CN115109740A - 肝细胞调控制剂及其制备方法和应用 - Google Patents
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Abstract
本发明提供了肝细胞调控制剂,包含人肝来源细胞的培养上清,所述人肝来源细胞具有前体细胞特征,所述人肝来源细胞的培养上清包含培养分泌物,能够有效促进肝细胞增殖。本发明还提供了所述肝细胞调控制剂的制备方法和应用。
Description
技术领域
本发明涉及生物技术领域,尤其涉及肝细胞调控制剂及其制备方法和应用。
背景技术
急性肝衰竭(Acute Liver Failure,ALF)是一种以广泛性肝细胞坏死,肝功能急性恶化及继发多功能脏器衰竭为特征的疾病,具有高发病率和死亡率。目前,原位肝移植是最有效的治疗手段,但由于供体缺乏,手术禁忌症以及严重并发症等原因导致等待肝移植的患者死亡。因此,寻找治疗急性肝衰竭的有效措施是亟待解决的临床问题。
因此,有必要开发肝细胞调控制剂以解决现有技术中存在的上述问题。
发明内容
本发明的目的在于提供肝细胞调控制剂及其制备方法和应用,以有效促进肝细胞再生。
为实现上述目的,本发明的肝细胞调控制剂包含人肝来源细胞的培养上清,所述人肝来源细胞具有前体细胞特征,所述人肝来源细胞的培养上清包含培养分泌物,能够有效促进肝细胞增殖。
优选的,所述培养分泌物包括至少一种miRNA,所述至少一种miRNA为miRNA-182、miRNA-183和miRNA-574的至少一种。
优选的,所述至少一种miRNA来源于人肝细胞来源外泌体。
优选的,所述人肝细胞来源外泌体来源于人原代肝细胞、人肝前体细胞和人肝前体样细胞的任意一种。
优选的,还包含稀释剂,以混匀所述至少一种miRNA。
进一步优选的,所述稀释剂为生理盐水、复方电解质溶液和PBS溶液的至少一种,以形成注射液。
进一步优选的,每微升所述注射液含1-200微克所述人肝细胞来源外泌体。
进一步优选的,还包含转染增强剂,以提高所述至少一种miRNA的稳定性和作用效率。
本发明的肝细胞调控制剂的制备方法包括:对人肝细胞进行体外培养得到培养上清,从所述培养上清中提取所述人肝细胞来源外泌体,以有效促进肝细胞增殖。
优选的,所述人肝细胞为人原代肝细胞、人肝前体细胞和人肝前体样细胞的任意一种。
优选的,对人肝细胞进行体外培养得到培养上清的步骤包括:使用由无血清类物质培养基和血清类物质组成的含血清类物质培养基对所述人肝细胞培养至融合度不低于90%后,使用所述无血清类物质培养基替代所述含血清类物质培养基继续培养以得到培养上清。
优选的,所述含血清类物质培养基由所述无血清类物质培养基和血清类物质组成,所述无血清类物质培养基包含基础培养基、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。
进一步优选的,所述无血清类物质培养基还包括N-乙酰-L-半胱氨酸、抗坏血酸的至少一种。
进一步优选的,所述人肝细胞为人原代肝细胞、人肝前体细胞和人肝前体样细胞的任意一种。
进一步优选的,所述血清类物质占所述含血清类物质培养基的体积含量为1-20%。
优选的,以占所述无血清类物质培养基的含量计:
优选的,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%。
优选的,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。
优选的,所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的至少一种。
优选的,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。
优选的,所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的至少一种。
本发明还提供了所述肝细胞调控制剂在体外培养方面的应用,所述肝细胞调控制剂与原代肝细胞共培养以促进肝细胞增殖。
本发明还提供了所述肝细胞调控制剂在制备肝衰竭治疗类药物方面的应用,使用所述肝细胞调控制剂干预肝衰竭体外类器官模型以促进肝组织再生。
附图说明
图1为实施例1的PHH Exo样本的透射电镜照片;
图2为实施例1的Hep Exo样本的透射电镜照片;
图3为实施例1的PHH Exo样本和Hep Exo样本中外泌体平均粒径对比图;
图4为实施例1通过流式分析检测不同细胞来源外泌体的CD63和CD81表达情况对比图;
图5为实施例1通过蛋白质印迹测试考察的不同细胞以及不同细胞来源外泌体的CD63和TSG101的表达情况照片;
图6为实施例2的PHH Exo样本和Hep Exo样本在标记后、染色后以及共培养后的免疫荧光共聚焦检测结果照片;
图7为实施例3的不同外泌体浓度的PHH Exo样本和Hep Exo样本共培养后得到的细胞的BrdU掺入作用对比图;
图8为实施例3对PHH Exo样本和Hep Exo样本分别与PHHs进行共培养后得到的细胞进行EdU荧光染色后得到的免疫荧光照片;
图9为实施例3对PHH Exo样本和Hep Exo样本分别与PHHs进行共培养后得到的细胞进行Ki67免疫荧光染色得到的免疫荧光照片;
图10为实施例4对PHH Exo-cell和Hep Exo-cell进行流式细胞周期分析得到的流式分析结果对比图;
图11为实施例4通过对对照组、PHH Exo-cell和Hep Exo-cell进行实时荧光定量PCR分析得到的细胞周期相关分子miRNA表达水平对比图;
图12为实施例4使用蛋白质印迹测试考察对照组、PHH Exo-cell和Hep Exo-cell表达细胞周期相关分子得到的印记照片对比图;
图13为实施例5的不同细胞来源外泌体miRNA测序表达聚类图;
图14为实施例5的原代肝细胞体外转染hsa-miR-182,hsa-miR-183,hsa-miR-149,hsa-miR-215,hsa-miR-574,hsa-miR-654和hsa-miR-675的48小时后使用ELISA检测各转染细胞的BrdU掺入作用对比图;
图15为实施例5的原代肝细胞分别转染hsa-miR-182,hsa-miR-183和hsa-miR-574的48小时后分别进行EdU染色后得到的EdU荧光显微照片;
图16为实施例5的原代肝细胞分别转染hsa-miR-182,hsa-miR-183和hsa-miR-574的48小时后,使用EdU荧光法检测得到的各转染细胞EdU掺入率对比情况。
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肝前体样细胞在肝再生微环境重塑方面的应用,包括:获得人肝来源细胞的培养上清并作用于至少一种肝病适应症的相关细胞,以对所述相关细胞起到调控作用。
一些具体的实施例中,所述肝病适应症为肝衰竭。
本发明实施例中,所述人肝来源细胞的培养上清包含培养分泌物,能够有效促进肝细胞增殖。
一些实施例中,所述培养分泌物为胞外囊泡和分泌蛋白的至少一种。
一些实施例中,所述胞外囊泡具体为外泌体。所述外泌体为人肝细胞来源的外泌体。
更具体的,所述人肝细胞为人原代肝细胞、人肝前体细胞和人肝前体样细胞的任意一种。
一些实施例中,所述外泌体包含至少一种microRNA(简记为miRNA),包括miRNA-182、miRNA-183、miRNA-149、miRNA-215、miRNA-574、miRNA-654和miRNA-675的至少一种。所述至少一种miRNA通过加快原代肝细胞由G1期向S期及G2/M期转化的细胞周期进程来促进肝细胞增殖。
本发明实施例提供了一种肝细胞调控制剂,包含稀释剂和所述至少一种miRNA。
一些实施例中,所述肝细胞调控制剂还包含转染增强剂,以提高所述至少一种miRNA的稳定性和作用效率。
一些实施例中,所述稀释剂为生理盐水、复方电解质溶液和PBS溶液的至少一种,使所述肝细胞调控制剂作为注射液使用,每微升所述肝细胞调控制剂含有1-200微克所述外泌体。
本发明实施例还提供了所述外泌体的制备方法,包括:使用含血清类物质培养基对所述人肝细胞培养至融合度不低于90%后,更换无血清类物质培养基继续培养至少24小时并得到培养上清,从所述培养上清中提取所述外泌体。
本发明实施例中,所述含血清类物质培养基由所述无血清类物质培养基和血清类物质组成,所述血清类物质占所述含血清类物质培养基的体积含量为1-20%。
本发明实施例中,所述无血清类物质培养基由基础培养基、无血清添加剂、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。所述无血清添加剂为N2和B27。
具体的,以占所述无血清类物质培养基的体积计: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,所述营养补充剂的含量为0.1-20%。
一些实施例中,所述含血清类物质培养基中血清类物质为动物源血清。
一些实施例中,所述动物源血清为胎牛血清。
一些实施例中,所述含血清类物质培养基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。
一些具体的实施例中,所述基础培养基为Hep-X基础培养基、DMEM-高糖培养基、DMEM-低糖培养基、DMEM/F12培养基、MEM培养基、William’sMedium E培养基、Ham’s F-10培养基、Ham’s F-12培养基、IMDM培养基、McCoy’5A培养基、RPMI-1640培养基、BME培养基、M-199 Medium培养基和Leibovitz Medium培养基的至少一种。
一些具体的实施例中,所述ROCK激酶抑制剂为Fasudil、Y-27632、Thiazovivin和SB-772077-B的至少一种。
一些具体的实施例中,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。所述糖原合成酶激酶3β抑制剂为CHIR99021、BIO和TWS119的任意一种。
一些具体的实施例中,所述TGF-β信号抑制剂为RepSox、SB431542和A83-01的至少一种。
本发明还提供了所述肝细胞调控制剂在体外培养方面的应用,所述肝细胞调控制剂与原代肝细胞共培养以促进肝细胞增殖。
本发明还提供了所述肝细胞调控制剂在制备肝衰竭治疗类药物方面的应用,使用所述肝细胞调控制剂干预肝衰竭体外类器官模型以促进肝组织再生。
本发明一些实施例中,所述外泌体使用标记物质进行标记,所述标记物质包括PKH67、PKH26、DiO、DiI、DiR、FM 4-64、DiD、萤火虫荧光素酶(Fluc)、GFP-荧光素酶(Gluc)和RFP-荧光素酶(Rluc)的任意一种。
本发明一些实施例中,体内诱导实验使用的回输液注射量按实验动物的体重计,每公斤注射量为0.1微克-100毫克。
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。
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。
以下通过具体的实施例进行详细说明:
实施例1
本实施例以人原代肝细胞(简记为PHHs)和人肝前体样细胞(简记为HepLPCs)作为种子细胞,采用含血清类物质培养基和无血清类物质培养基进行培养后成功分离出粒径100纳米左右,并表达外泌体标志蛋白TSG101、CD63和CD81的外泌体。
本实施例的PHHs购自广州深圳立沃科技有限公司,批号为Lot#201904001;HepLPCs来源于赛立维生物科技有限公司,批号为XLV-19006;Hep-X基础培养基来源于上海源培生物科技有限公司;胎牛血清、1%青霉素-链霉素溶液以及鼠尾胶原均来源于Gibco;肝细胞生长因子HGF来源于近岸生物;上皮细胞生长因子EGF来源于近岸生物;ROCK激酶抑制剂Y-27632来源于陶术生物;Wnt信号通路激动剂CHIR-99021来源于陶术生物;TGF-β信号抑制剂A-8301来源于陶术生物;CD63-FITC和CD81-PE流式抗体均来源于美国BDbioscience。
使用的含血清类物质培养基组成如下:Hep-X基础培养基,以及以占Hep-X基础培养基体积计,含量为1%的N2营养补充剂(100X),含量为1%的B27营养补充剂(50X)、10%胎牛血清FBS、含量为1%的1%青霉素-链霉素溶液;含量为20ng/mL的肝细胞生长因子HGF,含量为50ng/mL的上皮细胞生长因子EGF,含量为10μM的ROCK激酶抑制剂Y-27632,含量为3μM的Wnt信号通路激动剂CHIR-99021,含量为1μM的TGF-β信号抑制剂A-8301。
使用的无血清类物质培养基组成为所述含血清类物质培养基去除胎牛血清后的组成成分。
上述含血清类物质培养基和无血清类物质培养基使用前均经0.22微米滤器过滤以去除杂质。
本实施例提供了从两种种子细胞中分别获取包含外泌体的沉淀物质的过程,具体为:
以1×105个/平方厘米的接种密度将种子细胞接种于15cm培养皿中,每孔加2毫升含血清类物质培养基培养至细胞融合度不低于95%且生长状态良好,完成扩增培养。所述扩增培养的过程中,每2-3天更换一次含血清类物质培养基。
所述扩增培养完成后,将15cm培养皿中的培养基更换为无血清类物质培养基继续培养48小时后收取培养上清。使用美国System Biosciences公司的外泌体分离试剂盒ExoQuickULTRA EV Isolation从培养上清中分离出来源于的PHHs的沉淀物质以及来源于HepLPCs的沉淀物质。具体的操作步骤记载于外泌体分离试剂盒附带的说明中,在此不做赘述。
本实施例利用透射电镜、纳米颗粒追踪检测和流式分析对上述两种沉淀物质进行分析。来源于的PHHs的沉淀物质简记为PHH Exo样本,来源于HepLPCs的沉淀物质简记为HepExo样本。
将PHH Exo样本和Hep Exo样本稀释后使用含1%戊二醛,浓度为0.1M的磷酸盐缓冲液固定后滴加在铜网格上,然后使用1%的醋酸铀酰负染,室温干燥后用透射电镜观察拍照,得到图1和图2分别所示的PHH Exo和Hep Exo样本的透射电镜对比照片,磷酸盐缓冲液的pH为7.4。
利用德国Particle Metrix的PMX110纳米颗粒跟踪分析仪分别对PHH Exo样本和Hep Exo样本进行分析,得到图3所示的两种样本中颗粒平均粒径对比图。具体的检测和分析方法为本领域技术人员的常规技术手段,在此不做赘述。
参照图1和图2,PHH Exo样本和Hep Exo样本中颗粒直径大小约100纳米,且呈现形态规则的类圆形,进一步的参照图3,PHH Exo样本中颗粒平均粒径为135±9.103nm,HepExo样本中颗粒平均粒径为136.4±4.323nm,符合外泌体的形态特征。
使用PBS溶液对PHH Exo样本和Hep Exo样本分别稀释并混匀后,对一部分用CD63-FITC和CD81-PE流式抗体染色,另一部分未染色的PHH Exo样本和Hep Exo样本作为阴性对照,将上述样本在美国BD bioscience的Accuri C6flow cytomenter进行上机检测,得到图4所述的流式分析结果。具体操作和分析步骤为本领域技术人员的常规技术手段,在此不做赘述。
本实施例对经体外培养后得到的两种细胞产物(简记为PHH和Hep)、PHH Exo样本以及Hep Exo样本和培养上清产物通过BCA法进行蛋白定量分析,并进行蛋白质印迹(Western Blotting,WB)测试,得到图5所示的各样品的CD63、CD81、TSG101、EEA1、GRP78和β-actin的表达情况对比照片。其中,对细胞产物进行裂解的方法为:吸除培养上清后对细胞沉淀物使用PBS缓冲液洗涤入12孔板并加入适量RIPA裂解液后收集细胞,并在冰上裂解后于4摄氏度下12000rpm离心10min以收集上清作为测试样品。其中,蛋白定量使用碧云天生物技术有限公司的SDS-PAGE蛋白上样缓冲液(5×)以及美国Thermo Fisher的PierceTMBCAProteinAssay Kit试剂盒;使用南京诺唯赞生物科技有限公司的高敏型ECL化学发光检测试剂盒和美国BIO-RAD的ChemiDoc化学发光成像仪进行WB测试。具体操作和分析步骤为本领域技术人员的常规技术手段,在此不做赘述。
参照图4,PHH Exo样本和Hep Exo样本均阳性表达外泌体标志蛋白CD63和CD81,经统计PHH Exo样本和Hep Exo样本的CD63阳性率分别为61.85±3.465%和90.85±2.475%,PHH Exo样本和Hep Exo样本的CD81阳性率分别为69.90±4.95%和89.40±1.273%。参照图5,相较于细胞裂解产物Cell Iysate组的各样品,Exosome组的PHH Exo样本和Hep Exo样本均阳性表达了外泌体标志蛋白CD63和TSG101。
实施例2
本实施例对实施例1的PHH Exo样本和Hep Exo样本进行标记,然后与PHHs共培养,考察外泌体在肝细胞胞质内的表达情况,证明来源于PHHs和HepLPCs的外泌体可成功被肝细胞摄取。
以加入PBS缓冲溶液的PHHs为阴性对照,PHH Exo样本和Hep Exo样本使用PBS缓冲溶液进行稀释,使用美国Sigma的PKH26 Red Fluorescent Cell Linker Kit试剂盒分别标记稀释后的PHH Exo样本和不同浓度的Hep Exo样本,然后分别与PHHs共孵育24小时以完成共培养。共培养完毕后,取含细胞的培养物使用含1%戊二醛,浓度为0.1M的磷酸盐缓冲液固定后进行DAPI染色,然后在荧光显微镜下观察,得到图6所示的PHH Exo样本和Hep Exo样本在标记后、染色后以及共培养后的免疫荧光共聚焦检测结果。具体的标记步骤由试剂盒提供。图6显示,无论是PHHs来源还是HepLPCs来源的外泌体在肝细胞胞质内都有明显表达,即可以成功被肝细胞摄取。
实施例3
本实施例提供了包含外泌体的肝细胞调控制剂在体外培养方面的应用。具体的,将实施例1的PHH Exo样本和Hep Exo样本使用缓冲溶液稀释后与PHHs共培养,对得到的细胞通过BrdU ELISA检测和EdU荧光法进行增殖分析,以及通过免疫荧光检测Ki67阳性表达细胞的表达情况,证明PHHs来源和HepLPCs来源的外泌体均能够促进肝细胞增殖,且HepLPCs来源的外泌体促进肝细胞增殖的效果更为显著。
首先,将PHH Exo样本和Hep Exo样本分别用PBS缓冲液稀释为不同浓度,分别为0、1、10和100微克/毫升,分别与PHHs共培养24小时,PHHs接种密度为1×105个/平方厘米,培养容器使用12孔板。
共培养结束后,使用香港Abcam的BrdU Cell Proliferation ELISA Kit试剂盒通过ELISA检测获取波长为450纳米的OD值,进一步统计BrdU掺入作用,得到图7所示的不同外泌体浓度的PHH Exo样本和Hep Exo样本共培养后得到的细胞的BrdU掺入作用对比图,其中:每组浓度所对应的两个柱形图中,左侧为PHH Exo样本,右侧为Hep Exo样本。具体的检测步骤由试剂盒提供。ELISA检测和结果统计方法为本领域技术人员的常规技术手段。
共培养结束后,使用广州锐博生物技术公司的EdU Apollo 567 In VitroImaging Kit试剂盒对100微克/毫升外泌体浓度的样本经共培养得到的细胞进行EdU标记、细胞固定、Apollo染色以及DNA染色,DNA染色后使用荧光显微镜观察,并以PBS缓冲液作为阴性对照,得到图8所示的免疫荧光照片。具体的EdU标记、细胞固定、Apollo染色以及DNA染色步骤由试剂盒提供。
共培养结束后,对得到的各细胞进行Ki67免疫荧光染色得到图9所示的免疫荧光照片。
参照图7,浓度为100微克/毫升外泌体浓度的PHH Exo样本和Hep Exo样本对PHHs的增殖作用显著。进一步参照图8和图9,与阴性对照组相比,PHH Exo样本和Hep Exo样本均促进了肝细胞的增殖,从图8和图9中统计得到,Hep Exo样本和PHH Exo样本的EdU掺入率分别为19.89±1.049%和27.09±3.308%,Ki67阳性细胞百分比分别为38.7±2.406%和55.75±6.014%。
EdU和BrdU是胸腺嘧啶核苷类似物,在DNA复制时期代替胸腺嘧啶(T)渗入正在合成的DNA分子中,用来检测DNA复制活性。Ki67是增殖细胞的相关抗原,主要用于标记增殖周期中的细胞。从本实施例中可以看到,由于PHHs来源和HepLPCs来源的外泌体均能够促进肝细胞的增殖,由上述任意一种外泌体与稀释剂组合形成的肝细胞调控制剂能够作为促进肝细胞增殖的培养基使用。
一些实施例中,稀释剂为重悬液,外泌体的浓度为10-200微克/毫升。一些具体的实施例中,重悬液为PBS缓冲溶液、生理盐水和复方电解质溶液的任意一种。
实施例4
本实施例以实施例3中HepLPCs来源的外泌体经与PHHs共培养得到的细胞为例进行细胞周期分析和细胞周期相关分子的表达情况分析,证明了HepLPCs来源的外泌体通过加快细胞周期进程促进了肝细胞的增殖。
使用胰酶分别消化实施例3中PHH Exo样本和Hep Exo样本与PHHs共培养得到的细胞(分别简记为PHH Exo-cell和Hep Exo-cell),采用美国Abcam的Propidium Iodide FlowCytometry Kit试剂盒和美国BD bioscience的BD FACS Verse流式细胞仪进行细胞周期分析,检测激发波长488nm波长处检测红色荧光,同时检测光散射情况,用Flowjo软件进行细胞DNA含量和光散射分析,得到图10所示的细胞周期分析结果,其中的对照组为PHHs和等体积PBS缓冲液。具体的上机前操作步骤由试剂盒提供。
使用南京诺唯赞生物科技有限公司的HiScript III 1st Strand cDNASynthesis Kit以及ChamQ SYBR Color qPCR Master Mix在德国Roche的LightCycler480I实时荧光定量PCR仪对对照组、PHH Exo-cell和Hep Exo-cell进行实时荧光定量PCR分析,得到图11所示的细胞周期相关分子miRNA表达水平对比图,其中:每种细胞周期相关分子对应的三个柱状图,自左向右依次为对照组、PHH Exo样本和Hep Exo样本。
使用蛋白质印迹(Western Blotting,WB)测试考察对照组、PHH Exo-cell和HepExo-cell表达细胞周期相关分子的情况,得到图12所示的细胞周期相关分子miRNA表达情况对比图。具体操作步骤请参见实施例1论述的WB测试步骤。
参照图10,与对照组以及PHH Exo-cell相比,Hep Exo-cell的G0期和G1期细胞比例分别下降15.6±1.353%和10.733±0.874%(P<0.01),S期细胞比例分别增加6.47±0.97%和4.17±1.527%(P<0.01),G2/M期细胞比例分别增加14.9±1.413%和9.133±2.101%,表明Hep Exo样本加快了原代肝细胞G1期向S期及G2/M期进程,从而促进了细胞增殖。
参照图11和图12,细胞周期相关蛋白CyclinA2,Cyclin D1,Cyclin E表达明显上调,而p27 kip1表达明显下调,说明Hep Exo样本可能通过增加cyclin家族蛋白同时抑制p27蛋白表达,促进细胞周期进程,从而促进肝细胞增殖。
实施例5
本实施例对实施例1的PHH Exo和Hep Exo样本中外泌体的miRNA进行了提取,并通过外泌体miRNA高通量测序分析、测序生信分析以及miRNA mimic体外转染原代肝细胞后的BrdU ELISA测试和EdU增殖分析证明,外泌体中表达显著升高且能够有效促进肝细胞增殖的miRNA为hsa-miR-182、hsa-miR-183以及hsa-miR-574。
使用美国Invitrogen的Total Exosome RNA and Protein Isolation Kit进行miRNA的提取,得到PHH Exo来源的分析样本PHH-Exo-mi和Hep Exo来源的分析样本HepExo-mi,然后使用南京诺唯赞生物科技有限公司的miRNA 1st Strand cDNA SynthesisKit、miRNA Universal SYBR qPCR Master Mix以及HiScript III 1st Strand cDNASynthesis Kit和ChamQ SYBR Color qPCR Master Mix先后连接3’端和5’的接头,反转录成cDNA,再进行PCR扩增。PCR扩增后切胶回收目的片段文库,将质检合格的文库通过美国Illumina的Illumina HiSeqTM 2500高通量测序仪测序分析,得到图13所示的聚类热图,提供了上调或下调差异表达前15个miRNA。
本实施例选取了7个表达明显上调的miRNA,分别记为hsa-miR-182,hsa-miR-183,hsa-miR-149,hsa-miR-215,hsa-miR-574,hsa-miR-654和hsa-miR-675,通过原代肝细胞体外转染miRNAmimic增强miRNA表达,然后进行BrdU ELISA检测BrdU掺入作用,得到图14所示的转染各miRNA细胞的BrdU掺入作用对比图,其中NC为空白转染组。体外转染使用广州锐博生物技术公司的miRNAmimic/inhibitor),BrdU ELISA检测BrdU掺入作用的过程以及使用的试剂盒请参见实施例3。
本实施例进一步对转染hsa-miR-182,hsa-miR-183和hsa-miR-574的原代肝细胞分别进行EdU染色后,通过EdU荧光法检测EdU掺入率,得到图15所示的EdU荧光显微照片以及图16所示的EdU掺入率对比图。具体的检测用试剂盒请参见实施例3。
参照图14,7个表达明显上调的miRNA中,hsa-miR-182,hsa-miR-183和hsa-miR-574对原代肝细胞增殖具有明显的促进作用(p<0.05)。参照图15和图16,与NC mimic转染组相比,hsa-miR-183mimic体外转染的EdU掺入率明显增多,且明显高于hsa-miR-182和hsa-miR-574mimic转染组,NC mimic、hsa-miR-182、hsa-miR-183以及hsa-miR-574mimic转染组的EdU掺入率分别为10.04±2.946%,18.22±2.67%,29.46±4.799%和14.6±3.173%。
Claims (12)
1.一种肝细胞调控制剂,其特征在于,包含人肝来源细胞的培养上清,所述人肝来源细胞具有前体细胞特征,所述人肝来源细胞的培养上清包含培养分泌物,能够有效促进肝细胞增殖。
2.根据权利要求1所述的肝细胞调控制剂,其特征在于,所述培养分泌物包括至少一种miRNA,所述至少一种miRNA为miRNA-182、miRNA-183和miRNA-574的至少一种。
3.根据权利要求2所述的肝细胞调控制剂,其特征在于,每微升所述肝细胞调控制剂含有1-200微克所述至少一种miRNA。
4.根据权利要求2所述的肝细胞调控制剂,其特征在于,还包含稀释剂,所述稀释剂为生理盐水、复方电解质溶液和PBS溶液的至少一种。
5.根据权利要求2所述的肝细胞调控制剂,其特征在于,还包含转染增强剂,以提高所述至少一种miRNA的稳定性和作用效率。
6.根据权利要求2所述的肝细胞调控制剂,其特征在于,所述至少一种miRNA来源于人肝细胞来源外泌体。
7.一种肝细胞调控制剂的制备方法,其特征在于,包括:
使用含血清类物质培养基对人肝细胞进行体外培养至融合度不低于90%后,使用无血清类物质培养基替换所述含血清类物质培养基后继续体外培养得到培养上清;
所述含血清类物质培养基由所述无血清类物质培养基和血清类物质组成,所述无血清类物质培养基包含基础培养基、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。
8.根据权利要求7所述的肝细胞调控制剂的制备方法,其特征在于,所述无血清类物质培养基还包括N-乙酰-L-半胱氨酸、抗坏血酸的至少一种。
9.根据权利要求7所述的肝细胞调控制剂的制备方法,其特征在于,所述人肝细胞为人原代肝细胞、人肝前体细胞和人肝前体样细胞的任意一种。
10.根据权利要求7所述的肝细胞调控制剂的制备方法,其特征在于,所述血清类物质占所述含血清类物质培养基的体积含量为1-20%。
11.根据权利要求7所述的肝细胞调控制剂的制备方法,其特征在于,以占所述无血清类物质培养基的含量计:
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%。
12.如权利要求1所述的肝细胞调控制剂在体外培养方面的应用,其特征在于,将所述肝细胞调控制剂与原代肝细胞共培养以促进肝细胞增殖。
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CN115105530A (zh) | 2022-09-27 |
WO2022188788A1 (zh) | 2022-09-15 |
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