CN117025503A - 肝内胆管前体样细胞、细胞制剂及制备方法和应用 - Google Patents
肝内胆管前体样细胞、细胞制剂及制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种肝内胆管前体样细胞、细胞制剂及制备方法和应用,所述细胞能够在重编程培养基中扩增,所述细胞表面表达的标志物HNF1B、ONECUT1和SOX9中至少一种为阳性,其中,所述重编程培养基含有基础培养基和细胞增殖促进剂;本发明以使肝内胆管前体样细胞能够在体外连续传代以及能维持肝内胆管前体样细胞标志物。
Description
本申请要求2022年05月10日提交的申请号为2022105036786,发明名称为“生物制剂、细胞衍生物及制备方法和应用”的中国专利申请的优先权。上述申请的内容以引用方式被包含于此。
技术领域
本发明涉及生物技术领域,尤其涉及一种肝内胆管前体样细胞、细胞制剂及制备方法和应用。
背景技术
胆石病,胆管肿瘤,胆管梗阻,胆管感染是胆管疾病中的四大主题,通腹腔镜手术等是解决肝内胆管结石的行之有效的方法,手术操作中出现的不可避免的医源性胆管损伤是腹部手术尤其是肝内胆管切除术后的严重并发症,目前发生率有上升的趋势。胆管损伤会导致胆管狭窄,或出现胆漏,进一步会出现腹膜炎、肝功能不全的表现,甚至继发细菌性感染导致中毒性休克,严重者甚至危及生命。硬化性胆管炎同样也会导致肝内胆管的一系列损伤,如不加以控制,有导致胆管癌的可能性。
肝内前体样细胞分布在肝内胆管各处中,能够分化成胆管各种组织的细胞,恢复胆管功能,逆转胆管损伤,是胆管组织修复最重要的细胞。重编程来前体样细胞,来源广泛,是目前研究的热点,提高诱效率和增强功能是需要攻克的技术瓶颈。体外构建胆管前体样细胞培养体系,研究胆管前体样细胞对胆管损伤模型的治疗作用,对于研究胆管疾病的术后恢复等具有及其重要的意义。
目前肝内胆管前体样细胞已经被证明了,具有分化为膜层细胞和肌层细胞的功能,肝内胆管前体样细胞体外培养的来源为三种,1,成年肝内胆管;2,ESCs/iPSCs分化;3,肝内胆管细胞重编程;但是无论哪种方式来源的胆管前体样细胞,都存在体外培养困难,不能连续传代以及不能维持前体样细胞标志物的问题。为此亟需一种能够连续传代以及能维持肝内胆管前体样细胞标志物的肝内胆管前体样细胞。
发明内容
本发明的目的在于提供一种肝内胆管前体样细胞、细胞制剂及制备方法和应用,以使肝内胆管前体样细胞能够在体外连续传代以及能维持肝内胆管前体样细胞标志物。
为实现上述目的,本发明提供了一种肝内胆管前体样细胞,所述细胞能够在重编程培养基中扩增,所述细胞表面表达的标志物HNF1B、ONECUT1和SOX9中至少一种为阳性,其中,所述重编程培养基含有基础培养基和细胞增殖促进剂。
优选的,所述肝内胆管前体样细胞不表达MHC二类分子。
可选的,所述细胞来源于肝内胆管、肝内胆管小管上皮细胞、鲍曼囊和胚肝内胆管中的至少一种。
可选的,所述重编程培养基还包含无血清添加物、生长因子、TGF-β信号抑制剂、Wnt信号通路激动剂和ROCK激酶抑制剂。
优选的,所述无血清添加物包含N2和B27。以占所述基础培养基的含量计,所述N2的含量为0.5-4%;所述B27的含量为1-6%。
优选的,所述TGF-β信号抑制剂为A-8301,以占所述基础培养基的含量计,所述A-8301的含量为0.5-4μM。
优选的,所述ROCK激酶抑制剂为Y-27632,以占所述基础培养基的含量计,所述Y-27632的含量为7-14μM。
优选的,所述生长因子包含上皮细胞生长因子EGF和成纤维细胞生长因子bFGF,以占所述基础培养基的含量计,所述上皮细胞生长因子EGF的含量为15-30ng/mL。所述成纤维细胞生长因子bFGF的含量为45-65ng/mL。
可选的,所述细胞增殖促进剂为Netarsudil。优选地,以占所述基础培养基的含量计,所述Netarsudil的含量为1-4μM。
可选的,所述肝内胆管前体样细胞,在制备用于通过同种异体移植在人患者中提供保护和修复肝内胆管的急性损伤试剂中的用途。
本发明还提供了一种肝内胆管前体样细胞制剂,包括表达的标志物HNF1B、ONECUT1和SOX9中至少一种为阳性的肝内胆管前体样细胞,以及药学上可接受的载体。
可选的,所述载体包括生理盐水。
本发明还提供了一种肝内胆管前体样细胞制剂的应用,使用如所述的肝内胆管前体样细胞制剂干预体内动物模型,所述动物模型包括急性肝内胆管损伤模型。
本发明还提供了一种肝内胆管前体样细胞的制备方法,包括如下步骤:
S0:提供肝内胆管原代细胞;
S1:对所述S0中的肝内胆管原代细胞使用重编程培养基进行退分化培养以获得表达标志物HNF1B、ONECUT1和SOX9中至少一种为阳性的肝内胆管前体样细胞。
可选的,还包括如下步骤:
S2:对所述肝内胆管前体样细胞在所述重编程培养基中进行扩增培养,直至肝内胆管前体样细胞中的阳性标志物HNF1B、ONECUT1和SOX9中至少一种的表达率为50-99%之间后,使用胰酶消化液对所述肝内胆管前体样细胞进行消化处理,以得到初代肝内胆管前体样细胞。
本发明的有益效果在于:由于本发明中采用的重编程培养基内含有基础培养基和细胞增殖促进剂,因此肝内胆管前体样细胞在体外进行扩增时能够维持表达肝内胆管前体样细胞阳性标志物,不表达MHC二类分子,因此在移植肝内胆管前体样细胞后不需要服用抗排药,可以减少对人身体的伤害;同时,重编程培养基中含有的基础培养基和细胞增殖促进剂能够使得肝内胆管前体样细胞能够连续传代。
附图说明
图1为本发明提供的第二代肝内胆管前体样细胞的光学显微镜下的形态示意图;
图2为本发明提供的***肝内胆管前体样细胞的光学显微镜下的形态示意图;
图3为本发明提供的四种不同供体的肝内胆管前体样细胞的生长曲线图;
图4为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD34的情况示意图;
图5为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD45的情况示意图;
图6为本发明实施例的肝内胆管前体样细胞的基因表达标志物HLA-DR/DP/DQ的情况示意图;
图7为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD24的情况示意图;
图8为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD326的情况示意图;
图9为本发明实施例的肝内胆管前体样细胞的基因表达标志物SOX9的情况示意图;
图10为本发明实施例的肝内胆管前体样细胞的基因表达标志物CK19的情况示意图;
图11为本发明实施例的肝内胆管前体样细胞的基因表达标志物HNF1B的情况示意图;
图12为本发明实施例的肝内胆管前体样细胞的基因表达标志物ONECUT1的情况示意图;
图13为健康对照组、模型对照组和细胞治疗组中得到的细胞分别进行DAPI荧光染色后得到的免疫荧光照片;
图14为健康对照组、模型对照组和细胞治疗组中得到的细胞分别进行HE染色和天狼星红染色后得到的免疫荧光照片。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。除非另外定义,此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同,而不排除其他元件或者物件。
胆石病,胆管肿瘤,胆管梗阻,胆管感染是胆管疾病中的四大主题,我国胆石病的主要特点是胆管结石,胆管结石约占全部胆石病例的50%,肝内胆管结石的相对发病率约为38%,而通腹腔镜手术等是解决肝内胆管结石的行之有效的方法,手术操作中出现的不可避免的医源性胆管损伤是腹部手术尤其是肝内胆管切除术后的严重并发症,目前发生率有上升的趋势。胆管损伤会导致胆管狭窄,或出现胆漏,进一步会出现腹膜炎、肝功能不全的表现,甚至继发细菌性感染导致中毒性休克,严重者甚至危及生命。硬化性胆管炎同样也会导致肝内胆管的一系列损伤,如不加以控制,有导致胆管癌的可能性。
肝内前体样细胞分布在肝内胆管各处中,能够分化成胆管各种组织的细胞,恢复胆管功能,逆转胆管损伤,是胆管组织修复最重要的细胞。前体样细胞,来源广泛,是目前研究的热点,提高诱效率和增强功能是需要攻克的技术瓶颈。体外构建胆管前体样细胞培养体系,研究胆管前体样细胞对胆管损伤模型的治疗作用,对于研究胆管疾病的术后恢复等具有及其重要的意义。
目前肝内胆管前体样细胞已经被证明了,具有分化为膜层细胞和肌层细胞的功能,肝内胆管前体样细胞体外培养的来源为三种,1,成年肝内胆管;2,ESCs/iPSCs分化;3,肝内胆管细胞重编程;但是无论哪种方式来源的胆管前体样细胞,都存在体外培养困难,不能连续传代以及不能维持前体样细胞标志物的问题。应用前体样细胞或者干细胞尤其是来源于成年胆管的前体样细胞以修复受损胆管组织,是治疗胆管损伤颇具潜力的新型疗法。
肝内胆管前体样细胞体外培养的来源主要为以下几种方式:成年肝内胆管和胚肝内胆管:成人肝内胆管的不同位置均有肝内胆管前体样细胞的存在,包括近端肝内胆管小管上皮细胞、鲍曼囊等,胚肝内胆管组织中也大量存在肝内胆管前体样细胞。成人肝内胆管和胚肝内胆管,均可以通过肝内胆管脏组织中分离并且筛选的方法得到肝内胆管前体样细胞。获得的肝内胆管前体样细胞可以通过克隆性试验、使用细胞表面标记的流式细胞术等进行鉴定;
ESCs/iPSCs分化:ESCs是来自哺乳动物受精卵的多能干细胞,而iPSCs是通过重编程从体细胞转导的,均可通过诱导分化的方式(Yamanaka factors OCT3/4,SOX2,KLF4 andc-MYC),得到肝内胆管前体样细胞,使用肝内胆管脏发育研究中发现的关键转录因子作为分化的标记,如肝细胞核因子-1-β[HNF1B]、一切同源框1[ONECUT1]和SOX9;3,肝内胆管细胞重编程:成熟肝内胆管细胞可通细胞重编程的方式,通过6个转录因子的表达中(SIX1,SIX2,OSR1,EYA1,HOXA11和SNAI2)导致了与肝内胆管元祖细胞一致的基因网络的激活,得到肝内胆管前体样细胞。
肝内胆管细胞重编程:成熟肝内胆管细胞可通细胞重编程的方式,通过6个转录因子的表达中(SIX1,SIX2,OSR1,EYA1,HOXA11和SNAI2)导致了与肝内胆管元祖细胞一致的基因网络的激活,得到肝内胆管前体样细胞。
胚肝内胆管及成年肝内胆管组织分离得到肝内胆管前体样细胞,或者成熟肝内胆管细胞重编程,都需要足够的起始细胞,细胞连续传代能力有限,应用于治疗难度比较大。
而且通过血液***回输的肝内胆管脏细胞不能定植,肝内胆管包膜下注射需要开腹手术,胆管壁较薄,操作起来有一定难度,注射的干细胞很容易流失。需要通过手术切除部分肝内胆管脏组织后,注射肝内胆管前体样细胞内,才能够定植于肝内胆管组织中。
而目前胚胎干细胞或iPS细胞治疗肝内胆管脏疾病报道较少,主要是因为干细胞体外诱导分化为肝内胆管脏细胞的培养方案仍不成熟,缺乏统一规范的标准。经动脉或静脉注射的方式,能到达并留存在肝内胆管脏中的干细胞比例很低,而且有发展成畸胎瘤的风险。
针对现有技术存在的问题,本发明的实施例提供了一种肝内胆管前体样细胞,所述细胞能够在重编程培养基中扩增,所述细胞表面表达的标志物HNF1B、ONECUT1和SOX9中至少一种为阳性,其中,所述重编程培养基含有基础培养基和细胞增殖促进剂。
本发明一些实施例,所述细胞来源于肝内胆管、肝内胆管小管上皮细胞、鲍曼囊和胚肝内胆管中的至少一种。
本发明一些实施例,所述重编程培养基还含有无血清添加物、生长因子、TGF-β信号抑制剂、Wnt信号通路激动剂和ROCK激酶抑制剂。
本发明一些实施例,所述细胞增殖促进剂为Netarsudil。所述Netarsudil的设置,能够抑制ROCK1和ROCK2的信号通路,抑制细胞凋亡,促进细胞的贴壁生长。维持能够促进胆管前体样细胞的增殖。以占所述基础培养基的含量计,所述Netarsudil的含量为1-4μM,优选地,当所述Netarsudil的含量为1.5-2.2μM时达到最好的促进增殖的效果。其中,ROCK1和ROCK2为ROCK的两种同源异构体,ROCK是Rho分子下游靶效应分子。ROCK1和ROCK2具有一系列重要的生物学功能,可以在细胞调控过程中起“开关”作用并参与调控细胞增殖与分化、诱导细胞骨架重组、影响血管和组织通透性和应力纤维形成等,越来越多的研究表明Rho/ROCK信号通路可调控多种组织细胞的生理功能包括通过调控细胞增殖、上皮分化、细胞迁移和细胞黏附。
本发明一些实施例,所述生长因子包含上皮细胞生长因子EGF和成纤维细胞生长因子bFGF,以占所述基础培养基的含量计,所述上皮细胞生长因子EGF的含量为15-30ng/mL。所述成纤维细胞生长因子bFGF的含量为45-65ng/mL。
本发明的一些具体实施例,所述表皮生长因子(英文名称为Epidermal growthfactor,英文简称为EGF)来源于近岸生物,所述碱性成纤维生长因子(英文名称为basicfibrobast growth factor,英文简称为bFGF)来源于近岸生物。
本发明一些实施例中,所述肝内胆管前体样细胞不表达免疫排斥反应相关的MHC二类分子,即不表达蛋白HLA-DR/DP/DQ,由于肝内胆管前体样细胞表面不表达于免疫排斥反应相关的蛋白HLA-DR/DP/DQ,因此可以实现肝内胆管前体样细胞的异体回输,以临床上不能用于移植的捐献肝组织,取肝内胆管组织消化后得到的原代肝内胆管细胞作为种子细胞,使用重编程培养基进行体外培养,获得阳性表达HNF1B、ONECUT1和SOX9中至少一种,表达率具有上皮前体样细胞特性的肝内胆管前体样细胞,并且进行体外扩增建立细胞种子库。在用于治疗医源性胆管损伤时,可通过经肝内胆管动脉插管,直接回输肝内胆管前体样细胞,也可对肝内胆管前体样细胞进行分化培养成熟为肝内胆管上皮细胞,门静脉插管回输。
本发明一些实施例,所述肝内胆管前体样细胞在制备用于通过同种异体移植在人患者中提供保护和修复肝内胆管的急性损伤试剂中的用途。
本发明还提供了一种肝内胆管前体样细胞制剂,包括如所述的表达的标志物HNF1B、ONECUT1和SOX9中至少一种为阳性的肝内胆管前体样细胞,以及药学上可接受的载体。
本发明一些实施例,所述载体包括生理盐水。
本发明还提供了一种肝内胆管前体样细胞的制备方法,包括如下步骤:
S0:提供肝内胆管原代细胞;
其中,肝内胆管原代细胞的获得步骤如下:
S01:取成熟的肝内胆管组织;优选地,所述成熟的肝内胆管组织来源于4例肝切除病人、2例原发性胜汁性肝硬化或1例自体免疫性肝炎。
S02:将约30g新鲜肝组织切成1-2mm3大小碎片;用1mg/mL I型胶原酶在37℃消化45~90min,然后将消化液通过滤网;离心速度为600g,离心时间为10min收集细胞后用PBS清洗,获得细胞悬液;将细胞悬液置于等体积、密度分别为1104g/mL和1109g/mL的Percoll溶液内,离心速度为800g,离心时间为30min,收集下一界面细胞,然后使用PBS对下一界面细胞清洗;接着用15mLPBS溶液作用于清洗后的下一界面细胞悬浮细胞,加入215ugHBA125(一抗,鼠抗人),37C孵育30min;PBS清洗,细胞悬浮于含15mLPBS溶液的试管中,加入10μL磁株标记的羊抗鼠免疫球蛋白(一抗,4×108磁株/mL),4℃孵育30min;将试管放入磁株浓缩器内,2min后吸除液体,收集贴附在试管壁上的细胞,以得到肝内胆管原代细胞。其中,所述I型胶原酶来源于溶组织梭菌。(一抗,鼠抗人),可以理解为,人对于鼠IGG来说是异物,所以当鼠IGG接触人就会产生相应的抗体,称为鼠抗人IgG抗体,又由于自然存在的抗原大都存在多个抗原表位,会刺激机体产生多种针对同一抗原的不同抗原表位相应的不同抗体。(一抗,4×108磁株/mL),可以理解为第一抗体就是能和非抗体性抗原(特异性抗原)特异性结合的蛋白。
优选地,将得到的所述肝内胆管原代细胞接种在组织培养板上,并经过流式细胞仪检测胆管细胞角质素19(CK19),以对得到的所述肝内胆管原代细胞进行验证。
S1:对所述S0中的肝内胆管原代细胞使用重编程培养基进行退分化培养以获得表达标志物HNF1B、ONECUT1和SOX9中至少一种为阳性的肝内胆管前体样细胞。
本发明一些实施例,还包括如下步骤:
S2:对所述肝内胆管前体样细胞在所述重编程培养基中进行扩增培养,直至肝内胆管前体样细胞中的阳性标志物HNF1B、ONECUT1和SOX9中至少一种的表达率为50-99%之间后,使用胰酶消化液对所述肝内胆管前体样细胞进行消化处理,以得到初代肝内胆管前体样细胞。其中,所述胰酶消化液组成及来源于猪胰腺。
以下通过具体的实施例进行详细说明:
实施例
一、起始组织性质以及来源合法性声明:
以不能用于移植的正常肝内胆管组织作为起始原料获取阳性表达HNF1B/ONECUT1/SOX9的人肝内胆管前体样细胞。
具体的,所述肝内胆管组织经病理检查显示为正常肝内胆管组织。
具体的,所述不能用于移植的正常肝内胆管组织为来源于年龄不超过70岁的患者的手术样本,患者经医学检查无传染性病毒感染,患者在术前6个月内未使用过类固醇激素药物。患者在术前对手术样本的获取目的充分知情,并签署了知情同意书。
二、肝内胆管原代细胞的获取(胆管细胞分离)
将约30g新鲜肝组织切成1-2mm3大小碎片;
用1mg/mL I型胶原酶在37℃消化45~90min,然后将消化液通过滤网;
离心速度为600g,离心时间为10min,收集细胞后用PBS清洗,获得细胞悬液;
将细胞悬液置于等体积、密度分别为1104g/mL和1109g/mL的Percoll溶液内,离心速度为800g,离心时间为30min,收集下一界面细胞,然后使用PBS对下一界面细胞清洗;
接着用15mLPBS溶液作用于清洗后的下一界面细胞悬浮细胞,加入215ugHBA125(一抗,鼠抗人),37C孵育30min;
PBS清洗,细胞悬浮于含15mLPBS溶液的试管中,加入10μL磁株标记的羊抗鼠免疫球蛋白(一抗,4×108磁株/mL),4℃孵育30min;
将试管放入磁株浓缩器内,2min后吸除液体,收集贴附在试管壁上的细胞,以得到肝内胆管原代细胞。
三、肝内胆管前体样细胞体外培养
重编程培养基组分包括:以占重编程培养基的体积计,基础培养基DMEM/F12(来源于武汉普诺赛生命科技有限公司),含量为20ng/mL的上皮细胞生长因子EGF,含量为50ng/mL的碱性成纤维细胞生长因子bFGF,含量为0.5-4%的营养补充剂N2(1X),含量为1-6%的B27营养补充剂(1X),含量为7-14μM的ROCK激酶抑制剂Y-27632,含量为3μM的Wnt信号通路激动剂CHIR99021,含量为0.5-4μM的TGF-β信号抑制剂A8301,含量为1.5-22μM的促进胆管前体样细胞的增殖的Netarsudil。
将肝内胆管原代细胞以10000个/平方厘米的接种面积置于6孔板中,每孔加2毫升重编程培养基进行退分化培养,直至肝内胆管前体样细胞的融合度不低于80%后,使用胰酶消化液对肝内胆管前体样细胞进行1-5分钟的消化后,再继续使用重编程培养基进行扩增培养,扩增培养又可称为传代培养,肝内胆管前体样细胞可持续传代,使用重编程培养基对肝内胆管前体样细胞进行传代培养以得到***(P4)肝内胆管前体样细胞。
本申请还提供了肝内胆管前体样细胞在光学显微镜下的形态示意图,具体地,可参见图1和图2,其中,图1为本发明提供的第二代肝内胆管前体样细胞的光学显微镜下的形态示意图;图2为本发明提供的***肝内胆管前体样细胞的光学显微镜下的形态示意图。从图中可以看出,图2中的肝内胆管前体样细胞数量明显多于图1中的肝内胆管前体样细胞,可见通过该实施例中的重编程培养基能够体外扩增肝内胆管前体样细胞。
四、肝内胆管前体样细胞生长曲线图
取供体1的肝内胆管组织、取供体2的肝内胆管组织、取供体3的肝内胆管组织和取供体4的肝内胆管组织,并分别经过胆管细胞分离处理得到肝内胆管原代细胞,所述肝内胆管原代细胞重编程培养基进行退分化培养,直至肝内胆管前体样细胞的融合度不低于80%后,使用胰酶消化液对肝内胆管前体样细胞进行1-5分钟的消化后,再继续使用重编程培养基进行扩增培养以得到***(P4)肝内胆管前体样细胞,本实施例中四种供体采用重编程培养基扩增培养得到的肝内胆管前体样细胞生长曲线见图3,具体地,图3为本发明提供的四种不同供体的肝内胆管前体样细胞的生长曲线图。从图3中可以看出,采用该实施例中的重编程培养基培养扩增肝内胆管前体样细胞,随着扩增代数的增加,肝内胆管前体样细胞的数量也会逐渐增加,因此采用该重编程培养基对肝内胆管前体样细胞的扩增不会受到供体肝内胆管组织的来源影响。
其中,所述供体1、供体2、供体3和供体4的肝内胆管组织来源于4例肝切除病人。
五、对肝内胆管前体样细胞进行流式检测
图4为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD34的情况示意图;图5为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD45的情况示意图;图6为本发明实施例的肝内胆管前体样细胞的基因表达标志物HLA-DR/DP/DQ的情况示意图;图7为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD24的情况示意图;图8为本发明实施例的肝内胆管前体样细胞的基因表达标志物CD326的情况示意图;图9为本发明实施例的肝内胆管前体样细胞的基因表达标志物SOX9的情况示意图;图10为本发明实施例的肝内胆管前体样细胞的基因表达标志物CK19的情况示意图;图11为本发明实施例的肝内胆管前体样细胞的基因表达标志物HNF1B的情况示意图;图12为本发明实施例的肝内胆管前体样细胞的基因表达标志物ONECUT1的情况示意图。
使用重编程培养基对肝内胆管前体样细胞进行传代培养以得到***(P4)肝内胆管前体样细胞后,吸弃重编程培养基后用无菌PBS缓冲液润洗,然后用胰酶消化液对***胃肝内胆管前体样细胞进行消化处理后再进行离心处理,其中,离心处理的转速为200g,离心处理的时间为5分钟,离心处理结束后收集细胞沉淀物;向细胞沉淀物中加入100μL染色缓冲液重悬细胞至流式管中,再分别加入5μL待测流式抗体孵育20分钟后每管用400μL染色缓冲液重悬后进行表面抗体流式检测,流式检测结果见图4、图5、图6、图7、图8、图9、图10、图11和图12。
参照图4、图5、图6,标志物CD34的阴性峰的阳性率为0.75%,标志物CD45的阳性峰的阴性率为0.62%,标志物HLA-DR/DP/DQ的阴性峰的阳性率为2.34%,阴性峰的阴性率大于90%说明肝内胆管前体样细胞阴性表达该标志物,因此,本发明的胃粘膜上皮前体样细胞阴性表达CD34、CD45和HLA-DR/DP/DQ。图7、图8、图9、图10、图11和图12,标志物CD24的阳性峰的阳性率为86%,标志物CD326的阳性峰的阳性率为96.4%,标志物SOX9的阳性峰的阳性率为80.6%,标志物CK19的阳性峰的阳性率为91%,标志物HNF1B的阳性峰的阳性率为85.2%,标志物ONECUT1的阳性峰的阳性率为96.7%,阳性峰的阳性率介于50-99%说明肝内胆管前体样细胞阳性表达该标志物,因此,本发明的肝内胆管前体样细胞阳性表达CD24、CD326、SOX9、CK19、HNF1B和ONECUT1。
六、肝内胆管前体样细胞制剂的制备
将若干基质胶和肝内胆管前体样细胞的混合物从液氮罐中取出,然后放置在干冰上转运,并将混合物立即没于37℃水浴锅中,直至混合物完全融化至液体,得到混合液,摇晃水浴锅使混合液再水浴锅中分布均匀。将全部混合液用移液枪取出,加入50mL无菌离心管中,将离心管置于离心机中,以200g的转速离心处理5分钟。离心处理结束后,弃去离心管中的上清液,得到混合液沉淀,用生理盐水将混合液沉淀稀释至浓度为5×106/mL的肝内胆管前体样细胞溶液,然后再使用70μm的细胞滤网对肝内胆管前体样细胞溶液进行过滤处理得到肝内胆管前体样细胞的细胞制剂。肝内胆管前体样细胞制剂在动物适应症模型中的应用,并进行效果论证:在进行细胞治疗后,胆管增生明显消失,纤维化进程逆转,由此可见肝内胆管前体细胞对治疗急性肝内胆管损伤具有较为显著的效果。
本实施例提供了小鼠急性肝内胆管损伤模型的建模方法,并使用肝内胆管前体样细胞对小鼠急性肝内胆管损伤模型进行干预,考察肝内胆管前体样细胞对急性肝内胆管损伤的作用。
1、本实施例使用购自北京维通利华实验动物技术有限公司的健康雄性六至八周龄野生型(WT)C57BL/6小鼠进行建模。小鼠的体重平均达到18-20g。每组4只小鼠,分组情况如下:
组别 | 动物数 | 性别 | 供试品 | 剂量 | 给药途径 | 频次 |
造模组 | 4 | 雄性 | 生理盐水 | 0.2mL/只 | 肝内胆管 | 单次 |
治疗组 | 4 | 雄性 | 细胞制剂 | 1×106/只 | 肝内胆管 | 单次 |
2、小鼠急性肝内胆管损伤模型建立:在玉米油中稀释的CCl4浓度为0.7μL/g体重,但应在1至3天后处死小鼠。保证注射溶液处于室温(CCl4/玉米油);
注入恒定体积的50μL,其中含有CCl4在玉米油中的溶液。因此,用玉米油补足所需的CCl4体积(0.5-0.7μL/g小鼠体重)可达到50μL的总体积。例如,将溶于40μL玉米油中的10μLCCl4注入到玉米油中。体重为20g的小鼠,最终剂量为0.5μL/g体重。注射后一小时应检查动物是否异常,此后每24小时检查一次评估造模结果。健康对照组,随机选取四只健康小鼠不做任何处理。
模型对照组
随机选取四只小鼠,其中随机挑选3只小鼠用于造模,剩余1只小鼠作为正常对照。将放回笼盒饲养7天后的健康对照组、模型对照组的小鼠统一处死,具体操作步骤包括:从小鼠的尾静脉注射舒泰50,将小鼠深度麻醉后从小鼠腹主动脉放血,最后小鼠颈椎脱臼处死。其中,舒泰50购自维克,货号为785T。
将健康对照组、模型对照组的小鼠剖腹摘取肝脏,对健康对照组、模型对照组作肉眼和显微观察,并进行拍照,拍照和理化检测,造模组的小鼠示肝小叶结构紊乱,大量坏死的肝上皮细胞空泡样变,细胞核固缩、溶解,炎性细胞浸润,轻度脂肪变性,Kupffer细胞聚集,融合形成多核巨细胞,吞噬坏死细胞表征显示胆管纤维化严重,增生明显;但治疗组的小鼠肝内胆管纤维化较轻,胆管损伤相对较小,上皮无明显增生。
细胞治疗组:在24和48小时后肝内胆管前体样细胞注射3E+06 50μL+30μL基质胶两侧叶各注射50μL,建模同时注射一次,建模后1周,再次注射3E+06 50μL+30μL基质胶两侧叶各注射50μL。通过颈椎脱臼法处死动物后收集组织。在CCl4诱导2和4周后进行调查,而在诱导6周后对照组可以观察到严重的纤维化和胆管增生。
3、建模后的动物实现结果
(1)对细胞核DAPI染色
A.用1mL的ddH2O将DAPI进行溶解,制成为:2.9mM的DAPI溶液(1mg DAPI:1mL H2O)。
B.取适量DAPI水溶液加到PBS缓冲液中,制成50μM的DAPI溶液,推荐采用:PBS配制方法:使用8.00g,NaCl;0.20g KCl;2.9g Na2HPO4·12H2O以及0.2g KH2PO4溶解于1L纯水中。
C.将1/10体积培养基的DAPI溶液加入细胞培养基中,也可以采用1/10浓度的DAPI缓冲液替代培养基使用。
D.在37℃恒温条件下培养细胞约10~20分钟之间。
E.用PBS或者合适的缓冲液洗两次细胞。
F.用带有360nm激发波长和460nm发射波长的滤光片的荧光显微镜进行观察细胞。
图13为健康对照组、模型对照组和细胞治疗组中得到的细胞分别进行DAPI荧光染色后得到的免疫荧光照片。
如图13所示,模型对照组中的胆管增生明显,纤维化严重,而细胞治疗组在进行细胞治疗后,胆管增生明显消失,纤维化进程逆转,由此可见肝内胆管前体样细胞对治疗急性肝内胆管损伤具有较为显著的效果。
(2)对细胞进行HE染色和天狼星红染色
HE染色
A.石蜡切片脱蜡至水
依次将切片放入二甲苯Ⅰ10min,二甲苯Ⅱ10min,无水乙醇Ⅰ5min,无水乙醇Ⅱ5min,95%酒精5min,90%酒精5min,80%酒精5min,70%酒精5min,蒸馏水洗。
B.苏木素染细胞核
切片入Harris苏木素染3-8min,自来水洗,1%的盐酸酒精分化数秒,自来水冲洗,0.6%氨水返蓝,流水冲洗。
C.伊红染细胞质
切片入伊红染液中染色1-3min。
D.脱水封片
将切片依次放入95%酒精I 5min,95%酒精II 5min,无水乙醇Ⅰ5min,无水乙醇Ⅱ5min,二甲苯Ⅰ5min,二甲苯Ⅱ5min中脱水透明,将切片从二甲苯拿出来稍晾干,中性树胶封片。
E.显微镜镜检,图像采集分析
天狼星红染色
1)石蜡切片常规脱蜡至水。
2)天狼猩红染液滴染(浸染)8min,流水冲洗掉多余染液。
3)Mayer苏木素染液滴染(浸染)1分钟,流水冲洗掉多余染液。
4)切片经3缸无水乙醇脱水,二甲苯透明后,滴加中性树胶封片剂封片。
5)光学显微镜下观察。
图14为健康对照组、模型对照组和细胞治疗组中得到的细胞分别进行HE染色和天狼星红染色后得到的免疫荧光照片。
如图14所示,模型对照组中的急性炎症病变,胆管细胞变性、坏死,炎性细胞浸润,伴有明显肝细胞再生。小叶内许多细胞呈水样变性或气球样变性,使细胞肿大,胞浆疏松,淡染;其间常夹杂呈嗜酸性变的固缩肝细胞,并有凋亡小体形成散在的点、灶状坏死胆管增生明显,纤维化严重,而细胞治疗组细胞核大而圆,居中,异染色质少而着色浅,能清晰看到核仁。细胞胞质丰富,胞质内出现散在的嗜碱性物质。可知在进行细胞治疗后,胆管增生明显消失,纤维化进程逆转,由此可见肝内胆管前体样细胞对治疗急性肝内胆管损伤具有较为显著的效果。
七、肝内胆管前体样细胞分别通过重编程培养基和基础培养基培养,并对结果分析
1、扩增培养
重编程培养基培养组:将肝内胆管原代细胞以10000个/平方厘米的接种面积置于6孔板中,每孔加2毫升重编程培养基进行退分化培养,直至肝内胆管前体样细胞的融合度不低于80%后,使用胰酶消化液对肝内胆管前体样细胞进行1-5分钟的消化后,再继续使用重编程培养基进行扩增培养,扩增培养又可称为传代培养,肝内胆管前体样细胞可持续传代,使用重编程培养基对肝内胆管前体样细胞进行传代培养以得到***(P4)肝内胆管前体样细胞。
基础培养基培养组:将肝内胆管原代细胞以10000个/平方厘米的接种面积置于6孔板中,每孔加2毫升基础培养基进行退分化培养,直至肝内胆管前体样细胞的融合度不低于80%后,使用胰酶消化液对肝内胆管前体样细胞进行1-5分钟的消化后,再继续使用基础培养基进行扩增培养,扩增培养又可称为传代培养,肝内胆管前体样细胞可持续传代,使用基础培养基对肝内胆管前体样细胞进行传代培养以得到***(P4)肝内胆管前体样细胞。
3、扩增到***肝内胆管前体样细胞的数量
代数 | P1 | P2 | P3 | P4 |
重编程培养基 | 2.24E+05 | 3.36E+06 | 4.47E+06 | 6.65E+06 |
基础培养基 | 2.44E+05 | 3.34E+05 | 3.44E+05 | 3.21E+05 |
3、结果分析
从上述表格可以看出,基础培养基中的肝内胆管前体样细胞的数量少于重编程培养基中的肝内胆管前体样细胞的数量,具体地,基础培养基中的肝内胆管前体样细胞在第二代往后增殖代谢停滞,而重编程培养基随着扩增代数的增加肝内胆管前体样细胞迅速增殖,可见,重编程培养基中含有的Netarsudil,能够抑制ROCK1和ROCK2的信号通路,抑制细胞凋亡,促进细胞的贴壁生长,能够促进胆管前体样细胞的增殖。
细胞治疗目前已经被应用于多种人类疾病包括:胰岛细胞移植、自体软骨细胞软骨修复、B细胞治疗***性红斑狼疮、造血干细胞移植治疗癌症等。而通过对肝内胆管前体样细胞的培养和应用,通过细胞制剂或者细胞分泌因子对病变肝内胆管脏组织进行结构和功能修复、组织细胞再生的新疗法给肝内胆管脏损伤带来了新的希望。
中华医学会外科学分会胆道外科学组综合胆管损伤的解剖部位、致伤因素、病变特征和防治策略等多个方面,将胆管损伤分为3型4类。
依据损伤的部位分为3型:
1、Ⅰ型损伤,胰十二指肠区胆管损伤:根据胆管损伤部位以及是否合并胰腺和(或)十二指肠损伤可分为3个亚型。Ⅰ型,远段胆管单纯损伤;Ⅰ型,远段胆管损伤合并胰腺和/或十二指肠损伤;Ⅰ型,胆胰肠结合部损伤。
2、Ⅱ型损伤,肝外胆管损伤:指位于肝脏和胰十二指肠之间的肝外胆管损伤。依据损伤的解剖平面将Ⅱ型损伤分为4个亚型。Ⅱ1型,汇合部以下至十二指肠上缘的肝外胆管损伤;Ⅱ2型,左右肝管汇合部损伤;Ⅱ3型,一级肝管损伤(左和/或右肝管);Ⅱ4型,二级肝管损伤。
3、Ⅲ型损伤,肝内胆管损伤:指三级和三级以上肝管的损伤,包括在肝实质外异位汇入肝外胆管的副肝管和变异的三级肝管损伤以及来源于胆囊床的迷走肝管损伤。
依据胆管损伤的病变特征将其分为4类,即:
a类:非破裂伤,胆道管壁保持完整的损伤,包括胆管挫伤以及因缝扎、钛夹夹闭或其他原因造成的原发性损伤性胆管狭窄。
b类:裂伤。
c类:组织缺损。
d类:瘢痕性狭窄,指胆管损伤后因管壁纤维化而形成的继发性胆管狭窄。
实验证明,肝内胆管前体样细胞可以有效治疗I型和II型的胆管损伤对III型的胆管损伤也有一定的治疗效果
根据对肝内胆管前体样细胞培养后的表面标志物检测,发现该肝内胆管前体样细胞标志物为阳性表达CK19的细胞,并且在体外培养时外观呈现典型的上皮前体样细胞特征,在进行动物实验时,慢性肝内胆管纤维化模型细胞回输治疗后,可以显著降低肝内胆管脏纤维化的程度。并且,在急性肝内胆管损伤模型中,细胞回输后可以使胆管表面创面显著恢复。
虽然在上文中详细说明了本发明的实施方式,但是对于本领域的技术人员来说显而易见的是,能够对这些实施方式进行各种修改和变化。但是,应理解,这种修改和变化都属于权利要求书中所述的本发明的范围和精神之内。而且,在此说明的本发明可有其它的实施方式,并且可通过多种方式实施或实现。
Claims (10)
1.一种肝内胆管前体样细胞,其特征在于,所述细胞能够在重编程培养基中扩增,所述细胞表面表达的标志物HNF1B、ONECUT1和SOX9中至少一种为阳性,其中,所述重编程培养基含有基础培养基和细胞增殖促进剂。
2.根据权利要求1所述的肝内胆管前体样细胞,其特征在于,所述细胞来源于肝内胆管、肝内胆管小管上皮细胞、鲍曼囊和胚肝内胆管中的至少一种。
3.根据权利要求1所述的肝内胆管前体样细胞,其特征在于,所述重编程培养基还含有无血清添加物、生长因子、TGF-β信号抑制剂、Wnt信号通路激动剂和ROCK激酶抑制剂。
4.根据权利要求1所述的肝内胆管前体样细胞,其特征在于,所述细胞增殖促进剂为Netarsudil。
5.根据权利要求1所述的肝内胆管前体样细胞,其特征在于,所述肝内胆管前体样细胞,在制备用于通过同种异体移植在人患者中提供保护和修复肝内胆管的急性损伤试剂中的用途。
6.一种肝内胆管前体样细胞制剂,其特征在于,包括如权利要求1所述的表达的标志物HNF1B、ONECUT1和SOX9中至少一种为阳性的肝内胆管前体样细胞,以及药学上可接受的载体。
7.根据权利要求6所述的肝内胆管前体样细胞制剂,其特征在于,所述载体包括生理盐水。
8.一种肝内胆管前体样细胞制剂的应用,其特征在于,使用如权利要求7中所述的肝内胆管前体样细胞制剂干预体内动物模型,所述动物模型包括急性肝内胆管损伤模型。
9.一种肝内胆管前体样细胞的制备方法,其特征在于,包括如下步骤:
S0:提供肝内胆管原代细胞;
S1:对所述S0中的肝内胆管原代细胞使用重编程培养基进行退分化培养以获得表达标志物HNF1B、ONECUT1和SOX9中至少一种为阳性的肝内胆管前体样细胞。
10.根据权利要求9所述的肝内胆管前体样细胞的制备方法,其特征在于,还包括如下步骤:
S2:对所述肝内胆管前体样细胞在所述重编程培养基中进行扩增培养,直至肝内胆管前体样细胞中的标志物HNF1B、ONECUT1和SOX9中至少一种的表达率为50-99%之间后,使用胰酶消化液对所述肝内胆管前体样细胞进行消化处理,以得到初代肝内胆管前体样细胞。
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