活化肝星状细胞促进干细胞定向分化和肝损伤修复
|
摘要
【研究背景及目的】
骨髓组织中存在有多种类型的干细胞,其中关于造血干细胞和间充质干细胞(mesenchymal stem cells,MSCs)的研究报道较多。MSCs是一种较原始的细胞,常维持在低分化状态。MSCs除参与构成造血微环境、支持造血细胞生长外,还具有多向分化潜能。最初只发现MSCs具有生成成骨细胞、软骨细胞、脂肪细胞等间质组织细胞的能力。近年来发现骨髓间质干细胞在特定的条件下还可以向心肌细胞、神经细胞、血管内皮细胞、肝细胞等分化。MSCs具有取材容易、体外增殖能力强、可分化为不同类型的成熟细胞和用于多种疾病治疗等特点,可以避免用胚胎干细胞和异基因干细胞治疗的伦理和免疫排斥等问题,因此它在临床治疗和人体生物组织工程研究方面比其它来源的干细胞更有优势,可能成为多种器官功能老化、组织损伤修复、功能细胞退行性变治疗以及人体生物组织构建的理想种子细胞。
关于骨髓干细胞对肝组织修复的体内研究存在诸多争论:肝大部切除等急性肝损伤动物或患者有“外周血干细胞动员(peripheral blood stem cell mobilization)”现象。有文献证实骨髓干细胞能在体内分化为肝细胞参与组织修复,并可通过骨髓干细胞移植或外周血干细胞动员的途径促进肝损动物或病人的肝功能改善。但最近也有文献报道骨髓干细胞在体内并不能分化为肝细胞参与肝脏修复,却分化为纤维细胞而加剧肝纤维化的进展。这截然相反的两种结果的原因很可能是各组研究中宿主肝组织促分化微环境的不同所致。因而研究生理及病理条件下肝组织对干细胞增殖及分化的影响,以及阐明调控干细胞分化及成熟的适宜微环境对肝再生医学有重大意义。干细胞本身具有分化的多向性潜能和可塑性,其分化受主要由间质细胞和分泌的细胞因子和细胞外基质构成的微环境所调控。肝星状细胞(hepatic stellate cells,HSCs)主要位于Disse间隙,是肝脏的主要间质细胞之一,并且具有树突状突起并嵌入相邻肝细胞的凹陷内,其突起包绕肝窦边界的内皮细胞。在损伤因子作用下,HSC能由静息状态活化增殖,分泌细胞外基质并分泌细胞因子对周围细胞产生强大调控作用,因而很有可能对干细胞的分化产生影响。不同状态的HSCs的调控作用可能是造成骨髓干细胞不同分化方向的原因。为证实该假设,我们将MSCs与静息状态及不同活化状态的HSCs共培养,对比观察不同状态的HSCs对MSCs分化的调控作用。
为了进一步在体内环境下HSCs活化状态对肝再生及干细胞增殖分化的影响,我们采用2-乙酰氨基芴/肝大部切除(2-AAF/PH)模型诱导肝卵圆细胞(肝祖细胞)活化增殖,用免疫荧光方法验证卵圆细胞增殖过程中HSCs活化状态的变化及差异。并用Gliotoxin清除体内活化HSCs,对比研究清除活化HSCs对肝卵圆细胞活化增殖及肝脏再生的影响,进一步证实HSCs活化状态对干细胞分化增殖及肝再生的影响。
【实验方法】
一、原代分离大鼠骨髓MSCs并验证其多向分化潜能性
我们采用梯度离心并培养贴壁的方法分离纯化骨髓MSCs,检测分离细胞活力及传代次数对细胞增值及分化潜能影响,诱导MSCs分别向脂肪细胞既成骨细胞分化,检测MSCs的多向分化潜能,挑选适合本试验诱导分化的细胞。
二、原代分离获取大鼠HSCs、Kupffer细胞,构建不同激活程度的HSCs
1.采用原位灌注消化获得肝非实质细胞,根据细胞密度差异及贴壁速度差别采用梯度离心获得原代HSCs和原代大鼠Kupffer细胞。免疫荧光标记Desmin检测HSCs纯度,吞噬试验检测Kupffer细胞纯度和活力;
2.构建不同活化程度的HSCs,采用贴壁培养来获得半活化状态的HSCs,将HSCs与Kupffer细胞共培养的方式获得完全活化的HSCs。并采用Real-Time PCR检测肝细胞生长因子(hepatic growth factor,HGF)mRNA在各组HSCs的表达,以此研究不同状态HSCs对MSCs调控作用的差异。
三、构建双层细胞共培养系统,检测不同活化状态的HSCs对MSCs分化的影响
1.构建共培养系统,将MSCs分别与下列细胞五组细胞共培养:1)HSC-T6细胞;2)静息状态HSCs,为原代分离培养不多于5天的HSCs;3)贴壁活化的HSCs,为原代贴壁培养7-20天的HSCs;4)Kupffer细胞活化的HSCs:为按1:1比例与Kupffer细胞共培养7-20天的HSCs;5)原代分离的Kupffer细胞。
2.镜下观察不同时间段各组MSCs形态学变化。
3.检测肝细胞特异基因在诱导分化不同时段的各组MSCs中表达,包括肝细胞早期表达基因甲胎蛋白(AFP)、白蛋白(ALB)、细胞角蛋白18(CK-18)、磷酸化糖原合成酶(GS)及成熟肝细胞表达基因磷酸烯醇式丙酮酸羧基酶(PEPCK)、葡萄糖6磷酸酶(G6P),判断各组MSCs是否向肝细胞分化及分化成熟过程。
4.共培养第14天,免疫细胞荧光检测肝细胞特异蛋白在分化的MSCs中表达,包括ALB、AFP、CK-18及间质细胞标记α-平滑肌肌动蛋白(α-SMA),同时激光共聚焦法检测各组细胞在不同时间段是否存在共表达肝细胞标记白蛋白和胆管上皮标记细胞角蛋白19(CK-19)的细胞。
5.用过碘酸-希夫氏液染色(PAS染色)检测各组MSCs在不同时间段的糖原合成,判断细胞是否具有成熟肝细胞的部分功能。
四、对比研究肝卵圆细胞活化增殖过程HSCs活化状态的差异
1.采用2-AAF/PH模型诱导肝卵圆细胞(肝祖细胞)增殖。
2.荧光标记活化HSCs标记蛋白desmin,对比研究卵圆细胞活化增殖模型肝脏组织与对照组肝组织中HSCs活化状态的差别。
五、Gliotoxin清除活化HSCs对干细胞增殖活化及肝再生影响
1.2-AAF/PH造模,术后2天注射Gliotoxin,desmin免疫荧光染色证实活化HSCs的清除效果。
2.对比研究清除活化HSCs后肝再生及卵圆细胞增殖所受影响。
3.清除活化HSCs后,补充体外培养的活化HSCs培养上清,对比研究是否能抵消或部分抵消体内活化HSCs被清除对卵圆细胞增殖及肝再生的影响。
【实验结果】
一、原代分离骨髓MSCs,具有多向分化潜能
采用梯度离心,贴壁培养及适时消化,每只大鼠可分离、纯化获得2-8×10~7 MSCs,细胞存活率在95%以上,细胞均一性好纯度高。在适当诱导培养条件下,MSCs成功分化为脂肪细胞及成骨细胞。
二、原代分离HSCs及Kupffer细胞及不同活化状态HSCs的获取
1.原位灌注消化获得大鼠肝非实质细胞,梯度离心纯化获得大鼠的HSCs得率为2-8×10~7/肝。细胞存活率在90%以上,HSCs特异标记Desmin免疫荧光染色显示分离的细胞纯度)95%。
2.通过梯度离心机及差异贴壁,成功获得大鼠Kupffer细胞,台盼蓝染色检查细胞存活率＞90%,吞噬墨汁实验检测细胞纯度＞95%。
3.获取不同活化状态的HSCs,Real-Time PCR结果显示Kupffer细胞活化的HSCs及HSC-T6细胞的肝细胞生长因子mRNA表达明显高于静息状态HSCs的表达,而贴壁活化HSCs的HGF mRNA表达与静息状态HSCs比较无统计学差异。
三、不同活化状态HSCs诱导MSCs定向分化的差异
1.形态学上,与HSC-T6细胞共培养的MSCs,7天时观察到MSCs呈三角形或不规则形,14天左右分化为多边形细胞与Kuppfer细胞活化HSCs共培养的MSCs也观察到同样现象。而与静息HSC或贴壁活化HSCs共培养的MSCs逐渐展开呈纤维状细胞分化发展,未见到呈多边性细胞形成。单独与Kupffer细胞共培养的MSCs也呈纤维样细胞生长。
2.与Kupffer细胞活化HSCs共培养的MSCs,7天时检测到AFP、ALB、CK-18、GS、TAT的mRNA表达,第14天表达程度无变化,到28天减弱。共培养28天的MSCs检测到成熟肝细胞特异表达基因PEPCK、G6P的表达。而与静息HSC或贴壁活化HSCs共培养的MSCs未检测到肝细胞系基因的表达。
3.共培养第14天,与Kupffer细胞活化HSCs共培养的MSCs用免疫细胞荧光法检测到albumin、AFP和CK-18的表达,未检测到间质细胞标志α-SMA。而与静息状态HSCs共培养的MSCs及与贴壁活化HSCs共培养的MSCs未检测到肝细胞特异蛋白的表达,α-SMA强阳性表达。
4.在与Kupffer细胞活化HSCs共培养14天的MSCs中,有12.4±3.7%的细胞同时表达肝细胞标志蛋白ALB和胆管上皮标志蛋白CK-19,表明该细胞具有肝细胞和胆管细胞的双向分化潜能性。随着时间迁移这种双标记细胞所占比例逐渐减少,至第4周已完全检测不到该双标细胞。
5.与HSC-T6共培养MSCs在第4周用PAS染色检测到糖原沉积,到第6周达到最高峰,有55.4%±8.7%细胞检测到糖原沉积。与Kupffer细胞活化HSCs共培养的MSCs同样检测到糖原沉积,而与静息状态HSCs共培养的MSCs及与贴壁活化HSCs共培养的MSCs未检测到糖原沉积。
四、卵圆细胞活化增殖过程中HSCs活化状态的变化
1.2-AAF/PH模型成功诱导肝卵圆细胞活化增殖。
2.卵圆细胞活化增殖过程中肝脏HSCs大量活化增殖,表达desmin,而对照正常肝脏及单纯肝部分切除小鼠肝脏仅见少量desmin阳性的活化HSCs。
五、清除活化HSCs对卵圆细胞增殖及肝再生影响
1.Gliotoxin有效清除体内活化的HSCs。
2.Gliotoxin清除活化HSCs后,肝重指数明显小于单纯2-AAF/PH组,肝干/祖细胞标志基因CK-18、CD90、CD133、SOX-2表达水平明显低于单纯2-AAF/PH组,补充体外培养的活化HSCs培养上清后,肝重指数明显高于清除活化HSCs组,肝干/祖细胞标志基因的表达也得到部分恢复。
【结论】
1.密度梯度离心,贴壁培养和消化时间控制相结合,具有操作简单、快速、实用等优点,是一种较为有效的分离纯化MSCs方法。
2.充分活化的HSCs能诱导MSCs向肝细胞分化,具有类似肝细胞形态,表达肝细胞特异基因和蛋白,具有肝细胞特有的糖原合成功能。
3.活化HSCs诱导的MSCs定向分化是一个经肝细胞、胆管细胞双潜能细胞向成熟肝细胞逐渐分化和成熟的过程。
4.肝卵圆细胞活化增殖过程伴随着HSCs的活化;
5.HSCs的活化是肝再生及肝干/祖细胞增殖的必要环节。
【Background and Objective】
Bone marrow mesenchymal stem cells(MSC) are nonhematopoietic cells that reside within the bone marrow stroma.These cells are multipotent and serve as precursors for various cells including osteoblast,smooth muscle cells,chondrocytes,adipocytes and hematopoietic supportive cells.The generation of hepatocytes from bone marrow derived stem cells is a compelling concept that has provoked much interest in the liver transplant field.Petersen et al.reported that some liver oval cells were derived from transplanted bone marrow.Hepatic oval cells are hepatic progenitor cells which are characterized by an ovoid nucleus,small size,and scant basophilic cytoplasm.Oval cells express phenotypical markers of both the biliary epithelium and hepatocyte lineages.After that,there are both positive and negative reports concerning the potency of bone marrow derived stem cells to differentiate into hepatocytes in vivo.The conflict results may attribute to the different stem cell microenvironment(or "niche") which is composed of non-parenchymal cells and secreted factors.
The concept of the microenvironment was first developed in hematopoiesis,where other cells secrete factors to support proliferation,differentiation,and survival of distinct progenitor populations.The effect of niche cells on stem/progenitor cells in proliferation and differentiation was also demonstrated in other organs/tissues such as epidermal,heart, brain,etc.To date,the microenvironment which modulate differentiation of hepatic stem cells and control transdifferentiation of bone marrow derived stem cells migrated into liver are still unknown.
Hepatic stellate cell(HSC) is one of major hepatic nonparenchymal cells which are located in the Disse space between the endothelium and the hepatocytes.Under conditions of stress or liver injury,quiescent HSCs are activated and acquire a myofibroblastic phenotype,contributing to excessive extracellular matrix deposition which gives rise to development of liver fibrosis and cirrhosis.On the other hand,activated HSCs were recently demonstrated to secrete factors to stimulate hepatocyte proliferation.Anatomically, bone marrow derived stem cells must circumvent the Disse space during translocation form liver sinusoids to the parenchyma and may be regulated by HSCs.Therefore,we assume that HSCs may present as a modulator for the differentiation of MSCs into hepatic cells and different states of HSCs may play variant roles.
To prove this assumption,bone marrow MSCs were indirectly cocultured with quiescent HSCs or activated HSCs.Primary isolated quiescent HSCs were activated by being in vitro cultured alone or cocultured with Kupffer cells.Expression of hepatic specific markers and hepatocyte function of glycogen deposition were detected on differentiated MSCs.The variant effects of HSCs in different states on the differentiation of MSCs were compared.
【Methods】
1.MSCs Preparation and Delivery
Rat bone marrow MSCs were harvested from mononuclear cells which were separated by Percoll density gradient.MSCs were plastic-adherent mononuclear bone marrow cells after expansion in culture.The resulting MSCs(passage 6-10) were used for our studies. To confirm the identity of bone marrow MSCs,the cells were grown in medium that is conducive to differentiation into osteoclasts and adipocytes.Adipocytes containing lipid droplets were observed 2 weeks later by Oil Red O(Sigma-Aldrich) staining.The deposition of bone mineral by osteoclasts differentitated from MSCs was observed 2 weeks later by Alizarin red(pH 4.1;Sigma-Aldrich) staining.
2.Preparation of HSCs and Kupffer Cells
Primary HSCs and Kupffer cells were freshly isolated from rat liver nonparenchymal cells.The liver was perfused with collagenase and pronase solution.The supernatant was centrifuged at 50×g for 2 min for several rounds to collect and nonparenchymal cells containing the HSCs and Kupffer cells.HSCs were isolated by density gradient centrifugation.Kupffer cells were separated by density gradient and adhesion culture.
3.Establishment of coculture system and investigation of the effects of HSCs in different activation state on the MSCs
3.1 MSCs were coculture with HSCs at different states:HSC-T6 cells,the immortalized activated HSCs cells;quiescent HSCs:freshly isolated HSCs within 5 days;culture activated HSCs:primarily isolated HSCs cultured for 7 to 20 days;Kupffer cell activated HSCs:primarily isolated HSCs cocultured with Kupffer cells for 7 to 20 days.MSCs were also cocultured with Kupffer cells as control group for Kupffer cells activated HSCs group. Culture medium was half changed every 3 days.
3.2 Morphological changes of MSCs cocultured with HSCs were observed under microscopy.
3.3 The hepatic specific mRNA expression of MSCs cocultured with HSCs was analyzed by reverse transcription-polymerase chain reaction(RT-PCR),including unmature hepatocyte marker alpha-fetoprotein(AFP),albumin,cytokeratin 18(CK18), glutamine synthetase(GS) and mature hepatocyte marker phosphoenolpyruvate carboxykinase(PEPCK),glucose 6 phosphate dehydrogenase(G6P).
3.4 The MSCs were detected for expression of specific proteins after coculture with HSCs for 2 weeks with immunofluorescence,including biliary epithelial cells marker CK-19,hepatocyte marker CK-18,albumin and AFP,MSCs marker alpha-smooth muscle actin(α-SMA).
3.5 Glycogen deposition was detected by Periodic Acid-Schiff Stain.
4.Detection of HSCs activation state compared with hepatic oval cells proliferation.
4.1 Establish 2-AAF/PH model to stimulate hepatic stem cells differentiation into hepatic oval cells(hepatic progenitor cells).
4.2 Activated HSCs were detected by desmin staining.
5.Activated HSCs were eliminated by gliotoxin administration.The number of hepatic oval cells and expression of hepatic stem/progenitor cells specific genes was evaluated.
5.1 Gliotoxin were administrated 2 day after partial hepatectomy,the effects of eliminating activated HSCs was proved by desmin staining.
5.2 Effect of eliminating activated HSCs on liver regeneration and hepatic oval cells proliferation was evaluated.
5.3 After gliotoxin administration,conditioned HSCs culture medium was supplied by intraperitoneal injection.Liver regeneration and hepatic oval cells proliferation of mouse were compared among experimental groups.
【Results】
1.Characteristics of MSCs Derived from Bone Marrow
MSC cultures were initiated using mononuclear cells isolated from normal bone marrow samples and maintained for up to 10 passages.It took around 12 and 20 days to obtain a homogeneous adherent monolayer and establish primary culture of MSCs.Under adipogenic induction conditions for 3 weeks,the formation of intracellular microdroplets was noted,and stained positive for Oil Red O.While under osteogenic induction conditions for 3 weeks,bone marrow derived MSCs could differentiate into osteoblasts as proved by staining with Alizarin-Red-S for calciumphosphate precipitates.
2.HSC Isolation and activation
2.1 The purity of our HSC preparation was estimated to be greater than 95%according to desmin positive staining.Quiescent HSCs were negative forα-SMA staining.Cells cultured alone for 7 days acquired strongα-SMA staining with a typical filamentous distribution,indicating that HSCs were activated.HSCs cocultured with Kupffer cells were fully activated within 5 days,with strong expression ofα-SMA.
2.2 The purity of our Kupffer cells preparation was estimated to be greater than 95% according to phagocytize assay.
2.3 HGF mRNA expression in HSCs of different states were detected with RT-PCR. HGF was found to be either minimal or negligible in the quiescent HSCs and culture activated HSCs,whereas expressed by HSC-T6 cells and Kupffer cell activated HSCs.
3.Hepatic Differentiation of Bone Marrow-Derived MSC In Vitro
3.1 For HSC-T6 group and Kupffer cell activated HSCs group,the fibroblastic morphology of MSCs was lost and the cells developed a broadened and flattened morphology 1 week post-induction.A retraction of elongated ends was observed 2 weeks post-induction,and the cuboidal morphology of hepatocytes developed with increasing incubation which was retained up to 8 weeks.MSCs remained fibroblastic morphology in the groups of MSC cultured alone and MSCs co-cultured with Kupffer cells,quiescent HSCs or culture activated HSCs up to 8 weeks.
3.2 Hepatocyte-specific genes,such as albumin,AFP,CK-18,GS,tyrosine aminotransferase(TAT),G6P were found to be either minimal or negligible in the undifferentiated MSCs,whereas they could be significantly up-regulated during differentiation into hepatocyte-like cells.In the HSC-T6 and Kupffer cell activated HSCs group,albumin,CK-18,AFP and GS appeared to be expressed at the first or second week of the initiation,whereas the mRNAs of PEPCK and G6P genes was significantly induced at 4 weeks
3.3 On day 14,the majority of the differentiated MSCs in HSC-T6 group and Kupffer cell activated HSCs group expressed the hepatocyte markers,including CK-18,AFP,and albumin.Double-staining revealed that 12.4±3.7%(n=3) of the cells were positive for both albumin and biliary cell marker CK-19 two weeks post-induction,which decreased gradually and then disappeared 4 weeks post-induction.Meanwhile,the gain of hepatic function was also indicated by the gradual loss ofα-SMA,which was positive in nearly all cells up to day 7 and then gradually declined with ongoing culture to barely detectable levels at day 14.
3.4 No glycogen was detected in the primarily isolated MSCs,neither in MSCs cocultured with Kupffer cells,quiescent HSCs or culture activated HSCs.In the differentiated MSCs cultured with HSC-T6 and Kupffer cell activated HSCs group, glycogen was negative at 2 weeks post-induction,but was visualized since 4 weeks post-induction.By 6 weeks,55.4±8.7%(n=4) differentiated cells revealed stored glycogen.
4.Hepatic oval cells proliferation are companied with hepatic stellate cells activation.
4.1 After 2-AAF/PH,hepatic oval cells were detected in liver tissue sections.
4.2 Desmin positive activated HSCs were detected companied with hepatic oval cells proliferation.
5.Activated HSCs elimination defects liver regeneration and hepatic oval cells proliferation.
5.1 Gliotoxin administration eliminated activated HSCs effectively.
5.2 After eliminating activated HSCs,liver index and expression level of hepatic stem/progenitor cells specific genes were lower than that of control group,and were rebound after conditioned HSCs culture medium administration.
【Conclusion】
1.We had successfully isolated the MSCs with multipotency by density gradient and adhesion selection which is quick,simple and practical isolation method.
2.The fully activated HSCs can induce the differentiation of bone marrow derived MSCs into hepatocyte-like cells,which was demonstrated by morphological changes and expression of hepatic specific mRNA and protein.
3.Hepatic differentiation induced by activated HSCs was properly progressed from early to late stages of hepatogenesis.
4.Hepatic oval cells proliferation were companied with hepatic stellate cells activation.
5.Hepatic stellate cells activation is crucial for liver regeneration and hepatic oval cells proliferation.
骨髓组织中存在有多种类型的干细胞,其中关于造血干细胞和间充质干细胞(mesenchymal stem cells,MSCs)的研究报道较多。MSCs是一种较原始的细胞,常维持在低分化状态。MSCs除参与构成造血微环境、支持造血细胞生长外,还具有多向分化潜能。最初只发现MSCs具有生成成骨细胞、软骨细胞、脂肪细胞等间质组织细胞的能力。近年来发现骨髓间质干细胞在特定的条件下还可以向心肌细胞、神经细胞、血管内皮细胞、肝细胞等分化。MSCs具有取材容易、体外增殖能力强、可分化为不同类型的成熟细胞和用于多种疾病治疗等特点,可以避免用胚胎干细胞和异基因干细胞治疗的伦理和免疫排斥等问题,因此它在临床治疗和人体生物组织工程研究方面比其它来源的干细胞更有优势,可能成为多种器官功能老化、组织损伤修复、功能细胞退行性变治疗以及人体生物组织构建的理想种子细胞。
关于骨髓干细胞对肝组织修复的体内研究存在诸多争论:肝大部切除等急性肝损伤动物或患者有“外周血干细胞动员(peripheral blood stem cell mobilization)”现象。有文献证实骨髓干细胞能在体内分化为肝细胞参与组织修复,并可通过骨髓干细胞移植或外周血干细胞动员的途径促进肝损动物或病人的肝功能改善。但最近也有文献报道骨髓干细胞在体内并不能分化为肝细胞参与肝脏修复,却分化为纤维细胞而加剧肝纤维化的进展。这截然相反的两种结果的原因很可能是各组研究中宿主肝组织促分化微环境的不同所致。因而研究生理及病理条件下肝组织对干细胞增殖及分化的影响,以及阐明调控干细胞分化及成熟的适宜微环境对肝再生医学有重大意义。干细胞本身具有分化的多向性潜能和可塑性,其分化受主要由间质细胞和分泌的细胞因子和细胞外基质构成的微环境所调控。肝星状细胞(hepatic stellate cells,HSCs)主要位于Disse间隙,是肝脏的主要间质细胞之一,并且具有树突状突起并嵌入相邻肝细胞的凹陷内,其突起包绕肝窦边界的内皮细胞。在损伤因子作用下,HSC能由静息状态活化增殖,分泌细胞外基质并分泌细胞因子对周围细胞产生强大调控作用,因而很有可能对干细胞的分化产生影响。不同状态的HSCs的调控作用可能是造成骨髓干细胞不同分化方向的原因。为证实该假设,我们将MSCs与静息状态及不同活化状态的HSCs共培养,对比观察不同状态的HSCs对MSCs分化的调控作用。
为了进一步在体内环境下HSCs活化状态对肝再生及干细胞增殖分化的影响,我们采用2-乙酰氨基芴/肝大部切除(2-AAF/PH)模型诱导肝卵圆细胞(肝祖细胞)活化增殖,用免疫荧光方法验证卵圆细胞增殖过程中HSCs活化状态的变化及差异。并用Gliotoxin清除体内活化HSCs,对比研究清除活化HSCs对肝卵圆细胞活化增殖及肝脏再生的影响,进一步证实HSCs活化状态对干细胞分化增殖及肝再生的影响。
【实验方法】
一、原代分离大鼠骨髓MSCs并验证其多向分化潜能性
我们采用梯度离心并培养贴壁的方法分离纯化骨髓MSCs,检测分离细胞活力及传代次数对细胞增值及分化潜能影响,诱导MSCs分别向脂肪细胞既成骨细胞分化,检测MSCs的多向分化潜能,挑选适合本试验诱导分化的细胞。
二、原代分离获取大鼠HSCs、Kupffer细胞,构建不同激活程度的HSCs
1.采用原位灌注消化获得肝非实质细胞,根据细胞密度差异及贴壁速度差别采用梯度离心获得原代HSCs和原代大鼠Kupffer细胞。免疫荧光标记Desmin检测HSCs纯度,吞噬试验检测Kupffer细胞纯度和活力;
2.构建不同活化程度的HSCs,采用贴壁培养来获得半活化状态的HSCs,将HSCs与Kupffer细胞共培养的方式获得完全活化的HSCs。并采用Real-Time PCR检测肝细胞生长因子(hepatic growth factor,HGF)mRNA在各组HSCs的表达,以此研究不同状态HSCs对MSCs调控作用的差异。
三、构建双层细胞共培养系统,检测不同活化状态的HSCs对MSCs分化的影响
1.构建共培养系统,将MSCs分别与下列细胞五组细胞共培养:1)HSC-T6细胞;2)静息状态HSCs,为原代分离培养不多于5天的HSCs;3)贴壁活化的HSCs,为原代贴壁培养7-20天的HSCs;4)Kupffer细胞活化的HSCs:为按1:1比例与Kupffer细胞共培养7-20天的HSCs;5)原代分离的Kupffer细胞。
2.镜下观察不同时间段各组MSCs形态学变化。
3.检测肝细胞特异基因在诱导分化不同时段的各组MSCs中表达,包括肝细胞早期表达基因甲胎蛋白(AFP)、白蛋白(ALB)、细胞角蛋白18(CK-18)、磷酸化糖原合成酶(GS)及成熟肝细胞表达基因磷酸烯醇式丙酮酸羧基酶(PEPCK)、葡萄糖6磷酸酶(G6P),判断各组MSCs是否向肝细胞分化及分化成熟过程。
4.共培养第14天,免疫细胞荧光检测肝细胞特异蛋白在分化的MSCs中表达,包括ALB、AFP、CK-18及间质细胞标记α-平滑肌肌动蛋白(α-SMA),同时激光共聚焦法检测各组细胞在不同时间段是否存在共表达肝细胞标记白蛋白和胆管上皮标记细胞角蛋白19(CK-19)的细胞。
5.用过碘酸-希夫氏液染色(PAS染色)检测各组MSCs在不同时间段的糖原合成,判断细胞是否具有成熟肝细胞的部分功能。
四、对比研究肝卵圆细胞活化增殖过程HSCs活化状态的差异
1.采用2-AAF/PH模型诱导肝卵圆细胞(肝祖细胞)增殖。
2.荧光标记活化HSCs标记蛋白desmin,对比研究卵圆细胞活化增殖模型肝脏组织与对照组肝组织中HSCs活化状态的差别。
五、Gliotoxin清除活化HSCs对干细胞增殖活化及肝再生影响
1.2-AAF/PH造模,术后2天注射Gliotoxin,desmin免疫荧光染色证实活化HSCs的清除效果。
2.对比研究清除活化HSCs后肝再生及卵圆细胞增殖所受影响。
3.清除活化HSCs后,补充体外培养的活化HSCs培养上清,对比研究是否能抵消或部分抵消体内活化HSCs被清除对卵圆细胞增殖及肝再生的影响。
【实验结果】
一、原代分离骨髓MSCs,具有多向分化潜能
采用梯度离心,贴壁培养及适时消化,每只大鼠可分离、纯化获得2-8×10~7 MSCs,细胞存活率在95%以上,细胞均一性好纯度高。在适当诱导培养条件下,MSCs成功分化为脂肪细胞及成骨细胞。
二、原代分离HSCs及Kupffer细胞及不同活化状态HSCs的获取
1.原位灌注消化获得大鼠肝非实质细胞,梯度离心纯化获得大鼠的HSCs得率为2-8×10~7/肝。细胞存活率在90%以上,HSCs特异标记Desmin免疫荧光染色显示分离的细胞纯度)95%。
2.通过梯度离心机及差异贴壁,成功获得大鼠Kupffer细胞,台盼蓝染色检查细胞存活率＞90%,吞噬墨汁实验检测细胞纯度＞95%。
3.获取不同活化状态的HSCs,Real-Time PCR结果显示Kupffer细胞活化的HSCs及HSC-T6细胞的肝细胞生长因子mRNA表达明显高于静息状态HSCs的表达,而贴壁活化HSCs的HGF mRNA表达与静息状态HSCs比较无统计学差异。
三、不同活化状态HSCs诱导MSCs定向分化的差异
1.形态学上,与HSC-T6细胞共培养的MSCs,7天时观察到MSCs呈三角形或不规则形,14天左右分化为多边形细胞与Kuppfer细胞活化HSCs共培养的MSCs也观察到同样现象。而与静息HSC或贴壁活化HSCs共培养的MSCs逐渐展开呈纤维状细胞分化发展,未见到呈多边性细胞形成。单独与Kupffer细胞共培养的MSCs也呈纤维样细胞生长。
2.与Kupffer细胞活化HSCs共培养的MSCs,7天时检测到AFP、ALB、CK-18、GS、TAT的mRNA表达,第14天表达程度无变化,到28天减弱。共培养28天的MSCs检测到成熟肝细胞特异表达基因PEPCK、G6P的表达。而与静息HSC或贴壁活化HSCs共培养的MSCs未检测到肝细胞系基因的表达。
3.共培养第14天,与Kupffer细胞活化HSCs共培养的MSCs用免疫细胞荧光法检测到albumin、AFP和CK-18的表达,未检测到间质细胞标志α-SMA。而与静息状态HSCs共培养的MSCs及与贴壁活化HSCs共培养的MSCs未检测到肝细胞特异蛋白的表达,α-SMA强阳性表达。
4.在与Kupffer细胞活化HSCs共培养14天的MSCs中,有12.4±3.7%的细胞同时表达肝细胞标志蛋白ALB和胆管上皮标志蛋白CK-19,表明该细胞具有肝细胞和胆管细胞的双向分化潜能性。随着时间迁移这种双标记细胞所占比例逐渐减少,至第4周已完全检测不到该双标细胞。
5.与HSC-T6共培养MSCs在第4周用PAS染色检测到糖原沉积,到第6周达到最高峰,有55.4%±8.7%细胞检测到糖原沉积。与Kupffer细胞活化HSCs共培养的MSCs同样检测到糖原沉积,而与静息状态HSCs共培养的MSCs及与贴壁活化HSCs共培养的MSCs未检测到糖原沉积。
四、卵圆细胞活化增殖过程中HSCs活化状态的变化
1.2-AAF/PH模型成功诱导肝卵圆细胞活化增殖。
2.卵圆细胞活化增殖过程中肝脏HSCs大量活化增殖,表达desmin,而对照正常肝脏及单纯肝部分切除小鼠肝脏仅见少量desmin阳性的活化HSCs。
五、清除活化HSCs对卵圆细胞增殖及肝再生影响
1.Gliotoxin有效清除体内活化的HSCs。
2.Gliotoxin清除活化HSCs后,肝重指数明显小于单纯2-AAF/PH组,肝干/祖细胞标志基因CK-18、CD90、CD133、SOX-2表达水平明显低于单纯2-AAF/PH组,补充体外培养的活化HSCs培养上清后,肝重指数明显高于清除活化HSCs组,肝干/祖细胞标志基因的表达也得到部分恢复。
【结论】
1.密度梯度离心,贴壁培养和消化时间控制相结合,具有操作简单、快速、实用等优点,是一种较为有效的分离纯化MSCs方法。
2.充分活化的HSCs能诱导MSCs向肝细胞分化,具有类似肝细胞形态,表达肝细胞特异基因和蛋白,具有肝细胞特有的糖原合成功能。
3.活化HSCs诱导的MSCs定向分化是一个经肝细胞、胆管细胞双潜能细胞向成熟肝细胞逐渐分化和成熟的过程。
4.肝卵圆细胞活化增殖过程伴随着HSCs的活化;
5.HSCs的活化是肝再生及肝干/祖细胞增殖的必要环节。
【Background and Objective】
Bone marrow mesenchymal stem cells(MSC) are nonhematopoietic cells that reside within the bone marrow stroma.These cells are multipotent and serve as precursors for various cells including osteoblast,smooth muscle cells,chondrocytes,adipocytes and hematopoietic supportive cells.The generation of hepatocytes from bone marrow derived stem cells is a compelling concept that has provoked much interest in the liver transplant field.Petersen et al.reported that some liver oval cells were derived from transplanted bone marrow.Hepatic oval cells are hepatic progenitor cells which are characterized by an ovoid nucleus,small size,and scant basophilic cytoplasm.Oval cells express phenotypical markers of both the biliary epithelium and hepatocyte lineages.After that,there are both positive and negative reports concerning the potency of bone marrow derived stem cells to differentiate into hepatocytes in vivo.The conflict results may attribute to the different stem cell microenvironment(or "niche") which is composed of non-parenchymal cells and secreted factors.
The concept of the microenvironment was first developed in hematopoiesis,where other cells secrete factors to support proliferation,differentiation,and survival of distinct progenitor populations.The effect of niche cells on stem/progenitor cells in proliferation and differentiation was also demonstrated in other organs/tissues such as epidermal,heart, brain,etc.To date,the microenvironment which modulate differentiation of hepatic stem cells and control transdifferentiation of bone marrow derived stem cells migrated into liver are still unknown.
Hepatic stellate cell(HSC) is one of major hepatic nonparenchymal cells which are located in the Disse space between the endothelium and the hepatocytes.Under conditions of stress or liver injury,quiescent HSCs are activated and acquire a myofibroblastic phenotype,contributing to excessive extracellular matrix deposition which gives rise to development of liver fibrosis and cirrhosis.On the other hand,activated HSCs were recently demonstrated to secrete factors to stimulate hepatocyte proliferation.Anatomically, bone marrow derived stem cells must circumvent the Disse space during translocation form liver sinusoids to the parenchyma and may be regulated by HSCs.Therefore,we assume that HSCs may present as a modulator for the differentiation of MSCs into hepatic cells and different states of HSCs may play variant roles.
To prove this assumption,bone marrow MSCs were indirectly cocultured with quiescent HSCs or activated HSCs.Primary isolated quiescent HSCs were activated by being in vitro cultured alone or cocultured with Kupffer cells.Expression of hepatic specific markers and hepatocyte function of glycogen deposition were detected on differentiated MSCs.The variant effects of HSCs in different states on the differentiation of MSCs were compared.
【Methods】
1.MSCs Preparation and Delivery
Rat bone marrow MSCs were harvested from mononuclear cells which were separated by Percoll density gradient.MSCs were plastic-adherent mononuclear bone marrow cells after expansion in culture.The resulting MSCs(passage 6-10) were used for our studies. To confirm the identity of bone marrow MSCs,the cells were grown in medium that is conducive to differentiation into osteoclasts and adipocytes.Adipocytes containing lipid droplets were observed 2 weeks later by Oil Red O(Sigma-Aldrich) staining.The deposition of bone mineral by osteoclasts differentitated from MSCs was observed 2 weeks later by Alizarin red(pH 4.1;Sigma-Aldrich) staining.
2.Preparation of HSCs and Kupffer Cells
Primary HSCs and Kupffer cells were freshly isolated from rat liver nonparenchymal cells.The liver was perfused with collagenase and pronase solution.The supernatant was centrifuged at 50×g for 2 min for several rounds to collect and nonparenchymal cells containing the HSCs and Kupffer cells.HSCs were isolated by density gradient centrifugation.Kupffer cells were separated by density gradient and adhesion culture.
3.Establishment of coculture system and investigation of the effects of HSCs in different activation state on the MSCs
3.1 MSCs were coculture with HSCs at different states:HSC-T6 cells,the immortalized activated HSCs cells;quiescent HSCs:freshly isolated HSCs within 5 days;culture activated HSCs:primarily isolated HSCs cultured for 7 to 20 days;Kupffer cell activated HSCs:primarily isolated HSCs cocultured with Kupffer cells for 7 to 20 days.MSCs were also cocultured with Kupffer cells as control group for Kupffer cells activated HSCs group. Culture medium was half changed every 3 days.
3.2 Morphological changes of MSCs cocultured with HSCs were observed under microscopy.
3.3 The hepatic specific mRNA expression of MSCs cocultured with HSCs was analyzed by reverse transcription-polymerase chain reaction(RT-PCR),including unmature hepatocyte marker alpha-fetoprotein(AFP),albumin,cytokeratin 18(CK18), glutamine synthetase(GS) and mature hepatocyte marker phosphoenolpyruvate carboxykinase(PEPCK),glucose 6 phosphate dehydrogenase(G6P).
3.4 The MSCs were detected for expression of specific proteins after coculture with HSCs for 2 weeks with immunofluorescence,including biliary epithelial cells marker CK-19,hepatocyte marker CK-18,albumin and AFP,MSCs marker alpha-smooth muscle actin(α-SMA).
3.5 Glycogen deposition was detected by Periodic Acid-Schiff Stain.
4.Detection of HSCs activation state compared with hepatic oval cells proliferation.
4.1 Establish 2-AAF/PH model to stimulate hepatic stem cells differentiation into hepatic oval cells(hepatic progenitor cells).
4.2 Activated HSCs were detected by desmin staining.
5.Activated HSCs were eliminated by gliotoxin administration.The number of hepatic oval cells and expression of hepatic stem/progenitor cells specific genes was evaluated.
5.1 Gliotoxin were administrated 2 day after partial hepatectomy,the effects of eliminating activated HSCs was proved by desmin staining.
5.2 Effect of eliminating activated HSCs on liver regeneration and hepatic oval cells proliferation was evaluated.
5.3 After gliotoxin administration,conditioned HSCs culture medium was supplied by intraperitoneal injection.Liver regeneration and hepatic oval cells proliferation of mouse were compared among experimental groups.
【Results】
1.Characteristics of MSCs Derived from Bone Marrow
MSC cultures were initiated using mononuclear cells isolated from normal bone marrow samples and maintained for up to 10 passages.It took around 12 and 20 days to obtain a homogeneous adherent monolayer and establish primary culture of MSCs.Under adipogenic induction conditions for 3 weeks,the formation of intracellular microdroplets was noted,and stained positive for Oil Red O.While under osteogenic induction conditions for 3 weeks,bone marrow derived MSCs could differentiate into osteoblasts as proved by staining with Alizarin-Red-S for calciumphosphate precipitates.
2.HSC Isolation and activation
2.1 The purity of our HSC preparation was estimated to be greater than 95%according to desmin positive staining.Quiescent HSCs were negative forα-SMA staining.Cells cultured alone for 7 days acquired strongα-SMA staining with a typical filamentous distribution,indicating that HSCs were activated.HSCs cocultured with Kupffer cells were fully activated within 5 days,with strong expression ofα-SMA.
2.2 The purity of our Kupffer cells preparation was estimated to be greater than 95% according to phagocytize assay.
2.3 HGF mRNA expression in HSCs of different states were detected with RT-PCR. HGF was found to be either minimal or negligible in the quiescent HSCs and culture activated HSCs,whereas expressed by HSC-T6 cells and Kupffer cell activated HSCs.
3.Hepatic Differentiation of Bone Marrow-Derived MSC In Vitro
3.1 For HSC-T6 group and Kupffer cell activated HSCs group,the fibroblastic morphology of MSCs was lost and the cells developed a broadened and flattened morphology 1 week post-induction.A retraction of elongated ends was observed 2 weeks post-induction,and the cuboidal morphology of hepatocytes developed with increasing incubation which was retained up to 8 weeks.MSCs remained fibroblastic morphology in the groups of MSC cultured alone and MSCs co-cultured with Kupffer cells,quiescent HSCs or culture activated HSCs up to 8 weeks.
3.2 Hepatocyte-specific genes,such as albumin,AFP,CK-18,GS,tyrosine aminotransferase(TAT),G6P were found to be either minimal or negligible in the undifferentiated MSCs,whereas they could be significantly up-regulated during differentiation into hepatocyte-like cells.In the HSC-T6 and Kupffer cell activated HSCs group,albumin,CK-18,AFP and GS appeared to be expressed at the first or second week of the initiation,whereas the mRNAs of PEPCK and G6P genes was significantly induced at 4 weeks
3.3 On day 14,the majority of the differentiated MSCs in HSC-T6 group and Kupffer cell activated HSCs group expressed the hepatocyte markers,including CK-18,AFP,and albumin.Double-staining revealed that 12.4±3.7%(n=3) of the cells were positive for both albumin and biliary cell marker CK-19 two weeks post-induction,which decreased gradually and then disappeared 4 weeks post-induction.Meanwhile,the gain of hepatic function was also indicated by the gradual loss ofα-SMA,which was positive in nearly all cells up to day 7 and then gradually declined with ongoing culture to barely detectable levels at day 14.
3.4 No glycogen was detected in the primarily isolated MSCs,neither in MSCs cocultured with Kupffer cells,quiescent HSCs or culture activated HSCs.In the differentiated MSCs cultured with HSC-T6 and Kupffer cell activated HSCs group, glycogen was negative at 2 weeks post-induction,but was visualized since 4 weeks post-induction.By 6 weeks,55.4±8.7%(n=4) differentiated cells revealed stored glycogen.
4.Hepatic oval cells proliferation are companied with hepatic stellate cells activation.
4.1 After 2-AAF/PH,hepatic oval cells were detected in liver tissue sections.
4.2 Desmin positive activated HSCs were detected companied with hepatic oval cells proliferation.
5.Activated HSCs elimination defects liver regeneration and hepatic oval cells proliferation.
5.1 Gliotoxin administration eliminated activated HSCs effectively.
5.2 After eliminating activated HSCs,liver index and expression level of hepatic stem/progenitor cells specific genes were lower than that of control group,and were rebound after conditioned HSCs culture medium administration.
【Conclusion】
1.We had successfully isolated the MSCs with multipotency by density gradient and adhesion selection which is quick,simple and practical isolation method.
2.The fully activated HSCs can induce the differentiation of bone marrow derived MSCs into hepatocyte-like cells,which was demonstrated by morphological changes and expression of hepatic specific mRNA and protein.
3.Hepatic differentiation induced by activated HSCs was properly progressed from early to late stages of hepatogenesis.
4.Hepatic oval cells proliferation were companied with hepatic stellate cells activation.
5.Hepatic stellate cells activation is crucial for liver regeneration and hepatic oval cells proliferation.
引文
1. Shafritz DA, Oertel M, Menthena A, et al. Liver stem cells and prospects for liver reconstitution by transplanted cells. Hepatology. 2006;43(2 Suppl 1):S89-98. Review.
2. Oh SH, Witek RP, Bae SH, et al. Bone marrow-derived hepatic oval cells differentiate into hepatocytes in 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration. Gastroenterology. 2007; 132(3): 1077-1087.
3. Agarwal S, Holton KL, Lanza R. Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells. 2008;26(5):1117-1127.
4. Lemaigre F, Zaret KS. Liver development update: New embryo models, cell lineage control, and morphogenesis. Curr Opin Genet Dev 2004; 14:582-590.
5. Alison MR, Poulsom R, Forbes SJ. Update on hepatic stem cells. Liver 2001;21:367-373.
6. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276:60-66.
7. Michalopoulos GK, DeFrances M. Liver regeneration. Adv Biochem Eng Biotechnol 2005;93:101-134.
8. Rabes HM, Wirsching R, Tuczek HV, Iseler G. Analysis of cell cycle compartments of hepatocytes after partial hepatecomy. Cell Tissue Kinet. 1976;9:517-532.
9. Ankoma-Sey V. Hepatic regeneration-revisiting the myth of Prometheus. News Physiol Sci.1999;14:149-155.
10. Borowiak M, Alistair NG, Torsten WS, et al. C-Met provides essential signals for liver regeneration. PNAS 2004;101:10608-10613.
11. Yamada Y, Kirillova T, Peschon JJ, Fausto N. Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. PNAS 1997;94:1441-1446.
12. Cosgrove BD, Cheng C, Pritchard JR, et al. An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-alpha.Hepatology. 2008 Mar 24. [Epub ahead of print]
13. Yasukawa H, Sasaki A, Yoshimura A. Negative regulation of cytokine Signaling pathways. Ann Rev Immunol.2000;l:143-146.
14. Koniaris LG, McKillop IH, Schwartz SI, et al. Liver regeneration. J Am Coll Surg. 2003;197:634-659.
15. Malik R, Selden C, Hodgson H. The role of non-parenchymal cells in liver growth. Semin Cell Dev Biol. 2002; 13:425-431.
16. Sigal SH, Rajvanshi P, Gorla GR, et al. Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. A int physiol. 1997;276:1260-1272.
17. Wilson JW, Leduc EH. Role of cholangioles in restoration of the liver of the mouse after dietary injury. J Pathol Ticteriol 1958;76:441-449.
18．立野知世,吉理胜利。肝干细胞．蛋白质核酸酵素2000；45：2085-2091．
19. Thorgeirsson SS. Hepatic stem cells in liver regeneration. FASEB J. 1996; 10:1249-1256.
20. Yang L, Li S, Hatch H, et al. In vitro transdifferentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci USA 2002;99:8078-8083.
21. Grompe M. The role of bone marrow stem cells in liver regeneration. Semin Liver Dis 2003 Nov;23:363-72.
22. Petersen BE, Grossbard B, Hatch H, et al. Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology. 2003;37:632-640.
23. Mitaka T, MikamiM, Sattler GL, et al. Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor. Hepatology, 1992; 16(2):440-447.
24. Xu CS, Zhao LF, Yang KJ. The origination and action of the hepatic stems cells. Shi Yan Sheng Wu Xue Bao 2004;37:72-77.
25. Malcolm RA. Liver regeneration with reference to stem cells. Cell Develo Bio. 2002;13:385-387.
26.Overturf K,AL-Dhalimy M,Finegold M,et al.The repopulation potential of hepalocyte populations differing in size and prior mitotic expansion.Am J Pathol.1999;150:2135-2143.
27.Rudolph KL,Chang S,Millard M,et al.Inhibition of experimental liver cirrhosis in mice by LSL gene deliver.Science 2000;287:1253-1258.
28.He ZP,Tan WQ,Tang YF,et al.Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor.Differentiation,2003;71(425):281-290.
29.David A,Mariana D,Dabeva H,et al.Liver stem cell and model system for liver repopulation[J].J Hepatol,2002;36:552-564.
30.Braun KM,Sandgren EP.Cellular origin of regenerating parenchyma in a mouse model of severe hepatic injury.Am J Pathol 2000;157:5687-5691.
31.龚加庆,方驰华,李雅,田伏洲.卵圆细胞参与实验性肝癌形成过程的研究.中华外科杂志 2004;42:291-295.
32.Grisham JW,Gordon GJ,Coleman WB,et al.Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.Am J Pathol.2000;156:607-619.
33.Zimmermann A.Regulation of liver regeneration.Nephrol Dial Transplant.2004;19:6-10.
34.Michalopoulos GK,Khan Z.Liver regeneration,growth factors,and amphiregulin.Gastroenterology 2005;128:503-506.
35.Akatsugu Y,Masahide Y,Sei ji K,et al.In Vitro Differentiation of Embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green.Stem Cells 2002;20:146-154.
36.Hu A,Cai I,Zheng Q,et al.Hepatic differentiation from embryonic stem cells in vitro.Chin Med.116:1893-1897.
37.Yin J,Yew KL,Manuel ST,et al.AFP~+ ESC-derived Cells engraft and differentiate into hepatocytes in Vivo.Stem Cells.2002;20:338-346.
38. Yamada T, Yoshikawa M, Kanda S, et al. In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells.2002;20:146-154.
39. Ruhnke M, Ungefroren H, Zehle G, et al. Long-term culture and differentiation of rat embryonic stem cell like cells into neuronalglial, endothelial, and hepatic lineages. Stem Cells. 2003;21:428-436.
40. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science. 1999;284:1168-1170.
41. Alison MR, Poulsom R, Jeffery R, et al. Hepatocytes from non-hepatic adult stem cells.Nature 2000;406:257.
42. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med.2000;6:1229-1234.
43. Ramsey MR, Butz GM, Grisham JW, et al. Hepatocytic differentiation of bone marrow-derived progenitor cells in vivo. FASEB Journal, 2001;17:665-671.
44. Avital I, Inderbitzin D, Aoki T, et al. Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells. Biochem Biophys Res Commun.2001;288:156-164.
45. Zuleuski H , Abraham EJ , Gerlach MJ , et al . Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine , exocrine and hepatic phenotype. Diabetes , 2001 ;58:231-251.
46. Goodell MA. Stem cell plasticity: befuddled by the muddle. Curr Opin Hematol.2003;10:208-213.
47. Kodama S, Kuhtreiber W, Fujimura S, et al. Islet regeneration during the reversal of auto immune diabetes in NOD mice. Science.2003;302:1223-1227.
48. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003;425:968-973.
49. Lagasse E, Connors H, Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med.2000;6:1229-1234.
50. Wang X, Willenbring H, Akkari Y, et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature.2003;422:897-901.
51. Vassilopoulos G, Wang PR, Russell DW, et al. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901-904.
52. Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood.2003;102:3483-3493.
53. Omori K, Terai S, Ishikawa T, et al. Mollecular signature associated with plasticity of bone marrow cell under persistent liver damage by self-organizing-map-based gene expression. FEBS Lett. 2004;578:10-20.
54. Forbes SJ. Myelomonocytic cells are sufficient for therapeutic cell fusion in the liver. J Hepatol.2005;42:285-286.
55. Scott EW. Stem cell plasticity or fusion: two approaches to targeted Cell therapy. Blood Cells Mol Dis 2004;32:65-67.
56. Krause DS, Theise ND, Collector M. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369-377.
57. Min AD, Theise ND. Prospects for cell-based therapies for liver disease. Pan Minerva Med.2004;46:43-48.
58. Alison MR, Poulsom R, Jeffery R. Hepatocyte from non-hepatic adult stem cells. Nature 2000;406:257.
59. Linda A. Lee. Advances in hepatocyte transplantation: a myth becomes reality. Chem Invest.2001;108:367-369.
2. Oh SH, Witek RP, Bae SH, et al. Bone marrow-derived hepatic oval cells differentiate into hepatocytes in 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration. Gastroenterology. 2007; 132(3): 1077-1087.
3. Agarwal S, Holton KL, Lanza R. Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells. 2008;26(5):1117-1127.
4. Lemaigre F, Zaret KS. Liver development update: New embryo models, cell lineage control, and morphogenesis. Curr Opin Genet Dev 2004; 14:582-590.
5. Alison MR, Poulsom R, Forbes SJ. Update on hepatic stem cells. Liver 2001;21:367-373.
6. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276:60-66.
7. Michalopoulos GK, DeFrances M. Liver regeneration. Adv Biochem Eng Biotechnol 2005;93:101-134.
8. Rabes HM, Wirsching R, Tuczek HV, Iseler G. Analysis of cell cycle compartments of hepatocytes after partial hepatecomy. Cell Tissue Kinet. 1976;9:517-532.
9. Ankoma-Sey V. Hepatic regeneration-revisiting the myth of Prometheus. News Physiol Sci.1999;14:149-155.
10. Borowiak M, Alistair NG, Torsten WS, et al. C-Met provides essential signals for liver regeneration. PNAS 2004;101:10608-10613.
11. Yamada Y, Kirillova T, Peschon JJ, Fausto N. Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. PNAS 1997;94:1441-1446.
12. Cosgrove BD, Cheng C, Pritchard JR, et al. An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-alpha.Hepatology. 2008 Mar 24. [Epub ahead of print]
13. Yasukawa H, Sasaki A, Yoshimura A. Negative regulation of cytokine Signaling pathways. Ann Rev Immunol.2000;l:143-146.
14. Koniaris LG, McKillop IH, Schwartz SI, et al. Liver regeneration. J Am Coll Surg. 2003;197:634-659.
15. Malik R, Selden C, Hodgson H. The role of non-parenchymal cells in liver growth. Semin Cell Dev Biol. 2002; 13:425-431.
16. Sigal SH, Rajvanshi P, Gorla GR, et al. Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. A int physiol. 1997;276:1260-1272.
17. Wilson JW, Leduc EH. Role of cholangioles in restoration of the liver of the mouse after dietary injury. J Pathol Ticteriol 1958;76:441-449.
18．立野知世,吉理胜利。肝干细胞．蛋白质核酸酵素2000；45：2085-2091．
19. Thorgeirsson SS. Hepatic stem cells in liver regeneration. FASEB J. 1996; 10:1249-1256.
20. Yang L, Li S, Hatch H, et al. In vitro transdifferentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci USA 2002;99:8078-8083.
21. Grompe M. The role of bone marrow stem cells in liver regeneration. Semin Liver Dis 2003 Nov;23:363-72.
22. Petersen BE, Grossbard B, Hatch H, et al. Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology. 2003;37:632-640.
23. Mitaka T, MikamiM, Sattler GL, et al. Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor. Hepatology, 1992; 16(2):440-447.
24. Xu CS, Zhao LF, Yang KJ. The origination and action of the hepatic stems cells. Shi Yan Sheng Wu Xue Bao 2004;37:72-77.
25. Malcolm RA. Liver regeneration with reference to stem cells. Cell Develo Bio. 2002;13:385-387.
26.Overturf K,AL-Dhalimy M,Finegold M,et al.The repopulation potential of hepalocyte populations differing in size and prior mitotic expansion.Am J Pathol.1999;150:2135-2143.
27.Rudolph KL,Chang S,Millard M,et al.Inhibition of experimental liver cirrhosis in mice by LSL gene deliver.Science 2000;287:1253-1258.
28.He ZP,Tan WQ,Tang YF,et al.Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor.Differentiation,2003;71(425):281-290.
29.David A,Mariana D,Dabeva H,et al.Liver stem cell and model system for liver repopulation[J].J Hepatol,2002;36:552-564.
30.Braun KM,Sandgren EP.Cellular origin of regenerating parenchyma in a mouse model of severe hepatic injury.Am J Pathol 2000;157:5687-5691.
31.龚加庆,方驰华,李雅,田伏洲.卵圆细胞参与实验性肝癌形成过程的研究.中华外科杂志 2004;42:291-295.
32.Grisham JW,Gordon GJ,Coleman WB,et al.Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.Am J Pathol.2000;156:607-619.
33.Zimmermann A.Regulation of liver regeneration.Nephrol Dial Transplant.2004;19:6-10.
34.Michalopoulos GK,Khan Z.Liver regeneration,growth factors,and amphiregulin.Gastroenterology 2005;128:503-506.
35.Akatsugu Y,Masahide Y,Sei ji K,et al.In Vitro Differentiation of Embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green.Stem Cells 2002;20:146-154.
36.Hu A,Cai I,Zheng Q,et al.Hepatic differentiation from embryonic stem cells in vitro.Chin Med.116:1893-1897.
37.Yin J,Yew KL,Manuel ST,et al.AFP~+ ESC-derived Cells engraft and differentiate into hepatocytes in Vivo.Stem Cells.2002;20:338-346.
38. Yamada T, Yoshikawa M, Kanda S, et al. In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells.2002;20:146-154.
39. Ruhnke M, Ungefroren H, Zehle G, et al. Long-term culture and differentiation of rat embryonic stem cell like cells into neuronalglial, endothelial, and hepatic lineages. Stem Cells. 2003;21:428-436.
40. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science. 1999;284:1168-1170.
41. Alison MR, Poulsom R, Jeffery R, et al. Hepatocytes from non-hepatic adult stem cells.Nature 2000;406:257.
42. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med.2000;6:1229-1234.
43. Ramsey MR, Butz GM, Grisham JW, et al. Hepatocytic differentiation of bone marrow-derived progenitor cells in vivo. FASEB Journal, 2001;17:665-671.
44. Avital I, Inderbitzin D, Aoki T, et al. Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells. Biochem Biophys Res Commun.2001;288:156-164.
45. Zuleuski H , Abraham EJ , Gerlach MJ , et al . Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine , exocrine and hepatic phenotype. Diabetes , 2001 ;58:231-251.
46. Goodell MA. Stem cell plasticity: befuddled by the muddle. Curr Opin Hematol.2003;10:208-213.
47. Kodama S, Kuhtreiber W, Fujimura S, et al. Islet regeneration during the reversal of auto immune diabetes in NOD mice. Science.2003;302:1223-1227.
48. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003;425:968-973.
49. Lagasse E, Connors H, Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med.2000;6:1229-1234.
50. Wang X, Willenbring H, Akkari Y, et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature.2003;422:897-901.
51. Vassilopoulos G, Wang PR, Russell DW, et al. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901-904.
52. Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood.2003;102:3483-3493.
53. Omori K, Terai S, Ishikawa T, et al. Mollecular signature associated with plasticity of bone marrow cell under persistent liver damage by self-organizing-map-based gene expression. FEBS Lett. 2004;578:10-20.
54. Forbes SJ. Myelomonocytic cells are sufficient for therapeutic cell fusion in the liver. J Hepatol.2005;42:285-286.
55. Scott EW. Stem cell plasticity or fusion: two approaches to targeted Cell therapy. Blood Cells Mol Dis 2004;32:65-67.
56. Krause DS, Theise ND, Collector M. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369-377.
57. Min AD, Theise ND. Prospects for cell-based therapies for liver disease. Pan Minerva Med.2004;46:43-48.
58. Alison MR, Poulsom R, Jeffery R. Hepatocyte from non-hepatic adult stem cells. Nature 2000;406:257.
59. Linda A. Lee. Advances in hepatocyte transplantation: a myth becomes reality. Chem Invest.2001;108:367-369.