红景天苷诱导骨髓间充质干细胞分化为肝细胞以及治疗免疫性肝纤维化的体外、体内实验研究
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摘要
第一章绪论
肝脏是体内最大的消化腺和“化学工厂”,在糖、脂肪和蛋白质的合成、分解与代谢,形成以及分泌胆汁,调节内分泌,参与凝血以及机体的免疫以及降解机体代谢产物和解毒等多个方面发挥着重要的作用,所以肝脏一旦发病就严重威胁人类的身体健康,也是人类因疾病死亡的主要原因之一。
根据世界卫生组织截止到2005年的统计数据,全球乙型肝炎病毒感染者总数超过20亿,其中3.5亿人会成为慢性乙肝患者,每年约有100万人死于乙型肝炎感染所致的肝衰竭、肝硬化和原发性肝细胞癌。按照2008年调查乙肝表面抗原携带率7.18%推算,我国仍然有乙肝表面抗原携带者约9300万人,仅乙肝所致的肝纤维化和肝硬化的病人估计已超过3000万。所以,每年还需要投入大量的财力物力用于肝纤维化和肝硬化的治疗,给国家和个人造成了沉重的经济负担。
从治疗角度来讲,肝脏疾病的治疗不外乎以下几个方面:对因治疗(如抗病毒)、对症治疗(如抗纤维化、机体免疫调节)、保肝及营养支持、基因治疗、肝移植、人工肝支持治疗以及细胞(肝细胞或者干细胞)移植等等。但是,肝移植是目前临床上治疗肝脏疾病的最后手段,该技术日臻成熟,挽救了不少肝功能衰竭患者的生命。但由于供肝来源困难,免疫抑制剂的终生应用,手术费用昂贵等诸多因素,大大限制了肝移植在临床上的普及应用。肝细胞移植是新兴起的一种先进疗法,可部分克服肝脏移植的难题,与肝移植相比有以下优点:手术简单、创伤小,费用低廉;来源相对丰富;细胞可冻存备用,不会因供体短缺造成患者死亡;与肝移植相比,免疫原性弱,无需终生使用免疫抑制剂。
但肝细胞移植技术至今没有在临床上推广应用,其原因在于许多关键技术尚未解决,如制约肝细胞移植发展的两大瓶颈问题:细胞供应和免疫排斥。目前用于细胞移植的是同种异体成熟肝细胞,而同种异体成熟肝细胞难以在临床上大量获取;且肝细胞分离后会失去细胞间连接与信号,在体外难以培养扩增,体内移植后难以增殖并逐渐凋亡,造成长期存活的细胞量不足,难以达到预期的治疗效果。尽管同种异体细胞移植的免疫排斥反应比器官移植小,但有资料显示细胞移植后不使用免疫抑制剂,7天后移植细胞也所剩无几。
解决免疫排斥最好的办法是使用自体细胞;解决增殖问题是使用干细胞。目前的医学伦理基本上已经排除了使用胎肝细胞和胚胎干细胞的可能。最近有文献报道,作为人体重要成体干细胞之一,MSCs能在体内外分化成肝细胞,且MSCs来源丰富,这提示自体MSCs治疗肝脏疾病是一种非常有前途的新疗法。纵观目前研究MSCs的现状,无论是基础研究还是动物及临床实验,尚未有突出性的研究成果。在MSCs的分离、鉴定、定向分化及其机制、体外培养条件下MSCs性状的维持、体内移植的生物安全性问题以及MSCs疗效的综合评价等许多方面还需要深入研究。
我们课题组从体外诱导MSCs分化为肝细胞到移植MSCs治疗肝脏疾病实验,也只是试图从一个侧面继续探求MSCs体外分化的机理、新药(定向诱导作用)研发的新途径;验证MSCs治疗慢性免疫性肝纤维化的作用,建立由体外实验到体内实验的桥梁以及探讨MSCs逆转/缓解肝纤维化、改善肝脏功能的具体作用机制;也寄希望将MSCs迅速推向临床,为肝脏疾病患者带来新的福音,同时发展干细胞和中药相关产业,为医药经济开辟新的增长点。
第二章MSCs的分离、培养及鉴定
目的:优化大鼠MSCs体外分离、培养、扩增的有效方法,探讨其部分生物学特性以及分化潜能。
方法:在无菌条件下快速处死SD大鼠,收集骨髓细胞,快速悬浮于预冷的NH_4Cl低渗盐溶液中裂解红细胞,离心去上清,收集沉淀的细胞,D-Hank's充分洗涤后,用含10%的低糖型DMEM培养基进行扩增培养;传代时,采用胶原酶Ⅳ预先孵育1～2小时,再用胰蛋白酶-EDTA处理。倒置显微镜下观察细胞的一般形态结构;绘制生长曲线了解其生长特性;流式细胞学检测CD45、CD44、CD90等表面抗原的表达情况;用成骨、软骨以及脂肪方向分化的诱导液诱导MSCs向这三个方向分化,检测其多向分化潜能。
结果:原代培养的细胞接种4-6小时后有细胞贴壁,4～6天后细胞增殖速度明显增快,7～8天原代细胞汇合达80%-90%,原代及传代细胞形态学观察均成典型的成纤维样形态特征,旋涡状生长态势;传代培养的细胞在3天时增殖速度最快,5～6天时达到平台期;细胞可以连续传代20代而未见衰老现象,单个细胞可以扩增30倍左右。流式细胞仪检测显示该细胞表达MSCs表面抗原CD44和CD90等,不表达造血系干细胞的表面抗原CD45等等;经过诱导后,MSCs可以分化为成骨细胞、骨细胞和脂肪细胞,显示了该细胞具有多向分化潜能。
结论:采用低渗盐溶液裂解红细胞,离心收集MSCs,再进行常规贴壁培养可以分离更为纯化的MSCs;在含10%FBS的低糖DMEM培养基中可稳定传代20代左右,细胞的性状没有发生改变;根据细胞鉴定结果,该细胞具有以下特征:贴附塑料器皿生长的特性;表达MSCs特殊的表面抗原如CD44、CD90等;不表达造血系细胞的表面抗原如CD45等;具有向成骨细胞、脂肪细胞和成软骨细胞等胚层细胞方向分化的能力。所以,该细胞是MSCs;结合低渗裂解红细胞及常规贴壁培养方法,使得分离、培养及扩增MSCs更为方便。
第三章红景天苷诱导MSCs分化为肝细胞的体外实验研究
目的:建立并优化诱导MSCs分化为肝细胞的体外诱导系统;筛选能够诱导MSCs分化为肝细胞的中药小分子化合物;对诱导分化的肝细胞进行综合评价,为后续的实验奠定基础;探索由体外实验到体内实验的衔接过程。
方法:采用第五代的MSCs(P5)进行实验。
(1)红景天苷和成纤维生长因子(fibroblast growth factor,FGF)4联合应用对MSCs肝向分化的诱导作用
将MSCs接种到含10%胎牛血清的普通高糖型DMEM培养液中,细胞汇合后,换用含2%胎牛血清的预诱导培养液维持24小时;随后改用含2%FBS、红景天苷2μM及FGF4 10ng/ml的高糖型DMEM分化诱导培养液进行诱导。HGF及FGF4联合应用组作为阳性对照;FGF4单独诱导组作为阴性对照。倒置显微镜下每日观察诱导后细胞形态的变化;在每个诱导阶段末(第7、14、21天),收获诱导细胞,提取RNA和蛋白,检测肝细胞特异性相关基因以及蛋白的表达情况;在每个诱导阶段结束后,检测细胞的糖原合成及贮存能力、诱导性P450活性、低密度脂蛋白的摄取以及AFP、ALB以及CK18的表达情况。
(2)红景天苷对MSCs肝向分化的单独诱导作用
接种及预诱导方法如上所述。诱导液为在基础诱导液的基础上添加红景天苷至终浓度为10μM、20μM以及50μM。HGF及FGF4联合应用组作为阳性对照;FGF4单独诱导组作为阴性对照。诱导后的第7、14天分别收获细胞,检测肝特异性蛋白AFP和白蛋白的表达。
(3)红景天苷诱导MSCs肝向分化机制探讨
接种及预诱导方法如上所述。诱导液为在基础诱导液的基础上添加红景天苷至终浓度为20μM,在此基础上,依照添加的抑制剂的不同而分为三组(PI3K抑制剂组、P38抑制剂组以及ERK1/2抑制剂组)。红景天苷组(20μM)作为阳性对照;基础诱导液组作为阴性对照。诱导后的第7、14天分别收获细胞,检测肝特异性蛋白AFP和白蛋白的表达。
结果:(1)未诱导细胞传代后仍呈成纤维样,但是由漩涡状生长的特征逐步演变成过度生长的状态。被诱导的细胞密集生长,逐渐呈现菊花样的生长特征,形态趋向圆形或不规则形,有些类似于密集生长的肝细胞。RT-PCR检测显示肝细胞的相关基因如肝细胞生长因子(hepatocyte growth factor,HGF)、肝细胞生长因子受体(hepatocyte growth factor receptor,HGFR)、肝细胞核因子(hepatocyte nuclearfactors,HNF)-3β、甲胎蛋白(alpha fetoprotein,AFP)、白蛋白(Albumin,ALB)、细胞角蛋白(cytokeratin,CK)18和转甲状腺素蛋白(transthyretin,TTR)等在诱导过程中表达逐渐产生或被激活,在同一时期的表达量(或活性)均明显高于FGF-4组。Western Blot检测结果显示肝细胞特异性蛋白AFP(在诱导的早、初中期表达)、ALB以及CK18在诱导过程中表达逐渐产生或激活,在同一时期的表达量均明显高于FGF-4组。诱导性P450活性、摄取低密度脂蛋白(low-density lipoprotein,LDL)的能力等也在诱导过程中表达逐渐产生或激活,同FGF-4组相比具有统计学意义。(2)红景天苷在10μM及其以上具有单独诱导MSCs分化为肝细胞的作用,细胞在分化早、中期可以表达肝特异性蛋白AFP,在诱导中、晚期细胞表达蛋白ALB,且表达呈剂量依赖性的特点;但是在20μM以上则不具有更强的诱导能力,但对细胞也无明显的毒副作用。(3)在阻滞剂的作用下,MSCs整体生长不佳,表达AFP以及ALB的能力也不一样,总的来讲,ERK1/2和PI3K信号转导通路在MSCs的肝向分化过程中起着主要的作用。
结论:红景天苷联合诱导启动因子FGF4可以诱导MSCs分化为肝细胞,分化而来的肝细胞具有合成AFP(分化的早、中期)、白蛋白,贮存糖原、分泌尿素、吸收LDL和诱导性P450活性等一系列肝细胞所具有的功能;红景天苷可以单独诱导MSCs分化为肝细胞。红景天苷在10μM以上具有单独诱导MSCs分化为肝细胞的作用,呈剂量依赖性的特点;但是在20μM以上则不具有更强的诱导能力,但也无明显的毒副作用;红景天苷在单独诱导MSCs分化为肝细胞的过程中,ERK1/2和PI3K信号转导通路在MSCs的肝向分化过程中起着主要的作用。
第四章MSCs移植治疗慢性免疫性肝纤维化的实验研究
目的:检测MSCs移植对慢性免疫性肝纤维化大鼠的肝纤维化治疗情况以及肝脏功能的恢复程度,评价MSCs移植对慢性免疫性肝纤维化的治疗效果。
方法:通过腹腔注射猪血清(0.5ml/只,每周2次,连续8周)制造大鼠免疫性肝纤维化模型,注射等量生理盐水组大鼠作为对照。在第五周初,将成模大鼠随机分6组:模型对照组、MSCs移植组(5×10~6/只,尾静脉注射,后同)、MSCs移植组(1×10~6/只)、MSCs移植组(5×10~5/只)、MSCs移植组(5×10~6/只)结合红景天苷(腹腔注射,40mg/kg体重)组以及红景天苷组(腹腔注射,40mg/kg体重)等6组,以后继续腹腔注射猪血清持续至第八周。实验结束后,杀死大鼠,采血取血清,称肝重,计算肝脏指数;检测血清各项生化指标,包括总蛋白(total protein,TP),ALB,碱性磷酸酶(alkaline phosphatase,ALP),天冬氨酸氨基转移酶,或称谷草转氨酶(aspartate aminotransferase,AST),丙氨酸氨基转移酶,或称谷草转氨酶(alanine aminotransferase,ALT)和转化生长因子(transforming growth factor,TGF)-β1等;测定血清和肝组织中的羟脯氨酸以及还原型谷胱甘肽浓度;进行肝脏组织病理学检查(HE常规染色及偶氮卡红纤维特殊染色)等。
结果:从大体解剖学上来看,正常组肝脏呈红褐色,表面光滑,质地柔软,色泽明亮;模型组肝脏表面粗糙,肿大,有大量纤维状点,色泽发暗,质地较硬;MSCs移植能改善肝脏功能状态,且改善程度呈剂量依赖性,红景天苷MSCs对改善肝脏功能具有协同作用。从生化指标上来看,MSCs移植对肝脏具有保护作用,肝脏功能得到明显改善,红景天苷具有协同作用。HE常规染色以及偶氮卡红染色显示,正常组肝脏组织切片肝小叶结构清晰,肝细胞排列整齐,色泽均匀明亮,汇管区基本无胶原分布;模型组可见大量肝纤维化组织增生,细胞排列紊乱,色泽明显发暗,部分肝组织形成假小叶;MSCs移植能纠正肝细胞排列的紊乱状态,减轻肝纤维化程度,且改善程度基本呈移植细胞数量依赖性,红景天苷对肝纤维化具有一定的缓解及逆转作用,在与MSCs协同作用下,肝纤维化程度明显降低,肝功能改善也更为明显。从免疫荧光检测结果来看,部分移植的MSCs分化成为肝细胞,至于其治疗作用是取决于MSCs的分化还是MSCs分泌的生长因子,还需要进一步的实验支持。
结论:(1)中药单体红景天苷具有极其广泛的治疗作用。在肝脏疾病治疗方面,红景天苷能保护、增强、恢复损伤的肝细胞,缩小局部坏死区域,抑制胶原合成或者促进ECM的降解,表现出明确的抗肝纤维化作用。(2)MSCs移植能够在一定程度上改善肝脏功能,减少胶原的沉积,缓解或逆转肝纤维化状态。(3)红景天苷对MSCs移植治疗肝脏纤维化具有协同作用。中药单体化合物红景天苷与MSCs移植联合应用,两者可协同增效,优势互补,既可提高移植MSCs的成活、增殖和分化能力,又可明显改善肝功能,对抗肝损伤,逆转肝纤维化,为以后推广到临床应用奠定良好的基础。(4)移植的MSCs能分化为肝细胞,至于其治疗作用取决于MSCs的分化还是MSCs分泌的生长因子,还需要进一步的实验支持。
Chapter one:Introduction
The liver is the largest digestive gland in the body and it has a multitude of important and complex functions,such as synthesizing proteins,synthesizing,storing, and metabolizing fats,metabolizing and storing carbohydrates,forming and secreting bile,regulation of the endocrine,blood coagulation,as well as participating in the immune system,eliminating the potentially harmful biochemical products produced by the body,and detoxifying.Once liver is de-function or function failure,serious threat to human health may be caused.Therefore,liver diseases are the main causes of death in the world.
The data published by the World Health Statistics in 2005 showed that the total quantity of hepatitis B virus(HBV) in the world has infected more than two billion people,of which 350 million more will become chronic hepatitis B patients. Approximately 100 million people each year die from liver failure,liver cirrhosis, hepatocellular carcinoma and infections caused by hepatitis B.
According to the survey in 2008,about 7.18 percent of the total population are hepatitis B surface antigen(HBsAg) positive,and there are 93 million hepatitis B surface antigen carriers in China,while more than 30 million patients suffered from HBV-induced liver fibrosis and cirrhosis.Therefore substantial financial and material resources are required to pay for treatment every year,and the state and individuals bear this heavy financial burden.
From the treatment point of view,there are many methods to treat liver diseases, such as treatment according to a cause of disease(such as anti-virus),symptomatic treatment(e.g.anti-fibrosis and immune regulation),liver and nutritional support,gene therapy,liver transplantation,liver support treatment and cells(liver cells,or stem cell) transplantation and so on.
However,liver transplantation is currently ultimate method for the clinical treatment of liver disease,while a transplant operation is getting more advanced,and saves many patients of liver failure.However,due to donor source,graft versus host reaction,costly surgery,and many other factors,liver transplantation in clinical application is restricted.Liver cell transplantation is an advanced therapy,which can partially overcome the problems of liver transplantation,and has the following advantages:simple operation,minimally invasion,low-cost;abundant source,a donor liver for more patients;cells can be frozen,will not result in a shortage and the weak graft versus host reaction.
However,liver cell transplantation has not been applied in clinical practice because many problems are not yet resolved.Bottlenecks of liver cell transplantation are cell supply and immune rejection.Presently allogeneic mature liver cells are used for clinical application,but it is difficult to obtain many mature cells and to amplificate in vitro because of the loss of cross-talking in cells;what is more,liver cells transplanted may gradually apoptosis and fail to produce the desired therapeutic effect due to immune rejection.
How to solve these questions? Now fetal liver cells and embryonic stem cells are ruled out of the clinical application due to medical ethics,and then we applied to stem cells of one's own because autologous cells cannot cause immune rejection and stem cells proliferate faster.It has recently been reported bone marrow derived mesenchymal stem cells(BM-MSCs or MSCs),one of adult stem cells,can differentiate towards liver cells in vitro and in vivo.As a rich source of stem cells,an application of autologous MSCs for liver disease is very promising.Taking an overview of the studies of MSCs presently,both basic research and pre-clinical trials have not yet had outstanding results. Separation,identification,and differentiation of MSCs,maintenance of character, biological security and comprehensive evaluation of efficacy are required the in-depth studies.
Goals of our group are to explore the mechanism and controllable conditions of differentiation of MSCs,to research new drugs of induced differentiation;to verify the therapeutic efficacy and mechanism of MSCs in the treatment of autoimmune liver fibrosis.We wish MSCs transplantation could apply in clinic,a simultaneous development of stem cells and Chinese medicine-related industries,which will open up new economic growth point.
Chapter two:Isolation,cultivation and Characterization
Aim:To optimize the isolation,cultivation and amplification in vitro and to explore biological characteristics and differentiation potential of rMSCs.
Methods:Under sterile conditions,Sprague Dawley(SD) rats(one month old) rats were sacrificed,bone marrow cells were collected,suspended in the NH_4Cl hypotonic salt solution(erythrocyte lysis buffer) for erythrocyte lysis,and centrifuged to collect the cells for cultivating.Low glucose DMEM(LG-DMEM),supplemented with 10% fetal bovine serum(FBS) was applied.When the culture reached 80%confluence,it was pre-incubated with collagenase for 1～2 hours,cells were trypsinized and a serial subcultivation began.To determine growth characteristics of the cells,their morphology was observed under the inverted microscope and the cells growth curve was made.To determine the antigen expression profiles of the cells,surface antigens such as CD45, cluster of differentiation(CD) 44,CD90,were analyzed using a FACScan flow cytometer.To verify the multipotential mesenchymal characteristics,cells of passage 3 were analyzed for adipogenic,chondrogenic and osteogenic differentiation.
Result:Without interference from erythrocytes,the adherent spindle-shaped cells were observed in 4-6h.Cells rapidly grew colonies on day 4-6 and the culture reached 80%confluence 7-8 days later.Primary cells and passage cells both exhibited homogeneous fibroblast-like morphology and grew as a whirlpool.Passage cells proliferated quickly on day 3 and reached a plateau of confluence on day 5-6.They were subcultured for more than 20 times stably.Flow cytometry analyses revealed that the adherent fibroblastic cells of passage 3 were negative for CD45 while they were positive for CD44 and CD90,known as cell surface antigens of rMSCs.Differentiation assays of rMSCs revealed that the cultured cells could differentiate into osteoblastic, chondrogenic and adipocytic lineage and have the corresponding characteristics when cultured in osteoblastic,chondrogenic and adipocytic induction medium,respectively.
Conclusion:The more pure rMSCs could be isolated by lysing erythrocytes associated with adherent culture system.The adherent cells were subcultured in low glucose-Dulbecco's Minimum Essential Medium(DMEM) supplemented with 10% FBS for more than 20 times and their characters were stable.Immunophenotypic analysis revealed expression of CD44 and CD90 and no expression of CD45,which belongs to hematopoietic cell surface antigens.Differentiation assays of rMSCs revealed that the cultured cells could differentiate into osteoblastic,chondrogenic and adipocytic lineage and have the corresponding characteristics when cultured in osteoblastic,chondrogenic and adipocytic induction medium,respectively.Therefore, we adopted this method to isolate the cells is exactly bone marrow-derived mesenchymal stem cells.It is easy to obtain the pure rMSCs by lyzing erythrocyte with hypotonic salt solution in combination with adherent culture system.The method will make isolation,cultivation and amplification easy.
Chapter three:Salidroside differentiates rat MSCs towards hepatocytes in vitro
Aim:To establish and optimize the special induction system of differentiating rMSCs towards hepatocytes in vitro;to screen small-molecule compounds which come from traditional Chinese medicine and can induce rMSCs to differentiate towards hepatocytes;to evaluate the hepatocytes of hepatic differentiation and to underlay the follow-up experiments,induction of differentiation from comprehensive evaluation of the liver cells,in order to lay the foundation for and to bridge findings in vitro and application in vivo.
Methods:(1) salidroside in association with fibroblast growth factor(FGF)-4 could differentiate rMSCs towards hepatocytes,rMSCs of passage 3 were incubated in basic differentiation DMEM medium(high glycose),supplemented with 2%FBS.When reaching confluence,rMSCs were serum deprived for 2 days,in DMEM supplemented with FGF4,prior to induction.Differentiation was induced by treating rMSCs with differentiation DMEM medium,rMSCs were divided into three groups according to supplement:Group A(negative control),B(positive control) and C were supplemented with FGF4,FGF 4 & hepatocyte growth factor(HGF),and FGF 4 & salidroside, respectively.Cell morphology was observed under inverted microscope every day after the induction,rMSCs,induced for day 0,7,14 and 21 days,were harvested to analyze the typical hepatic characteristics,such as gene expression of HGF、hepatocyte growth factor receptor(HGFR)、hepatocyte nuclear factors(HNF)-3β、cytochrome P450 (CYP)2B1 & 1A1、alpha fetoprotein(AFP)、Albumin(ALB)、cytokeratin(CK)18 and transthyretin(TTR),protein expression of AFP,ALB and CK18,CYP450 (CYP450)-dependent activity and inducibility,cellular uptake of low density lipoprotein (LDL) and urea synthesis.(2) salidroside could alone differentiate rMSCs towards hepatocytes.Methods of incubated and pre-induced were carried out as described above. rMSCs were divided into several groups according to supplement:Group A(negative control),B(positive control),C,D and E were supplemented with FGF4,FGF 4 & HGF, 10μM salidroside,20μM salidroside and 50μM salidroside,respectively,rMSCs, induced for day 0,7,14,were harvested to analyze the expression of AFP and ALB, which are hepatic proteins.(3) The mechanism by which salidroside differentiates rMSCs towards hepatocytes.Methods of incubated and pre-induced were carried out as described above,rMSCs were divided into several groups according to supplement: Group A(positive control),B(negative control),C,D,E and F were supplemented with 20μM salidroside and salidroside & LY294002,salidroside & SB203580,salidroside & U0126,respectively,rMSCs induced for day 7 and day 14,were harvested to analyze the expression of AFP and ALB,which are hepatic proteins.
Result:(1)rMSCs of negative control still showed fibroblast-like grew as a whirlpool and gradually evolved into a state of overgrowth,rMSCs induced showed the state of intensive growth and tended to form circular or irregular shapes which were similar to hepatocytes of intensive growth.RT-PCR detection showed that hepatocyte-related genes such as HGF,c-MET,HNF-3β,CYP2B1,AFP,Albumin, CK18 and transferring were expressed in a time-dependent manner and each production of group C was significantly higher than that of group A.Western Blot analysis showed that hepatocyte-specific protein AFP(only expressed in earlier period),ALB,and CK18 were expressed in a time-dependent manner and the level of each protein was significantly higher than that of group A.The levels of aminotransferase,induction of P450 activity,cellular uptake of LDL and urea also increased in a time-dependent manner,which were significantly increased in comparison to group A.(2) Salidroside (10μM and more for salidroside) alone could differentiate towards hepatocytes whereas the differentiation rate of rMSCs presents a platform in the concentration range(20μM more for salidroside).The liver-specific proteins AFP and ALB were expressed in a dose-dependent manner,but 20μM more for salidroside did not possess a stronger induction capacity,but no obvious cell toxicity.(3) rMSCs all grew badly when medium was supplemented with each inhibitor but expression level of AFP and ALB among groups had a statistical significance:rMSCs of group C expressed AFP and ALB protein to some extent;ones of group D expressed more AFP and ALB than that of group C,while ones of group E did not express AFP and ALB protein.
Conclusion:salidroside in association with FGF-4 could differentiate rMSCs towards hepatocytes.Differentiated rMSCs have typical functional hepatic characteristics,such as gene expression of HGF、c-MET、HNF-3β、CYP2B1、AFP、Albumin、CK18 and TTR,protein expression of alpha fetoprotein(AFP),albumin(ALB) and CK18,CYP450-dependent activity and inducibility,cellular uptake of LDL and urea synthesis.Salidroside(10μM and more for salidroside) alone could differentiate towards hepatocytes whereas the differentiation rate of rMSCs presents a platform in the concentration range(20μM more for salidroside).Liver-specific proteins,AFP and ALB were expressed in a dose-dependent manner,but 20μM more for salidroside did not possess a stronger induction capacity.Overall,the ERK1/2 and PI3K signaling pathway plays an important role in the regulatory effects of salidroside on hepatic differentiation of rMSCs and involved in cell fate determinations.
Chapter four:MSCs could reduce fibrosis or improve function in a rat model of severe chronic liver injury and salidroside has synergistic effects in vivo
Aim:to investigate rMSCs and rMSCs in associate with salidroside for use in the reversal of experimental liver fibrosis in rat.
Methods:Hepatic fibrosis of rats was induced by intraperitoneal administration of porcine serum twice a week for 8 weeks.Control rats received saline instead of porcine serum all the time(8 wks),the others intraperitoneally received porcine serum all the time.rMSCs were administrated by tail vein injection on day 29 while salidroside was intraperitoneally injected once a day from day 29 to day 56.Seven groups were designed:A and B represent group control and model received porcine serum;C,D and E represent group received porcine serum and 5~*10~6,1~*10~6 and 5~*10~5 rMSCs, respectively;F represents group received porcine serum,5~*10~6 rMSCs and salidroside (40mg/kg);G represents group received porcine serum and salidroside(40mg/kg).All rats were sacrificed on day 56,and then many analyses,such as morphologic and histopathologic analyses,immunofluorescence and fluorescence fluorescence detection of liver sections,plasma levels of total protein(TP),ALB,alkaline phosphatase(ALP), aspartate aminotransferase(AST),alanine aminotransferase(ALT) and transforming growth factor(TGF)-β1,hydroxyproline content assay,were carried out.
Results:The amount of collagen deposition in liver of rats of administration of rMSCs(in associate with salidroside or not) was lower than that of rats without administration of MSCs or salidroside,and salidroside has the synergistic effect.Liver sections stained with HE and azan revealed large areas of fibrosis and hepatocyte denaturation in the group B when in comparison to ones of group A.The amelioration was found in the group C,D,E,F and G while liver fibrosis and hepatocyte denaturation in group F were significantly suppressed when in comparison to ones in group C,D,E or G,and salidroside has the synergism effect.Some positive rMSCs were found in group C,D,E and G after transplantation for 28 days,part of which had differentiated towards hepatocytes,but it was difficult to measure the basal rate of cell proliferation and differentiation because rMSCs transplanted were small probability events.Serum ALT and AST activity of group C,D,E,F and G decreased,and ones of group F did to the normal levels when coadministration of salidroside and rMSCs, which suggested salidroside had a synergistic effect.Hepatic hydroxyproline contents in group B were two-fold higher than in normal untreated rats,which indicates the onset of liver fibrosis,rMSC administration could decrease the hydroxyproline content of livers and salidroside had the synergism effect.The levels of serum TGF-β1 increased significantly after intraperitoneal administration of porcine serum for 8 wk.rMSCs transplanted could decrease the level of serum TGF-β1 and,what is best of all, co-administration of rMSCs and salidroside could did it.
Conclusion:rMSCs could ameliorate experimental liver fibrosis,and decrease the serum ALT and AST activity and levels of serum TGF-β1 and,what is best of all, rMSCs in association with salidroside could do the same.Suppression of TGF-β1 played an important role in the process of recovery from hepatic fibrosis.
肝脏是体内最大的消化腺和“化学工厂”,在糖、脂肪和蛋白质的合成、分解与代谢,形成以及分泌胆汁,调节内分泌,参与凝血以及机体的免疫以及降解机体代谢产物和解毒等多个方面发挥着重要的作用,所以肝脏一旦发病就严重威胁人类的身体健康,也是人类因疾病死亡的主要原因之一。
根据世界卫生组织截止到2005年的统计数据,全球乙型肝炎病毒感染者总数超过20亿,其中3.5亿人会成为慢性乙肝患者,每年约有100万人死于乙型肝炎感染所致的肝衰竭、肝硬化和原发性肝细胞癌。按照2008年调查乙肝表面抗原携带率7.18%推算,我国仍然有乙肝表面抗原携带者约9300万人,仅乙肝所致的肝纤维化和肝硬化的病人估计已超过3000万。所以,每年还需要投入大量的财力物力用于肝纤维化和肝硬化的治疗,给国家和个人造成了沉重的经济负担。
从治疗角度来讲,肝脏疾病的治疗不外乎以下几个方面:对因治疗(如抗病毒)、对症治疗(如抗纤维化、机体免疫调节)、保肝及营养支持、基因治疗、肝移植、人工肝支持治疗以及细胞(肝细胞或者干细胞)移植等等。但是,肝移植是目前临床上治疗肝脏疾病的最后手段,该技术日臻成熟,挽救了不少肝功能衰竭患者的生命。但由于供肝来源困难,免疫抑制剂的终生应用,手术费用昂贵等诸多因素,大大限制了肝移植在临床上的普及应用。肝细胞移植是新兴起的一种先进疗法,可部分克服肝脏移植的难题,与肝移植相比有以下优点:手术简单、创伤小,费用低廉;来源相对丰富;细胞可冻存备用,不会因供体短缺造成患者死亡;与肝移植相比,免疫原性弱,无需终生使用免疫抑制剂。
但肝细胞移植技术至今没有在临床上推广应用,其原因在于许多关键技术尚未解决,如制约肝细胞移植发展的两大瓶颈问题:细胞供应和免疫排斥。目前用于细胞移植的是同种异体成熟肝细胞,而同种异体成熟肝细胞难以在临床上大量获取;且肝细胞分离后会失去细胞间连接与信号,在体外难以培养扩增,体内移植后难以增殖并逐渐凋亡,造成长期存活的细胞量不足,难以达到预期的治疗效果。尽管同种异体细胞移植的免疫排斥反应比器官移植小,但有资料显示细胞移植后不使用免疫抑制剂,7天后移植细胞也所剩无几。
解决免疫排斥最好的办法是使用自体细胞;解决增殖问题是使用干细胞。目前的医学伦理基本上已经排除了使用胎肝细胞和胚胎干细胞的可能。最近有文献报道,作为人体重要成体干细胞之一,MSCs能在体内外分化成肝细胞,且MSCs来源丰富,这提示自体MSCs治疗肝脏疾病是一种非常有前途的新疗法。纵观目前研究MSCs的现状,无论是基础研究还是动物及临床实验,尚未有突出性的研究成果。在MSCs的分离、鉴定、定向分化及其机制、体外培养条件下MSCs性状的维持、体内移植的生物安全性问题以及MSCs疗效的综合评价等许多方面还需要深入研究。
我们课题组从体外诱导MSCs分化为肝细胞到移植MSCs治疗肝脏疾病实验,也只是试图从一个侧面继续探求MSCs体外分化的机理、新药(定向诱导作用)研发的新途径;验证MSCs治疗慢性免疫性肝纤维化的作用,建立由体外实验到体内实验的桥梁以及探讨MSCs逆转/缓解肝纤维化、改善肝脏功能的具体作用机制;也寄希望将MSCs迅速推向临床,为肝脏疾病患者带来新的福音,同时发展干细胞和中药相关产业,为医药经济开辟新的增长点。
第二章MSCs的分离、培养及鉴定
目的:优化大鼠MSCs体外分离、培养、扩增的有效方法,探讨其部分生物学特性以及分化潜能。
方法:在无菌条件下快速处死SD大鼠,收集骨髓细胞,快速悬浮于预冷的NH_4Cl低渗盐溶液中裂解红细胞,离心去上清,收集沉淀的细胞,D-Hank's充分洗涤后,用含10%的低糖型DMEM培养基进行扩增培养;传代时,采用胶原酶Ⅳ预先孵育1～2小时,再用胰蛋白酶-EDTA处理。倒置显微镜下观察细胞的一般形态结构;绘制生长曲线了解其生长特性;流式细胞学检测CD45、CD44、CD90等表面抗原的表达情况;用成骨、软骨以及脂肪方向分化的诱导液诱导MSCs向这三个方向分化,检测其多向分化潜能。
结果:原代培养的细胞接种4-6小时后有细胞贴壁,4～6天后细胞增殖速度明显增快,7～8天原代细胞汇合达80%-90%,原代及传代细胞形态学观察均成典型的成纤维样形态特征,旋涡状生长态势;传代培养的细胞在3天时增殖速度最快,5～6天时达到平台期;细胞可以连续传代20代而未见衰老现象,单个细胞可以扩增30倍左右。流式细胞仪检测显示该细胞表达MSCs表面抗原CD44和CD90等,不表达造血系干细胞的表面抗原CD45等等;经过诱导后,MSCs可以分化为成骨细胞、骨细胞和脂肪细胞,显示了该细胞具有多向分化潜能。
结论:采用低渗盐溶液裂解红细胞,离心收集MSCs,再进行常规贴壁培养可以分离更为纯化的MSCs;在含10%FBS的低糖DMEM培养基中可稳定传代20代左右,细胞的性状没有发生改变;根据细胞鉴定结果,该细胞具有以下特征:贴附塑料器皿生长的特性;表达MSCs特殊的表面抗原如CD44、CD90等;不表达造血系细胞的表面抗原如CD45等;具有向成骨细胞、脂肪细胞和成软骨细胞等胚层细胞方向分化的能力。所以,该细胞是MSCs;结合低渗裂解红细胞及常规贴壁培养方法,使得分离、培养及扩增MSCs更为方便。
第三章红景天苷诱导MSCs分化为肝细胞的体外实验研究
目的:建立并优化诱导MSCs分化为肝细胞的体外诱导系统;筛选能够诱导MSCs分化为肝细胞的中药小分子化合物;对诱导分化的肝细胞进行综合评价,为后续的实验奠定基础;探索由体外实验到体内实验的衔接过程。
方法:采用第五代的MSCs(P5)进行实验。
(1)红景天苷和成纤维生长因子(fibroblast growth factor,FGF)4联合应用对MSCs肝向分化的诱导作用
将MSCs接种到含10%胎牛血清的普通高糖型DMEM培养液中,细胞汇合后,换用含2%胎牛血清的预诱导培养液维持24小时;随后改用含2%FBS、红景天苷2μM及FGF4 10ng/ml的高糖型DMEM分化诱导培养液进行诱导。HGF及FGF4联合应用组作为阳性对照;FGF4单独诱导组作为阴性对照。倒置显微镜下每日观察诱导后细胞形态的变化;在每个诱导阶段末(第7、14、21天),收获诱导细胞,提取RNA和蛋白,检测肝细胞特异性相关基因以及蛋白的表达情况;在每个诱导阶段结束后,检测细胞的糖原合成及贮存能力、诱导性P450活性、低密度脂蛋白的摄取以及AFP、ALB以及CK18的表达情况。
(2)红景天苷对MSCs肝向分化的单独诱导作用
接种及预诱导方法如上所述。诱导液为在基础诱导液的基础上添加红景天苷至终浓度为10μM、20μM以及50μM。HGF及FGF4联合应用组作为阳性对照;FGF4单独诱导组作为阴性对照。诱导后的第7、14天分别收获细胞,检测肝特异性蛋白AFP和白蛋白的表达。
(3)红景天苷诱导MSCs肝向分化机制探讨
接种及预诱导方法如上所述。诱导液为在基础诱导液的基础上添加红景天苷至终浓度为20μM,在此基础上,依照添加的抑制剂的不同而分为三组(PI3K抑制剂组、P38抑制剂组以及ERK1/2抑制剂组)。红景天苷组(20μM)作为阳性对照;基础诱导液组作为阴性对照。诱导后的第7、14天分别收获细胞,检测肝特异性蛋白AFP和白蛋白的表达。
结果:(1)未诱导细胞传代后仍呈成纤维样,但是由漩涡状生长的特征逐步演变成过度生长的状态。被诱导的细胞密集生长,逐渐呈现菊花样的生长特征,形态趋向圆形或不规则形,有些类似于密集生长的肝细胞。RT-PCR检测显示肝细胞的相关基因如肝细胞生长因子(hepatocyte growth factor,HGF)、肝细胞生长因子受体(hepatocyte growth factor receptor,HGFR)、肝细胞核因子(hepatocyte nuclearfactors,HNF)-3β、甲胎蛋白(alpha fetoprotein,AFP)、白蛋白(Albumin,ALB)、细胞角蛋白(cytokeratin,CK)18和转甲状腺素蛋白(transthyretin,TTR)等在诱导过程中表达逐渐产生或被激活,在同一时期的表达量(或活性)均明显高于FGF-4组。Western Blot检测结果显示肝细胞特异性蛋白AFP(在诱导的早、初中期表达)、ALB以及CK18在诱导过程中表达逐渐产生或激活,在同一时期的表达量均明显高于FGF-4组。诱导性P450活性、摄取低密度脂蛋白(low-density lipoprotein,LDL)的能力等也在诱导过程中表达逐渐产生或激活,同FGF-4组相比具有统计学意义。(2)红景天苷在10μM及其以上具有单独诱导MSCs分化为肝细胞的作用,细胞在分化早、中期可以表达肝特异性蛋白AFP,在诱导中、晚期细胞表达蛋白ALB,且表达呈剂量依赖性的特点;但是在20μM以上则不具有更强的诱导能力,但对细胞也无明显的毒副作用。(3)在阻滞剂的作用下,MSCs整体生长不佳,表达AFP以及ALB的能力也不一样,总的来讲,ERK1/2和PI3K信号转导通路在MSCs的肝向分化过程中起着主要的作用。
结论:红景天苷联合诱导启动因子FGF4可以诱导MSCs分化为肝细胞,分化而来的肝细胞具有合成AFP(分化的早、中期)、白蛋白,贮存糖原、分泌尿素、吸收LDL和诱导性P450活性等一系列肝细胞所具有的功能;红景天苷可以单独诱导MSCs分化为肝细胞。红景天苷在10μM以上具有单独诱导MSCs分化为肝细胞的作用,呈剂量依赖性的特点;但是在20μM以上则不具有更强的诱导能力,但也无明显的毒副作用;红景天苷在单独诱导MSCs分化为肝细胞的过程中,ERK1/2和PI3K信号转导通路在MSCs的肝向分化过程中起着主要的作用。
第四章MSCs移植治疗慢性免疫性肝纤维化的实验研究
目的:检测MSCs移植对慢性免疫性肝纤维化大鼠的肝纤维化治疗情况以及肝脏功能的恢复程度,评价MSCs移植对慢性免疫性肝纤维化的治疗效果。
方法:通过腹腔注射猪血清(0.5ml/只,每周2次,连续8周)制造大鼠免疫性肝纤维化模型,注射等量生理盐水组大鼠作为对照。在第五周初,将成模大鼠随机分6组:模型对照组、MSCs移植组(5×10~6/只,尾静脉注射,后同)、MSCs移植组(1×10~6/只)、MSCs移植组(5×10~5/只)、MSCs移植组(5×10~6/只)结合红景天苷(腹腔注射,40mg/kg体重)组以及红景天苷组(腹腔注射,40mg/kg体重)等6组,以后继续腹腔注射猪血清持续至第八周。实验结束后,杀死大鼠,采血取血清,称肝重,计算肝脏指数;检测血清各项生化指标,包括总蛋白(total protein,TP),ALB,碱性磷酸酶(alkaline phosphatase,ALP),天冬氨酸氨基转移酶,或称谷草转氨酶(aspartate aminotransferase,AST),丙氨酸氨基转移酶,或称谷草转氨酶(alanine aminotransferase,ALT)和转化生长因子(transforming growth factor,TGF)-β1等;测定血清和肝组织中的羟脯氨酸以及还原型谷胱甘肽浓度;进行肝脏组织病理学检查(HE常规染色及偶氮卡红纤维特殊染色)等。
结果:从大体解剖学上来看,正常组肝脏呈红褐色,表面光滑,质地柔软,色泽明亮;模型组肝脏表面粗糙,肿大,有大量纤维状点,色泽发暗,质地较硬;MSCs移植能改善肝脏功能状态,且改善程度呈剂量依赖性,红景天苷MSCs对改善肝脏功能具有协同作用。从生化指标上来看,MSCs移植对肝脏具有保护作用,肝脏功能得到明显改善,红景天苷具有协同作用。HE常规染色以及偶氮卡红染色显示,正常组肝脏组织切片肝小叶结构清晰,肝细胞排列整齐,色泽均匀明亮,汇管区基本无胶原分布;模型组可见大量肝纤维化组织增生,细胞排列紊乱,色泽明显发暗,部分肝组织形成假小叶;MSCs移植能纠正肝细胞排列的紊乱状态,减轻肝纤维化程度,且改善程度基本呈移植细胞数量依赖性,红景天苷对肝纤维化具有一定的缓解及逆转作用,在与MSCs协同作用下,肝纤维化程度明显降低,肝功能改善也更为明显。从免疫荧光检测结果来看,部分移植的MSCs分化成为肝细胞,至于其治疗作用是取决于MSCs的分化还是MSCs分泌的生长因子,还需要进一步的实验支持。
结论:(1)中药单体红景天苷具有极其广泛的治疗作用。在肝脏疾病治疗方面,红景天苷能保护、增强、恢复损伤的肝细胞,缩小局部坏死区域,抑制胶原合成或者促进ECM的降解,表现出明确的抗肝纤维化作用。(2)MSCs移植能够在一定程度上改善肝脏功能,减少胶原的沉积,缓解或逆转肝纤维化状态。(3)红景天苷对MSCs移植治疗肝脏纤维化具有协同作用。中药单体化合物红景天苷与MSCs移植联合应用,两者可协同增效,优势互补,既可提高移植MSCs的成活、增殖和分化能力,又可明显改善肝功能,对抗肝损伤,逆转肝纤维化,为以后推广到临床应用奠定良好的基础。(4)移植的MSCs能分化为肝细胞,至于其治疗作用取决于MSCs的分化还是MSCs分泌的生长因子,还需要进一步的实验支持。
Chapter one:Introduction
The liver is the largest digestive gland in the body and it has a multitude of important and complex functions,such as synthesizing proteins,synthesizing,storing, and metabolizing fats,metabolizing and storing carbohydrates,forming and secreting bile,regulation of the endocrine,blood coagulation,as well as participating in the immune system,eliminating the potentially harmful biochemical products produced by the body,and detoxifying.Once liver is de-function or function failure,serious threat to human health may be caused.Therefore,liver diseases are the main causes of death in the world.
The data published by the World Health Statistics in 2005 showed that the total quantity of hepatitis B virus(HBV) in the world has infected more than two billion people,of which 350 million more will become chronic hepatitis B patients. Approximately 100 million people each year die from liver failure,liver cirrhosis, hepatocellular carcinoma and infections caused by hepatitis B.
According to the survey in 2008,about 7.18 percent of the total population are hepatitis B surface antigen(HBsAg) positive,and there are 93 million hepatitis B surface antigen carriers in China,while more than 30 million patients suffered from HBV-induced liver fibrosis and cirrhosis.Therefore substantial financial and material resources are required to pay for treatment every year,and the state and individuals bear this heavy financial burden.
From the treatment point of view,there are many methods to treat liver diseases, such as treatment according to a cause of disease(such as anti-virus),symptomatic treatment(e.g.anti-fibrosis and immune regulation),liver and nutritional support,gene therapy,liver transplantation,liver support treatment and cells(liver cells,or stem cell) transplantation and so on.
However,liver transplantation is currently ultimate method for the clinical treatment of liver disease,while a transplant operation is getting more advanced,and saves many patients of liver failure.However,due to donor source,graft versus host reaction,costly surgery,and many other factors,liver transplantation in clinical application is restricted.Liver cell transplantation is an advanced therapy,which can partially overcome the problems of liver transplantation,and has the following advantages:simple operation,minimally invasion,low-cost;abundant source,a donor liver for more patients;cells can be frozen,will not result in a shortage and the weak graft versus host reaction.
However,liver cell transplantation has not been applied in clinical practice because many problems are not yet resolved.Bottlenecks of liver cell transplantation are cell supply and immune rejection.Presently allogeneic mature liver cells are used for clinical application,but it is difficult to obtain many mature cells and to amplificate in vitro because of the loss of cross-talking in cells;what is more,liver cells transplanted may gradually apoptosis and fail to produce the desired therapeutic effect due to immune rejection.
How to solve these questions? Now fetal liver cells and embryonic stem cells are ruled out of the clinical application due to medical ethics,and then we applied to stem cells of one's own because autologous cells cannot cause immune rejection and stem cells proliferate faster.It has recently been reported bone marrow derived mesenchymal stem cells(BM-MSCs or MSCs),one of adult stem cells,can differentiate towards liver cells in vitro and in vivo.As a rich source of stem cells,an application of autologous MSCs for liver disease is very promising.Taking an overview of the studies of MSCs presently,both basic research and pre-clinical trials have not yet had outstanding results. Separation,identification,and differentiation of MSCs,maintenance of character, biological security and comprehensive evaluation of efficacy are required the in-depth studies.
Goals of our group are to explore the mechanism and controllable conditions of differentiation of MSCs,to research new drugs of induced differentiation;to verify the therapeutic efficacy and mechanism of MSCs in the treatment of autoimmune liver fibrosis.We wish MSCs transplantation could apply in clinic,a simultaneous development of stem cells and Chinese medicine-related industries,which will open up new economic growth point.
Chapter two:Isolation,cultivation and Characterization
Aim:To optimize the isolation,cultivation and amplification in vitro and to explore biological characteristics and differentiation potential of rMSCs.
Methods:Under sterile conditions,Sprague Dawley(SD) rats(one month old) rats were sacrificed,bone marrow cells were collected,suspended in the NH_4Cl hypotonic salt solution(erythrocyte lysis buffer) for erythrocyte lysis,and centrifuged to collect the cells for cultivating.Low glucose DMEM(LG-DMEM),supplemented with 10% fetal bovine serum(FBS) was applied.When the culture reached 80%confluence,it was pre-incubated with collagenase for 1～2 hours,cells were trypsinized and a serial subcultivation began.To determine growth characteristics of the cells,their morphology was observed under the inverted microscope and the cells growth curve was made.To determine the antigen expression profiles of the cells,surface antigens such as CD45, cluster of differentiation(CD) 44,CD90,were analyzed using a FACScan flow cytometer.To verify the multipotential mesenchymal characteristics,cells of passage 3 were analyzed for adipogenic,chondrogenic and osteogenic differentiation.
Result:Without interference from erythrocytes,the adherent spindle-shaped cells were observed in 4-6h.Cells rapidly grew colonies on day 4-6 and the culture reached 80%confluence 7-8 days later.Primary cells and passage cells both exhibited homogeneous fibroblast-like morphology and grew as a whirlpool.Passage cells proliferated quickly on day 3 and reached a plateau of confluence on day 5-6.They were subcultured for more than 20 times stably.Flow cytometry analyses revealed that the adherent fibroblastic cells of passage 3 were negative for CD45 while they were positive for CD44 and CD90,known as cell surface antigens of rMSCs.Differentiation assays of rMSCs revealed that the cultured cells could differentiate into osteoblastic, chondrogenic and adipocytic lineage and have the corresponding characteristics when cultured in osteoblastic,chondrogenic and adipocytic induction medium,respectively.
Conclusion:The more pure rMSCs could be isolated by lysing erythrocytes associated with adherent culture system.The adherent cells were subcultured in low glucose-Dulbecco's Minimum Essential Medium(DMEM) supplemented with 10% FBS for more than 20 times and their characters were stable.Immunophenotypic analysis revealed expression of CD44 and CD90 and no expression of CD45,which belongs to hematopoietic cell surface antigens.Differentiation assays of rMSCs revealed that the cultured cells could differentiate into osteoblastic,chondrogenic and adipocytic lineage and have the corresponding characteristics when cultured in osteoblastic,chondrogenic and adipocytic induction medium,respectively.Therefore, we adopted this method to isolate the cells is exactly bone marrow-derived mesenchymal stem cells.It is easy to obtain the pure rMSCs by lyzing erythrocyte with hypotonic salt solution in combination with adherent culture system.The method will make isolation,cultivation and amplification easy.
Chapter three:Salidroside differentiates rat MSCs towards hepatocytes in vitro
Aim:To establish and optimize the special induction system of differentiating rMSCs towards hepatocytes in vitro;to screen small-molecule compounds which come from traditional Chinese medicine and can induce rMSCs to differentiate towards hepatocytes;to evaluate the hepatocytes of hepatic differentiation and to underlay the follow-up experiments,induction of differentiation from comprehensive evaluation of the liver cells,in order to lay the foundation for and to bridge findings in vitro and application in vivo.
Methods:(1) salidroside in association with fibroblast growth factor(FGF)-4 could differentiate rMSCs towards hepatocytes,rMSCs of passage 3 were incubated in basic differentiation DMEM medium(high glycose),supplemented with 2%FBS.When reaching confluence,rMSCs were serum deprived for 2 days,in DMEM supplemented with FGF4,prior to induction.Differentiation was induced by treating rMSCs with differentiation DMEM medium,rMSCs were divided into three groups according to supplement:Group A(negative control),B(positive control) and C were supplemented with FGF4,FGF 4 & hepatocyte growth factor(HGF),and FGF 4 & salidroside, respectively.Cell morphology was observed under inverted microscope every day after the induction,rMSCs,induced for day 0,7,14 and 21 days,were harvested to analyze the typical hepatic characteristics,such as gene expression of HGF、hepatocyte growth factor receptor(HGFR)、hepatocyte nuclear factors(HNF)-3β、cytochrome P450 (CYP)2B1 & 1A1、alpha fetoprotein(AFP)、Albumin(ALB)、cytokeratin(CK)18 and transthyretin(TTR),protein expression of AFP,ALB and CK18,CYP450 (CYP450)-dependent activity and inducibility,cellular uptake of low density lipoprotein (LDL) and urea synthesis.(2) salidroside could alone differentiate rMSCs towards hepatocytes.Methods of incubated and pre-induced were carried out as described above. rMSCs were divided into several groups according to supplement:Group A(negative control),B(positive control),C,D and E were supplemented with FGF4,FGF 4 & HGF, 10μM salidroside,20μM salidroside and 50μM salidroside,respectively,rMSCs, induced for day 0,7,14,were harvested to analyze the expression of AFP and ALB, which are hepatic proteins.(3) The mechanism by which salidroside differentiates rMSCs towards hepatocytes.Methods of incubated and pre-induced were carried out as described above,rMSCs were divided into several groups according to supplement: Group A(positive control),B(negative control),C,D,E and F were supplemented with 20μM salidroside and salidroside & LY294002,salidroside & SB203580,salidroside & U0126,respectively,rMSCs induced for day 7 and day 14,were harvested to analyze the expression of AFP and ALB,which are hepatic proteins.
Result:(1)rMSCs of negative control still showed fibroblast-like grew as a whirlpool and gradually evolved into a state of overgrowth,rMSCs induced showed the state of intensive growth and tended to form circular or irregular shapes which were similar to hepatocytes of intensive growth.RT-PCR detection showed that hepatocyte-related genes such as HGF,c-MET,HNF-3β,CYP2B1,AFP,Albumin, CK18 and transferring were expressed in a time-dependent manner and each production of group C was significantly higher than that of group A.Western Blot analysis showed that hepatocyte-specific protein AFP(only expressed in earlier period),ALB,and CK18 were expressed in a time-dependent manner and the level of each protein was significantly higher than that of group A.The levels of aminotransferase,induction of P450 activity,cellular uptake of LDL and urea also increased in a time-dependent manner,which were significantly increased in comparison to group A.(2) Salidroside (10μM and more for salidroside) alone could differentiate towards hepatocytes whereas the differentiation rate of rMSCs presents a platform in the concentration range(20μM more for salidroside).The liver-specific proteins AFP and ALB were expressed in a dose-dependent manner,but 20μM more for salidroside did not possess a stronger induction capacity,but no obvious cell toxicity.(3) rMSCs all grew badly when medium was supplemented with each inhibitor but expression level of AFP and ALB among groups had a statistical significance:rMSCs of group C expressed AFP and ALB protein to some extent;ones of group D expressed more AFP and ALB than that of group C,while ones of group E did not express AFP and ALB protein.
Conclusion:salidroside in association with FGF-4 could differentiate rMSCs towards hepatocytes.Differentiated rMSCs have typical functional hepatic characteristics,such as gene expression of HGF、c-MET、HNF-3β、CYP2B1、AFP、Albumin、CK18 and TTR,protein expression of alpha fetoprotein(AFP),albumin(ALB) and CK18,CYP450-dependent activity and inducibility,cellular uptake of LDL and urea synthesis.Salidroside(10μM and more for salidroside) alone could differentiate towards hepatocytes whereas the differentiation rate of rMSCs presents a platform in the concentration range(20μM more for salidroside).Liver-specific proteins,AFP and ALB were expressed in a dose-dependent manner,but 20μM more for salidroside did not possess a stronger induction capacity.Overall,the ERK1/2 and PI3K signaling pathway plays an important role in the regulatory effects of salidroside on hepatic differentiation of rMSCs and involved in cell fate determinations.
Chapter four:MSCs could reduce fibrosis or improve function in a rat model of severe chronic liver injury and salidroside has synergistic effects in vivo
Aim:to investigate rMSCs and rMSCs in associate with salidroside for use in the reversal of experimental liver fibrosis in rat.
Methods:Hepatic fibrosis of rats was induced by intraperitoneal administration of porcine serum twice a week for 8 weeks.Control rats received saline instead of porcine serum all the time(8 wks),the others intraperitoneally received porcine serum all the time.rMSCs were administrated by tail vein injection on day 29 while salidroside was intraperitoneally injected once a day from day 29 to day 56.Seven groups were designed:A and B represent group control and model received porcine serum;C,D and E represent group received porcine serum and 5~*10~6,1~*10~6 and 5~*10~5 rMSCs, respectively;F represents group received porcine serum,5~*10~6 rMSCs and salidroside (40mg/kg);G represents group received porcine serum and salidroside(40mg/kg).All rats were sacrificed on day 56,and then many analyses,such as morphologic and histopathologic analyses,immunofluorescence and fluorescence fluorescence detection of liver sections,plasma levels of total protein(TP),ALB,alkaline phosphatase(ALP), aspartate aminotransferase(AST),alanine aminotransferase(ALT) and transforming growth factor(TGF)-β1,hydroxyproline content assay,were carried out.
Results:The amount of collagen deposition in liver of rats of administration of rMSCs(in associate with salidroside or not) was lower than that of rats without administration of MSCs or salidroside,and salidroside has the synergistic effect.Liver sections stained with HE and azan revealed large areas of fibrosis and hepatocyte denaturation in the group B when in comparison to ones of group A.The amelioration was found in the group C,D,E,F and G while liver fibrosis and hepatocyte denaturation in group F were significantly suppressed when in comparison to ones in group C,D,E or G,and salidroside has the synergism effect.Some positive rMSCs were found in group C,D,E and G after transplantation for 28 days,part of which had differentiated towards hepatocytes,but it was difficult to measure the basal rate of cell proliferation and differentiation because rMSCs transplanted were small probability events.Serum ALT and AST activity of group C,D,E,F and G decreased,and ones of group F did to the normal levels when coadministration of salidroside and rMSCs, which suggested salidroside had a synergistic effect.Hepatic hydroxyproline contents in group B were two-fold higher than in normal untreated rats,which indicates the onset of liver fibrosis,rMSC administration could decrease the hydroxyproline content of livers and salidroside had the synergism effect.The levels of serum TGF-β1 increased significantly after intraperitoneal administration of porcine serum for 8 wk.rMSCs transplanted could decrease the level of serum TGF-β1 and,what is best of all, co-administration of rMSCs and salidroside could did it.
Conclusion:rMSCs could ameliorate experimental liver fibrosis,and decrease the serum ALT and AST activity and levels of serum TGF-β1 and,what is best of all, rMSCs in association with salidroside could do the same.Suppression of TGF-β1 played an important role in the process of recovery from hepatic fibrosis.
引文
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