CN115094020A - 抗肝纤维化制剂及其制备方法和应用 - Google Patents
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Abstract
本发明提供了抗肝纤维化制剂及其制备方法和应用。所述抗肝纤维化制剂包含肝脏来源前体细胞的分泌上清,能够实现对肝纤维化的抑制作用。
Description
技术领域
本发明涉及生物技术领域,尤其涉及抗肝纤维化制剂及其制备方法和应用。
背景技术
肝星状细胞(Hepatic stellate cells,HSCs)位于Disse间隙内,紧贴着肝窦内皮细胞和肝细胞。正常肝脏中的肝星状细胞处于静止状态;当肝脏受到炎症或机械刺激等损伤时,肝星状细胞被激活并转分化为肌成纤维细胞样细胞(MFC)。肝星状细胞的持续激活是肝纤维化发生发展过程中的关键环节,能够加重肝损伤。
因此,有必要开发新型的抗肝纤维化制剂以解决现有技术中存在的上问题。
发明内容
本发明的目的在于提供抗肝纤维化制剂及其制备方法和应用,以对肝纤维化起到抑制作用。
为实现上述目的,本发明的抗肝纤维化制剂包含肝脏来源前体细胞的分泌上清,能够对肝纤维化起到抑制作用。
优选的,所述肝脏来源前体细胞为肝前体细胞或肝前体样细胞。
优选的,所述分泌上清包含作用于JAK-STAT通路的起效成分,以抑制肝星状细胞活化或诱导所述肝星状细胞死亡。
优选的,所述分泌上清包含白血病抑制因子、内皮素、集落刺激因子、双调蛋白和成纤维细胞生长因子的至少一种。
优选的,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。
优选的,还包含辅助成分,所述辅助成分包括免疫抑制成分、血清、抗生素的至少一种。
本发明所述抗肝纤维化制剂的制备方法包括:使用分泌培养基对肝脏来源前体细胞进行体外培养后收集体外培养上清,从所述体外培养上清中获取所述分泌上清。
优选的,所述分泌培养基包含基础培养基,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。
本发明所述抗肝纤维化制剂的体外应用包括对所述抗肝纤维化制剂与肝星状细胞进行共培养,以抑制肝星状细胞活化或诱导所述肝星状细胞死亡。
优选的,使用共培养基对所述抗肝纤维化制剂与肝星状细胞进行共培养,以占所述共培养基的体积百分比计,所述抗肝纤维化制剂的含量不低于1%。
进一步优选的,所述共培养基包括肝星状细胞活化剂。
本发明所述抗肝纤维化制剂在制备抗肝纤维化药物方面的应用包括:
使用所述抗肝纤维化制剂干预肝纤维化体外类器官模型后获取离体样本;
通过所述离体样本考察所述抗肝纤维化制剂对肝纤维化的抑制作用。
优选的,所述肝纤维化体内动物模型为硫代乙酰胺诱导或四氯化碳诱导的哺乳动物肝硬化模型。
附图说明
图1为实施例1的人原代肝细胞的基因表达情况示意图;
图2为实施例1的人肝前体样细胞的基因表达情况示意图;
图3为实施例2的实验组、控制组和对照组中各肝星状细胞的微观形貌对比照片;
图4为实施例2的实验组、控制组和对照组中与HSCs活化相关基因的相对mRNA表达情况对比图;
图5为实施例2的实验组细胞聚集体在透射显微镜观察下的微观形貌照片;
图6为针对实施例2的对照组细胞聚集体进行流式细胞术测定得到的分析结果;
图7为针对实施例2的实验组细胞聚集体进行流式细胞术测定得到的分析结果;
图8为针对实施例2的控制组、对照组和实验组的细胞聚集体进行蛋白质印记分析得到的各组细胞的纤维化相关蛋白表达和关键纤维化信号的表达情况对比图;
图9为实施例3的鼠肝原代细胞和鼠肝前体样细胞的肝祖细胞基因和肝实质细胞标志物的表达情况对比图;
图10为实施例4的控制组、对照组和实验组共培养48小时后,各组HSCs-T6细胞的微观形貌照片对比图;
图11为实施例4的控制组、对照组和实验组的HSCs-T6中与HSCs活化相关基因的相对mRNA表达情况对比图;
图12为实施例4的实验组中细胞微观形貌照片;
图13为针对实施例4的对照组的细胞进行流式细胞术测定得到的分析结果;
图14为针对实施例4的实验组的细胞进行流式细胞术测定得到的分析结果;
图15为实施例5的第一种抗肝纤维化制剂中,JAK-STAT通路与第一种抗肝纤维化制剂中参与生长因子活性、细胞因子活性和受体-配体活性的蛋白质之间的可视化网络示意图;
图16为实施例6的控制组、对照组和实验组进行蛋白质印记分析后得到图16所示的各组细胞的pSTAT1信号的表达情况对比图;
图17为针对实施例7的控制组、对照组和实验组各组的细胞聚集体进行流式分析的分析结果对比图;
图18为根据图17统计得到的各组凋亡细胞数量百分比对比图;
图19为实施例8的正常组、PBS注射组和抗肝纤维化制剂干预组小鼠的肝组织切片微观形貌照片对比图;
图20为实施例8的正常组、PBS注射组和抗肝纤维化制剂干预组小鼠肝脏内活化的肝星状细胞分布状态对比照片。
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抗肝纤维化制剂及其制备方法和应用,以通过抑制HSCs的活化来实现对肝纤维化的抑制作用。
本发明实施例的抗肝纤维化制剂包含肝脏来源前体细胞的分泌上清。
一些实施例中,所述肝脏来源前体细胞为肝前体细胞。
一些实施例中,所述肝脏来源前体细胞为肝前体样细胞。
一些实施例中,所述肝脏来源前体细胞为人肝来源前体细胞。
一些实施例中,所述人肝来源前体细胞为人肝前体细胞。
一些实施例中,所述人肝来源前体细胞为人肝前体样细胞。
一些实施例中,所述分泌上清包含作用于JAK-STAT通路的起效成分,以抑制肝星状细胞活化。
一些实施例中,所述分泌上清包含白血病抑制因子、内皮素、集落刺激因子、双调蛋白和成纤维细胞生长因子的至少一种。
一些实施例中,所述成纤维生长因子为FGF19。
一些实施例中,所述抗肝纤维化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。
一些实施例中,所述抗肝纤维化制剂还包括辅助成分,所述辅助成分包括免疫抑制成分、血清、抗生素和协同起效成分的至少一种。
本发明实施例提供了所述抗肝纤维化制剂的制备方法,包括:使用分泌培养基对肝脏来源前体细胞进行体外培养后收集体外培养上清,从所述体外培养上清中获取所述分泌上清。
一些实施例中,所述分泌培养基为基础培养基,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。
一些实施例中,所述分泌培养基由所述基础培养基,以及血清类物质和双抗的至少一种组成。
一些实施例中,所述分泌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,血清类物质的含量不超过20%,双抗的含量不超过2%。
一些实施例中,所述肝脏来源前体细胞经肝细胞扩增转化培养基(TEM培养基)对原代肝细胞进行体外培养得到。
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培养基还含有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,TEM培养基由所述血清类物质、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信号通路激动剂和TGF-β信号抑制剂组成。
一些实施例中,所述分泌培养基和所述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,所述分泌培养基和所述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。
一些实施例的TEM培养基中,以占基础培养基的含量计,所述丙酮酸钠的含量为0.5-1.5毫摩尔/升,所述抗坏血酸的含量为5-50微克/毫升,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%。另一些实施例的TEM培养基中,使用胎牛血清替代一磷酸鞘氨酸和吲哚乙酸。
本发明实施例提供了抗肝纤维化制剂的体外应用,包括:对所述抗肝纤维化制剂与肝星状细胞进行共培养,以抑制肝星状细胞活化或诱导所述肝星状细胞死亡。
一些实施例中,使用共培养基对所述抗肝纤维化制剂与肝星状细胞进行共培养,以占所述共培养基的体积百分比计,所述抗肝纤维化制剂的含量不低于1%。
一些实施例的共培养过程中,所述肝星状细胞的铺板密度为1×104个/平方厘米。
一些实施例中,所述共培养基由基础培养基、血清和抗生素组成。
一些实施例中,所述血清为胎牛血清。
一些实施例中,所述共培养基中,以占所述基础培养基的体积百分比计,血清的含量不超过15%,抗生素的含量不超过2%。
一些实施例中,所述共培养基还含有肝星状细胞活化剂。
一些实施例中,所述肝星状细胞活化剂为肝星状细胞活化因子。
一些实施例中,所述肝星状细胞活化因子为TGF-β1。
本发明还提供了抗肝纤维化制剂在制备肝纤维化治疗药物方面的应用,包括:
使用所述抗肝纤维化制剂干预肝纤维化体外类器官模型后获取离体样本;
通过所述离体样本考察所述抗肝纤维化制剂对肝纤维化的抑制作用。
一些实施例中,所述肝纤维化体内动物模型为硫代乙酰胺诱导或四氯化碳诱导的哺乳动物肝硬化模型。
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,每组实验至少重复3次,实验结果数据利用GraphPad Prism 8.0软件进行统计学分析。两组数据间比较使用双尾非配对t检验来计算统计学差异,多组数据间差异的比较使用用ANOVA方差分析计算统计学差异。p<0.05被认为具有统计学差异,说明书附图中:*代表P<0.05;**代表P<0.005;***代表P<0.001;****代表P<0.0001。
以下通过具体的实施例进行详细说明:
实施例1
本实施例提供了第一种抗肝纤维化制剂,其制备方法如下:
S0:提供汇合度不低于60%的人肝前体样细胞Human-HepLPCs作为种子细胞;
S1:使用无血清的DMEM培养基对Human-HepLPCs进行24小时的体外培养;
S2:收集体外培养上清,去除所述体外培养上清中的细胞碎片后进行25倍的过滤浓缩,得到的分泌上清作为抗肝纤维化制剂。具体的,所述步骤S2中,在3000g的离心力下去除培养上清中的细胞碎片;使用10kDa的Amicon Ultra超滤器进行过滤浓缩。
本实施例采用BCA蛋白定量试剂盒(来源于上海碧云天生物技术有限公司)按说明书提供的检测方法检测上述分泌上清中的总蛋白含量为2.2毫克/毫升。
本实施例所述步骤S0的Human-HepLPCs由人原代肝细胞Human-primaryhepatocytes经TEM培养基进行7-9天的转化扩增培养后,再进行1:(3-6)的传代培养至第2-5代得到。具体的,TEM培养基由以下成分组成:DMEM/F12基础培养基,以及以占DMEM/F12基础培养基的含量计:含量为1%的N2营养补充剂(100X),含量为1%的B27营养补充剂(50X),1mM的丙酮酸钠,10μg/mL的抗坏血酸,20ng/mL的肝细胞生长因子HGF,20ng/mL的上皮细胞生长因子EGF,10μM的ROCK激酶抑制剂Y27632,3μM的Wnt信号通路激动剂CHIR99021,1μM的TGF-β信号抑制剂A8301,1μM的鞘氨醇-1-磷酸S1P and 5μM的吲哚乙酸LPA。其中的DMEM/F12、N2营养补充剂、B27营养补充剂和丙酮酸钠来源于Invitrogen;抗坏血酸来源于Sigma-Aldrich;HGF和EGF来源于Novoprotein;Y27632、CHIR99021、A8301、s1p和LPA均来源于TargetMol。
本实施例分别使用qPCR和流式细胞术分析了人原代肝细胞和人肝前体样细胞的基因表达情况,结果如图1和图2所示。参照图1,箭头所指的柱状图代表人肝前体样细胞的基因相对表达情况。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,人肝前体样细胞的肝细胞标志物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%。人肝前体样细胞不表达MHC II类抗原HLA-DP,HLA-DQ和HLA-DR,表现出低免疫原性。
实施例2
本实施例对实施例1的第一种抗肝纤维化制剂与人永生化肝星状细胞系LX-2进行共培养,考察抗肝纤维化制剂对LX-2的促凋亡作用。本实施例的LX-2购自Procell。
实施例1的第一种抗肝纤维化制剂与LX-2进行共培养的过程如下:LX-2固定于含10%FBS、100U/mL的青霉素和100mg/mL的链霉素的DMEM培养基中,加入2.5ng/mL的TGF-β1活化LX-2;再加入实施例1的抗肝纤维化制剂混匀后静置48小时。共培养混合物中,第一种抗肝纤维化制剂的体积百分比含量为1%、2.5%和5%。其中,第一种抗肝纤维化制剂在使用前先加入10μg/mL的抗FGF19抗体(兔单克隆抗体,来源于R&D Systems)和10μg/mL的抗AREG抗体(兔多克隆抗体,来源于R&D Systems)孵育2小时。
本实施例将上述共培养得到的细胞作为实验组,LX-2和DMEM培养基共培养48小时得到的细胞作为控制组,加入TGF-β1活化LX-2后与DMEM培养基共培养48小时得到的细胞聚集体作为TGF-β1活化组,三组制样后使用透射电子显微镜观察形态,得到图3所示的各组LX-2细胞的微观形貌照片。参照图3,TGF-β1激活LX-2细胞导致LX-2细胞形态改变为细长的树突状,而加入第一种抗肝纤维化制剂共培养则能够逆转这种变化。
本实施例通过实时聚合酶链反应(RT-PCR)考察了TGF-β1活化组和实验组(第一种抗肝纤维化制剂的体积百分比含量为1%)的LX-2中与HSCs活化相关基因Col1a1、Col3a1、TGF-β1、Desmin、α-SMA和Pdgfb的相对mRNA表达情况,将数据标准化为GAPDH表达,并与对照组相比统计差异化程度,得到图4所示的各组与HSCs活化相关基因的相对mRNA表达情况对比图。从图4可以看到,TGF-β1激活了LX-2细胞,使得上述HSCs活化相关基因的表达水平上调;而第一种抗肝纤维化制剂的引入显著抑制了上述HSCs活化相关基因的表达水平,可见第一种抗肝纤维化制剂对HSCs活化具有显著的抑制作用。
本实施例对实验组(第一种抗肝纤维化制剂的体积百分比含量为1%)的细胞制样后进行透射电子显微镜观察,得到图5所示的微观照片。参见图5所示,第一种抗肝纤维化制剂与LX-2共培养得到的细胞聚集体中观察到箭头所示的凋亡小体。进一步对实验组和TGF-β1活化组得到的细胞进行膜联蛋白V/PI染色后进行流式细胞术测定,得到图6所示的对照组流式细胞术图和图7所示的实验组的流式细胞术图。参照图6和图7,第一种抗肝纤维化制剂诱导了HSC凋亡。
本实施例分别对控制组、TGF-β1活化组以及不同抗肝纤维化制剂的质量百分比含量的实验组的细胞聚集体进行蛋白质印记分析(Westernblot),得到图8所示的各组细胞的纤维化相关蛋白表达和关键纤维化信号的表达情况对比图。可见,在存在TGF-β1的情况下,第一种抗肝纤维化制剂诱导纤维化相关蛋白表达和关键纤维化信号TGF-β-SMAD途径的剂量依赖性降低。
实施例3
本实施例提供了第二种抗肝纤维化制剂,其制备方法以汇合度不低于60%的鼠肝前体样细胞Rat-HepLPCs作为种子细胞,其余制备过程请参见实施例1。
Rat-HepLPCs以鼠肝原代细胞Ratprimary hepatocytes为种子细胞,通过TEM培养基培养得到。具体的培养过程请参见实施例1。
本实施例分别使用qPCR和流式细胞术分析了Rat primary hepatocytes和Rat-HepLPCs的基因表达情况,结果如图9所示。参照图9,鼠肝前体样细胞在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。
实施例4
本实施例对实施例3的第二种抗肝纤维化制剂与鼠肝星状细胞系HSCs-T6进行共培养,考察抗肝纤维化制剂对HSCs-T6的促凋亡作用。本实施例的HSCs-T6购自Procell。
实施例3的第二种抗肝纤维化制剂与HSCs-T6进行共培养的过程请参见实施例2。其中共培养混合物中第二种抗肝纤维化制剂的体积百分比含量为1%。
本实施例将第二种抗肝纤维化制剂与HSCs-T6进行共培养得到的细胞作为实验组,HSCs-T6和DMEM培养基共培养48小时得到的细胞作为控制组,加入TGF-β1活化HSCs-T6后与DMEM培养基共培养48小时得到的细胞作为TGF-β1活化组,三组制样后使用透射电子显微镜观察形态,得到图10所示的各组HSCs-T6细胞的微观形貌照片。参照图10,TGF-β1激活HSCs-T6细胞导致HSCs-T6细胞形态改变为细长的树突状,而加入第一种抗肝纤维化制剂共培养则能够逆转这种变化。
本实施例通过实时聚合酶链反应(RT-PCR)考察了控制组、TGF-β1活化组和实验组的HSCs-T6中与HSCs活化相关基因Col1a1、Col3a1、TGF-β1、Desmin、α-SMA和Pdgfb的相对mRNA表达情况,将数据标准化为GAPDH表达,并与对照组相比统计差异化程度,得到图11所示的各组与HSCs活化相关基因的相对mRNA表达情况对比图。从图11可以看到TGF-β1激活了HSCs-T6细胞,使得上述HSCs活化相关基因的表达水平上调;而第二种抗肝纤维化制剂的引入显著抑制了上述HSCs活化相关基因的表达水平,可见第二种抗肝纤维化制剂对HSCs活化具有显著的抑制作用。
本实施例对实验组的细胞制样后进行透射电子显微镜观察,得到图12所示的微观照片。参见图12所示,第二种抗肝纤维化制剂与HSCs-T6共培养得到的细胞中也观察到了箭头所示的凋亡小体。进一步对实验组和TGF-β1活化组得到的细胞进行膜联蛋白V/PI染色后进行流式细胞术测定,得到图13所示的TGF-β1活化组流式细胞术图和图14所示的实验组的流式细胞术图。参照图13和图14,第二种抗肝纤维化制剂诱导了HSC凋亡。
实施例5
JAK/STAT通路在肝纤维化的形成进程中具有重要的调控作用。本实施例对实施例1的由人肝前体样细胞Human-HepLPCs体外培养得到的第一种抗肝纤维化制剂使用串联质谱标签(TMT)分析蛋白质组学组成,通过蛋白质-蛋白质相互作用(PPI)分析构建得到图15所示的第一种抗肝纤维化制剂中JAK-STAT通路与第一种抗肝纤维化制剂中参与生长因子活性、细胞因子活性和受体-配体活性的蛋白质之间的可视化网络示意图。
参照图15,白血病抑制因子(LIF)、内皮素1(EDN1)、集落刺激因子1(CSF1)、双调蛋白(AREG)、成纤维细胞生长因子19(FGF19)直接或间接与JAK-STAT通路的中间分子相互作用。
实施例6
本实施例提供了第三种抗肝纤维化制剂,包含重组人FGF19(rhFGF19)和重组人AREG(rhAREG)。本实施例参照实施例2的方法将所述第三种抗肝纤维化制剂与LX-2共培养,形成TGF-β1+rhFGF19+rhAREG组,考察其对LX-2的作用,区别在于:rhFGF19的浓度分别为0.1ng/mL、1ng/mL、10ng/mL、100ng/mL和1000ng/mL,rhAREG的浓度分别为0.1ng/mL、1ng/mL、10ng/mL、100ng/mL和1000ng/mL。不同实验组的rhFGF19浓度和rhAREG浓度不同。
作为公知的是,p-STAT1在肝纤维化过程中发挥重要作用,主要通过抑制肝星状细胞的功能实现抗纤维化作用。本实施例控制组、对照组和不同实验组进行蛋白质印记分析(Westernblot),得到图16所示的各组细胞的pSTAT1信号的表达情况对比图。参照图16可知,每种重组蛋白的浓度不低于10ng/mL时,p-STAT1水平增加。
实施例7
本实施例将FGF19的中和抗体FGF19 Ab和AREG的中和抗体AREGAb加入到实施例2的第一种抗肝纤维化制剂与活化的LX-2的共培养体系中形成TGF-β1+scrtms+FGF19 Ab+AREG Ab组,与实施例6的TGF-β1+rhFGF19+rhAREG组,以及实施例2的TGF-β1活化组以及实验组一同考察rhFGF19和rhAREG的协同作用。rhFGF19和rhFGF19在共培养体系中的浓度均为100纳克/毫升,FGF19的中和抗体FGF19 Ab和AREG的中和抗体AREGAb在共培养体系中的浓度均为10微克/毫升。
共培养48小时后,对各组细胞进行流式分析,根据图17所示的各组细胞计数情况对比图。根据图17统计凋亡细胞的数量百分比,得到图18所示的各种各组凋亡细胞数量百分比对比图。参照图17和图18,在TGF-β1存在的情况下,相较于TGF-β1和LX-2的共培养体系,rhFGF19和rhAREG的联合使用在一定程度上诱导了LX-2的凋亡;而rhFGF19 Ab和rhAREGAb的联合使用则降低了上述的促LX-2凋亡作用,说明rhFGF19和rhAREG的联合使用有助于诱导STAT1介导的HSC凋亡。
实施例8
本实施例提供了抗肝纤维化制剂在制备抗肝纤维化药物方面的应用。
首先,为了诱发肝纤维化,使用硫代乙酰胺(Thioacetamide,TAA)诱导肝纤维化。具体的,由硫代乙酰胺(TAA)诱导5~6周龄雌性老鼠(C57BL/C)造成肝纤维化模型,TAA稀释于生理盐水中,采用200mg/kg剂量腹腔注射,1周3次,共7周。动物共三个组,正常对照组、假手术组(PBS注射组)、抗肝纤维化制剂干预组、动物数量分别为:8只、8只、8只。
采用PBS将实施例1的作为抗肝纤维化制剂的分泌上清稀释至总蛋白浓度为2mg/ml,得到抗肝纤维化制剂注射液。TAA注射7周后,正常对照组不做处理,假手术组通过脾脏注射予以250ulPBS溶液,抗肝纤维化制剂干预组通过脾脏注射250ul的抗肝纤维化制剂注射液。注射完毕的7日后,取小鼠肝脏用福尔马林溶液浸泡进行固定后包埋和切片后用于HE染色检测,马森三色检测和天狼星检测,对小鼠肝纤维化程度进行综合分析。
如图19所示,经过7周TAA药物的诱导,PBS注射组小鼠肝脏表面不平整,呈粗糙质地,免疫形态学显示胶原广泛存在,纤维之间相互连接将正常肝组织间隔。而经过抗肝纤维化制剂治疗后,抗肝纤维化制剂干预组小鼠肝脏质地更接近正常组,免疫形态学显示纤维组织明显在肝脏中减少,纤维组织呈现更为纤细的结构,这提示抗肝纤维化制剂干预明显改善了TAA诱导的小鼠肝纤维化程度。
如图20所示,经过7周TAA药物的诱导,PBS注射组大鼠肝脏内活化的肝星状细胞(α-SMA阳性细胞)数量明显增多,而抗肝纤维化制剂干预组中,活化肝星状细胞(α-SMA阳性细胞)数量较假手术组明显减少,这说明抗肝纤维化制剂可抑制体内肝星状细胞的活化。
本发明实施例的qPCR测试过程如下:使用Eastep SuperRNA提取试剂盒(货号为LS1040,来源于Promega)提取总mRNA。使用第一链cDNA合成试剂盒(货号为R211-01,来源于Vazyme)进行反转录。然后,使用AceQ qPCR SYBR Green Master Mix(货号为Q131-02,来源于Vazyme)和Life TechnologyABI 7500系统开发实时PCR。GAPDH表达用作内部对照,确定阈值周期(CT),并使用Δ(ΔCT)方法计算基因表达的相对变化。本发明实施例使用透射电子显微镜(型号为Jem 1200ex II,来源于JEOL)观察细胞的形态。将细胞用2.5%戊二醛和2%锇酸固定,然后脱水并包埋在环氧树脂中,切成80nm厚的切片,然后用醋酸铀酰和醋酸铅双重染色后观察。
本发明实施例的RNA测序和生物信息学分析使用试剂从肝组织中分离总RNA;使用DNA酶I(来源于TaKara)去除基因组DNA;RNA样品的浓度和纯度由2100生物分析仪(来源于安捷伦)测定,并使用ND-2000进行定量。文库制备和Illumina Hiseq xten/Novaseq 6000测序RNA seq转录组文库按照来自Illumina的TruSeqTM RNA样品制备试剂盒制备,使用1μg总RNA;根据Illumina的文库构建方案的说明,片段RNA经过第一链和第二链cDNA合成,然后以低周期进行接头连接和富集;量化后,使用Illumina HiSeq xten/NovaSeq6000测序仪在广州RiboBio有限公司对配对末端RNA seq测序文库进行测序。
本发明实施例的基因表达通过EDASEQ标准化。使用版本1.10.1的DESeq2获得差异表达基因,Q值的截止值<0.05和log2(折叠变化)>1用于识别差异表达基因。选择所有差异表达的mRNA进行GO分析clusterProfiler。使用glbase进行其他分析。
本发明实施例使用含有蛋白酶抑制剂混合物(P1010,来源于Beyotime)的RIPA缓冲液(P0013B,来源于Beyotime)提取细胞或分泌体的总蛋白。样品在冰上超声处理30秒,然后在4℃下以12000xg离心15分钟。收集上清液并用BCA蛋白质分析试剂盒(ZJ101,来源于Epizyme)定量。定量蛋白质样品通过5x SDS-PAGE(P0015,来源于Beyotime)进行解析,并转移至疏水性PVDF转移膜(IPVH00010,来源于默克密理博)。将膜在TBST中的5%BSA中封闭1.5小时,并在4℃下与一级抗体孵育过夜。然后用TBST洗涤膜三次,并在室温下与二级抗体孵育2小时。使用增强ECL化学发光检测试剂盒(E411-04,来源于Vazyme和数字发光图像分析仪(BioRad)检测印迹。使用ImageJ软件或QingXiang软件确定每个波段的密度分析。使用的一级和二级抗体见表1。
表1
本发明实施例使用来源于Beyotime的膜联蛋白V-FITC凋亡检测试剂盒以及来源于Biogems的膜联蛋白V-APC凋亡检测试剂盒通过膜联蛋白V/碘化丙啶(PI)或膜联蛋白V/7-AAD分析检测LX-2中的凋亡。具体的,收集细胞并重新悬浮在结合缓冲液中,然后根据试剂盒说明用膜联蛋白V和PI或7-AAD染色。0.5μM的蛋白激酶抑制剂Staurosporine用作促凋亡对照(阳性对照)。采用BD-facverse流式细胞仪检测荧光,并用FlowJo软件进行数据分析。
本发明实施例使用细胞因子抗体阵列(AAH-INF-G3,系列)测量培养上清液中40种细胞因子的表达。用激光扫描仪检测阳性信号。用于显著性分析的基本统计数据是折叠变化。差异表达蛋白(DEP)被定义为折叠变化超过1.2或小于0.83(绝对对数fc>0.263)的蛋白质。细胞因子的功能通过基因本体(GO)注释进行注释。
原始质谱(MS)数据文件使用蛋白质组发现(PD)软件(版本2.4.0.305)和内置的Sequest HT搜索引擎进行处理。根据智人UniProt FASTA数据库(UniProt-Human-9606-2020-10.FASTA)搜索MS光谱列表,其中氨基甲基[C]、TMT 6复合物(K)和TMT 6复合物(N-术语)作为固定修饰,氧化(M)和乙酰基(蛋白质N-术语)作为可变修饰。用于鉴定肽的参数为:10ppm前体离子质量耐受性,0.02Da片段质量耐受性,最多2次缺失裂解。PSM和肽水平的错误发现率(FDR)均设置为0.01。通过基因本体(GO)注释对蛋白质的功能进行了注释(http://www.geneontology.org/).京都基因和基因组百科全书(KEGG)数据库用于分析富集途径。GO-KEGG富集分析中使用了双尾Fisher精确检验。P<0.05被认为是显著的。Cytoscape 2.6版(www.Cytoscape.org)用于可视化和分析分子和蛋白质相互作用网络。差异表达的蛋白质通过层次聚类进行排列,并以热图表示。热图由R软件生成(http://www.r-project.org).。
Claims (13)
1.一种抗肝纤维化制剂,其特征在于,包含肝脏来源前体细胞的分泌上清。
2.根据权利要求1所述的抗肝纤维化制剂,其特征在于,所述肝脏来源前体细胞为肝前体细胞或肝前体样细胞。
3.根据权利要求1所述的抗肝纤维化制剂,其特征在于,所述分泌上清包含作用于JAK-STAT通路的起效成分,以抑制肝星状细胞活化或诱导所述肝星状细胞死亡。
4.根据权利要求1所述的抗肝纤维化制剂,其特征在于,所述分泌上清包含白血病抑制因子、内皮素、集落刺激因子、双调蛋白和成纤维细胞生长因子的至少一种。
5.根据权利要求1所述的抗肝纤维化制剂,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。
6.根据权利要求1所述的抗肝纤维化制剂,其特征在于,还包含辅助成分,所述辅助成分包括免疫抑制成分、血清、抗生素的至少一种。
7.根据权利要求1所述的抗肝纤维化制剂的制备方法,其特征在于,包括:
使用分泌培养基对肝脏来源前体细胞进行体外培养后收集体外培养上清,从所述体外培养上清中获取所述分泌上清。
8.根据权利要求7所述的抗肝纤维化制剂的制备方法,其特征在于,所述分泌培养基包括基础培养基,所述基础培养基为HepX培养基、DMEM/F12细胞培养基、William’s E细胞培养基、NeurobasalMedium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。
9.根据权利要求1所述的抗肝纤维化制剂的体外应用,其特征在于,对所述抗肝纤维化制剂与肝星状细胞进行共培养,以抑制肝星状细胞活化或诱导所述肝星状细胞死亡。
10.根据权利要求9所述的抗肝纤维化制剂的体外应用,其特征在于,使用共培养基对所述抗肝纤维化制剂与肝星状细胞进行共培养,以占所述共培养基的体积百分比计,所述抗肝纤维化制剂的含量不低于1%。
11.根据权利要求10所述的抗肝纤维化制剂的体外应用,其特征在于,所述共培养基包括肝星状细胞活化剂。
12.根据权利要求1所述的抗肝纤维化制剂在制备抗肝纤维化药物方面的应用,其特征在于,包括:
使用所述抗肝纤维化制剂干预肝纤维化体外类器官模型后获取离体样本;
通过所述离体样本考察所述抗肝纤维化制剂对肝纤维化的抑制作用。
13.根据权利要求12所述的抗肝纤维化制剂在制备抗肝纤维化药物方面的应用,其特征在于,所述肝纤维化体内动物模型为硫代乙酰胺诱导或四氯化碳诱导的哺乳动物肝硬化模型。
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