髓样抑制细胞在小鼠肝纤维化发生中的影响机制研究
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摘要
肝纤维化是各种慢性肝病向肝硬化发展的必经阶段，是所有慢性肝病的共同病理基础。虽然肝纤维化发病率高，但目前对其发病机制尚不清楚，且无有效的根本的抗纤维化治疗药物。现在正兴起以肝脏星形细胞（hepatic stellate cells, HSC）为靶点的治疗模式，但其具体的抗纤维化作用机理尚待进一步研究。
正常情况下HSC处于静止状态,在各种肝损伤因素刺激下，HSC可经历从静止到具有增殖性、成纤维性和收缩性的肌成纤维样细胞的转化过程。在病毒性肝炎、铁铜代谢异常、自身免疫疾病等因素作用下，相关细胞分泌多种细胞因子，这些细胞因子首先与HSC膜上受体结合，然后通过不同或相同的胞内信号转导系统传递，在某些转录因子的作用下，转导信号入核，从而启动DNA复制、转录及翻译表达过程，实现HSC活化、增殖、转型并分泌细胞外基质，最终导致肝纤维化的发生。
髓样抑制细胞（Myeloid-derived suppressor cells，MDSC）是一群多种多样的幼稚骨髓细胞。生理状态下，这些细胞在骨髓产生，并进一步分化为成熟的巨噬细胞，树突状细胞（dendritic cells，DCs）和粒细胞，这些细胞中一小部分存在于脾脏中。形成这一群体的祖细胞的多样性显示它们具有多种多样的形态性和生理功能。在病理情况下，在多种组织中具有相同的表型和免疫抑制活性的细胞大量扩增，从而在炎症，感染和自身免疫疾病中，乃至促肿瘤血管生成和肿瘤转移中都发挥一定的抑制作用。这些具有潜在的免疫抑制功能的幼稚细胞群通过介导其它免疫细胞，如T细胞，巨噬细胞，NK细胞和树突状细胞（dendritic cells，DCs）和调节性T细胞（Tregs）从而负性调节免疫反应。对于小鼠，MDSCs一般被定义为Gr-1+CD11b+细胞。Gr-1抗原被认为是粒细胞分化的标志物，抗体结合于Ly6G和Ly6C两个表位。应用这些表位特异性的抗体已经鉴定了具有不同的形态特征和功能的两种MDSC亚群，单核细胞亚群和粒细胞亚群。
目前，据文献报道巨噬细胞可分为两型，M1和M2。M1型，或者叫经典型巨噬细胞，它可表达高水平的iNOS和肿瘤坏死因子,而M2型，也叫选择活化型巨噬细胞，它可产生Arg-1和IL-10。M1型巨噬细胞主要介导急性炎症反应，而M2型巨噬细胞在促进急性炎症转变为慢性炎症和组织修复中发挥重要作用。在肿瘤微环境中，M2型巨噬细胞可抑制T细胞介导的抗肿瘤反应，并促进肿瘤发展，转移和血管形成。相反，M1型巨噬细胞可发挥抗肿瘤作用。
我们知道肝脏是人体最大的免疫器官，多个研究证实肝脏纤维化的形成与免疫系统具有相关性。且有研究证实HSC可诱导MDSC从骨髓大量聚集到肝脏。故我们利用小鼠肝脏纤维化和肝癌模型，检测MDSC在肝脏组织中的分布情况，并将MDSC与HSC体外共培养，观察MDSC是否对HSC的增殖有影响，进而研究HSC和MDSC的交互作用在肝脏纤维化发生中的影响机制，为肝纤维化的治疗寻找有效的靶点。主要研究结果如下：
1、成功构建了肝脏纤维化动物模型，并用免疫组织化学鉴定。
2、成功从纤维化肝脏组织中分离HSC细胞，并培养了不同活化状态的HSC细胞，
3、分析肝纤维化模型肝脏组织中MDSC的情况，在注射CCl42周的早期肝脏纤维化模型中，组织学可见肝脏细胞大量变性坏死;与对照组相比，纤维化肝脏组织内MDSC及巨噬细胞均明显增加。而骨髓中CCl4组和橄榄油组MDSC及两种巨噬细胞的比例和数量无统计学差别，P>0.05。这提示在纤维化早期骨髓内MDSC并无明显变化。在脾脏中实验组的Treg的细胞比例和数量，与对照组相比，无显著性差异，P>0.05。这提示Treg在早期肝纤维化的发生中未发挥促进作用。而在注射CCl44周的肝脏纤维化组织中，M2巨噬细胞的数量和比例明显增加，而且M2巨噬细胞相关蛋白Arg-1,IL-10和CD206的表达量明显高于对照组，而肝脏组织学切片提示肝脏细胞大量再生，纤维化组织结构消失，这提示M2巨噬细胞促进了肝脏纤维化的组织修复，而这一结果正好与目前认为的M2巨噬细胞主要介导组织修复的生理功能相吻合。
4、成功构建了转癌基因肝癌动物模型，根据饮食分为高脂肪饮食组（HFD）和低脂肪饮食组（LFD），用B超可见肝脏从注射后4周即开始生长肉眼可见肿瘤，肿瘤成弥漫性。而且HFD组的生存率低于LFD组。可见高脂肪饮食在肝癌的发生中起到了一定的促进作用。此外，HFD组的肿瘤平均重量明显高于LFD组。
5、分析肝癌组织中MDSC的数量及性质。结果发现肿瘤中CD11b+Ly6C+Ly6G+MDSC的数量及比例，与对照组比较明显增加，有显著性差异，P<0.001。这提示MDSC参与了肝癌的发生和发展，且M2型巨噬细胞在肝癌的发生中发挥着重要的作用。类似的，骨髓内M2型巨噬细胞也明显增多。脾脏中CD4+CD25+FoxP3+Treg细胞也明显增加，这提示MDSC通过诱导Treg的聚集增多介导肝癌的发生发展。
6、小鼠来源的HSC与MDSC共培养后，发现MDSC可明显抑制HSC的增殖,最高抑制率达75%，同时HSC可促进MDSC定向分化为M2巨噬细胞的能力。人类来源的HSC与MDSC共培养后，也发现HSC的增生明显受到抑制，最高抑制率达90%。这可能是未来逆转肝脏纤维化的有效靶点之一。结论：
通过本课题的研究，证实在肝脏纤维化和肝癌的发生中MDSC发挥一定的免疫抑制作用。在肝脏纤维化早期MDSC分化而来的M1巨噬细胞和M2巨噬细胞均明显增加，提示M1巨噬细胞参与了肝脏的急性炎症反应，而M2巨噬细胞通过增加试图抑制急性炎症反应，进行组织修复。而骨髓中MDSC和脾脏中的Treg并没有明显的变化。但当肝纤维化进一步发展，甚至转变为肝癌时，MDSC多定向分化为M2型巨噬细胞，并在纤维化组织和肿瘤中大量浸润，在肿瘤的发生发展中发挥重要的作用。而Treg在肝癌模型的脾脏内也明显增加，促进了肿瘤细胞的免疫逃逸。且高脂肪饮食对肝癌的发生有促进作用。MDSC与HSC共培养后，MDSC可明显抑制HSC的增殖，HSC可促进MDSC定向分化为M2巨噬细胞的能力。这可能是未来寻找抗纤维化治疗有效靶点的思路之一。
Liver fibrosis is the same outcome of all kinds of chronic liver diseases, and is the commonpathological basis of all chronic liver diseases. Although it could be reversed at early stage bycontrolling potential factors, the only treatment for severe cirrhosis nowadays is livertransplantation. Even if liver fibrosis has a high incidence, the pathogenic mechanism is stillunclear. In addition, currently there is no treatable-radically drug. Now hepatic stellate cell(HSC) is being considered as a new treatment target, but the anti-fibrosis mechanism is stillunclear.
Under physical condition, HSC is in quiescent state. Under diversity liver injury factors,HSC can be activated into myofibroblasts which has properties of proliferation, fibrogenesisand contractility. Under the effect of virus, abnormal metal metabolism and autoimmunologicaldiseases, HSC can excrete several cytokines, which bind with the receptors locating at themembrane of HSC, then transmit the cell signal through the same or different intracellularsignal transduction pathways. With the help of some transcriptional factors, transductionsignals are transmitted into nuclear, then promots DNA replication, transcription and translationprocess. Finally HSC could be activated into a proliferation state, and excrete extracellularmatrix. In the end the liver becomes fibrosis.
Myeloid-derived suppressor cells (MDSC) are identified as a heterogeneous cellpopulation in mice and humans. Under physiological conditions, these cells are generated in thebone marrow and differentiated into mature macrophages, dendritic cells (DCs), andgranulocytes, and are present to a lesser extent in the spleen.. The different progenitor cells thatform this population demonstrate a broad range of morphology and functional capacity. Incontrast, in pathological conditions, there is a dramatic expansion of cells with the samephenotype and immunosuppressive activity in various tissues. These immature cell populationswith potent immunosuppressive activity are important as negative regulators of immune responses. During chronic inflammation and cancer, immature cell populations significantlyexpand owing to a partial block in their differentiation into mature myeloid cells. MDSCssuppress proliferation and cytokine secretion in both T lymphocytes and Natural Killer (NK)cells, as well as induce apoptosis in T cell subsets. MDSCs inhibit immune responses bymediating other immune cells, such as T cells, macrophages, NK cells, dendritic cells, andTregs. In mice, MDSCs are generally characterized as Gr-1+CD11b+cells. Gr-1antigen isconsidered to be a marker of granulocytic differentiation, and the antibodies bind to twoepitopes, Ly6G and Ly6C. The use of these epitope-specific antibodies has led to theidentification of two MDSC subsets, monocytic, and granulocytic subsets, which have differentmorphological and functional features. As both Ly6C and Ly6G epitopes are bound byanti-Gr-1antibodies, the relative intensity of Gr-1staining is indicative of monocytic (low orintermediate) and granulocytic (high) subsets. In both humans and mice, MDSCs are markedby high expression of CD11b (a common myeloid cell surface marker).
Macrophages, on the other hand, display a remarkable plasticity and can differentiate intofunctionally diverse subtypes, e.g.'classically activated' M1and 'alternatively activated' M2macrophages. Experimental animal models indicate that macrophages are not only critical forfibrosis progression, but also for fibrosis regression, because macrophages can also degradeextracellular matrix proteins and exert anti-inflammatory actions. M1macrophage can expresshigh level of iNOS and TNF-α, which mediates acute inflammation, and M2macrophagesexpress Arg-1and IL-10, which plays an important role in transfering acute inflammation intochronic inflammation and promoting liver fibrosis. Similarly, in tumor microenvironment, M2macrophages promote tumor progression, metastasis and angiogenesis by inhibiting T cellmediation anti-tumor response, On the contrary, M1macrophage has an effect of inhibitingtumor growth.
Liver belongs to human immunological organ. Many researchers suggest that liver fibrosisrelates to immunological abnormal. Although chronic liver disease has many etiologies,including chronic viral hepatitis, alcohol abuse, metabolic syndrome, and autoimmune disorders, the cellular and pathological mechanisms leading to hepatic fibrosis, and as anend-stage cirrhosis are relatively common and uniform. Liver fibrosis is characterized by anaccumulation of extracellular matrix proteins and activated HSC. Portal fibroblasts andmyofibroblasts have been identified as major collagen-producing cells in the injured liver.Experimental models of liver fibrosis highlight the importance of hepatic macrophages,so-called Kupffer cells, for perpetuating an inflammatory phase resulting in the massive releaseof proinflammatory cytokines and chemokines as well as activation of HSC. Recent studiesdemonstrate that these actions are only partially conducted by liver-resident macrophages, butlargely depend on recruitment of monocytes into the liver, namely of the inflammatory Gr1+(Ly6C+) monocyte subset as precursors of tissue macrophages. As a result, we want toinvestigate if there is cross-talk between HSC and MDSC through mice models and co-culturetwo cells. Finally a new treatment target could be found to control liver fibrosis and liver cancer.Our main results are as follows:
1.Liver fibrosis animal model was established successfully, and identified withimmunohistochemistry staining.
2. HSC was isolated from liver fibrosis tissues, and cultured to get different activationstatus with different morphology and function.
3. We analysed MDSC and Tregs in liver tissues, bone marrow and spleen. It was shownthat in two weeks model, liver histology presented that a majority of hepatocytes becamenecrosis. MDSC in liver tissue increased dramatically, and numbers of M1and M2in CCl4group were higher than those of oil groups, and the difference was significant statistically. Thepercentage of MDSC from bone marrow and Tregs from spleen in two groups both had nodifference statistically. This suggested that M1macrophage induced acute inflammation ofliver, and M2macrophage simultaneously increased, which suggested that M2macrophageswanted to transfer acute inflammation into chronic inflammation. In addition, in four weeksmodel, the percentage of M2macrophages in CCl4group was much higher than that of oilgroup，but the percentage of M1macrophages in CCl4group decreased. And the expression of surface and intracellular marker of M2macrophages, such as Arginase-1, IL-10and CD206,were up-regulated. All this indicated that M2macrophages plays an important role in liverfibrosis reversion.
4. HCC animal model by transgenic method was established successfully. From the fourthweek after injection the tumor could be detected by ultrasound from animal imagingdepartment. The tumor grew with a diffuse manner. The mice were divided into two groups bydiet: low fat diet group(LFD) and high fat diet group(HFD). Finally we found that the survivalrate of HFD group was lower than that of LFD group. This indicated that HFD can promotetumorigenesis. Moreover, the average weight of tumors in HFD group was higher than that ofLFD.
5. MDSC in liver cancer mice was analysed by flow cytometry. In liver cancer tissue, thenumber of CD11b+Ly6C+Ly6G+MDSC was much higher than that of control group. Thissuggested that MDSC regulated the tumorigenesis and development of liver cancer. In addition,the percentage of M2macrophage increased, which demonstrated that M2macrophages playsan crucial importance in tumor formation. Similarly, the percentage of M2macrophages inbone marrow of CCl4group increased as well. Moreover, CD4+CD25+FoxP3+Treg in spleenalso increased compared with control group, which suggested that MDSC presentedimmunological inhibitory effect through propagation of Tregs.
6. MDSC could inhibit HSC proliferation after co-culture of HSC and MDSC，and HSCcould induce MDSC to differentiate into M2macrophages. Especially MDSC from humancould inhibit HSC proliferation by90%. This is the possible mechanism to resolute or reverseliver fibrosis in the future.Conclusion
In conclusion, we find firstly that MDSC plays an important role in promoting liverfibrogenesis and liver tumorigenesis. Meantime, at acute inflammation stage MDSC andMDSC-derived M1and M2macrophages both increase. Moreover, M2macrophage is verycrucial in fibrosis reversion and tumorigenesis. And MDSC promotes liver tumorigenesis through inducing propagation of Tregs. In addition, high fat diet is very important for tumordevelopment. Moreover, HSC proliferation could be inhibited by MDSC dramatically afterco-culture24hours, which suggests a possible target to inhibit fibrogenesis, and MDSC candifferentiate into M2macrophages after co-culture probably.
正常情况下HSC处于静止状态,在各种肝损伤因素刺激下，HSC可经历从静止到具有增殖性、成纤维性和收缩性的肌成纤维样细胞的转化过程。在病毒性肝炎、铁铜代谢异常、自身免疫疾病等因素作用下，相关细胞分泌多种细胞因子，这些细胞因子首先与HSC膜上受体结合，然后通过不同或相同的胞内信号转导系统传递，在某些转录因子的作用下，转导信号入核，从而启动DNA复制、转录及翻译表达过程，实现HSC活化、增殖、转型并分泌细胞外基质，最终导致肝纤维化的发生。
髓样抑制细胞（Myeloid-derived suppressor cells，MDSC）是一群多种多样的幼稚骨髓细胞。生理状态下，这些细胞在骨髓产生，并进一步分化为成熟的巨噬细胞，树突状细胞（dendritic cells，DCs）和粒细胞，这些细胞中一小部分存在于脾脏中。形成这一群体的祖细胞的多样性显示它们具有多种多样的形态性和生理功能。在病理情况下，在多种组织中具有相同的表型和免疫抑制活性的细胞大量扩增，从而在炎症，感染和自身免疫疾病中，乃至促肿瘤血管生成和肿瘤转移中都发挥一定的抑制作用。这些具有潜在的免疫抑制功能的幼稚细胞群通过介导其它免疫细胞，如T细胞，巨噬细胞，NK细胞和树突状细胞（dendritic cells，DCs）和调节性T细胞（Tregs）从而负性调节免疫反应。对于小鼠，MDSCs一般被定义为Gr-1+CD11b+细胞。Gr-1抗原被认为是粒细胞分化的标志物，抗体结合于Ly6G和Ly6C两个表位。应用这些表位特异性的抗体已经鉴定了具有不同的形态特征和功能的两种MDSC亚群，单核细胞亚群和粒细胞亚群。
目前，据文献报道巨噬细胞可分为两型，M1和M2。M1型，或者叫经典型巨噬细胞，它可表达高水平的iNOS和肿瘤坏死因子,而M2型，也叫选择活化型巨噬细胞，它可产生Arg-1和IL-10。M1型巨噬细胞主要介导急性炎症反应，而M2型巨噬细胞在促进急性炎症转变为慢性炎症和组织修复中发挥重要作用。在肿瘤微环境中，M2型巨噬细胞可抑制T细胞介导的抗肿瘤反应，并促进肿瘤发展，转移和血管形成。相反，M1型巨噬细胞可发挥抗肿瘤作用。
我们知道肝脏是人体最大的免疫器官，多个研究证实肝脏纤维化的形成与免疫系统具有相关性。且有研究证实HSC可诱导MDSC从骨髓大量聚集到肝脏。故我们利用小鼠肝脏纤维化和肝癌模型，检测MDSC在肝脏组织中的分布情况，并将MDSC与HSC体外共培养，观察MDSC是否对HSC的增殖有影响，进而研究HSC和MDSC的交互作用在肝脏纤维化发生中的影响机制，为肝纤维化的治疗寻找有效的靶点。主要研究结果如下：
1、成功构建了肝脏纤维化动物模型，并用免疫组织化学鉴定。
2、成功从纤维化肝脏组织中分离HSC细胞，并培养了不同活化状态的HSC细胞，
3、分析肝纤维化模型肝脏组织中MDSC的情况，在注射CCl42周的早期肝脏纤维化模型中，组织学可见肝脏细胞大量变性坏死;与对照组相比，纤维化肝脏组织内MDSC及巨噬细胞均明显增加。而骨髓中CCl4组和橄榄油组MDSC及两种巨噬细胞的比例和数量无统计学差别，P>0.05。这提示在纤维化早期骨髓内MDSC并无明显变化。在脾脏中实验组的Treg的细胞比例和数量，与对照组相比，无显著性差异，P>0.05。这提示Treg在早期肝纤维化的发生中未发挥促进作用。而在注射CCl44周的肝脏纤维化组织中，M2巨噬细胞的数量和比例明显增加，而且M2巨噬细胞相关蛋白Arg-1,IL-10和CD206的表达量明显高于对照组，而肝脏组织学切片提示肝脏细胞大量再生，纤维化组织结构消失，这提示M2巨噬细胞促进了肝脏纤维化的组织修复，而这一结果正好与目前认为的M2巨噬细胞主要介导组织修复的生理功能相吻合。
4、成功构建了转癌基因肝癌动物模型，根据饮食分为高脂肪饮食组（HFD）和低脂肪饮食组（LFD），用B超可见肝脏从注射后4周即开始生长肉眼可见肿瘤，肿瘤成弥漫性。而且HFD组的生存率低于LFD组。可见高脂肪饮食在肝癌的发生中起到了一定的促进作用。此外，HFD组的肿瘤平均重量明显高于LFD组。
5、分析肝癌组织中MDSC的数量及性质。结果发现肿瘤中CD11b+Ly6C+Ly6G+MDSC的数量及比例，与对照组比较明显增加，有显著性差异，P<0.001。这提示MDSC参与了肝癌的发生和发展，且M2型巨噬细胞在肝癌的发生中发挥着重要的作用。类似的，骨髓内M2型巨噬细胞也明显增多。脾脏中CD4+CD25+FoxP3+Treg细胞也明显增加，这提示MDSC通过诱导Treg的聚集增多介导肝癌的发生发展。
6、小鼠来源的HSC与MDSC共培养后，发现MDSC可明显抑制HSC的增殖,最高抑制率达75%，同时HSC可促进MDSC定向分化为M2巨噬细胞的能力。人类来源的HSC与MDSC共培养后，也发现HSC的增生明显受到抑制，最高抑制率达90%。这可能是未来逆转肝脏纤维化的有效靶点之一。结论：
通过本课题的研究，证实在肝脏纤维化和肝癌的发生中MDSC发挥一定的免疫抑制作用。在肝脏纤维化早期MDSC分化而来的M1巨噬细胞和M2巨噬细胞均明显增加，提示M1巨噬细胞参与了肝脏的急性炎症反应，而M2巨噬细胞通过增加试图抑制急性炎症反应，进行组织修复。而骨髓中MDSC和脾脏中的Treg并没有明显的变化。但当肝纤维化进一步发展，甚至转变为肝癌时，MDSC多定向分化为M2型巨噬细胞，并在纤维化组织和肿瘤中大量浸润，在肿瘤的发生发展中发挥重要的作用。而Treg在肝癌模型的脾脏内也明显增加，促进了肿瘤细胞的免疫逃逸。且高脂肪饮食对肝癌的发生有促进作用。MDSC与HSC共培养后，MDSC可明显抑制HSC的增殖，HSC可促进MDSC定向分化为M2巨噬细胞的能力。这可能是未来寻找抗纤维化治疗有效靶点的思路之一。
Liver fibrosis is the same outcome of all kinds of chronic liver diseases, and is the commonpathological basis of all chronic liver diseases. Although it could be reversed at early stage bycontrolling potential factors, the only treatment for severe cirrhosis nowadays is livertransplantation. Even if liver fibrosis has a high incidence, the pathogenic mechanism is stillunclear. In addition, currently there is no treatable-radically drug. Now hepatic stellate cell(HSC) is being considered as a new treatment target, but the anti-fibrosis mechanism is stillunclear.
Under physical condition, HSC is in quiescent state. Under diversity liver injury factors,HSC can be activated into myofibroblasts which has properties of proliferation, fibrogenesisand contractility. Under the effect of virus, abnormal metal metabolism and autoimmunologicaldiseases, HSC can excrete several cytokines, which bind with the receptors locating at themembrane of HSC, then transmit the cell signal through the same or different intracellularsignal transduction pathways. With the help of some transcriptional factors, transductionsignals are transmitted into nuclear, then promots DNA replication, transcription and translationprocess. Finally HSC could be activated into a proliferation state, and excrete extracellularmatrix. In the end the liver becomes fibrosis.
Myeloid-derived suppressor cells (MDSC) are identified as a heterogeneous cellpopulation in mice and humans. Under physiological conditions, these cells are generated in thebone marrow and differentiated into mature macrophages, dendritic cells (DCs), andgranulocytes, and are present to a lesser extent in the spleen.. The different progenitor cells thatform this population demonstrate a broad range of morphology and functional capacity. Incontrast, in pathological conditions, there is a dramatic expansion of cells with the samephenotype and immunosuppressive activity in various tissues. These immature cell populationswith potent immunosuppressive activity are important as negative regulators of immune responses. During chronic inflammation and cancer, immature cell populations significantlyexpand owing to a partial block in their differentiation into mature myeloid cells. MDSCssuppress proliferation and cytokine secretion in both T lymphocytes and Natural Killer (NK)cells, as well as induce apoptosis in T cell subsets. MDSCs inhibit immune responses bymediating other immune cells, such as T cells, macrophages, NK cells, dendritic cells, andTregs. In mice, MDSCs are generally characterized as Gr-1+CD11b+cells. Gr-1antigen isconsidered to be a marker of granulocytic differentiation, and the antibodies bind to twoepitopes, Ly6G and Ly6C. The use of these epitope-specific antibodies has led to theidentification of two MDSC subsets, monocytic, and granulocytic subsets, which have differentmorphological and functional features. As both Ly6C and Ly6G epitopes are bound byanti-Gr-1antibodies, the relative intensity of Gr-1staining is indicative of monocytic (low orintermediate) and granulocytic (high) subsets. In both humans and mice, MDSCs are markedby high expression of CD11b (a common myeloid cell surface marker).
Macrophages, on the other hand, display a remarkable plasticity and can differentiate intofunctionally diverse subtypes, e.g.'classically activated' M1and 'alternatively activated' M2macrophages. Experimental animal models indicate that macrophages are not only critical forfibrosis progression, but also for fibrosis regression, because macrophages can also degradeextracellular matrix proteins and exert anti-inflammatory actions. M1macrophage can expresshigh level of iNOS and TNF-α, which mediates acute inflammation, and M2macrophagesexpress Arg-1and IL-10, which plays an important role in transfering acute inflammation intochronic inflammation and promoting liver fibrosis. Similarly, in tumor microenvironment, M2macrophages promote tumor progression, metastasis and angiogenesis by inhibiting T cellmediation anti-tumor response, On the contrary, M1macrophage has an effect of inhibitingtumor growth.
Liver belongs to human immunological organ. Many researchers suggest that liver fibrosisrelates to immunological abnormal. Although chronic liver disease has many etiologies,including chronic viral hepatitis, alcohol abuse, metabolic syndrome, and autoimmune disorders, the cellular and pathological mechanisms leading to hepatic fibrosis, and as anend-stage cirrhosis are relatively common and uniform. Liver fibrosis is characterized by anaccumulation of extracellular matrix proteins and activated HSC. Portal fibroblasts andmyofibroblasts have been identified as major collagen-producing cells in the injured liver.Experimental models of liver fibrosis highlight the importance of hepatic macrophages,so-called Kupffer cells, for perpetuating an inflammatory phase resulting in the massive releaseof proinflammatory cytokines and chemokines as well as activation of HSC. Recent studiesdemonstrate that these actions are only partially conducted by liver-resident macrophages, butlargely depend on recruitment of monocytes into the liver, namely of the inflammatory Gr1+(Ly6C+) monocyte subset as precursors of tissue macrophages. As a result, we want toinvestigate if there is cross-talk between HSC and MDSC through mice models and co-culturetwo cells. Finally a new treatment target could be found to control liver fibrosis and liver cancer.Our main results are as follows:
1.Liver fibrosis animal model was established successfully, and identified withimmunohistochemistry staining.
2. HSC was isolated from liver fibrosis tissues, and cultured to get different activationstatus with different morphology and function.
3. We analysed MDSC and Tregs in liver tissues, bone marrow and spleen. It was shownthat in two weeks model, liver histology presented that a majority of hepatocytes becamenecrosis. MDSC in liver tissue increased dramatically, and numbers of M1and M2in CCl4group were higher than those of oil groups, and the difference was significant statistically. Thepercentage of MDSC from bone marrow and Tregs from spleen in two groups both had nodifference statistically. This suggested that M1macrophage induced acute inflammation ofliver, and M2macrophage simultaneously increased, which suggested that M2macrophageswanted to transfer acute inflammation into chronic inflammation. In addition, in four weeksmodel, the percentage of M2macrophages in CCl4group was much higher than that of oilgroup，but the percentage of M1macrophages in CCl4group decreased. And the expression of surface and intracellular marker of M2macrophages, such as Arginase-1, IL-10and CD206,were up-regulated. All this indicated that M2macrophages plays an important role in liverfibrosis reversion.
4. HCC animal model by transgenic method was established successfully. From the fourthweek after injection the tumor could be detected by ultrasound from animal imagingdepartment. The tumor grew with a diffuse manner. The mice were divided into two groups bydiet: low fat diet group(LFD) and high fat diet group(HFD). Finally we found that the survivalrate of HFD group was lower than that of LFD group. This indicated that HFD can promotetumorigenesis. Moreover, the average weight of tumors in HFD group was higher than that ofLFD.
5. MDSC in liver cancer mice was analysed by flow cytometry. In liver cancer tissue, thenumber of CD11b+Ly6C+Ly6G+MDSC was much higher than that of control group. Thissuggested that MDSC regulated the tumorigenesis and development of liver cancer. In addition,the percentage of M2macrophage increased, which demonstrated that M2macrophages playsan crucial importance in tumor formation. Similarly, the percentage of M2macrophages inbone marrow of CCl4group increased as well. Moreover, CD4+CD25+FoxP3+Treg in spleenalso increased compared with control group, which suggested that MDSC presentedimmunological inhibitory effect through propagation of Tregs.
6. MDSC could inhibit HSC proliferation after co-culture of HSC and MDSC，and HSCcould induce MDSC to differentiate into M2macrophages. Especially MDSC from humancould inhibit HSC proliferation by90%. This is the possible mechanism to resolute or reverseliver fibrosis in the future.Conclusion
In conclusion, we find firstly that MDSC plays an important role in promoting liverfibrogenesis and liver tumorigenesis. Meantime, at acute inflammation stage MDSC andMDSC-derived M1and M2macrophages both increase. Moreover, M2macrophage is verycrucial in fibrosis reversion and tumorigenesis. And MDSC promotes liver tumorigenesis through inducing propagation of Tregs. In addition, high fat diet is very important for tumordevelopment. Moreover, HSC proliferation could be inhibited by MDSC dramatically afterco-culture24hours, which suggests a possible target to inhibit fibrogenesis, and MDSC candifferentiate into M2macrophages after co-culture probably.
引文
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