CN110013544A - 小分子组合在制备治疗慢性肝损伤的药物中的应用 - Google Patents
小分子组合在制备治疗慢性肝损伤的药物中的应用 Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及医药技术领域,具体是小分子组合在制备治疗慢性肝损伤的药物中的应用。本发明首次发现联合培养基中的小分子HGF、Y‑27632、A‑83‑01和CHIR99021(HACY),可有效增加小鼠肝脏体内的CD24+肝前体细胞的数量,并具有逆转由CCL4诱导的肝纤维化,恢复受损的肝功能。与肝前体细胞移植治疗相比,小分子组合HACY的体内应用将更具有临床实用价值,为干细胞移植治疗慢性肝损伤提供了新的思路和替代治疗方案,对中止慢性肝纤维化进一步恶化演变为肝硬化肝癌进程具备一定治疗应用意义。
Description
技术领域
本发明涉及医药技术领域,具体地说,是联用培养基中的可溶性小分子组合:HGF、Y-27632、A-83-01和CHIR99021(HACY),诱导内源性肝细胞来源的肝前体样细胞(HepLPCs),逆转肝纤维化进程,为干细胞移植治疗慢性肝损伤提供新的思路和替代治疗方案。
背景技术
在慢性损伤过程中,肝细胞通过逐渐转化为肝前体样细胞去逃避损伤,并在损伤停止后重新分化为成熟的肝细胞,这是一个可逆的过程。那我们是否可以利用这个可逆转化过程,通过诱导肝细胞转化为肝前体样细胞方式,帮助肝脏迅速修复损伤?已有研究表明,通过体外分离扩增EpCAM+CD24+CD133+肝前体细胞并回输慢性纤维化肝脏,不但可以补充肝实质细胞,更能有效缓解肝脏纤维化进程。这提示了我们通过补充肝干细胞/前体细胞的治疗方式,将有助于缓解慢性肝纤维化损伤,逆转肝脏失代偿。2017年,我们团队发现体外通过小分子组合可诱导成熟肝细胞转化为具有增殖能力的肝前体样细胞,并具有分化为成熟肝细胞的潜能。这种体外通过小分子组合高效活化肝细胞重编程相关信号通路所产生的前体细胞,安全性及均一性高,在慢性肝脏纤维化中或具有一定临床应用价值。(参考文献:Lu,W.Y.,T.G.Bird,L.Boulter,A.Tsuchiya,A.M.Cole,T.Hay,R.V.Guest,D.Wojtacha,T.Y.Man,A.Mackinnon,R.A.Ridgway,T.Kendall,M.J.Williams,T.Jamieson,A.Raven,D.C.Hay,J.P.Iredale,A.R.Clarke,O.J.Sansom,and S.J.Forbes,Hepatic progenitorcells of biliary origin with liver repopulation capacity.Nat Cell Biol,2015.17(8):p.971-983;Wu,H.,X.Zhou,G.B.Fu,Z.Y.He,H.P.Wu,P.You,C.Ashton,X.Wang,H.Y.Wang,and H.X.Yan,Reversible transition between hepatocytes and liverprogenitors for in vitro hepatocyte expansion.Cell Res,2017.27(5):p.709-712.。)
发明内容
本发明的目的在于提供可溶性小分子组合(HGF、Y-27632、A-83-01和CHIR99021,本发明中简称HACY)联用在制备治疗慢性肝损伤的药物中的应用。
Ira J.Fox在研究中发现,通过腺相关病毒表达肝细胞特异的转录因子HNF4α,可在体内重设肝功能相关信号通路,实现对终末慢性肝病的逆转。这提示通过模拟正常肝再生信号通路,有可能使再生功能被抑制的肝细胞重新获得再生潜能。因此本发明推测通过直接体内注射特定小分子组合,活化肝再生相关信号通路,提高肝细胞向肝前体细胞的转化效率,有助于减轻甚至逆转肝病进程。与本发明的推测相符,周伟杰团队近期发现通过鼠尾注射HGF/R-spondin1可诱导小鼠体内肝脏产生Lgr5+标志的肝前体细胞(参考文献:Nishikawa,T.,A.Bell,J.M.Brooks,K.Setoyama,M.Melis,B.Han,K.Fukumitsu,K.Handa,J.Tian,K.H.Kaestner,Y.Vodovotz,J.Locker,A.Soto-Gutierrez,and I.J.Fox,Resetting the transcription factor network reverses terminal chronic hepaticfailure.J Clin Invest,2015.125(4):p.1533-44.;Lin,Y.,Z.P.Fang,H.J.Liu,L.J.Wang,Z.Cheng,N.Tang,T.Li,T.Liu,H.X.Han,G.Cao,L.Liang,Y.Q.Ding,andW.J.Zhou,HGF/R-spondin1 rescues liver dysfunction through the induction ofLgr5(+)liver stem cells.Nat Commun,2017.8(1):p.1175.。)
基于在小鼠体内注射HGF可减缓BDL和CCL4诱导的肝纤维化发展进程,本发明发现联合培养基中的小分子HGF、Y-27632、A-83-01和CHIR99021(HACY),可有效增加小鼠肝脏体内的CD24+肝前体细胞的数量,并具有逆转由CCL4诱导的肝纤维化,恢复受损的肝功能。与肝前体细胞移植治疗相比,小分子组合HACY的体内应用将更具有临床实用价值,对中止慢性肝纤维化进一步恶化演变为肝硬化肝癌进程具备一定治疗应用意义。
为了实现上述目的,本发明的第一方面,提供HGF、Y-27632、A-83-01和CHIR99021联用在制备治疗慢性肝损伤的药物中的应用。
进一步的,所述的HGF为肝细胞生长因子;所述的Y-27632、A-83-01和CHIR99021为常用的用于肝细胞体外培养扩增的小分子添加物。
在本发明的优选实施方式中,所述的HGF购于Peprotech公司;而其余的所述的Y27632、A83-01和CHIR99021均购于TargetMol公司。
进一步的,所述的治疗慢性肝损伤的药物为逆转或缓解肝纤维化进程的药物。
进一步的,所述的治疗慢性肝损伤的药物为促进肝脏再生修复的药物。
进一步的,所述的治疗慢性肝损伤的药物为诱导内源性肝细胞来源的肝前体样细胞的药物。
进一步的,所述的治疗慢性肝损伤的药物是以HGF、Y-27632、A-83-01和CHIR99021为活性成分的药物组合物,或是包含HGF、Y-27632、A-83-01和CHIR99021的药物组合物。
进一步的,所述的药物组合物中HGF、Y-27632、A-83-01和CHIR99021的重量比为1:1:1:1。
进一步的,所述的治疗慢性肝损伤的药物中HGF、Y-27632、A-83-01和CHIR99021的给药剂量分别为5-50μg/kg。
本发明的第二方面,提供一种治疗慢性肝损伤的药物,所述的药物是以HGF、Y-27632、A-83-01和CHIR99021为活性成分的药物组合物,或是包含HGF、Y-27632、A-83-01和CHIR99021的药物组合物。
进一步的,所述的药物组合物中HGF、Y-27632、A-83-01和CHIR99021的重量比为1:1:1:1。
所述的药物中还包括常用的药物辅料。所述的药物辅料为稀释剂、赋形剂、粘合剂、填充剂、崩裂剂、香味剂、甜味剂中的一种或两种以上。所述的药物的剂型为注射剂。
本发明优点在于:
本发明首次发现联合培养基中的小分子HGF、Y-27632、A-83-01和CHIR99021(HACY),可有效增加小鼠肝脏体内的CD24+肝前体细胞的数量,并具有逆转由CCL4诱导的肝纤维化,恢复受损的肝功能。与肝前体细胞移植治疗相比,小分子组合HACY的体内应用将更具有临床实用价值,为干细胞移植治疗慢性肝损伤提供了新的思路和替代治疗方案,对中止慢性肝纤维化进一步恶化演变为肝硬化肝癌进程具备一定治疗应用意义。
附图说明
图1:CD24+HepLPCs细胞移植促进肝纤维化修复;A:HepLPCs稳定转染CAG–GFP表达载体,并通过流式分选GFP阳性细胞建立GFP-HepLPCs细胞系,其中bar=50μm;B:慢性肝纤维化损伤及移植治疗实验原理图概述;C:慢性肝纤维化损伤及移植治疗6周后小鼠肝脏组织标本的形态学相关检测,包括天狼星红染色、H&E染色、GFP染色及α-SMA染色的免疫组化图,显示GFP-HepLPCs移植组较两实验对照组具有较显著的逆转肝纤维化的功能,其中bar=600μm;D:天狼星红染色及α-SMA免疫组化染色的阳性肝脏区域统计通过ImageJ计算,一张扫描片子随机选取3个视野;E:q-PCR检测损伤肝脏标本中α-SMA及COL1α1转录水平;F:小鼠血清中检测AST、ALT等血生化学指标。
图2:小分子组合体内注射可有效增加肝脏CD24+肝前体细胞;A:CCL4损伤及HACY小分子组合治疗实验原理图概述,40天后麻醉处死进行原代肝细胞灌注分离;B、C:流式检测橄榄油空白对照组、CCL4损伤组及高低剂量HACY治疗组中小鼠原代肝细胞中CD24阳性细胞的比例情况,C图为B图的流式检测中细胞CD24阳性率统计情况;D:q-PCR检测不同组别中分离的原代肝细胞的CD24转录水平;E:为橄榄油空白对照组、单纯CCL4组和实验组中HGF组、ACY组及HACY组中肝脏CD24阳性细胞率的流式检测结果及肝脏标本的天狼星红染色结果,其中bar=600μm。
图3:小分子组合体内注射可有效缓解肝纤维化;A:通过抗体标记免疫荧光实验检测空白对照组、单纯CCL4损伤组及HACY治疗组肝脏组织中肝细胞CD24的表达情况,其中bar=100μm;B,C:流式检测上述三组中肝脏分离的原代肝细胞中CD24阳性率;D,E:H&E染色及天狼星红染色检测上述三组小鼠中肝脏的纤维化程度,bar=600μm;其中天狼星红染色的阳性肝脏区域统计通过ImageJ计算;F,G:分析血清中AST、ALT含量;H:上述3组肝脏样本中羟脯胺酸的含量检测;I:Western Blot检测上述3组肝脏样本中α-SMA蛋白水平,其中GAPDH用作内参,数字1、2、3分别代表同一实验组中随机3只不同小鼠肝脏样本。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例:
一、实验材料
本发明所用的重组蛋白HGF购于Peprotech公司;而其余的小分子化合物如Y-27632、A-83-01和CHIR99021均购于TargetMol公司;所用的细胞皆来源于新鲜肝脏组织分离而来原代肝细胞,并使用特定的细胞培养基(即TEM增殖培养基)培养,分离原代细胞中所用IV型胶原酶:购于Sigma公司;青霉素与链霉素双联合抗生素购买自博锐生物医药公司;William's E培养基及HANKs溶液购于上海源培生物公司;用于细胞的冲洗及重悬所用PBS缓冲液(Phosphate Buffer Solution),购于Gibco公司;所用的冻存细胞的生物试剂购于新赛美公司;后续用于原代肝细胞分离实验所用的培养基Dulbecco’s modified Eaglemedium(DMEM)以及胎牛血清(FBS)均购于Gibco公司;0.5M EGTA购买于碧云天(Beyotime)生物科技公司;分离原代细胞实验中用于滤过未被消化的肝脏组织的40um细胞过滤网以及培养的原代肝细胞及肝前体细胞所用的基质胶Matrigel均购于BD公司。
二、实验方法
本发明采用Graphpad7生物统计软件对相应的实验数据进行分析,采用双尾非配对t检验和one-way ANOVA方差分析比较两组或多组不同处理之间的差异。所有的统计数据,至少使用了三个独立的样品或重复实验的数据,数据均以means±s.e.m.的形式表示,其中ns为无显着性,*P<0.05被认为存在统计学差异,其中**,P<0.01,***,P<0.0001。
1、慢性肝纤维化及小分子治疗模型:
实验中所使用的6-8周,体重约为18-20g的雄性野生型C57BL/6小鼠从第二军医大学动物实验中心取回2周后开始建模。CCL4用量为2ml/kg,与橄榄油以1:4的比例配好置于常温暗处放置备用。小鼠随机分为3组,每组5只,进行每周2次的腹腔注射用橄榄油稀释后的CCL4或等量橄榄油,共构建6周,形成慢性肝纤维化损伤模型。小分子治疗模型中各小分子先分别用对应溶剂以储备浓度溶解好,并分装好置于-80℃冰箱保存,待使用前置于4℃冰箱或冰上溶解,以生理盐水按1μg/per molecule/200μl/mice的量准备好置于冰上准备腹腔注射,现配现用,并以等量生理盐水作为对照。
2、细胞移植:
细胞准备:将小鼠HepLPCs或贴壁4小时的小鼠原代肝细胞用Accutase消化后,先用生理盐水清洗2遍,按1×107/ml的浓度用William's E培养基重悬细胞,置于冰上保存。小鼠的肝细胞脾脏移植模型步骤具体如下:先同配置好的三溴乙醇将小鼠麻醉,以左侧卧位在腹部寻找脾脏位置,并通过剃毛机剔除干净脾脏的位置周围的毛发,然后剪刀在皮层、结缔组织层、黏膜层剪开一小切口,寻找并暴露脾脏,用镊子轻轻将脾头抽出后,手术线环绕脾头部打一松结。在冰上取出细胞,轻轻混匀细胞悬液,再通过胰岛素针去吸取200μl悬液,通过镊子夹紧位于松结下的脾头处,平行推进针尖,缓慢注射细胞。待悬液完全注入脾脏后,退针并扎紧脾头处线结防止血液及细胞外渗,同时去除线头,用棉签轻轻将脾推回腹部。最后分层将切口缝合。手术结束后观察小鼠状态,对照组以同样手术方式注射等量的William's E培养基。术后连续3天密切观察并记录好小鼠伤口恢复情况及健康活动状态。
3、流式检测及分选:
细胞膜上抗体孵育方法:新鲜分离的小鼠原代肝细胞或HepLPCs用PBS清洗2-3次后,用PBS以1×106/100μl浓度重悬并按比例加入CD24、CD45、CD90及Ter119抗体,置于4℃孵育30分钟;细胞质内抗体孵育方法:按照Invitrogen公司的流式间标试剂盒的操作步骤,先用细胞固定液常温避光孵育细胞20分钟后,用细胞穿膜液清洗2次,并以1×106/100μl的浓度重悬细胞,按比例加入Alb及CK19抗体后置于4℃孵育30分钟,清洗2-3次后再重悬细胞,按比例加入对应的荧光二抗,室温或37℃避光孵育细胞20分钟;流式分选步骤同上。
4、免疫组化:
免疫组织切片放置于60℃烘烤60分钟后,放入自动化免疫组化脱水机器进行脱蜡至水,结束后放置于双蒸水浸泡5分钟。然后取出组织切片,并放置于3%过氧化氢溶液中浸泡20min,以灭活内源性的过氧化物酶。后双蒸水浸泡清洗3次,进行酸性抗原修复。将配置好酸修缓冲液倒入高压锅中,约1000ml,再将组织切片连同架字平放于高压锅内,注意酸修液没过所有切片,后调节高压锅至大功率模式,随着水温升高,锅内气压上升,待冒出大量白色水蒸汽,调至低功率维持高压2分钟后,关闭,打开高压锅盖待锅内液体自然冷却。然后取出切片,并用配置好的PBS缓冲液浸泡清洗3-4次,每次分钟,后准备用将1%BSA溶液滴在组织片上,37℃孵育进行封闭,提高抗体特异性。30分钟后吸走封闭液,根据抗体说明书稀释抗体,小心滴加到切片上至完全覆盖,置于4℃孵育。8-10小时后从4℃冰箱取出组织切片,为防止温度骤变脱片,置于室温下约20-30分钟后,PBS重复清洗4次,同时准备孵育二抗。滴加与一抗种属来源一致的二抗,并置于37℃,30分钟后PBS重复浸泡清洗4次,同时按1:50比例配制DAB混合液并滴加在切片组织上,可见1-5分钟后可见组织部分呈现砖红色,此时可放入双蒸水以终止进一步显色。注意等待时间不宜不超过10分钟n,以防止染色过深,进一步用后双蒸水清洗2次后,将切片放入苏木素溶液中浸泡。10分钟后在放入盐酸乙醇分化液前,用双蒸水洗去多余苏木素,切片放置于分化液约1至2s后即可取出,至自来水通过流水返蓝20-30分钟,重新放入自动化免疫组化脱水机器中脱水,最后用中性树脂封片,待风干后可在镜下观察。
5、天狼星红染色及H&E染色:
天狼星红染色及H&E染色切片的烘烤、脱蜡至水、苏木素复染及组织脱水步骤参考免疫组化,其中天狼星红染色在烘烤、脱蜡至水后,其具体处理方法主要试剂盒内说明书操作(Chondred公司);H&E按照实验室常规操作,在烘烤、脱蜡至水、苏木素复染后,放置于伊红溶液1-2分钟,然后二者均进行组织脱水,最后中性树脂封片,镜下观察。
6、细胞及组织切片免疫荧光:
首先PBS溶液冲洗两遍,快速加入常温的4%多聚甲醛溶液至完全覆盖培养皿,常温固定10分钟后,PBS清洗3次,每次5分钟,然后加入0.4%Triton穿膜液至完全覆盖培养皿,放入37℃孵箱,15分钟后用PBS清洗三次,然后加入3%BSA封闭液至完全覆盖培养皿,放入37℃孵箱孵育30分钟。然后吸走封闭液,加入稀释好的一抗,注意要完全覆盖避免干片,4℃孵育8-10小时后取出,用PBS清洗三次,然后添加一抗相对应种属的荧光二抗,常温或37℃孵育30-60分钟后用DAPI进行核复染,最后用甘油或含防止荧光淬灭的专用封片剂封片固定,可在4℃保存或直接在共聚焦显微镜下观察。
7、肝功能障碍评价
肝功能障碍根据标准肝脏生化测试确定。使用IFCC方法在自动生化分析仪上分析小鼠血清中天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)含量。另外,肝脏中羟脯氨酸含量通过使用购自南京建成生物科技有限公司市售的羟脯氨酸检测试剂盒,根据制造商的说明书测量肝组织中羟脯氨酸的量来测试总胶原蛋白含量。
三、实验结果:
1、CD24+HepLPCs细胞移植促进肝纤维化修复
首先本发明通过慢病毒荧光标记系统转染HepLPCs,使其带上GFP荧光蛋白(GFP-HepLPCs),有利于示踪体内变化,同时通过流式分选GFP阳性细胞后扩增建立GFP-HepLPCs细胞系(图1A)。然后按照图1B建立慢性肝损伤模型。而在首次注射完CCL4后2-4小时,对各组小鼠实施脾脏细胞移植治疗术,其中GFP-HepLPCs移植为实验组,等量体积的生理盐水和等量的小鼠原代肝细胞为对照组。术中小鼠以腹腔注射麻药三溴乙醇维持麻醉状态,在小鼠腹部左侧切开暴露脾脏,将约1-2×106的GFP-HepLPCs或小鼠原代肝细胞经脾脏移植进小鼠肝脏内。40天后麻醉处死小鼠,取出肝脏进行相关检测。如图1C所示,移植GFP-HepLPCs后通过免疫组化可探寻到标记GFP细胞的肝脏区域,同时,从天狼星红染色和H&E染色可见肝脏纤维化程度较原代肝细胞移植及生理盐水组两组对照组明显缓解。而通过免疫组化检测胶原纤维相关的标志物α-SMA,结果与上述形态学检测相符。进一步本发明通过统计天狼星红染色剂α-SMA的肝脏阳性区域,其中与无细胞移植组相比,GFP-HepLPCs移植组的肝脏纤维化程度减轻具有统计学意义,相反的是,原代肝细胞移植无统计学差异。进一步我们取不同组的肝脏样本进行α-SMA及COL1α1转录水平的检测比较,结果也与上述形态学实验一致。最后本发明检测小鼠血清中AST、ALT等血生化学指标,结果显示两种转移酶在GFP-HepLPCs移植组均明显下降,反映其肝脏损伤有较明显缓解。以上结果均显示GFP-HepLPCs较原代肝细胞在CCL4诱导的肝脏纤维化中具有损伤修复乃至逆转纤维化的功能。
图1:CD24+HepLPCs细胞移植促进肝纤维化修复;A:HepLPCs稳定转染CAG–GFP表达载体,并通过流式分选GFP阳性细胞建立GFP-HepLPCs细胞系,其中bar=50μm;B:慢性肝纤维化损伤及移植治疗实验原理图概述;C:慢性肝纤维化损伤及移植治疗6周后小鼠肝脏组织标本的形态学相关检测,包括天狼星红染色、H&E染色、GFP染色及α-SMA染色的免疫组化图,显示GFP-HepLPCs移植组较两实验对照组具有较显著的逆转肝纤维化的功能,其中bar=600μm;D:天狼星红染色及α-SMA免疫组化染色的阳性肝脏区域统计通过ImageJ计算,一张扫描片子随机选取3个视野;E:q-PCR检测损伤肝脏标本中α-SMA及COL1α1转录水平;F:小鼠血清中检测AST、ALT等血生化学指标。
2、小分子组合体内注射可有效增加肝脏CD24+肝前体细胞
下一步本发明探索是否可以通过运用TEM培养基中的小分子组合基础(HGF,CHIR99021,A83-01及Y27632,以下简称为HACY)直接诱导肝脏内产生更多的CD24+的肝前体细胞,促进肝脏损伤修复。
首先本发明先检测HACY这一小分子组合,是否可以有效地体内产生CD24+的肝前体细胞。为了这个目标,本发明设计了以下实验方案:建立C57小鼠肝纤维化慢性损伤模型,以橄榄油注射组为空白对照,每周注射2次2ml/kg CCL4或等体积的橄榄油。同时设立高低剂量的HACY处理组(其中高剂量组为4种成分每种注射1μg/只,而低剂量组为4种成分每种注射0.1μg/只),每周经腹腔注射三次,连续3天注射以保证其体内浓度维持,建模6周后对4组小鼠麻醉后进行原代肝细胞灌注分离并进行流式检测CD24阳性率细胞量及CD24的转录水平。如图2B、2C,HACY处理组中流式检测CD24阳性率比例及CD24转录水平均较空白组及CCL4处理组上升,而高剂量组(1μg/只)升高更为显著,具有统计学意义。
由于HGF为肝脏生长因子,具一定的肝脏保护修复作用,为探究其单用或联合ACY运用哪种方案具有更高的效率诱导CD24+肝前体细胞,本发明进一步以小分子注射1μg/permolecule/200μl/mice的剂量设计3种组合:注射HGF(H)组、注射ACY组及注射HACY组(图2E)。6周CCL4慢性肝纤维化建模及小分子治疗后,分离不同组小鼠的原代肝细胞进行流式检测CD24阳性细胞的含量,结果显示,实验组中HGF组、ACY组及HACY组中肝脏CD24阳性细胞均有不同程度升高,而以HACY组最为显著,为13.81%。可见联用HACY小分子组合,具有更高效的诱导效果,而另一方面,本发明取小鼠肝脏标本进行天狼星红染色发现,HACY组中的小鼠肝纤维化较单纯CCL4组有明显缓解,这提示我们,体内运用HACY可能有助于逆转慢性肝脏损伤,促进肝脏再生修复。
图2:小分子组合体内注射可有效增加肝脏CD24+肝前体细胞;A:CCL4损伤及HACY小分子组合治疗实验原理图概述,40天后麻醉处死进行原代肝细胞灌注分离;B、C:流式检测橄榄油空白对照组、CCL4损伤组及高低剂量HACY治疗组中小鼠原代肝细胞中CD24阳性细胞的比例情况,C图为B图的流式检测中细胞CD24阳性率统计情况;D:q-PCR检测不同组别中分离的原代肝细胞的CD24转录水平;E:为橄榄油空白对照组、单纯CCL4组和实验组中HGF组、ACY组及HACY组中肝脏CD24阳性细胞率的流式检测结果及肝脏标本的天狼星红染色结果,其中bar=600μm。
3、小分子组合体内注射可有效缓解肝纤维化
进一步,本发明深入探索小分子组合HACY的体内运用诱导CD24+肝前体细胞对慢性肝纤维化损伤模型的治疗作用。基于上述结果,本发明运用1μg/只的HACY治疗剂量探求对CCL4肝损伤模型的作用,如图3,无论是流式检测还是免疫荧光结果显示,HACY治疗组中CD24阳性率均有明显提高,同时从天狼星红染色剂HE染色中可见伴有纤维化明显的减轻。然后,本发明通过收集小鼠血清和肝脏样本,进行相关的血生化学检测及肝纤维程度的比较,与空白对照组结果相似,血清中AST及ALT水平均较单纯CCL4组下降,同时本发明观察到,羟脯胺酸水平及α-SMA蛋白水平,这两个可灵敏地反应肝脏样本中胶原纤维含量的指标,也较CCL4损伤组显著降低,与血清学损伤指标结果一致。上述结果显示,若从纤维化进展前开始小分子HACY治疗,可有效通过诱导CD24+肝前体细胞,逆转肝脏纤维化,缓解肝脏损伤程度,有助于肝脏的再生修复。
图3:小分子组合体内注射可有效缓解肝纤维化;A:通过抗体标记免疫荧光实验检测空白对照组、单纯CCL4损伤组及HACY治疗组肝脏组织中肝细胞CD24的表达情况,其中bar=100μm;B,C:流式检测上述三组中肝脏分离的原代肝细胞中CD24阳性率;D,E:H&E染色及天狼星红染色检测上述三组小鼠中肝脏的纤维化程度,bar=600μm;其中天狼星红染色的阳性肝脏区域统计通过ImageJ计算;F,G:分析血清中AST、ALT含量;H:上述3组肝脏样本中羟脯胺酸的含量检测;I:Western Blot检测上述3组肝脏样本中α-SMA蛋白水平,其中GAPDH用作内参,数字1、2、3分别代表同一实验组中随机3只不同小鼠肝脏样本。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (9)
1.HGF、Y-27632、A-83-01和CHIR99021联用在制备治疗慢性肝损伤的药物中的应用。
2.根据权利要求1所述的HGF、Y-27632、A-83-01和CHIR99021联用在制备治疗慢性肝损伤的药物中的应用,其特征在于,所述的治疗慢性肝损伤的药物为逆转或缓解肝纤维化进程的药物。
3.根据权利要求1所述的HGF、Y-27632、A-83-01和CHIR99021联用在制备治疗慢性肝损伤的药物中的应用,其特征在于,所述的治疗慢性肝损伤的药物为促进肝脏再生修复的药物。
4.根据权利要求1所述的HGF、Y-27632、A-83-01和CHIR99021联用在制备治疗慢性肝损伤的药物中的应用,其特征在于,所述的治疗慢性肝损伤的药物为诱导内源性肝细胞来源的肝前体样细胞的药物。
5.根据权利要求1所述的HGF、Y-27632、A-83-01和CHIR99021联用在制备治疗慢性肝损伤的药物中的应用,其特征在于,所述的治疗慢性肝损伤的药物是以HGF、Y-27632、A-83-01和CHIR99021为活性成分的药物组合物,或是包含HGF、Y-27632、A-83-01和CHIR99021的药物组合物。
6.根据权利要求5所述的HGF、Y-27632、A-83-01和CHIR99021联用在制备治疗慢性肝损伤的药物中的应用,其特征在于,所述的药物组合物中HGF、Y-27632、A-83-01和CHIR99021的重量比为1:1:1:1。
7.一种治疗慢性肝损伤的药物,其特征在于,所述的药物是以HGF、Y-27632、A-83-01和CHIR99021为活性成分的药物组合物,或是包含HGF、Y-27632、A-83-01和CHIR99021的药物组合物。
8.根据权利要求7所述的治疗慢性肝损伤的药物,其特征在于,所述的药物组合物中HGF、Y-27632、A-83-01和CHIR99021的重量比为1:1:1:1。
9.根据权利要求7所述的治疗慢性肝损伤的药物,其特征在于,所述的药物中还包括常用的药物辅料。
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