功后,固定动物,酒精消毒2次,取腹部正中偏左切口暴露肝脏。暴露左肝脏面,PBS重悬后的肿瘤细胞悬液 70ul,细胞密度1×107个/mL,胰岛素注射器沿肝门方向进针,深度0.5cm。注射后压迫入针部位的肝实质,压迫时间2分钟。观察注射部位的肝实质血运情况,无渗血可立即关腹,消毒皮肤,纱布包扎,电热毯保持动物体温,直至苏醒为止。
(2)造模后3周、4周和4周以上给予药物过量麻醉(10%水合氯醛, 0.1-0.2mL/10g)后联合颈椎脱臼处死动物。病理发现肝脏存在低分化肝癌和胆管癌栓形成。而阴性对照组WB-F344-NC未发现肝内肿瘤,无胆管转移灶形成。
本实施例的实验结果说明如下:
步骤S1实施Fisher 344系大鼠WB-F344成体肝干细胞的扩增及培养
Fisher 344大鼠来源的WB-F344系成体肝卵圆细胞,属于成体肝干细胞,来源于上海东方肝胆医院馈赠。
步骤S2稳定过表达细胞WB-F344-Bmi1构建
(1)大鼠Bmi1基因已成功插入到慢病毒载体中,测序序列正确。Blast 结果比对显示已经测出来的部分序列与PubMed上序列100%一致。
(2)如图1所示,PCR和Western blot证实WB-F344-Bmi1细胞的Bmi1 表达增加。
步骤S3稳定过表达细胞WB-F344-Bmi1的选择连续培养法
(1)本实施例中使用的细胞培养液:
选择连续培养的改良Richter培养液配方为:以改良的Richter培养基 (改良的MEM含Zn)为基础培养基:含L-谷氨酰胺,2mg/IL-脯氨酸及 50ug/mL庆大霉素,不含胰岛素、HEPES及酚红。在前者基础上添加碳酸氢钠、HEPES、胰岛素,胎牛血清。各添加物的终浓度为:2.6mM碳酸氢钠,20mM HEPES,4.0mg/L胰岛素,10%胎牛血清。0.22μm过滤器除菌,4℃保存。细胞冻存液由改良Richter培养液或者DMEM培养基 7mL,胎牛血清2mL和DMSO 1mL组成,使用前现配。
(2)稳定过表达细胞WB-F344-Bmi1的细胞形态变化。
如图2所示,通过慢病毒上调Bmi1后,WB-F344细胞形态发生变化,长梭形细胞包绕融合,形成类似结节,但细胞边界仍清晰,细胞大小均一。无法在裸鼠皮下形成肿瘤。经过选择连续培养方法后,选择连续培养第1周期后WB-F344-Bmi1细胞形态不再变化,呈现为长梭型,长满后呈颗粒状,细胞边界模糊不清,融合形成边界不清晰的结节,少量出现透亮的细胞团块。
(3)裸鼠皮下成瘤及组织学类型变化
每个周期选择连续培养后的细胞,通过裸鼠皮下成瘤评估致瘤性,然后通过免疫组织化学方法确定肿瘤类型,发现WB-F344-Bmi1细胞的裸鼠皮下肿瘤大小随着选择连续培养周期增多而逐步增加,致瘤性为3/3, 100%。选择连续培养1周期后的细胞,形态学逐步稳定,同时皮下成瘤的肿瘤类型为低分化肝癌,稳定表达CK19-,AFP-,ALB+,Bmi1+(图2所示)。而WB-F344-NC对照组始终无法在裸鼠皮下形成肿瘤。
步骤S4裸鼠原位肝脏移植瘤模型
如图3所示,裸鼠原位肝脏移植瘤模型,裸鼠外观皮肤变黄,肝脏MR 检查提示胆管扩张,胆囊增大;处死动物检查肝脏发现:肝内多发病灶形成,胆管癌栓逐渐蔓延至肝外胆管。图4所示肝脏形成肝癌,并胆管癌栓形成。图5A为HE染色显示肝内胆管存在癌栓,且癌细胞浸润包绕胆管;图5B为通过CK19标记胆管上皮细胞。
发生胆管癌栓的判断方法:
1、外形皮肤发黄;
2、肝脏外表黄色,个别肝外胆管存在癌栓,检测外周血胆红素水平显著升高,以直接胆红素为主;
3、病理切片可显示胆管癌栓形成。
本实施例所述的胆管转移肝癌细胞已提交保藏,保藏信息如下:
收到日(保藏日):2019.12.25
培养物名称(分类命名):大鼠恶性转化株C1 WB-F344-Bmi1
保藏编号:CCTCC NO:C202002
保藏单位:中国典型培养物保藏中心
保藏单位地址:湖北省武汉市武昌区八一路299号武汉大学校内 (武汉大学第一附小对面)。
虽然本发明以较佳实施例揭露如上,但并非用以限定本发明实施的范围。任何本领域的普通技术人员,在不脱离本发明的发明范围内,当可作些许的改进,即凡是依照本发明所做的同等改进,应为本发明的范围所涵盖。
Claims (8)
1.一种具有胆管转移特性的肝癌模型构建方法,其包括如下步骤:
S1:对大鼠成体肝干细胞进行扩增、培养并保存;
S2:采用基因转染技术将含有Bmi1基因的慢病毒载体转入大鼠成体肝干细胞,获得稳定过表达Bmi1的大鼠成体肝干细胞;
S3:利用稳定过表达Bmi1的大鼠成体肝干细胞,采用选择连续培养方法进行培养,使其在体外进一步分化,筛选出致瘤能力强及稳定的肝癌细胞;
S4:采用筛选的肝癌细胞,通过动物体内原位肝脏移植瘤模型,可在肝脏形成肝癌且伴胆管癌栓,具有胆管转移特性。
2.如权利要求1所述的方法,其中,步骤S2中,通过基因测序确认所述Bmi1基因插入所述慢病毒载体。
3.如权利要求2所述的方法,其中,步骤S2中,通过PCR和Western blot检测Bmi1过表达效果,建立稳定过表达Bmi1的大鼠成体肝干细胞。
4.如权利要求3所述的方法,其中,步骤S3包括多次重复的选择连续培养方法,每一周期所述选择连续培养内容包括:
a:反复更换细胞培养液,持续时间为4周;
b:细胞培养液采用改良的Richter培养液;
c:每周期培养后需观察细胞形态并记录;
d:每周期培养后将细胞分为三组,一组为用于验证致瘤性的评估组,一组为用于冷冻存样的冻存组,一组为用于下一培养周期的继续培养组。
5.如权利要求4所述的方法,其中,通过观察形态,筛选出形态学上稳定的肿瘤细胞。
6.如权利要求4所述的方法,其中,通过裸鼠皮下成瘤,评估所述评估组细胞的致瘤性,筛选出致瘤能力强的细胞;所述致瘤性包括成瘤率和肿瘤大小。
7.如权利要求4所述的方法,其中,步骤S3中,通过免疫组织化学方法确定裸鼠皮下肿瘤类型为肝癌细胞。
8.一种采用权利要求1-7之一所述方法制备的肝癌细胞。
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