CN115105530A - 免疫细胞的增殖抑制剂及其制备方法和应用 - Google Patents
免疫细胞的增殖抑制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种免疫细胞的增殖抑制剂及其制备方法和应用。本发明的所述免疫细胞的增殖抑制剂包含肝源细胞的条件培养上清,所述肝源细胞的条件培养上清能够通过抑制所述免疫细胞的增殖来诱导受体建立有效的免疫耐受。
Description
技术领域
本发明涉及生物技术领域,尤其涉及免疫细胞的增殖抑制剂及其制备方法和应用。
背景技术
肝脏是个免疫特惠器官,由于肝脏内各种细胞表达低水平的MHC抗原,故肝脏的免疫原性较低。实验和临床研究表明,肝脏对排斥反应的敏感性低于皮肤,心脏,肺或肾脏,这是肝脏产生致耐受力的结果。
虽然肝脏移植的免疫排斥反应相对于其他器官移植较弱,但是在标准的免疫抑制剂使用下,仍然有10%-40%的肝移植患者会发生急性排斥反应,5%左右会发生慢性排斥反应,1%患者会发生移植物抗宿主病并导致移植失败。
随着移植后存活率的提高,改善移植患者的长期生活质量并减少免疫抑制的副作用变得越来越重要。患者长期服用免疫排斥药物会增加患有多种疾病的风险。若能够诱导免疫耐受现象,撤除免疫抑制剂的使用,将极大地改善肝移植患者预后和生存情况。
因此,有必要开发新型的免疫细胞的增殖抑制剂以解决现有技术中存在的上问题。
发明内容
本发明的目的在于一种免疫细胞的增殖抑制剂及其制备方法和应用,以通过抑制免疫细胞的增殖诱导受体建立有效的免疫耐受。
为实现上述目的,本发明的所述一种免疫细胞的增殖抑制剂包含肝源细胞的条件培养上清,所述肝源细胞的条件培养上清能够通过抑制所述免疫细胞的增殖来诱导受体建立有效的免疫耐受。
优选的,所述免疫细胞为巨噬细胞、B细胞、T细胞、NK细胞和NKT细胞的任意一种。
优选的,所述肝源细胞为肝前体细胞或肝前体样细胞。
优选的,所述免疫细胞的增殖抑制剂还包括重悬成分,所述重悬成分包括生理盐水、复方电解质溶液、缓冲溶液和基础培养基的至少一种。
本发明所述免疫细胞的增殖抑制剂在制备免疫耐受相关药物方面的应用包括,将所述增殖抑制剂与所述免疫相关细胞共培养,并使用刺激剂诱导所述免疫相关细胞的增殖,考察所述增殖抑制剂对所述免疫相关细胞的增殖抑制作用。
优选的,所述免疫相关细胞为外周血单个核细胞和脾脏细胞的任意一种。
优选的,将所述增殖抑制剂与所述免疫相关细胞共培养的步骤包括,使用共培养基对所述增殖抑制剂进行重悬,并控制所述增殖抑制剂占所述共培养基的体积浓度不低于5%,以使所述增殖抑制剂对所述免疫相关细胞的增殖抑制率不低于30%。
优选的,将所述增殖抑制剂与所述免疫相关细胞共培养的步骤包括,使用不同增殖抑制剂与所述免疫相关细胞共培养,所述不同增殖抑制剂中所含有的肝源细胞的培养上清来源于不同供体的肝源细胞。
本发明所述免疫细胞的增殖抑制剂的制备方法包括:使用体外培养基对所述肝源细胞进行体外培养后,从得到的培养产物中获取所述肝源细胞的条件培养上清。
优选的,所述体外培养基包括基础培养基,所述基础培养基为DMEM/F12细胞培养基、HepX培养基、William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基和F12细胞培养基的至少一种。
附图说明
图1为实施例1的各肝前体相关标志物在HepLPCs细胞中的表达情况对比图;
图2为实施例1的经TEM培养基培养10天后得到的细胞进行明场拍照得到的HepLPCs的明场照片;
图3为实施例1采用流式细胞术考察阳性对照组以及各共培养组中HepLPC-CM对脾脏细胞的增殖抑制情况得到的对比图;
图4为实施例2采用流式细胞术考察供体1来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图;
图5为实施例2采用流式细胞术考察供体2来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图;
图6为实施例2采用流式细胞术考察供体3来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图;
图7为实施例2采用流式细胞术考察供体4来源HepLPC-CM对脾脏细胞增殖的抑制情况以及对应的阳性对照组中脾脏细胞增殖情况得到的对比图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将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。
本发明实施例提供了免疫细胞的增殖抑制剂及其制备方法和应用,以通过抑制免疫细胞的增殖诱导受体建立有效的免疫耐受。
本发明实施例的所述免疫细胞的增殖抑制剂包含肝源细胞的条件培养上清,所述肝源细胞的条件培养上清能够通过抑制所述免疫细胞的增殖来诱导受体建立有效的免疫耐受。
一些实施例中,所述肝源细胞为肝前体细胞。
一些实施例中,所述肝源细胞为肝前体样细胞。
一些实施例中,所述肝源细胞为人肝来源前体细胞或鼠肝来源前体细胞。
一些实施例中,所述肝源细胞为人肝前体细胞或鼠肝前体细胞。
一些实施例中,所述肝源细胞为人肝前体样细胞或鼠肝前体样细胞。
一些实施例中,所述免疫细胞为巨噬细胞、B细胞、T细胞、NK细胞和NKT细胞的任意一种。
一些实施例中,所述免疫细胞的增殖抑制剂还包括重悬成分。
一些实施例中,所述重悬成分为生理盐水、复方电解质溶液、缓冲溶液和基础培养基的至少一种。
本发明实施例提供了所述免疫细胞的增殖抑制剂的制备方法包括:使用体外培养基对所述肝源细胞进行体外培养后,从得到的培养产物中获取所述肝源细胞的条件培养上清。
一些实施例中,所述体外培养基包括基础培养基,所述基础培养基为DMEM/F12细胞培养基、William’s E细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基和F12细胞培养基、HepX培养基的至少一种。
一些实施例中,所述体外培养基包括基础培养基和血清类物质。
一些实施例中,所述体外培养基由基础培养基和血清类物质组成。以占所述基础培养基的体积含量计,血清类物质的含量不超过20%。
一些实施例中,所述体外培养基由所述基础培养基、血清类物质和双抗组成。其中,以占所述基础培养基的体积含量计,双抗的含量不超过2%。
一些实施例中,所述肝源细胞的制备方法包括,使用转化扩增培养基(TEM培养基)对原代肝细胞进行体外培养。
一些实施例中,TEM培养基包括基础培养基、无血清添加物、血清类物质、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。
以占所述基础培养基的含量计,所述生长因子的含量为0.1-100纳克/毫升,所述ROCK激酶抑制剂的含量为0.1-100微摩尔,所述Wnt信号通路激动剂的含量为0.1-50微摩尔,所述TGF-β信号抑制剂的含量为0.1-100微摩尔,所述血清类物质的含量不超过20%,所述无血清添加物的体积含量不超过2%。
一些实施例中,TEM培养基还包括N-乙酰-L-半胱氨酸、抗坏血酸的至少一种。
一些实施例中,所述生长因子为表皮生长因子、成纤维细胞生长因子2、血管内皮生长因子、血小板衍生生长因子、肝细胞生长因子、白介素-6和抑瘤素的至少一种。
一些实施例中,所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的至少一种。
一些实施例中,所述Wnt信号通路激动剂为重组Wnt蛋白、重组R-spondin蛋白和糖原合成酶激酶3β抑制剂的至少一种。
一些实施例中,所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的至少一种。
一些实施例中,所述转化扩增培养基由基础培养基、N2添加剂、B27添加剂、丙酮酸钠、抗坏血酸、表皮细胞生长因子、肝细胞生长因子、ROCK激酶抑制剂、Wnt信号通路激动剂、TGF-β信号抑制剂和血清类物质组成。
一些实施例中,所述体外培养基和所述TEM培养基中任意一种的血清类物质为动物源血清。
一些实施例中,所述体外培养基和所述TEM培养基中任意一种中的动物源血清可以用血清替代物替换。
一些实施例中,所述血清替代物为无动物源成份的血小板及其衍生物。
一些实施例中,所述血清替代物为一磷酸鞘氨酸和吲哚乙酸。
一些实施例中,所述动物源血清为胎牛血清。
一些实施例中,所述转化扩增培养基由基础培养基、N2添加剂、B27添加剂、丙酮酸钠、抗坏血酸、表皮细胞生长因子、肝细胞生长因子、ROCK激酶抑制剂、Wnt信号通路激动剂、TGF-β信号抑制剂,以及血清类物质组成。
一些实施例中,所述体外培养基和所述TEM培养基任意一种中的血清类物质为动物源血清。
一些实施例中,所述体外培养基和所述TEM培养基任意一种中的动物源血清可以用血清替代物替换。
一些实施例中,所述血清替代物为无动物源成份的血小板及其衍生物。
一些实施例中,所述血清替代物为一磷酸鞘氨酸和吲哚乙酸。
一些实施例中,所述动物源血清为胎牛血清。
一些实施例的所述转化扩增培养基中,以占基础培养基的含量计,所述表皮细胞生长因子的含量为5-25纳克/毫升,所述肝细胞生长因子的含量为5-25纳克/毫升,所述ROCK激酶抑制剂的含量为5-20微摩尔/升,所述Wnt信号通路激动剂的含量为1-5微摩尔/升,所述TGF-β信号抑制剂的含量为0.5-2微摩尔/升,所述一磷酸鞘氨酸的含量为0.5-2微摩尔/升,所述吲哚乙酸的含量为2-10微摩尔/升,所述N2添加剂和所述B27添加剂的体积百分比不超过1%。
一些实施例的肝源细胞培养方法中,控制所述原代肝细胞的接种密度为0.5-2×104/cm2。
一些实施例的肝源细胞培养方法中,将原代肝细胞接种于胶原蛋白I或Matrigel基质包被的培养支持物中,经含血清的WE培养基培养至贴壁后更换为TEM培养基进行7-10天的培养,且每2-3天更换一次TEM培养基。
一些实施例的肝源细胞培养方法中,所述原代肝细胞在经TEM培养前经Percoll密度梯度离心联合流式分选排除掉CD24阳性和EpCAM阳性的前体细胞。
本发明实施例提供了所述免疫细胞的增殖抑制剂在制备免疫耐受相关药物方面的应用,包括,将所述增殖抑制剂与所述免疫相关细胞共培养,并使用刺激剂诱导所述免疫相关细胞的增殖,考察所述增殖抑制剂对所述免疫相关细胞的增殖抑制作用。
一些实施例中,所述免疫相关细胞为外周血单个核细胞PBMC。
一些具体的实施例中,所述免疫相关细胞为人PBMC。人PBMC主要由淋巴细胞组成,其中淋巴细胞包括B细胞和T细胞。
一些实施例中,所述免疫相关细胞为脾脏细胞。脾脏细胞中的大量细胞为淋巴细胞,主要是B细胞和T细胞。
一些实施例中,将所述增殖抑制剂与所述免疫相关细胞共培养的步骤包括,使用共培养基对所述增殖抑制剂进行重悬,并控制所述增殖抑制剂占所述共培养基的体积浓度不低于5%,以使所述增殖抑制剂对所述免疫相关细胞的增殖抑制率不低于30%。
一些实施例中,将所述增殖抑制剂与所述免疫相关细胞共培养的步骤包括,使用不同增殖抑制剂与所述免疫相关细胞共培养,所述不同增殖抑制剂中所含有的肝源细胞的培养上清来源于不同供体的肝源细胞。
本发明各实施例中,如无特别说明,细胞培养均在37摄氏度环境下且二氧化碳浓度为5%的细胞培养箱中进行。细胞培养使用的培养基以及处理细胞所使用的各类试剂,例如缓冲液使用前均经无菌化处理以及0.22微米滤器过滤以去除杂质。
本发明各实施例中涉及统计学分析的数据均为三次独立重复,实验数据以均值±标准差表示。正态分布数据,对两组数据间差异分析使用Student’s t-test统计法;多组数据比较分析使用one-way ANOVA,和Dunnett校正进行多重比较,必要时选用Tukey校正方法。偏态分布的数据,采用两组间Mann-Whitney U test统计法进行非参数统计分析。P值小于0.05为差异有统计学意义。所有数据均通过SPSS软件(25.0版)选取合适的统计分析方法进行分析。
以下通过具体的实施例进行详细说明:
实施例1
本实施例将来源于Invitrogen公司的供体1来源人原代肝细胞分别体外培养为肝前体样细胞HepLPC,并获取条件培养上清(HepLPC-CM),然后将HepLPC-CM与ConA刺激的小鼠脾脏细胞共培养,考察HepLPC-CM对脾脏细胞的增殖抑制作用。
HepLPC的体外培养方法包括:将人原代肝细胞经Percoll密度梯度离心联合流式分选排除掉CD24阳性和EpCAM阳性的前体细胞后,先以2×104个细胞/cm2的密度接种于Matrigel(Corning公司)包被的细胞培养6孔板中经含10%血清的WE培养基(Invitrogen公司)培养至贴壁,然后以2×104/cm2的接种密度转移至TEM培养基中进行10天的培养,且每隔一天更换一次TEM培养基。
本实施例的TEM培养基组成和各组成成分的来源如下:DMEM/F12基础培养基(Invitrogen公司),以及以占DMEM/F12基础培养基的含量计:体积含量1%的N2添加剂和体积含量1%的B27添加剂(Invitrogen公司),1mmol/L丙酮酸钠(Invitrogen公司),10μg/mL抗坏血酸(Sigma-Aldrich公司),20ng/mL肝细胞生长因子HGF(Peprotech公司),20ng/mL表皮细胞生长因子EGF(Peprotech公司),10μmol/L ROCK激酶抑制剂Y27632(TargetMol公司),3μmol/L Wnt信号通路激动剂CHIR99021(TargetMol公司),1μmol/L TGF-β信号抑制剂A83-01(TargetMol公司),1μmol/L一磷酸鞘氨酸S1P(Santa Cruz公司)以及5μmol/L吲哚乙酸LPA(Santa Cruz公司)。
HepLPC-CM的制备方法包括:消化使用0.25%的Trypsin-EDTA(来源于美国Gibco公司)对经TEM培养10天得到的前体样细胞进行消化,并按照1:3的比例接种入新的培养皿中扩增培养到第三代并直至细胞汇合率达80%,然后将TEM培养基更换为无血清的DMEM培养基进行24小时的体外培养;体外培养结束后收集细胞上清,对所述细胞上清在300g离心力下离心10分钟以去除细胞碎片,得到条件培养上清HepLPC-CM。
加入刺激剂的小鼠脾脏细胞悬液的制备方法为:断颈处死1周龄的C57BL/6小鼠后取脾脏在200目筛网研磨后使用淋巴分离液冲洗,收集包含淋巴细胞的分离液并使用含10%胎牛血清的RPMI-1640完全培养基重悬后在800g离心力下低温离心30分钟;离心结束后吸取淋巴细胞层后使用含10%胎牛血清的RPMI-1640完全培养基重悬后在250g离心力下低温离心10分钟,使用含10%胎牛血清的RPMI-1640完全培养基再次重悬得到的淋巴细胞层并加入含10%胎牛血清的RPMI-1640完全培养基重悬的刀豆蛋白A(ConA)悬液作为刺激剂。
共培养步骤包括:将前述加入刺激剂的小鼠脾脏细胞悬液以2×104个细胞/cm2的密度接种于6孔板;使用HepLPC-CM以及使用含10%胎牛血清的RPMI-1640完全培养基重悬来自供体1的HepLPC-CM,得到HepLPC-CM浓度分别为100%、50%、25%、12.5%以及6.25%的HepLPC-CM悬液,将上述HepLPC-CM悬液分别加入到接种有小鼠脾脏细胞的6孔板中,控制ConA的终浓度为2.5微克/毫升,形成以下共培养组:100%-CM组、50%-CM组、25%-CM组、12.5%-CM组以及6.25%-CM组。另外,以不添加HepLPC-CM的共培养组作为阳性对照组。
本实施例分别使用qPCR和流式细胞术分析了供体1的人原代肝细胞和经TEM培养得到的HepLPC的基因表达情况,结果如图1和图2所示。参照图1,人原代肝细胞在TEM培养基作用下,肝祖细胞基因Ck7、Ck19和Sox9的表达显著增加,而Alb、Cyp3a4和Hnf4α这类肝实质细胞标志物表达显著下降。参照图2,Human-HepLPCs的肝细胞标志物HNF4α以及肝干细胞/肝祖细胞标志物CD24 and CK19显著表达,造血干细胞抗原CD34、白细胞共同抗原CD45以及肝胎儿细胞标志物AFP的表达水平均小于2%。Human-HepLPCs不表达MHC II类抗原HLA-DP,HLA-DQ和HLA-DR,表现出低免疫原性。
本实施例中,共培养72小时后,使用CFSE细胞增殖检测试剂盒对阳性对照组以及各共培养组进行处理后上流式仪检测,考察各共培养组中HepLPC-CM对脾脏细胞的增殖抑制情况,结果如图3所示。根据图3所示阳性对照组和各共培养组的增殖脾脏细胞占比情况可知,100%-CM组、50%-CM组、25%-CM组、12.5%-CM组以及6.25%-CM组中,HepLPC-CM对脾脏细胞增殖的抑制率分别为47%、50%、40%、37%和34%。可见,HepLPC-CM能够显著抑制小鼠脾脏细胞的增殖,且呈现剂量依赖性。
实施例2
本实施例将来源于Invitrogen公司的不同供体来源人原代肝细胞分别体外培养为肝前体样细胞HepLPC,并获取不同供体来源的条件培养上清,然后将不同供体来源的条件培养上清分别与ConA刺激的小鼠脾脏细胞共培养,考察HepLPC-CM对脾脏细胞的增殖抑制作用。
人原代肝细胞体外培养方法、条件培养上清制备方法、加入刺激剂的小鼠脾脏细胞悬液制备方法请参见实施例1。共培养步骤与实施例1的区别在于:每种供体来源的HepLPC不使用含10%胎牛血清的RPMI-1640完全培养基重悬,而是直接与加入刺激剂的小鼠脾脏细胞共培养。
本实施例中,共培养72小时后,使用CFSE细胞增殖检测试剂盒对阳性对照组以及含不同供体来源HepLPC-CM的共培养组进行处理后上流式仪检测,考察各供体来源HepLPC-CM的共培养组对脾脏细胞的增殖抑制情况与对应的的阳性对照组中脾脏细胞的增殖情况,结果如图4至图7所示。参照图4至图7所示的阳性对照组和各共培养组的增殖脾脏细胞占比情况可知,供体1、供体2、供体3和供体4来源的HepLPC-CM对脾脏细胞增殖的抑制率分别为42%、69%、59%和56%。可见,不同供体来源HepLPC-CM对脾脏细胞增殖的抑制效果体现出差异性,有利于后续开展免疫耐受药物的异质性研究。
Claims (10)
1.一种免疫细胞的增殖抑制剂,其特征在于,包含肝源细胞的条件培养上清,所述肝源细胞的条件培养上清能够通过抑制所述免疫细胞的增殖来诱导受体建立有效的免疫耐受。
2.根据权利要求1所述的免疫细胞的增殖抑制剂,其特征在于,所述免疫细胞为巨噬细胞、B细胞、T细胞、NK细胞和NKT细胞的任意一种。
3.根据权利要求1所述的免疫细胞的增殖抑制剂,其特征在于,所述肝源细胞为肝前体细胞或肝前体样细胞。
4.根据权利要求1所述的免疫细胞的增殖抑制剂,其特征在于,还包括重悬成分,所述重悬成分包括生理盐水、复方电解质溶液、缓冲溶液和基础培养基的至少一种。
5.根据权利要求1所述的免疫细胞的增殖抑制剂在制备免疫耐受相关药物方面的应用,其特征在于,包括将所述增殖抑制剂与所述免疫相关细胞共培养,并使用刺激剂诱导所述免疫相关细胞的增殖,考察所述增殖抑制剂对所述免疫相关细胞的增殖抑制作用。
6.根据权利要求5所述的免疫细胞的增殖抑制剂在制备免疫耐受相关药物方面的应用,其特征在于,所述免疫相关细胞为外周血单个核细胞和脾脏细胞的任意一种。
7.根据权利要求5所述的免疫细胞的增殖抑制剂在制备免疫耐受相关药物方面的应用,其特征在于,将所述增殖抑制剂与所述免疫相关细胞共培养的步骤包括,使用共培养基对所述增殖抑制剂进行重悬,并控制所述增殖抑制剂占所述共培养基的体积浓度不低于5%,以使所述增殖抑制剂对所述免疫相关细胞的增殖抑制率不低于30%。
8.根据权利要求5所述的免疫细胞的增殖抑制剂在制备免疫耐受相关药物方面的应用,其特征在于,将所述增殖抑制剂与所述免疫相关细胞共培养的步骤包括,使用不同增殖抑制剂与所述免疫相关细胞共培养,所述不同增殖抑制剂中所含有的肝源细胞的培养上清来源于不同供体的肝源细胞。
9.根据权利要求1所述的免疫细胞的增殖抑制剂的制备方法,其特征在于,包括:
使用体外培养基对所述肝源细胞进行体外培养后,从得到的培养产物中获取所述肝源细胞的条件培养上清。
10.根据权利要求9所述的免疫细胞的增殖抑制剂的制备方法,其特征在于,所述体外培养基包括基础培养基,所述基础培养基为DMEM/F12细胞培养基、HepX培养基、William’sE细胞培养基、Neurobasal Medium细胞培养基、MEM细胞培养基、DMEM细胞培养基、1640RPMI细胞培养基和F12细胞培养基的至少一种。
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