间充质干细胞在肝纤维化形成中的作用及机制研究
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摘要
【研究背景及目的】
肝纤维化(hepatic fibrosis)是肝脏对慢性损伤的一种修复反应,以细胞外基质(extracellular matrix, ECM)在肝内过多沉积为特征。肝纤维化为一动态过程,属可逆性病变,因此,阻断、抑制或逆转肝纤维化是治疗慢性肝病的一个重要目标。既往认为,肝纤维化发生的中心环节是肝星状细胞(hepatic stellate cells,HSCs)激活并向肌成纤维样细胞(myofibroblasts, MFs)转化,抑制HSC激活、增殖与迁移、诱导凋亡是肝纤维化治疗的重要策略。近年来研究发现,在体内环境中通过单纯抑制HSCs来治疗肝纤维化的效果并不理想。而更多的研究者也逐渐认识到HSC也不是唯一重要的影响肝纤维化形成的细胞。那么探索其他参与肝纤维化形成的细胞亚群,并深入研究其作用机制想法就越来越受到研究者的重视。
肝纤维化的发生、发展始终伴随着慢性炎症反应。其过程中受损的肝脏会分泌大量的细胞因子,生长因子,趋化因子参与纤维化的形成。与此同时多种肝外细胞群也会向受损部位趋化,发挥其作用。已有研究显示骨髓间充质干细胞(mesenchymal stem cells, MSCs)也是这些肝外细胞种群之一,在肝脏损伤的过程中它们会趋化到受损部位参与了肝纤维化的形成。MSCs是一种多潜能分化的非造血干细胞,主要存在于骨髓中。以往对MSCs的了解主要集中在它有向损伤部位趋化并修复损伤的功能,因此将其作为基因治疗药物进行开发,并在某些疾病,如结缔组织类疾病以及骨损伤的治疗中取得了良好的效果。但随着研究的深入,研究者又发现MSCs在某些疾病的发生、发展中还可能发挥了某些不利的作用,如它可以促进多种肿瘤的生长。对很多疾病特别是慢性疾病来说MSCs可能是一把双刃剑。这些结果提示研究者应该重新严格的审视MSCs在疾病当中起到的作用。对于肝脏损伤来说,MSCs在纤维化形成中的作用目前也存在着较大的争议。MSCs到底是促进还是抑制纤维化的发展是我们研究的目的之一。
肝纤维化形成的一个重要的特点是肝脉管系统被大肆重建。血管系统重建伴随着肝纤维化发展的始终。在此过程中肝脏必然分泌大量的与血管生成有关的细胞因子,如VEGF,IL-6,IL-8等。而某些研究者也证实在体外实验中VEGF,PDGF等某些细胞因子或生长因子可以促进MSCs的增殖。MSCs向受损部位趋化是一个持续的过程,只要病因存在,炎症环境充分的建立起来,MSCs就会源源不断向受损部位募集。为了满足大量的、持续不断的细胞需要,就必然有某种原因或机制促使骨髓中的MSCs自我增殖补充。在此基础上我们提出假设,肝纤维化形成过程中分泌的大量的促血管生成的细胞因子如VEGF可能就是体内骨髓MSCs不断增殖的重要原因之一。当然,体内MSCs的趋化动员,不单单需要骨髓中MSCs的不断增殖,还需要有某些机制调节BM-MSCs转移出骨髓,进而随着外周血趋化募集到受损的部位。目前研究显示MSCs的迁移与趋化因子家族成员及其受体密切相关,其中最为重要的是CC-趋化家族和CXC-趋化家族的成员。但也有研究证明不同器官或组织损伤时,影响MSCs的迁移的趋化因子及其受体是不尽相同的,因此明确何种趋化因子在肝脏损伤,肝纤维化形成过程中影响BM-MSCs向肝脏的趋化募集,就可以为控制纤维化的发展寻找新的治疗靶点。
本课题在建立多种小鼠动物模型的基础上,使用了荧光染色,免疫组化,PCR等多种分子生物学技术,探讨MSCs在肝纤维化形成过程中发挥的作用及其对肝功能的影响。并同时深入的探讨了BM-MSCs在体内增殖、迁移的主要原因及其作用机制。同时结合肝硬化临床标本的检测,以期进一步深入了解肝纤维化的发病机制,为肝纤维化的治疗提供新的思路。
【实验方法】
一、骨髓间充质干细胞的分离、培养和鉴定
1.分别使用雄性WT-BALB/c小鼠和EGFP-BALB/c小鼠分离培养WT-MSCs和EGFP-MSCs细胞。采用密度梯度离心与贴壁培养相结合的方法来分离MSCs,但此时也混有少量的单核或局势细胞以及成纤维细胞。利用这些细胞与MSCs对塑料培养瓶的贴壁性强弱不同,细胞传代时用0.05%胰蛋白酶+0.02%EDTA静止消化2-3min后,反复多次的消化传代后可逐渐剔除其它种类细胞。
2. MSCs的鉴定
倒置相差显微镜动态观察小鼠WT-MSCs原代培养的生长过程;荧光显微镜下观察小鼠EGFP-MSCs细胞的生长过程。流式细胞仪技术、以及细胞免疫荧光染色技术检测小鼠MSCs上表面标记物CD34,CD90,CD45,CD90,CD105的表达情况。成骨诱导分化Von Kossa染色,以及ALP活性定量,检测MSCs成骨定向分化的能力,成脂诱导分化Oil Red O染色检测MSCs成脂定向分化的能力。
二、间充质干细胞在肝纤维化形成中的作用
构建小鼠CCL4肝损伤模型(模型1),以及小鼠骨髓移植模型+CCL4肝损伤模型(模型2)。确定骨髓移植模型构建成功后受体wt小鼠骨髓内有供体小鼠的EGFP-MSCs存在。通过尾静脉注射外源性的EGFP-MSCs给模型1的小鼠,3d后,冰冻切片免疫荧光显微镜下观察外源性的MSCs的趋化部位,以及肝纤维化的程度;通过模型2示踪内源性的EGFP-MSCs在肝脏损伤后的趋化部位;同时检测不同时间点AST,ALT的含量,检测肝纤维化形成过程中肝功能的情况。
三、纤维化形成过程中骨髓间充质干细胞的动员
使用模型1和模型2的小鼠,每周末处死部分小鼠,连续5周。模型1的每周处死的小鼠,于处死前3天注射外源性的EGFP-MSCs进行示踪。冰冻切片后荧光显微镜下观察,确定内、外源性的MSCs在小鼠动物模型中向肝脏趋化募集的时间。Realtime-PCR法筛选MSCs表面重要的趋化因子受体,以及对应的损伤肝脏中主要的发挥作用的趋化因子。然后观察此趋化因子在小鼠肝脏以及骨髓中的表达情况,以及体内外试验中对BM-MSCs的趋化能力。
四、肝纤维化形成过程中VEGF促进骨髓间充质干细胞增殖的机制研究
用免疫组化,realtime-PCR,ELISA等方法检测小鼠肝脏,血清中VEGF mRNA和/或蛋白质的表达情况。体外细胞增殖试验和单克隆形成试验检测VEGF对MSCs的增殖的影响,流式细胞仪技术检测小鼠体内VEGF对BM-MSCs增殖的影响,以及使用Avastin是否可以逆转这种影响。同时运用Transwell实验及划痕试验检测VEGF是否具有促进MSCs趋化迁移的能力。
五、统计学处理
数据采用SPSS 11.0以及Prism GraphPad5统计软件包进行分析、作图。P<0.05为具有显著性差异,P<0.01为具有非常显著性差异。
【实验结果】
一、骨髓间充质干细胞的分离、培养和鉴定
1. MSCs的分离培养:小鼠BM-MSCs原代细胞进行培养后,24h后出现较多贴壁细胞,48-72h后成纺锤丝形或梭形生长,第4-5天开始形成典型的均匀分布的簇状增生灶,7-10天非贴壁细胞经反复换液而消失,细胞80%以上融合。再经过多次传代培养,细胞都呈梭形。人MSCs细胞系的培养从形态学上观察细胞呈典型的长梭形,细胞形态趋于一致,紧密排列类似旋涡状。
2.小鼠BM-MSCs表面抗原的鉴定:运用流式细胞仪技术记忆细胞免疫荧光染色技术,检测上述分离培养的MSCs的表面标记物的表达情况,结果显示其表面表达粘附分子CD29,CD105,CD90的阳性率在95%以上,而造血干细胞的表面标志CD34,CD45几乎不表达。表明这些分离培养的细胞具有MSCs特征的比例非常高。
3.小鼠MSCs的多向分化潜能:成骨诱导分化后14d,我们对细胞进行ALP阳性的检测,发现与对照组相比,诱导组ALP活性定量增高。第21d,Von Kossa染色,细胞表现为黑色的致密结节。成脂诱导分化后8d,光镜下可见细胞形态发生改变,胞内不乏圆形透亮脂质空泡,应用Oil Red-O原位染色检测脂质沉积,染色后可见红色深染的脂滴
二、间充质干细胞在肝纤维化形成中的作用
1. MSCs参与肝纤维化的形成:野生型小鼠尾静脉注射外源性的EGFP-MSCs随血液循环来到受损的肝脏,冰冻切片免疫荧光图显示有大量的绿色荧光蛋白分布于纤维间隔的附近。内源性的EGFP-MSCs在肝脏损伤后也趋化到受损伤的部位参与肝纤维化的形成。以SSEA4为人MSCs的标记物,检测MSCs在人肝纤维化组织中的表达,结果显示其主要表达的部位在纤维间隔与肝实质区的交界处,以此标记物流式细胞仪分离而得到的细胞,其中大部分的细胞仍然有成骨、成脂肪分化的能力,为MSCs细胞。这说明MSCs细胞也同样参与人肝纤维化的形成。
2.小鼠骨髓间充质干细胞促进纤维化的形成
天狼猩红染色,及Masson染色观察,以及羟脯氨酸定量检测发现注射大量的MSCs的小鼠其纤维化程度明显重于三个对照组(P<0.05)。但其肝功能(主要是AST)却有一定的改善,这说明MSC来到肝脏中加速纤维化的形成,是对损伤的一种修复反应,可以在一定程度上可以改善肝功能。
三、纤维化形成过程中骨髓间充质干细胞的动员
1.肝损过程MSCs向肝脏动员募集的时间
想了解BM-MSCs向肝脏趋化的具体机制,首先要知道MSCs趋化的时间,从两种动物模型冰冻切片的结果看,MSCs动员的时间在第3周左右。而随着时间的推移这种动员程度也在增加。
2.筛选肝纤维化形成过程中起主要作用的趋化因子及其受体。
realtime-PCR法检测到小鼠MSCs细胞主要表达CCR1,CCR2,CCR5,CCR7,CXCR4,CXCR6等6中趋化因子受体,而对应这些趋化因子受体,我们检测到肝损后肝脏主要分泌的趋化因子有CCR1的配体MIP1α,MIP1β;CCR5的配体RANTES,但升高最为明显的是CXCR4的配体SDF-1α,因此我们认为在肝纤维形成过程中促使MSCs趋化最为重要的是SDF-1α/CXCR4趋化轴。
3.验证SDF-1α/CXCR4趋化轴对MSCs的趋化募集作用
结果显示肝损过程中小鼠肝脏的SDF-1α的表达逐渐增高,而骨髓SDF-1α的表达略有降低,当肝脏中SDF-1α浓度高于骨髓形成浓度梯度时,才能从骨髓中动员出大量的MSCs细胞,这也解释了为什么到第3周时,才能看到大量的绿色荧光蛋白出现于肝脏中。而Transwell的结果也显示在体外SDF-1α的确对MSCs有很强的趋化作用。
四、肝纤维化形成过程中VEGF促进骨髓间充质干细胞增殖的机制研究
结果显示在肝损过程中,肝脏有大量的VEGF的产生和分泌,其可以随血液分布于全身各处。外源性注射VEGF可以增加受体小鼠骨髓中EGFP-MSCs的含量,而这种效应可以被VEGF的单克隆抗体Avastin逆转。Transwell试验和划痕试验的结果显示VEGF影响MSC的迁移趋化的能力不强(P>0.05)。这说明VEGF在MSC的动员中主要的作用是促进MSCs的增殖,而不是MSCs的迁移。
【结论】
1.肝损过程中,间充质干细胞促进纤维化的形成,至少是促进早期肝纤维化的形成。
2.早期的肝纤维化形成主要原因是肝损伤的修复,因此如果人为的加速肝脏修复促进早期肝纤维化的形成,可以相应改善肝功能。
3.肝损过程中,骨髓间充质干细胞的动员,主要依赖于SDF1α/CXCR4趋化轴,肝脏和骨髓SDF1α浓度的变化决定着MSCs的走向。此趋化轴是MSCs转移出骨随进入循环,向受损部位趋化,募集的主要影响因素之一。
4.肝纤维化中MSCs向受损部位趋化是一个持续的过程,在人肝硬化组织中也依然可以检测到MSCs的存在。VEGF是纤维化形成过程中骨髓MSCs增殖的主要影响因素之一。
【Background and Objective】
Hepatic fibrosis, characterized by deposition of extracellular matrix (ECM) in the Disse’s space, is a pathological response to a variety of chronic liver diseases, which is recognized as a dynamic and reversal process. Therefore, blocking, inhibition or even reversal of hepatic fibrosis is a major target for the treatment of chronic liver disease. Generally, it has been accepted that activated hepatic stellate cells (HSC), accompanying phenotypic transformation into myofibroblast-like cells, play the pivotal role in hepatic fibrogenesis. In the past decades, it is an important target on hepatic fibrosis therapy by repressing activation, proliferation and migration of HSC as well as inducing its apoptosis. Recently, several studies reported that in vivo environment by simply inhibiting the effect of the treatment of liver fibrosis is not ideal. And more researchers have come to realize that HSC is not the only important influence on the cells of liver fibrosis. So researchers take more attention to find other cell subsets that involve in hepatic fibrogenesis, and study the mechanism of these cells.
The development of Liver fibrosis is always accompanied with chronic inflammation. The cells in liver will secrete a large number of cytokines, growth factors, chemokines involved in fibrosis during liver damage. At the same time a variety of extrahepatic cells will also chemotactic to the injured parts to involve in fibrogenesis. Has been shown that mesenchymal stem cells (MSCs) are one of these extrahepatic cell populations, during liver injury they also chemotaxis to the site of injury involved in hepatic fibrogenesis. MSCs is a pluripotent differential non-hematopoietic stem cells, generally located in the bone marrow. The understanding to MSCs in the past mainly focused on the capability of chemotaxis to the injuried site and the function of repairing damage, so as to develop its as a gene drug. In some diseases, such as connective tissue diseases and bone injury, MSCs therapy achieved good results. But with further research, the investigators found that MSCs in some diseases, also may play some adverse effects, such as it can promote the growth and development of various tumors. For many diseases, MSCs may be play a role of double-edged sword. These results suggested that the researchers should be re-examine strictly the role of MSCs in the disease.Currently, there is a big controvery to the role of MSCs in hepatic fibrogenesis. The purposes of our study is to explore the effect of MSCs to fibrogenesis is whether promoting or inhibiting.
Hepatic fibrosis and other parts of the fibrosis is that the biggest difference between the vascular system reconstruction. Reconstruction of the vascular system associated with the development of liver fibrosis has always been. In this process must secrete large amounts of liver and angiogenesis-related cytokines, such as VEGF, IL-6, IL-8 and so on. And some researchers also confirmed in vitro VEGF, PDGF and other angiogenic some cytokines or growth factors can promote the proliferation of MSCs. MSCs to the damaged parts of the chemokine is a continuous process, as long as the cause of the existence of fully established inflammatory environment, MSCs will be a steady stream of chemotaxis to the liver, bone marrow, MSCs therefore have to supplement self-proliferation. On this basis, we hypothesize, the secretion of hepatic fibrosis in a large number of angiogenesis-promoting cytokines such as VEGF in vivo bone marrow MSCs may be a major reason for continued proliferation.
Of course, MSCs migrate from extrahepatic tissues to the liver, in addition to the proliferation of MSCs been added, but also the ability of MSCs with directional migration. Studies have shown that MSCs migration and chemokine receptor family members and their closely related, the most important is CC-chemokine family and CXC-chemokine family. But there are also studies have shown that different organs or tissue damage, the impact of the migration of MSCs chemokine and its receptor is not the same, so clear which chemokines in liver injury, liver fibrosis in the liver of MSCs the chemotactic recruitment, we can control the development of fibrosis to find new therapeutic targets. The issue in the establishment of a variety of animal models based on the mouse, using fluorescent staining, immunohistochemistry, PCR and other molecular biology techniques to explore the formation of MSCs in the liver (at least the early fiber formation) played the and its effect on liver function. While in-depth discussion of the BM-MSCs in vivo proliferation, migration, raising the main reason for its mechanism. Combined with the detection of liver cirrhosis clinical samples to further understand the pathogenesis of hepatic fibrosis, hepatic fibrosis is to provide a new way of thinking.
In this study ,we used a variety of animal models in mice, based on a clear process of liver fibrosis in MSCs in the presence of liver tissue and the formation of fiber (at least the early fiber formation) and liver function. And through a number of experiments in vivo testing, described BM-MSCs in the process of liver fibrosis in the liver migrate to the transfer of the bone marrow, raising the main factors, while exploring the causes of liver damage after the proliferation of BM-MSCs in vivo the main factors and mechanisms to the pathogenesis of hepatic fibrosis research and treatment of new ideas.
【Methods】
1. Isolation, culture and identification of mesenchymal stem cells
1.1 Isolation and culture of MSCs
Respectively male WT-BALB / c mice and EGFP-BALB / c mice were isolated and cultured WT-MSCs and EGFP-MSCs cells. By density gradient centrifugation and adherent culture methods to separate the combination of MSCs, but this time also mixed with a small amount of mononuclear cells or the situation, and fibroblasts. Use of these cells and MSCs cultured on plastic bottles of different strength of adherent cells when subcultured with 0.05% trypsin +0.02% EDTA digestion and 2-3min rest after repeated digestion can be gradually removed after the passage of other types of cells.
1.2 MSCs Identification
Dynamic inverted microscope WT-MSCs in mice the growth process of primary culture; fluorescence microscope mice EGFP-MSCs cell growth process. Flow cytometry, and immunofluorescence staining to detect surface markers on mouse MSCs CD34, CD90, CD45, CD90, CD105 expression. Osteogenic differentiation Von Kossa staining and ALP activity quantification, detection of directional differentiation of MSCs into the bone ability to adipogenic differentiation Oil Red O staining directional differentiation of MSCs into fat capacity.
2. Effects of MSCs on liver fibrogenesis
Construction of mouse CCL4 liver injury model (model 1), and bone marrow transplantation model + CCL4 liver injury model (model 2). Model determined after successful bone marrow transplant bone marrow receptor wt donor mice within the EGFP-MSCs exist. By tail vein injection of exogenous EGFP-MSCs in mice to model 1, 3d, the frozen section was observed under immunofluorescence microscopy exogenous chemotactic parts of MSCs, as well as the degree of liver fibrosis; trace through the model 2 Endogenous EGFP-MSCs in the liver after injury chemotactic parts; also detected at different time points AST, ALT levels, detection of liver fibrosis in the liver function of the situation
3. Mobilization of MSCs during hepatic fibrogenesis
Model 1 and Model 2 using mice, some mice were sacrificed every week for 5 weeks. Model mice were sacrificed 1 week, 3 days before injection in the death of the EGFP-MSCs exogenous tracer. Frozen sections were observed under fluorescence microscope to determine the intrinsic and extrinsic of MSCs in the mouse liver in animal models of chemotaxis to raise the time. Realtime-PCR Screening MSCs important chemokine surface receptors, and the corresponding damage the liver play a role in the major chemokines. Then observe this chemokine in the mouse liver and the expression of bone marrow and in vivo experiments on BM-MSCs of chemotaxis.
4. The proliferative effect and mechanism of VEGF on MSCs during liver fibrogenesis
By immunohistochemistry, realtime-PCR, ELISA used to detect liver and serum VEGF mRNA and / or protein expression. Vitro cell proliferation assay and monoclonal VEGF formation test the impact of the proliferation of MSCs, flow cytometry, mice, VEGF was detected on the proliferation of BM-MSCs, and the use of Avastin can reverse this effect. Transwell experiments and also the use of scratch test whether VEGF can promote the ability of MSCs chemotactic migration.
5. Statistical Analysis
Statistical analysis of values was performed with SPSS (11.0 version) and Prism GraphPad5 software, with a P value <0.05 considered significant and P <0.01 as very significant.
【Results】
1. Isolation, culture and identification of mesenchymal stem cells
(1) MSCs isolation and culture of: mouse BM-MSCs were cultured primary cells, 24h after more adherent cells ,48-72h after the spindle into spindle-shaped or growth, the first form of a typical 4-5 day proliferation of uniform distribution of clusters of lesions, 7-10 days non-adherent cells disappeared after repeated medium change, cell fusion more than 80%. After several passages and then cultured spindle cells were tested. MSCs cultured human cell lines cells were observed from the morphology typical spindle cell morphology consistent, tightly arranged similar to the whirlpool.
(2) Murine BM-MSCs Identification of surface antigen: memory cells by flow cytometry using immunofluorescence staining to detect the isolated and cultured MSCs, the expression of surface markers showed that the surface expression of adhesion molecules CD29, CD105 , CD90 positive rate of 95% or more, almost no expression of blood cell surface markers CD34, CD45. That these cells were isolated and cultured MSCs characteristics with a very high proportion.
(3) A number of differentiation potential of MSCs in mice: induction of differentiation into bone 14d, our ALP-positive cells were detected, compared with the control group found that the induction group quantitative ALP activity increased. Section 21d, Von Kossa staining, cells showed dense black nodules. Adipogenic differentiation 8d, light microscope, morphological changes, intracellular lipid vacuoles lack circular translucent, applied in situ Oil Red-O lipid staining results seen after staining of lipid droplets stained red
2. Effects of MSCs on liver fibrogenesis
(1) MSCs participate in the formation of hepatic fibrosis: wt mice were injected with exogenous EGFP-MSCs with the blood circulation to the damaged liver, frozen section immunofluorescence figure shows a large number of green fluorescent protein distribution in the fiber spacing nearby. Endogenous EGFP-MSCs in the liver after injury by chemotaxis to the site of injury in liver fibrosis. Human MSCs to SSEA4 markers to detect liver fibrosis in human MSCs tissue showed the expression of the main parts of the fiber spacing and the junction of the liver parenchyma, as markers by flow cytometry, cell separation , most of which are still successful bone cells, the ability to differentiate into fat, the MSCs cells. This indicates that MSCs cells are also involved in the formation of liver fibrosis.
(2) Murine bone marrow mesenchymal stem cells promote fibrosis: Sirius red staining, and Masson staining, and quantitative analysis of hydroxyproline found in mice injected with a large number of MSCs was more severe fibrosis in the three control groups (P <0.05). However, liver function (mainly AST) there is some improvement, indicating that MSC to accelerate liver fibrosis is a repair response to injury, to a certain extent, can improve liver function.
3. Mobilization of MSCs during hepatic fibrogenesis
(1) MSCs to the liver, liver damage during the time of mobilization to raise Want to know BM-MSCs to specific mechanisms of liver chemokine, the first time that MSCs chemotaxis, frozen sections from both the results of animal models to see, MSCs mobilization time in 3 weeks. With this mobilization level over time is also increasing.
(2) The process of liver fibrosis screening play a major role of chemokines and their receptors.
realtime-PCR assay to the mouse MSCs cells mainly expressed CCR1, CCR2, CCR5, CCR7, CXCR4, CXCR6 and other chemokine receptor 6, and the corresponding receptors of these chemokines, we detected the secretion of liver damage after major liver There CCR1 chemokine ligand MIP1α, MIP1β; CCR5 ligands RANTES, but increased the most obvious is the CXCR4 ligand SDF-1α, we consider the process of fibril formation in the liver to promote the most important chemokine MSCs is SDF-1α/CXCR4 chemotactic axis.
(3) Verify SDF-1α/CXCR4 chemotactic chemotactic axis raised on the role of MSCs
The results showed that liver damage the liver of mice during the expression of SDF-1αincreased gradually, while the expression of bone marrow SDF-1αdecreased slightly, when the liver was higher than in the bone marrow SDF-1αconcentration gradient formed only when a large number of mobilized from the bone marrow of the MSCs cells, which explains why the first 3 weeks, to see the large number of green fluorescent protein found in the liver. The Transwell The results also show that SDF-1αin vitro on MSCs do have a strong chemotactic effect.
4. The proliferative effect and mechanism of VEGF on MSCs during liver fibrogenesis
The results showed that the process of liver damage, liver and a large number of VEGF production and secretion, which can be distributed with the blood throughout the body. Exogenous VEGF can increase the recipient mice injected with bone marrow content of EGFP-MSCs, and this effect can be reversed VEGF monoclonal antibody Avastin. Transwell test and scratch test results showed that VEGF chemotactic migration of MSC's capability is not strong (P> 0.05). This shows that the mobilization of VEGF in the MSC is to promote a major role in the proliferation of MSCs, rather than the migration of MSCs.
【Conclusion】
All of our results revealed that:
1. The process of liver damage, mesenchymal stem cells promote the formation of fibrosis, at least for early liver fibrosis.
2. The early hepatic fibrosis is mainly due to the repair of liver injury, so if the acceleration of liver repair man for the formation of early stage of liver fibrosis, liver function can be improved correspondingly.
3. The process of liver damage, bone marrow mesenchymal stem cells mobilization, mainly depends on the SDF1α/CXCR4 chemotactic axis, the concentration of liver and bone marrow SDF1αMSCs determines the direction of change. This chemotactic axis is moved out of bone with MSCs into the circulation, to the damaged parts of the chemokines, raising one of the main factors.
4. MSCs liver fibrosis in chemotaxis to the site of injury is an ongoing process, but also in human liver tissue is still able to detect the presence of MSCs. VEGF is the formation of bone marrow fibrosis and proliferation of MSCs one of the main factors.
肝纤维化(hepatic fibrosis)是肝脏对慢性损伤的一种修复反应,以细胞外基质(extracellular matrix, ECM)在肝内过多沉积为特征。肝纤维化为一动态过程,属可逆性病变,因此,阻断、抑制或逆转肝纤维化是治疗慢性肝病的一个重要目标。既往认为,肝纤维化发生的中心环节是肝星状细胞(hepatic stellate cells,HSCs)激活并向肌成纤维样细胞(myofibroblasts, MFs)转化,抑制HSC激活、增殖与迁移、诱导凋亡是肝纤维化治疗的重要策略。近年来研究发现,在体内环境中通过单纯抑制HSCs来治疗肝纤维化的效果并不理想。而更多的研究者也逐渐认识到HSC也不是唯一重要的影响肝纤维化形成的细胞。那么探索其他参与肝纤维化形成的细胞亚群,并深入研究其作用机制想法就越来越受到研究者的重视。
肝纤维化的发生、发展始终伴随着慢性炎症反应。其过程中受损的肝脏会分泌大量的细胞因子,生长因子,趋化因子参与纤维化的形成。与此同时多种肝外细胞群也会向受损部位趋化,发挥其作用。已有研究显示骨髓间充质干细胞(mesenchymal stem cells, MSCs)也是这些肝外细胞种群之一,在肝脏损伤的过程中它们会趋化到受损部位参与了肝纤维化的形成。MSCs是一种多潜能分化的非造血干细胞,主要存在于骨髓中。以往对MSCs的了解主要集中在它有向损伤部位趋化并修复损伤的功能,因此将其作为基因治疗药物进行开发,并在某些疾病,如结缔组织类疾病以及骨损伤的治疗中取得了良好的效果。但随着研究的深入,研究者又发现MSCs在某些疾病的发生、发展中还可能发挥了某些不利的作用,如它可以促进多种肿瘤的生长。对很多疾病特别是慢性疾病来说MSCs可能是一把双刃剑。这些结果提示研究者应该重新严格的审视MSCs在疾病当中起到的作用。对于肝脏损伤来说,MSCs在纤维化形成中的作用目前也存在着较大的争议。MSCs到底是促进还是抑制纤维化的发展是我们研究的目的之一。
肝纤维化形成的一个重要的特点是肝脉管系统被大肆重建。血管系统重建伴随着肝纤维化发展的始终。在此过程中肝脏必然分泌大量的与血管生成有关的细胞因子,如VEGF,IL-6,IL-8等。而某些研究者也证实在体外实验中VEGF,PDGF等某些细胞因子或生长因子可以促进MSCs的增殖。MSCs向受损部位趋化是一个持续的过程,只要病因存在,炎症环境充分的建立起来,MSCs就会源源不断向受损部位募集。为了满足大量的、持续不断的细胞需要,就必然有某种原因或机制促使骨髓中的MSCs自我增殖补充。在此基础上我们提出假设,肝纤维化形成过程中分泌的大量的促血管生成的细胞因子如VEGF可能就是体内骨髓MSCs不断增殖的重要原因之一。当然,体内MSCs的趋化动员,不单单需要骨髓中MSCs的不断增殖,还需要有某些机制调节BM-MSCs转移出骨髓,进而随着外周血趋化募集到受损的部位。目前研究显示MSCs的迁移与趋化因子家族成员及其受体密切相关,其中最为重要的是CC-趋化家族和CXC-趋化家族的成员。但也有研究证明不同器官或组织损伤时,影响MSCs的迁移的趋化因子及其受体是不尽相同的,因此明确何种趋化因子在肝脏损伤,肝纤维化形成过程中影响BM-MSCs向肝脏的趋化募集,就可以为控制纤维化的发展寻找新的治疗靶点。
本课题在建立多种小鼠动物模型的基础上,使用了荧光染色,免疫组化,PCR等多种分子生物学技术,探讨MSCs在肝纤维化形成过程中发挥的作用及其对肝功能的影响。并同时深入的探讨了BM-MSCs在体内增殖、迁移的主要原因及其作用机制。同时结合肝硬化临床标本的检测,以期进一步深入了解肝纤维化的发病机制,为肝纤维化的治疗提供新的思路。
【实验方法】
一、骨髓间充质干细胞的分离、培养和鉴定
1.分别使用雄性WT-BALB/c小鼠和EGFP-BALB/c小鼠分离培养WT-MSCs和EGFP-MSCs细胞。采用密度梯度离心与贴壁培养相结合的方法来分离MSCs,但此时也混有少量的单核或局势细胞以及成纤维细胞。利用这些细胞与MSCs对塑料培养瓶的贴壁性强弱不同,细胞传代时用0.05%胰蛋白酶+0.02%EDTA静止消化2-3min后,反复多次的消化传代后可逐渐剔除其它种类细胞。
2. MSCs的鉴定
倒置相差显微镜动态观察小鼠WT-MSCs原代培养的生长过程;荧光显微镜下观察小鼠EGFP-MSCs细胞的生长过程。流式细胞仪技术、以及细胞免疫荧光染色技术检测小鼠MSCs上表面标记物CD34,CD90,CD45,CD90,CD105的表达情况。成骨诱导分化Von Kossa染色,以及ALP活性定量,检测MSCs成骨定向分化的能力,成脂诱导分化Oil Red O染色检测MSCs成脂定向分化的能力。
二、间充质干细胞在肝纤维化形成中的作用
构建小鼠CCL4肝损伤模型(模型1),以及小鼠骨髓移植模型+CCL4肝损伤模型(模型2)。确定骨髓移植模型构建成功后受体wt小鼠骨髓内有供体小鼠的EGFP-MSCs存在。通过尾静脉注射外源性的EGFP-MSCs给模型1的小鼠,3d后,冰冻切片免疫荧光显微镜下观察外源性的MSCs的趋化部位,以及肝纤维化的程度;通过模型2示踪内源性的EGFP-MSCs在肝脏损伤后的趋化部位;同时检测不同时间点AST,ALT的含量,检测肝纤维化形成过程中肝功能的情况。
三、纤维化形成过程中骨髓间充质干细胞的动员
使用模型1和模型2的小鼠,每周末处死部分小鼠,连续5周。模型1的每周处死的小鼠,于处死前3天注射外源性的EGFP-MSCs进行示踪。冰冻切片后荧光显微镜下观察,确定内、外源性的MSCs在小鼠动物模型中向肝脏趋化募集的时间。Realtime-PCR法筛选MSCs表面重要的趋化因子受体,以及对应的损伤肝脏中主要的发挥作用的趋化因子。然后观察此趋化因子在小鼠肝脏以及骨髓中的表达情况,以及体内外试验中对BM-MSCs的趋化能力。
四、肝纤维化形成过程中VEGF促进骨髓间充质干细胞增殖的机制研究
用免疫组化,realtime-PCR,ELISA等方法检测小鼠肝脏,血清中VEGF mRNA和/或蛋白质的表达情况。体外细胞增殖试验和单克隆形成试验检测VEGF对MSCs的增殖的影响,流式细胞仪技术检测小鼠体内VEGF对BM-MSCs增殖的影响,以及使用Avastin是否可以逆转这种影响。同时运用Transwell实验及划痕试验检测VEGF是否具有促进MSCs趋化迁移的能力。
五、统计学处理
数据采用SPSS 11.0以及Prism GraphPad5统计软件包进行分析、作图。P<0.05为具有显著性差异,P<0.01为具有非常显著性差异。
【实验结果】
一、骨髓间充质干细胞的分离、培养和鉴定
1. MSCs的分离培养:小鼠BM-MSCs原代细胞进行培养后,24h后出现较多贴壁细胞,48-72h后成纺锤丝形或梭形生长,第4-5天开始形成典型的均匀分布的簇状增生灶,7-10天非贴壁细胞经反复换液而消失,细胞80%以上融合。再经过多次传代培养,细胞都呈梭形。人MSCs细胞系的培养从形态学上观察细胞呈典型的长梭形,细胞形态趋于一致,紧密排列类似旋涡状。
2.小鼠BM-MSCs表面抗原的鉴定:运用流式细胞仪技术记忆细胞免疫荧光染色技术,检测上述分离培养的MSCs的表面标记物的表达情况,结果显示其表面表达粘附分子CD29,CD105,CD90的阳性率在95%以上,而造血干细胞的表面标志CD34,CD45几乎不表达。表明这些分离培养的细胞具有MSCs特征的比例非常高。
3.小鼠MSCs的多向分化潜能:成骨诱导分化后14d,我们对细胞进行ALP阳性的检测,发现与对照组相比,诱导组ALP活性定量增高。第21d,Von Kossa染色,细胞表现为黑色的致密结节。成脂诱导分化后8d,光镜下可见细胞形态发生改变,胞内不乏圆形透亮脂质空泡,应用Oil Red-O原位染色检测脂质沉积,染色后可见红色深染的脂滴
二、间充质干细胞在肝纤维化形成中的作用
1. MSCs参与肝纤维化的形成:野生型小鼠尾静脉注射外源性的EGFP-MSCs随血液循环来到受损的肝脏,冰冻切片免疫荧光图显示有大量的绿色荧光蛋白分布于纤维间隔的附近。内源性的EGFP-MSCs在肝脏损伤后也趋化到受损伤的部位参与肝纤维化的形成。以SSEA4为人MSCs的标记物,检测MSCs在人肝纤维化组织中的表达,结果显示其主要表达的部位在纤维间隔与肝实质区的交界处,以此标记物流式细胞仪分离而得到的细胞,其中大部分的细胞仍然有成骨、成脂肪分化的能力,为MSCs细胞。这说明MSCs细胞也同样参与人肝纤维化的形成。
2.小鼠骨髓间充质干细胞促进纤维化的形成
天狼猩红染色,及Masson染色观察,以及羟脯氨酸定量检测发现注射大量的MSCs的小鼠其纤维化程度明显重于三个对照组(P<0.05)。但其肝功能(主要是AST)却有一定的改善,这说明MSC来到肝脏中加速纤维化的形成,是对损伤的一种修复反应,可以在一定程度上可以改善肝功能。
三、纤维化形成过程中骨髓间充质干细胞的动员
1.肝损过程MSCs向肝脏动员募集的时间
想了解BM-MSCs向肝脏趋化的具体机制,首先要知道MSCs趋化的时间,从两种动物模型冰冻切片的结果看,MSCs动员的时间在第3周左右。而随着时间的推移这种动员程度也在增加。
2.筛选肝纤维化形成过程中起主要作用的趋化因子及其受体。
realtime-PCR法检测到小鼠MSCs细胞主要表达CCR1,CCR2,CCR5,CCR7,CXCR4,CXCR6等6中趋化因子受体,而对应这些趋化因子受体,我们检测到肝损后肝脏主要分泌的趋化因子有CCR1的配体MIP1α,MIP1β;CCR5的配体RANTES,但升高最为明显的是CXCR4的配体SDF-1α,因此我们认为在肝纤维形成过程中促使MSCs趋化最为重要的是SDF-1α/CXCR4趋化轴。
3.验证SDF-1α/CXCR4趋化轴对MSCs的趋化募集作用
结果显示肝损过程中小鼠肝脏的SDF-1α的表达逐渐增高,而骨髓SDF-1α的表达略有降低,当肝脏中SDF-1α浓度高于骨髓形成浓度梯度时,才能从骨髓中动员出大量的MSCs细胞,这也解释了为什么到第3周时,才能看到大量的绿色荧光蛋白出现于肝脏中。而Transwell的结果也显示在体外SDF-1α的确对MSCs有很强的趋化作用。
四、肝纤维化形成过程中VEGF促进骨髓间充质干细胞增殖的机制研究
结果显示在肝损过程中,肝脏有大量的VEGF的产生和分泌,其可以随血液分布于全身各处。外源性注射VEGF可以增加受体小鼠骨髓中EGFP-MSCs的含量,而这种效应可以被VEGF的单克隆抗体Avastin逆转。Transwell试验和划痕试验的结果显示VEGF影响MSC的迁移趋化的能力不强(P>0.05)。这说明VEGF在MSC的动员中主要的作用是促进MSCs的增殖,而不是MSCs的迁移。
【结论】
1.肝损过程中,间充质干细胞促进纤维化的形成,至少是促进早期肝纤维化的形成。
2.早期的肝纤维化形成主要原因是肝损伤的修复,因此如果人为的加速肝脏修复促进早期肝纤维化的形成,可以相应改善肝功能。
3.肝损过程中,骨髓间充质干细胞的动员,主要依赖于SDF1α/CXCR4趋化轴,肝脏和骨髓SDF1α浓度的变化决定着MSCs的走向。此趋化轴是MSCs转移出骨随进入循环,向受损部位趋化,募集的主要影响因素之一。
4.肝纤维化中MSCs向受损部位趋化是一个持续的过程,在人肝硬化组织中也依然可以检测到MSCs的存在。VEGF是纤维化形成过程中骨髓MSCs增殖的主要影响因素之一。
【Background and Objective】
Hepatic fibrosis, characterized by deposition of extracellular matrix (ECM) in the Disse’s space, is a pathological response to a variety of chronic liver diseases, which is recognized as a dynamic and reversal process. Therefore, blocking, inhibition or even reversal of hepatic fibrosis is a major target for the treatment of chronic liver disease. Generally, it has been accepted that activated hepatic stellate cells (HSC), accompanying phenotypic transformation into myofibroblast-like cells, play the pivotal role in hepatic fibrogenesis. In the past decades, it is an important target on hepatic fibrosis therapy by repressing activation, proliferation and migration of HSC as well as inducing its apoptosis. Recently, several studies reported that in vivo environment by simply inhibiting the effect of the treatment of liver fibrosis is not ideal. And more researchers have come to realize that HSC is not the only important influence on the cells of liver fibrosis. So researchers take more attention to find other cell subsets that involve in hepatic fibrogenesis, and study the mechanism of these cells.
The development of Liver fibrosis is always accompanied with chronic inflammation. The cells in liver will secrete a large number of cytokines, growth factors, chemokines involved in fibrosis during liver damage. At the same time a variety of extrahepatic cells will also chemotactic to the injured parts to involve in fibrogenesis. Has been shown that mesenchymal stem cells (MSCs) are one of these extrahepatic cell populations, during liver injury they also chemotaxis to the site of injury involved in hepatic fibrogenesis. MSCs is a pluripotent differential non-hematopoietic stem cells, generally located in the bone marrow. The understanding to MSCs in the past mainly focused on the capability of chemotaxis to the injuried site and the function of repairing damage, so as to develop its as a gene drug. In some diseases, such as connective tissue diseases and bone injury, MSCs therapy achieved good results. But with further research, the investigators found that MSCs in some diseases, also may play some adverse effects, such as it can promote the growth and development of various tumors. For many diseases, MSCs may be play a role of double-edged sword. These results suggested that the researchers should be re-examine strictly the role of MSCs in the disease.Currently, there is a big controvery to the role of MSCs in hepatic fibrogenesis. The purposes of our study is to explore the effect of MSCs to fibrogenesis is whether promoting or inhibiting.
Hepatic fibrosis and other parts of the fibrosis is that the biggest difference between the vascular system reconstruction. Reconstruction of the vascular system associated with the development of liver fibrosis has always been. In this process must secrete large amounts of liver and angiogenesis-related cytokines, such as VEGF, IL-6, IL-8 and so on. And some researchers also confirmed in vitro VEGF, PDGF and other angiogenic some cytokines or growth factors can promote the proliferation of MSCs. MSCs to the damaged parts of the chemokine is a continuous process, as long as the cause of the existence of fully established inflammatory environment, MSCs will be a steady stream of chemotaxis to the liver, bone marrow, MSCs therefore have to supplement self-proliferation. On this basis, we hypothesize, the secretion of hepatic fibrosis in a large number of angiogenesis-promoting cytokines such as VEGF in vivo bone marrow MSCs may be a major reason for continued proliferation.
Of course, MSCs migrate from extrahepatic tissues to the liver, in addition to the proliferation of MSCs been added, but also the ability of MSCs with directional migration. Studies have shown that MSCs migration and chemokine receptor family members and their closely related, the most important is CC-chemokine family and CXC-chemokine family. But there are also studies have shown that different organs or tissue damage, the impact of the migration of MSCs chemokine and its receptor is not the same, so clear which chemokines in liver injury, liver fibrosis in the liver of MSCs the chemotactic recruitment, we can control the development of fibrosis to find new therapeutic targets. The issue in the establishment of a variety of animal models based on the mouse, using fluorescent staining, immunohistochemistry, PCR and other molecular biology techniques to explore the formation of MSCs in the liver (at least the early fiber formation) played the and its effect on liver function. While in-depth discussion of the BM-MSCs in vivo proliferation, migration, raising the main reason for its mechanism. Combined with the detection of liver cirrhosis clinical samples to further understand the pathogenesis of hepatic fibrosis, hepatic fibrosis is to provide a new way of thinking.
In this study ,we used a variety of animal models in mice, based on a clear process of liver fibrosis in MSCs in the presence of liver tissue and the formation of fiber (at least the early fiber formation) and liver function. And through a number of experiments in vivo testing, described BM-MSCs in the process of liver fibrosis in the liver migrate to the transfer of the bone marrow, raising the main factors, while exploring the causes of liver damage after the proliferation of BM-MSCs in vivo the main factors and mechanisms to the pathogenesis of hepatic fibrosis research and treatment of new ideas.
【Methods】
1. Isolation, culture and identification of mesenchymal stem cells
1.1 Isolation and culture of MSCs
Respectively male WT-BALB / c mice and EGFP-BALB / c mice were isolated and cultured WT-MSCs and EGFP-MSCs cells. By density gradient centrifugation and adherent culture methods to separate the combination of MSCs, but this time also mixed with a small amount of mononuclear cells or the situation, and fibroblasts. Use of these cells and MSCs cultured on plastic bottles of different strength of adherent cells when subcultured with 0.05% trypsin +0.02% EDTA digestion and 2-3min rest after repeated digestion can be gradually removed after the passage of other types of cells.
1.2 MSCs Identification
Dynamic inverted microscope WT-MSCs in mice the growth process of primary culture; fluorescence microscope mice EGFP-MSCs cell growth process. Flow cytometry, and immunofluorescence staining to detect surface markers on mouse MSCs CD34, CD90, CD45, CD90, CD105 expression. Osteogenic differentiation Von Kossa staining and ALP activity quantification, detection of directional differentiation of MSCs into the bone ability to adipogenic differentiation Oil Red O staining directional differentiation of MSCs into fat capacity.
2. Effects of MSCs on liver fibrogenesis
Construction of mouse CCL4 liver injury model (model 1), and bone marrow transplantation model + CCL4 liver injury model (model 2). Model determined after successful bone marrow transplant bone marrow receptor wt donor mice within the EGFP-MSCs exist. By tail vein injection of exogenous EGFP-MSCs in mice to model 1, 3d, the frozen section was observed under immunofluorescence microscopy exogenous chemotactic parts of MSCs, as well as the degree of liver fibrosis; trace through the model 2 Endogenous EGFP-MSCs in the liver after injury chemotactic parts; also detected at different time points AST, ALT levels, detection of liver fibrosis in the liver function of the situation
3. Mobilization of MSCs during hepatic fibrogenesis
Model 1 and Model 2 using mice, some mice were sacrificed every week for 5 weeks. Model mice were sacrificed 1 week, 3 days before injection in the death of the EGFP-MSCs exogenous tracer. Frozen sections were observed under fluorescence microscope to determine the intrinsic and extrinsic of MSCs in the mouse liver in animal models of chemotaxis to raise the time. Realtime-PCR Screening MSCs important chemokine surface receptors, and the corresponding damage the liver play a role in the major chemokines. Then observe this chemokine in the mouse liver and the expression of bone marrow and in vivo experiments on BM-MSCs of chemotaxis.
4. The proliferative effect and mechanism of VEGF on MSCs during liver fibrogenesis
By immunohistochemistry, realtime-PCR, ELISA used to detect liver and serum VEGF mRNA and / or protein expression. Vitro cell proliferation assay and monoclonal VEGF formation test the impact of the proliferation of MSCs, flow cytometry, mice, VEGF was detected on the proliferation of BM-MSCs, and the use of Avastin can reverse this effect. Transwell experiments and also the use of scratch test whether VEGF can promote the ability of MSCs chemotactic migration.
5. Statistical Analysis
Statistical analysis of values was performed with SPSS (11.0 version) and Prism GraphPad5 software, with a P value <0.05 considered significant and P <0.01 as very significant.
【Results】
1. Isolation, culture and identification of mesenchymal stem cells
(1) MSCs isolation and culture of: mouse BM-MSCs were cultured primary cells, 24h after more adherent cells ,48-72h after the spindle into spindle-shaped or growth, the first form of a typical 4-5 day proliferation of uniform distribution of clusters of lesions, 7-10 days non-adherent cells disappeared after repeated medium change, cell fusion more than 80%. After several passages and then cultured spindle cells were tested. MSCs cultured human cell lines cells were observed from the morphology typical spindle cell morphology consistent, tightly arranged similar to the whirlpool.
(2) Murine BM-MSCs Identification of surface antigen: memory cells by flow cytometry using immunofluorescence staining to detect the isolated and cultured MSCs, the expression of surface markers showed that the surface expression of adhesion molecules CD29, CD105 , CD90 positive rate of 95% or more, almost no expression of blood cell surface markers CD34, CD45. That these cells were isolated and cultured MSCs characteristics with a very high proportion.
(3) A number of differentiation potential of MSCs in mice: induction of differentiation into bone 14d, our ALP-positive cells were detected, compared with the control group found that the induction group quantitative ALP activity increased. Section 21d, Von Kossa staining, cells showed dense black nodules. Adipogenic differentiation 8d, light microscope, morphological changes, intracellular lipid vacuoles lack circular translucent, applied in situ Oil Red-O lipid staining results seen after staining of lipid droplets stained red
2. Effects of MSCs on liver fibrogenesis
(1) MSCs participate in the formation of hepatic fibrosis: wt mice were injected with exogenous EGFP-MSCs with the blood circulation to the damaged liver, frozen section immunofluorescence figure shows a large number of green fluorescent protein distribution in the fiber spacing nearby. Endogenous EGFP-MSCs in the liver after injury by chemotaxis to the site of injury in liver fibrosis. Human MSCs to SSEA4 markers to detect liver fibrosis in human MSCs tissue showed the expression of the main parts of the fiber spacing and the junction of the liver parenchyma, as markers by flow cytometry, cell separation , most of which are still successful bone cells, the ability to differentiate into fat, the MSCs cells. This indicates that MSCs cells are also involved in the formation of liver fibrosis.
(2) Murine bone marrow mesenchymal stem cells promote fibrosis: Sirius red staining, and Masson staining, and quantitative analysis of hydroxyproline found in mice injected with a large number of MSCs was more severe fibrosis in the three control groups (P <0.05). However, liver function (mainly AST) there is some improvement, indicating that MSC to accelerate liver fibrosis is a repair response to injury, to a certain extent, can improve liver function.
3. Mobilization of MSCs during hepatic fibrogenesis
(1) MSCs to the liver, liver damage during the time of mobilization to raise Want to know BM-MSCs to specific mechanisms of liver chemokine, the first time that MSCs chemotaxis, frozen sections from both the results of animal models to see, MSCs mobilization time in 3 weeks. With this mobilization level over time is also increasing.
(2) The process of liver fibrosis screening play a major role of chemokines and their receptors.
realtime-PCR assay to the mouse MSCs cells mainly expressed CCR1, CCR2, CCR5, CCR7, CXCR4, CXCR6 and other chemokine receptor 6, and the corresponding receptors of these chemokines, we detected the secretion of liver damage after major liver There CCR1 chemokine ligand MIP1α, MIP1β; CCR5 ligands RANTES, but increased the most obvious is the CXCR4 ligand SDF-1α, we consider the process of fibril formation in the liver to promote the most important chemokine MSCs is SDF-1α/CXCR4 chemotactic axis.
(3) Verify SDF-1α/CXCR4 chemotactic chemotactic axis raised on the role of MSCs
The results showed that liver damage the liver of mice during the expression of SDF-1αincreased gradually, while the expression of bone marrow SDF-1αdecreased slightly, when the liver was higher than in the bone marrow SDF-1αconcentration gradient formed only when a large number of mobilized from the bone marrow of the MSCs cells, which explains why the first 3 weeks, to see the large number of green fluorescent protein found in the liver. The Transwell The results also show that SDF-1αin vitro on MSCs do have a strong chemotactic effect.
4. The proliferative effect and mechanism of VEGF on MSCs during liver fibrogenesis
The results showed that the process of liver damage, liver and a large number of VEGF production and secretion, which can be distributed with the blood throughout the body. Exogenous VEGF can increase the recipient mice injected with bone marrow content of EGFP-MSCs, and this effect can be reversed VEGF monoclonal antibody Avastin. Transwell test and scratch test results showed that VEGF chemotactic migration of MSC's capability is not strong (P> 0.05). This shows that the mobilization of VEGF in the MSC is to promote a major role in the proliferation of MSCs, rather than the migration of MSCs.
【Conclusion】
All of our results revealed that:
1. The process of liver damage, mesenchymal stem cells promote the formation of fibrosis, at least for early liver fibrosis.
2. The early hepatic fibrosis is mainly due to the repair of liver injury, so if the acceleration of liver repair man for the formation of early stage of liver fibrosis, liver function can be improved correspondingly.
3. The process of liver damage, bone marrow mesenchymal stem cells mobilization, mainly depends on the SDF1α/CXCR4 chemotactic axis, the concentration of liver and bone marrow SDF1αMSCs determines the direction of change. This chemotactic axis is moved out of bone with MSCs into the circulation, to the damaged parts of the chemokines, raising one of the main factors.
4. MSCs liver fibrosis in chemotaxis to the site of injury is an ongoing process, but also in human liver tissue is still able to detect the presence of MSCs. VEGF is the formation of bone marrow fibrosis and proliferation of MSCs one of the main factors.
引文
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