肝星状细胞的基因差异表达以及抑制T细胞功能与肝细胞癌转移的实验研究
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摘要
肝细胞肝癌(hepatocellular carcinoma,HCC)是发病率最高的肿瘤之一,细胞分子生物学的迅猛发展加深了人们对HCC形成机制的理解,目前普遍认为HCC的发生是由于在肝炎病毒和其它致癌因素的作用下,肝细胞发生了基因突变或表观遗传改变而导致癌变。然而这些认识却未能给如何控制HCC带来突破性的进展,原因之一是以往对HCC的研究大多只把重点放在肝细胞恶变上,而忽视了间质细胞在其中的重要作用。恶性肿瘤基因和细胞生物学的研究表明,实质细胞和间质细胞的共同作用是癌症形成和发展的重要基础。间质细胞包括肌成纤维细胞、炎症细胞、内皮细胞等,通过细胞因子、趋化因子、细胞外基质(extracellularmatrix,ECM)或细胞间的直接接触等途径和实质细胞相互作用,与实质细胞癌变、肿瘤中新生血管及结缔组织生成、肿瘤免疫等有关,在肿瘤发生发展中扮演重要角色。
肝星状细胞(hepatic stellate cell,HSC)是肝脏主要的间质细胞之一,HSC又被称为Ito细胞、贮脂细胞、窦周细胞,呈梭形或多边形,位于肝窦内皮与肝细胞之间的Disse间隙内,其特点是具有长的分枝状的细胞突起,并通过这些细胞突起与肝窦内皮细胞和肝细胞形成紧密接触,为HSC和肝细胞的相互作用奠定了生理解剖学基础。正常时HSC占肝脏固有细胞总数8%-12%,作为储脂细胞主要参与体内维生素A的代谢,处于静止状态,不表达a-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、低增殖活性、低合成胶原能力。在肝炎病毒和其它肝损因素的作用下,HSC可被激活,脂质渐消失,并发生形态学改变,转化为肌成纤维细胞。HSC活化的主要标志为细胞增殖、表达a-SMA、异常分泌一些细胞因子、趋化因子以及合成以Ⅰ、Ⅲ型胶原为主的多种ECM蛋白,结果造成肝脏纤维化并进一步发展为肝硬化。近来研究提示HSC活化在HCC的发生、发展和转移中可能起了至关重要的作用,然而具体的作用机制仍然不详。
目前认为,HSC基本的非免疫学功能包括维生素A平衡维持、肝纤维化和调节肝血流。HSC的深入研究证明其表达模式识别受体包括调节免疫应答所需要的Toll样受体(Toll-like receptors,TLRs),共刺激分子和细胞因子,提示其潜在的免疫学作用。近来大多研究证明HSC代表强大的APC,诱导CD-1、MHC-Ⅰ和MHC-Ⅱ限制性T细胞激活和介导抗感染免疫。此外,新的研究提示HSC在维甲酸(retinoic acid,RA)介导T细胞分化中起重要作用。而活化HSC能使T细胞凋亡,行使肝内的免疫保护反应,有利于肝内免疫平衡。HSC在HCC内的免疫学功能如何,与肝癌的复发转移是否相联系,目前尚无相关报道。
本课题首先通过比较大鼠肝癌细胞诱导活化的HSC与培养活化HSC间基因表达差异,证实了肝癌相关活化的HSC功能是不同于培养活化的HSC,为进一步研究HCC内HSC的免疫抑制与肝癌侵袭转移之间的关系提供理论基础;通过建立大鼠HCC肺转移模型为研究HCC中HSC的免疫学特征以及与HCC转移的关系提供实验平台;通过分离纯化大鼠HCC内的HSC和大鼠脾T细胞,在体外进行相互作用研究,并探索HSC对T细胞的功能抑制与肝癌侵袭、转移的可能机制,为体内研究和干预研究提供实验基础,进而为肝癌转移的免疫治疗提供新的靶点。
第一部分大鼠肝癌细胞诱导活化和体外培养活化HSC的基因差异表达
本部分实验目的是为了解肝癌内HSC和正常肝HSC有无区别,为后续的研究提供理论基础,比较了大鼠HCC细胞株C5F条件培养基(conditioned medium,CM)诱导的HSC和体外培养活化HSC的基因表达。通过链霉蛋白酶和胶原酶Ⅳ肝脏原位灌注,Nycodenz密度梯度离心分离大鼠HSC,制备大鼠C5F肝癌细胞株的CM,体外诱导HSC活化。应用cDNA微阵列比较了静止、体外培养活化和肝癌细胞CM诱导活化HSC之间27,100基因表达。应用Real Time RT-PCR和Western blot对芯片进行验证。结果表明同静止HSC相比,HCC细胞诱导活化HSC和体外培养HSC共有1967个基因差异表达,包括促炎症因子、细胞表面受体、细胞粘附分子、信号转导通路分子、免疫相关因子等。肿瘤诱导活化的HSC有一部分基因表达变化与体外培养相同如Ccr1、Vcam1、Tagln、Colla1、Xlkd1和Ccl24等,部分基因如cystatin F、MMP-9、Jun、IgG-2a、Il7r、IAP1和Igf1等表达发生特异性改变,提示HCC细胞可特异性驱动HSC的活化。RealTime RT-PCR和Western blot证实了基因芯片结果。以上研究表明C5F诱导HSC的基因表达部分特异于体外培养HSC的基因表达模式,提示诱导活化HSC在HCC细胞的复发转移中可能起重要作用。体内活化的HSC应作为研究HSC生物学功能的标准。
第二部分大鼠肝细胞癌肺转移模型的建立及肝星状细胞免疫功能的初步研究
本部分实验目的是为了解HCC组织有无T细胞凋亡以及与HCC转移的相关性,建立了Buffalo大鼠HCC肺转移模型。首先将大鼠肝癌细胞株McA-RH7777调整为1×10~6/100μl种入2只Buffalo大鼠胁腹皮下,待皮下瘤长到直径约1cm时取下,切成1mm~3大小植入大鼠肝左叶(n=20),B超及MRI追踪观察肝脏肿瘤的生长情况,做肿瘤生长曲线。后随机抽取1、2、3和4w的大鼠(n=3)处死,切取肝癌组织及肺脏。对肝和肺行HE染色,行肺转移灶计数。正常肝和肝癌组织行α-SMA和CD3免疫组化单染和双重染色及TUNEL和CD3双重染色,并行阳性细胞计数,表示为细胞数/mm~2。对肿瘤内HSC(intratumoral HSC,tHSC)数和T细胞凋亡数以及T细胞凋亡数和肺转移进行相关分析。分离HCC组织内的HSC和脾T淋巴细胞进行体外共培养。结果表明大鼠肝内肿瘤形成率100%,B超检查到1w时肿瘤清晰可见,此后进行性增大;MRI检查发现第4w时开始出现肺转移,肺转移率100%,病理证实第3w时开始出现肺转移。所有HCC内和癌旁组织均观察到α-SMA表达,正常肝组织很少或无。α-SMA数量随着时间延长逐渐增加,在正常肝、1、2、3、和4w的HCC内分别为2.3±0.8/mm~2、85±5.8/mm~(2、)、151±12/mm~2、239±14.7/mm~2和321±16.9/mm~2(P＜0.05)。CD3和Tunel双染法表明T细胞凋亡。正常肝凋亡细胞稀少,肝癌组织凋亡细胞数明显增多。在正常肝、1、2、3、和4w的HCC内分别为1.5±0.5/mm~2、43±2.4/mm~2、82±4.1/mm~2、99±4.6/mm~2和91±11/mm~2(P＜0.05)。α-SMA和CD3免疫组化双重染色表明T细胞凋亡在空间可能与活化的HSC相联系。tHSC数和T细胞凋亡数呈正相关(r=0.711,P＜0.01),T细胞凋亡数和肺转移呈正相关(r=0.561,P＜0.05)。同活化T细胞与qHSC或静止T细胞与tHSC共培养相比,活化的T细胞和iHSC共培养更频繁诱导T细胞凋亡(P＜0.05),且tHSC可能是通过直接与T淋巴细胞接触起作用。以上研究表明渗透和浸润到肝癌组织的T淋巴细胞可能通过与tHSC相互作用导致凋亡,tHSC致T细胞凋亡可能与HCC转移相关。这种模型具有高存活率、良好的肿瘤形成、癌内和癌周HSC的一致性活化以及组织学形态类似于人HSC-T细胞分布模式,是研究体内HSC免疫学功能与HCC转移相关可靠的动物模型。
第三部分大鼠肝癌内HSC抑制T淋巴细胞功能的体外研究
HCC中含有大量活化的HSC,与HCC的复发转移相关。但HSC在HCC免疫应答中的作用知之甚少。本部分实验目的为进一步了解tHSC在HCC的免疫学作用及其机制,在体外研究tHSC对T淋巴细胞的功能影响。主要方法是从Buffalo大鼠正常肝及肝癌中分离HSC,应用流式细胞术、荧光定量RT-PCR检测tHSC表面分子和部分基因的表达,~3H胸腺嘧啶脱氧核苷(~3H-TdR)掺入及释放实验观察tHSC对T细胞的增殖和杀伤活性的影响。应用ELISA检测T细胞的IL-2、IL-10和IFN-γ等表达水平,TUNEL法检测T细胞的凋亡。应用Transwell法观察tHSC抑制T细胞对肿瘤运动、侵袭能力的影响。以上研究结果表明qHSC表达较多的重要表面分子如CD40、CD80、CD54、RT1A和RT1D等,B7-H1少量表达。tHSCs表达CD40、CD80、CD54、RT1A和RT1D等下调,但B7-H1表达上调(P＜0.05),并产生各种免疫抑制相关的细胞因子。加入tHSC抑制通过同种异体抗原或抗CD3介导T细胞受体连接作用活化的T细胞~3H-TdR摄取,抑制特异性抗原活化的T细胞的杀伤活性,并且是剂量依赖的。加入tHSC的活化T细胞与未加tHSC相比,T细胞凋亡数量明显增多(P＜0.01),加入抗B7-H1抗体与未加入抗B7-H1抗体相比,T细胞凋亡数量减少(P＜0.05)。加入tHSC的活化T细胞与未加tHSC相比,McA-RH7777细胞的运动、侵袭能力下降(P＜0.05),加入抗B7-H1抗体与未加入抗B7-H1抗体相比,肿瘤细胞的侵袭能力有所增强,但弱于无HSC组(P＜0.05)。研究结果表明T细胞仍产生大量细胞因子,提示T细胞的活化未受到抑制。tHSC诱导的T细胞低反应性与增加的T细胞凋亡相关。tHSCs的活化与明显提高的B7-H1表达相联系。阻断B7-H1/PD-1连接明显降低tHSC免疫抑制活性,提示B7-H1的重要作用。tHSC对T淋巴细胞的抑制作用可能有助于HCC的复发和转移。为进一步进行体内实验和干预研究提供支持。
结论
1.肝癌细胞诱导活化的HSC与培养活化HSC的具有不同的基因表达谱,肿瘤诱导活化的HSC具有特异性表达的基因,为研究HCC内的HSC的生物学功能提供了理论基础。
2.建立的大鼠HCC肺转移模型,验证了tHSC与免疫抑制及HCC转移相关,为深入研究HCC内HSC的免疫抑制与HCC的转移关系提供了实验平台。
3.tHSC抑制活化T淋巴细胞的功能主要是通过促进T细胞的凋亡,而没有抑制T细胞的活化。tHSCs的活化与明显提高的B7-H1表达相联系。阻断B7-H1/PD-1连接明显降低tHSC免疫抑制活性,提示B7-H1的重要作用。tHSC对T淋巴细胞的抑制作用可能有助于HCC的复发和转移。
创新点
1.首次证实HCC诱导活化的HSC与体外培养活化的HSC具有不同的基因表达谱。
2.首次证明Morris肝癌模型可用于tHSC的免疫抑制与HCC转移关系的研究。
3.首次初步证明tHSC促进T淋巴细胞凋亡有助于肿瘤细胞的运动和侵袭,与HCC的转移相关。
潜在应用价值
1.tHSC是抑制肝癌复发转移的潜在治疗靶点。
2.为深入研究tHSC的免疫抑制参与肝癌侵袭转移的机制提供实验基础。
Hepatocellular carcinoma(HCC) is one of the malignant tumor of the highest incidence.The rapid development of cellular and molecular biology has deepened the understanding of the HCC formation mechanism.Currently,the most accepted hypothesis describes a step-by-step process of HCC in which external stimuli including hepatitis virus and other carcinogenic factors induce genetic or epigenetic alterations in mature hepatocytes resulting in the progression of normal cells through preneoplastic states into invasive cancers.However,none of the findings has turned into a breakthrough in the prevention and treatment for HCC.One reason is that the previous studies of HCC mostly focused on liver cells,and ignored the important role of mesenchymal cells in the malignant transformation.Gene and cell biology research for malignant tumor showed that interaction between parenchymal cells and stromal cells is important foundation of cancer formation and development.Mesenchymal cells include myofibroblasts,inflammatory cells,endothelial cells and so on, interacting with parenchymal cells by secreting cytokines,chemical factors, extracellular matrix(ECM) and direct cell-to-cell contiguity.The stroma cells are considered to be correlated with the angiogenesis,desmoplasia of the cancer and tumor immunity,and are therefore an important player in the progression,growth and spread of cancer.Studies about the role of tumor troma in the tumor development and progression are therefore becoming hot spot.
In the liver,hepatoeytes represent the major cell type of the parenchyma,whereas mesenchymal compartment is composed of various cell types including Kuffper cells and hepatic stellate cells(HSC).HSC,also referred as Ito cells,lipocytes,or perisinusoidal cells,comprise 5%-8%of the total number of resident liver cells.They localized in Disse and characterized by their long processes by which they closely contact with hepatic sinusoid endothelial cells and hepatocytes.These provide the anatomical basis for the interaction between HSC and hepatocytes.Activation of HSC represents a final common pathway of the hepatic response to liver injury.In response to both acute and chronic liver injury,the normally quiescent HSC undergo a progressive activation and transdifferentiate into proliferative,expressingα-SMA, fibrogenic,proinflammatory and contractile myofibroblasts.Through increased secretion of cytokines,chemokines,growth factors,and extracellular matrix proteins and decreased degradation of extracellular matrix,activated HSC are responsible for modification,deposition,and accumulation of the majority of the excess extracellular matrix,resulting in liver fibrosis.Recently,studies suggested that it is possible that HSC play a great role in the progression and metastasis of HCC.Yet,the knowledge about the mechanism of activated HSC involving in the pathological event of HCC is currently limited.
So far,a large body of knowledge has evolved regarding primary non-immunological functions of stellate cells including vitamin A homeostasis,liver fibrosis and regulation of hepatic blood flow.Profound studies extended the scope of stellate cell capacities and suggested a potential immune role in HSC.Accordingly, stellate cells were shown to express pattern recognition receptors including Toll-like receptors(TLRs),costimulatory molecules and cytokines required for modulation of immune responses.Most recent work demonstrated that hepatic stellate cells represented powerful antigen presenting cells(APC),which induce CD1-,MHC-Ⅰ-and MHC-Ⅱ-restricted T cell activation and mediate anti-infection immunity. Moreover,novel studies suggested a central role for stellate cells in retinoic acid mediated T cell differentiation.Activated HSC can also induce T cells apoptosis and assume the responsibility for immune equilibrium in liver.This overview takes you on a journey from non-immunological to immunological functions of stellate cells in the liver.At present,there are no related reports in what HSC immunological function is in HCC,and whether HSC immunological function is associated with the metastasis and recurrence of HCC.
This subject is chiefly to investigate defferentially expression genes between induction-activated HSC(iHSC) and culture-activated HSC(aHSC).We confirmed that iHSC are different from aHSC in the biological function,which provided theretical foundation for studying relationships between iHSC immunosuppression and HCC metastasis.Subsequently,establishment of rat HCC lung metastatic model offered an experimental platform by which we can study correlation between intratumoral HSC(tHSC) immunology and HCC metastasis.Eventually,isolation and purification of HSC and T lyphocytes paved a way for exploring interaction between tHSC and T cell as well as studying potential mechanism of HSC immunosuppression and invasive and metastasis,thus provided experimental basis for in vivo and intervention study.The aim is to provide new immunotherapy target for HCC metastasis.
Part one Gene different expression between culture-activated hepatic stellate cell and hepatocellular carcinoma cells induction-activated hepatic stellate cell
The main aim of this part was to study whether gene expression profiles are different between two kind of HSC in HCC and normal liver,and provide theoretical basis for follow-up investigation.We compared induced HSC by C5F cell lines conditioned medium with in vitro cultured HSC.Rat HSC were isolated from rat livers by perfusion of collagenase and pronase,followed by centrifugation over Nycodenz gradient.To prepare C5F conditioned medium induce HSC activation. 27100 gene expression was analyzed by cDNA microarray in quiescent, culture-activated and induction-activated HSC,and confirmed by real time polymerase chain reaction and Western blot analysis.Results demonstrated that 1967 genes were differentially expressed in iHSC and aHSC,including novel genes that encode proinflammatory mediators;cell adhesion moleculars;cell surface receptors; transduction molecules;and immunity factors.Some genes including Ccr1,Vcam1, Tagln,Colla1,Xlkd1 and Ccl24 were co-expressed in induction-activated and culture-activated HSC.Some genes including cystatin F,MMP-9,Jun,IgG-2a,Il7r, IAP1 and Igf1 were specifically expressed in induction-activated HSC.HCC cell induction-activated HSCs showed specific gene expression patterns,whereas culture activation only partially reproduced the gene expression changes observed during induced activation,suggesting that HCC cells can induce HSC activation. Induction-activated HSCs play a major role in HCC during invasion and metastasis. The above suggested that induce-activated HSC gene expression patterns are different from culture-activated HSC.Because culture activation does not properly regulate gene expression in HSC,in vivo activation should be considered the gold standard for the study of HSC biology.
Part two Establishment of rat hepatocellular carcinoma lung metastasis model and primary study of intratumoral HSC immunity
The main aim of this part was to study whether T cell apoptosis appeared in hepatocellular carcinoma organization as well as correlation with HCC metastasis.We established rat hepatocellular carcinoma lung metastatic model.First,we injected rat HCC cell line McA-RH7777 1×10~6 into flank subcutaneously in 2 Buffalo rat.When subcutaneous tumor diameter is chalking to 1cm,they were cut into 1mm3 size and implanted rat liver left lobe(n=20).We observed hepatic tumor growth situation by B ultrasonic and MRI and drawn tumor growth curve.Randomly selected 1,2,3 and 4 weeks rats(n=3) and executed.Liver and lung tissue were dyed by HE,and lung metastasis focus amounts were counted.Normal liver and HCC tissue were detected by a-SMA and CD3 immunohistochemical staining or double staining,Tunel and CD3 double staining,then positive cells were counted(cell numbers/mm~2). Relationship in intratumoral HSC(tHSC),T cell apoptosis and lung metastasis was investigated by correlation analysis.Intratumoral HSC and spleen T lymphocytes were cocultured in vitro.Results showed that rat tumor formation rate was 100%,and the tumor in liver was clearly visible to one week by B ultrasonic localization, thereafter growing gradually.Metastatic tumor focus in lung occurred in all rats to four weeks by MRI detection.These are demonstrated by HE staining and pathology. We observed a-SMA expression around and within all HCC,and little in normal liver tissue.A-SMA content gradually increased with prolonged time.In normal liver, 1,2,3 and 4 week HCC,the number of a-SMA positive cells are 2.3±0.8,85±5.8, 151±12,239±14.7 and 321±16.9/mm~2 respectively(P＜0.05).Double CD3 and TUNEL stained firmed T cell apoptosis.The apoptotic T cells in HCC were more frequent than in normal liver and may spatially associated to tHSC.In normal liver,1, 2,3 and 4 week HCC,the number of apoptotic T cell are 1.5±0.5,43±2.4,82±3.1, 99±6.5 and 91±11/mm~2 respectively(P＜0.05).T cell apoptosis directly correlated to tHSC(r=0.711,P＜0.01),and lung metastasis directly correlated to T cell apoptosis in HCC(r=0.561,P＜0.05).To compare with activation T cells and quiescent HSC or quiescent T cells and tHSC cocultivation,T cell apoptosis was more frequently induced in cocultivation of T cell and tHSC(P＜0.05),moreover,tHSC was associated with T lymphocytes through direct contact.The above showed that T lymphocytes by permeating and infiltrating into HCC tissue may be induced apoptosis by tHSC, consequently associated with HCC metastasis.This model has high rates of tumor formation and survival,the activation consistency of HSC around and within HCC, and the histologic form similar to human HSC-T cells patterns,therefore is a reliable animal models that studied HSC immunological function in HCC as well as HCC metastasis correlation.
Part three Experimental research of rat intratumoral HSC inhibit T lymphocyte function
A great deal of activated HSC in HCC tissue are associated with HCC metastasis. But little were known about intratumoral HSC(tHSC) immune response role in HCC. The main aim of this part was to further understand of tHSC immunosuppressive function and mechanism.To study tHSC inhibiting T lymphocyte function in vitro. The main methods are the isolation,culture and purification of HSC from Buffalo rat normal liver and HCC tissue.To detect tHSC surface molecules and gene expression by flow cytometry and fluorescence quantitative RT-PCR.To observe T cell proliferation and cytotoxic activity by ~3H thymine(~3H-TdR) incorporating and releasing experiment.To test IL-2,IL-10 and IFN-γexpression level in T cell by application of ELISA.TUNEL was used as T cell apoptosis detection method.To observe tHSC inhibition to T cell and then the influence of tumor invasion and movement ability by application of Transwell method.The above research results showed that quiescent HSC expressed many important surface molecules such as CD40,CD54,RT1A,RT1D,CD80 and expressed less B7-H1.Expression of CD40, CD54,RT1A,RT1D and CD80 in tHSC were downregulated,but B7-H1 upregulated (P＜0.05).Intratumoral HSC produced many immunosuppression related factors. Addition of the tHSC(but not quiescent) suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation and antigen-specific T cell killing activity in a dose-dependent manner.Addition of tHSC, compared with without addition,T cell apoptosis quantity increased significantly (P＜0.01).Addition of anti-B7-H1 antibodies decreased T cell apoptosis(P＜0.05). Addition of tHSC in activated T cells,compared to without addition,the movement and invasive ability of McA-RH7777 cells were significantly enhanced(P＜0.05). Addition of B7-H1 resistance antibodies,compared to without anti-B7-H1 antibody, weakened tumor cells movement and invasive ability(P＜0.05).Results indicated that high cytokine production by the T cells suggested that the inhibition was probably not a result of suppression of their activation.T-cell division was also found to be normal. The tHSC-induced T cell hyporesponsiveness was associated with enhanced T-cell apoptosis.Intratumoral HSC was associated with markedly enhanced expression of B7-H1.Blockade of B7-H1/PD-1 ligation significantly reduced tHSC immunomodulatory activity,suggesting an important role of B7-H1.In conclusion, the interactions between tHSCs and T cells may contribute to HCC recurrence and metastasis.The above in vitro study provide feasibility basis for contiguous in vivo experiment and interventional investigation.
Conclusions
1 Gene expression profiles of HCC cell induction-activated HSC are different from culture-activated HSC.HCC cell induction-activated HSC showed specific gene expression patterns.This study provided theoretical foundations for investigating biology of HSC in HCC.
2 The established rat HCC lung metastatic models were verified with correlation between intratumoral HSC immunosuppression and HCC metastasis,which provides an experimental platform for further research on relationships between intratumoral HSC immunosuppression and HCC metastasis.
3 Intratumoral HSC inhibit activated T lymphocyte function mainly through promoting T cell apoptosis,but not restraining T cell activation.The intratumoral HSC obviously improved the B7-H1 of expression.Blockade of B7-H1/PD-1 connection decreased intratumoral HSC immunosuppression activity,suggesting B7-H1 important role.Inhibition of T lymphocytes by intratumoral HSC may contribute to HCC recurrence and metastasis.
Novelty
1 To confirm that there are different gene expression patterns between HCC cell induction-activated HSC and culture-activated HSC first time.
2 To prove that Morris HCC model can be used for studying relationships between intratumoral HSC immunosuppression and HCC metastasis first time.
3 Preliminary proof suggest that intratumoral HSC promoted T lymphocytes apoptosis,then may contribute to HCC recurrence and metastasis first time.
Potential merits for clinical application
1 Intratumoral HSC was a potential therapeutic targets inhibiting HCC recurrence and metastasis.
2 This study provided experimental basis for further research the mechanisms of intratumoral HSC immunosuppression and the invasion and metastasis of HCC.
肝星状细胞(hepatic stellate cell,HSC)是肝脏主要的间质细胞之一,HSC又被称为Ito细胞、贮脂细胞、窦周细胞,呈梭形或多边形,位于肝窦内皮与肝细胞之间的Disse间隙内,其特点是具有长的分枝状的细胞突起,并通过这些细胞突起与肝窦内皮细胞和肝细胞形成紧密接触,为HSC和肝细胞的相互作用奠定了生理解剖学基础。正常时HSC占肝脏固有细胞总数8%-12%,作为储脂细胞主要参与体内维生素A的代谢,处于静止状态,不表达a-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、低增殖活性、低合成胶原能力。在肝炎病毒和其它肝损因素的作用下,HSC可被激活,脂质渐消失,并发生形态学改变,转化为肌成纤维细胞。HSC活化的主要标志为细胞增殖、表达a-SMA、异常分泌一些细胞因子、趋化因子以及合成以Ⅰ、Ⅲ型胶原为主的多种ECM蛋白,结果造成肝脏纤维化并进一步发展为肝硬化。近来研究提示HSC活化在HCC的发生、发展和转移中可能起了至关重要的作用,然而具体的作用机制仍然不详。
目前认为,HSC基本的非免疫学功能包括维生素A平衡维持、肝纤维化和调节肝血流。HSC的深入研究证明其表达模式识别受体包括调节免疫应答所需要的Toll样受体(Toll-like receptors,TLRs),共刺激分子和细胞因子,提示其潜在的免疫学作用。近来大多研究证明HSC代表强大的APC,诱导CD-1、MHC-Ⅰ和MHC-Ⅱ限制性T细胞激活和介导抗感染免疫。此外,新的研究提示HSC在维甲酸(retinoic acid,RA)介导T细胞分化中起重要作用。而活化HSC能使T细胞凋亡,行使肝内的免疫保护反应,有利于肝内免疫平衡。HSC在HCC内的免疫学功能如何,与肝癌的复发转移是否相联系,目前尚无相关报道。
本课题首先通过比较大鼠肝癌细胞诱导活化的HSC与培养活化HSC间基因表达差异,证实了肝癌相关活化的HSC功能是不同于培养活化的HSC,为进一步研究HCC内HSC的免疫抑制与肝癌侵袭转移之间的关系提供理论基础;通过建立大鼠HCC肺转移模型为研究HCC中HSC的免疫学特征以及与HCC转移的关系提供实验平台;通过分离纯化大鼠HCC内的HSC和大鼠脾T细胞,在体外进行相互作用研究,并探索HSC对T细胞的功能抑制与肝癌侵袭、转移的可能机制,为体内研究和干预研究提供实验基础,进而为肝癌转移的免疫治疗提供新的靶点。
第一部分大鼠肝癌细胞诱导活化和体外培养活化HSC的基因差异表达
本部分实验目的是为了解肝癌内HSC和正常肝HSC有无区别,为后续的研究提供理论基础,比较了大鼠HCC细胞株C5F条件培养基(conditioned medium,CM)诱导的HSC和体外培养活化HSC的基因表达。通过链霉蛋白酶和胶原酶Ⅳ肝脏原位灌注,Nycodenz密度梯度离心分离大鼠HSC,制备大鼠C5F肝癌细胞株的CM,体外诱导HSC活化。应用cDNA微阵列比较了静止、体外培养活化和肝癌细胞CM诱导活化HSC之间27,100基因表达。应用Real Time RT-PCR和Western blot对芯片进行验证。结果表明同静止HSC相比,HCC细胞诱导活化HSC和体外培养HSC共有1967个基因差异表达,包括促炎症因子、细胞表面受体、细胞粘附分子、信号转导通路分子、免疫相关因子等。肿瘤诱导活化的HSC有一部分基因表达变化与体外培养相同如Ccr1、Vcam1、Tagln、Colla1、Xlkd1和Ccl24等,部分基因如cystatin F、MMP-9、Jun、IgG-2a、Il7r、IAP1和Igf1等表达发生特异性改变,提示HCC细胞可特异性驱动HSC的活化。RealTime RT-PCR和Western blot证实了基因芯片结果。以上研究表明C5F诱导HSC的基因表达部分特异于体外培养HSC的基因表达模式,提示诱导活化HSC在HCC细胞的复发转移中可能起重要作用。体内活化的HSC应作为研究HSC生物学功能的标准。
第二部分大鼠肝细胞癌肺转移模型的建立及肝星状细胞免疫功能的初步研究
本部分实验目的是为了解HCC组织有无T细胞凋亡以及与HCC转移的相关性,建立了Buffalo大鼠HCC肺转移模型。首先将大鼠肝癌细胞株McA-RH7777调整为1×10~6/100μl种入2只Buffalo大鼠胁腹皮下,待皮下瘤长到直径约1cm时取下,切成1mm~3大小植入大鼠肝左叶(n=20),B超及MRI追踪观察肝脏肿瘤的生长情况,做肿瘤生长曲线。后随机抽取1、2、3和4w的大鼠(n=3)处死,切取肝癌组织及肺脏。对肝和肺行HE染色,行肺转移灶计数。正常肝和肝癌组织行α-SMA和CD3免疫组化单染和双重染色及TUNEL和CD3双重染色,并行阳性细胞计数,表示为细胞数/mm~2。对肿瘤内HSC(intratumoral HSC,tHSC)数和T细胞凋亡数以及T细胞凋亡数和肺转移进行相关分析。分离HCC组织内的HSC和脾T淋巴细胞进行体外共培养。结果表明大鼠肝内肿瘤形成率100%,B超检查到1w时肿瘤清晰可见,此后进行性增大;MRI检查发现第4w时开始出现肺转移,肺转移率100%,病理证实第3w时开始出现肺转移。所有HCC内和癌旁组织均观察到α-SMA表达,正常肝组织很少或无。α-SMA数量随着时间延长逐渐增加,在正常肝、1、2、3、和4w的HCC内分别为2.3±0.8/mm~2、85±5.8/mm~(2、)、151±12/mm~2、239±14.7/mm~2和321±16.9/mm~2(P＜0.05)。CD3和Tunel双染法表明T细胞凋亡。正常肝凋亡细胞稀少,肝癌组织凋亡细胞数明显增多。在正常肝、1、2、3、和4w的HCC内分别为1.5±0.5/mm~2、43±2.4/mm~2、82±4.1/mm~2、99±4.6/mm~2和91±11/mm~2(P＜0.05)。α-SMA和CD3免疫组化双重染色表明T细胞凋亡在空间可能与活化的HSC相联系。tHSC数和T细胞凋亡数呈正相关(r=0.711,P＜0.01),T细胞凋亡数和肺转移呈正相关(r=0.561,P＜0.05)。同活化T细胞与qHSC或静止T细胞与tHSC共培养相比,活化的T细胞和iHSC共培养更频繁诱导T细胞凋亡(P＜0.05),且tHSC可能是通过直接与T淋巴细胞接触起作用。以上研究表明渗透和浸润到肝癌组织的T淋巴细胞可能通过与tHSC相互作用导致凋亡,tHSC致T细胞凋亡可能与HCC转移相关。这种模型具有高存活率、良好的肿瘤形成、癌内和癌周HSC的一致性活化以及组织学形态类似于人HSC-T细胞分布模式,是研究体内HSC免疫学功能与HCC转移相关可靠的动物模型。
第三部分大鼠肝癌内HSC抑制T淋巴细胞功能的体外研究
HCC中含有大量活化的HSC,与HCC的复发转移相关。但HSC在HCC免疫应答中的作用知之甚少。本部分实验目的为进一步了解tHSC在HCC的免疫学作用及其机制,在体外研究tHSC对T淋巴细胞的功能影响。主要方法是从Buffalo大鼠正常肝及肝癌中分离HSC,应用流式细胞术、荧光定量RT-PCR检测tHSC表面分子和部分基因的表达,~3H胸腺嘧啶脱氧核苷(~3H-TdR)掺入及释放实验观察tHSC对T细胞的增殖和杀伤活性的影响。应用ELISA检测T细胞的IL-2、IL-10和IFN-γ等表达水平,TUNEL法检测T细胞的凋亡。应用Transwell法观察tHSC抑制T细胞对肿瘤运动、侵袭能力的影响。以上研究结果表明qHSC表达较多的重要表面分子如CD40、CD80、CD54、RT1A和RT1D等,B7-H1少量表达。tHSCs表达CD40、CD80、CD54、RT1A和RT1D等下调,但B7-H1表达上调(P＜0.05),并产生各种免疫抑制相关的细胞因子。加入tHSC抑制通过同种异体抗原或抗CD3介导T细胞受体连接作用活化的T细胞~3H-TdR摄取,抑制特异性抗原活化的T细胞的杀伤活性,并且是剂量依赖的。加入tHSC的活化T细胞与未加tHSC相比,T细胞凋亡数量明显增多(P＜0.01),加入抗B7-H1抗体与未加入抗B7-H1抗体相比,T细胞凋亡数量减少(P＜0.05)。加入tHSC的活化T细胞与未加tHSC相比,McA-RH7777细胞的运动、侵袭能力下降(P＜0.05),加入抗B7-H1抗体与未加入抗B7-H1抗体相比,肿瘤细胞的侵袭能力有所增强,但弱于无HSC组(P＜0.05)。研究结果表明T细胞仍产生大量细胞因子,提示T细胞的活化未受到抑制。tHSC诱导的T细胞低反应性与增加的T细胞凋亡相关。tHSCs的活化与明显提高的B7-H1表达相联系。阻断B7-H1/PD-1连接明显降低tHSC免疫抑制活性,提示B7-H1的重要作用。tHSC对T淋巴细胞的抑制作用可能有助于HCC的复发和转移。为进一步进行体内实验和干预研究提供支持。
结论
1.肝癌细胞诱导活化的HSC与培养活化HSC的具有不同的基因表达谱,肿瘤诱导活化的HSC具有特异性表达的基因,为研究HCC内的HSC的生物学功能提供了理论基础。
2.建立的大鼠HCC肺转移模型,验证了tHSC与免疫抑制及HCC转移相关,为深入研究HCC内HSC的免疫抑制与HCC的转移关系提供了实验平台。
3.tHSC抑制活化T淋巴细胞的功能主要是通过促进T细胞的凋亡,而没有抑制T细胞的活化。tHSCs的活化与明显提高的B7-H1表达相联系。阻断B7-H1/PD-1连接明显降低tHSC免疫抑制活性,提示B7-H1的重要作用。tHSC对T淋巴细胞的抑制作用可能有助于HCC的复发和转移。
创新点
1.首次证实HCC诱导活化的HSC与体外培养活化的HSC具有不同的基因表达谱。
2.首次证明Morris肝癌模型可用于tHSC的免疫抑制与HCC转移关系的研究。
3.首次初步证明tHSC促进T淋巴细胞凋亡有助于肿瘤细胞的运动和侵袭,与HCC的转移相关。
潜在应用价值
1.tHSC是抑制肝癌复发转移的潜在治疗靶点。
2.为深入研究tHSC的免疫抑制参与肝癌侵袭转移的机制提供实验基础。
Hepatocellular carcinoma(HCC) is one of the malignant tumor of the highest incidence.The rapid development of cellular and molecular biology has deepened the understanding of the HCC formation mechanism.Currently,the most accepted hypothesis describes a step-by-step process of HCC in which external stimuli including hepatitis virus and other carcinogenic factors induce genetic or epigenetic alterations in mature hepatocytes resulting in the progression of normal cells through preneoplastic states into invasive cancers.However,none of the findings has turned into a breakthrough in the prevention and treatment for HCC.One reason is that the previous studies of HCC mostly focused on liver cells,and ignored the important role of mesenchymal cells in the malignant transformation.Gene and cell biology research for malignant tumor showed that interaction between parenchymal cells and stromal cells is important foundation of cancer formation and development.Mesenchymal cells include myofibroblasts,inflammatory cells,endothelial cells and so on, interacting with parenchymal cells by secreting cytokines,chemical factors, extracellular matrix(ECM) and direct cell-to-cell contiguity.The stroma cells are considered to be correlated with the angiogenesis,desmoplasia of the cancer and tumor immunity,and are therefore an important player in the progression,growth and spread of cancer.Studies about the role of tumor troma in the tumor development and progression are therefore becoming hot spot.
In the liver,hepatoeytes represent the major cell type of the parenchyma,whereas mesenchymal compartment is composed of various cell types including Kuffper cells and hepatic stellate cells(HSC).HSC,also referred as Ito cells,lipocytes,or perisinusoidal cells,comprise 5%-8%of the total number of resident liver cells.They localized in Disse and characterized by their long processes by which they closely contact with hepatic sinusoid endothelial cells and hepatocytes.These provide the anatomical basis for the interaction between HSC and hepatocytes.Activation of HSC represents a final common pathway of the hepatic response to liver injury.In response to both acute and chronic liver injury,the normally quiescent HSC undergo a progressive activation and transdifferentiate into proliferative,expressingα-SMA, fibrogenic,proinflammatory and contractile myofibroblasts.Through increased secretion of cytokines,chemokines,growth factors,and extracellular matrix proteins and decreased degradation of extracellular matrix,activated HSC are responsible for modification,deposition,and accumulation of the majority of the excess extracellular matrix,resulting in liver fibrosis.Recently,studies suggested that it is possible that HSC play a great role in the progression and metastasis of HCC.Yet,the knowledge about the mechanism of activated HSC involving in the pathological event of HCC is currently limited.
So far,a large body of knowledge has evolved regarding primary non-immunological functions of stellate cells including vitamin A homeostasis,liver fibrosis and regulation of hepatic blood flow.Profound studies extended the scope of stellate cell capacities and suggested a potential immune role in HSC.Accordingly, stellate cells were shown to express pattern recognition receptors including Toll-like receptors(TLRs),costimulatory molecules and cytokines required for modulation of immune responses.Most recent work demonstrated that hepatic stellate cells represented powerful antigen presenting cells(APC),which induce CD1-,MHC-Ⅰ-and MHC-Ⅱ-restricted T cell activation and mediate anti-infection immunity. Moreover,novel studies suggested a central role for stellate cells in retinoic acid mediated T cell differentiation.Activated HSC can also induce T cells apoptosis and assume the responsibility for immune equilibrium in liver.This overview takes you on a journey from non-immunological to immunological functions of stellate cells in the liver.At present,there are no related reports in what HSC immunological function is in HCC,and whether HSC immunological function is associated with the metastasis and recurrence of HCC.
This subject is chiefly to investigate defferentially expression genes between induction-activated HSC(iHSC) and culture-activated HSC(aHSC).We confirmed that iHSC are different from aHSC in the biological function,which provided theretical foundation for studying relationships between iHSC immunosuppression and HCC metastasis.Subsequently,establishment of rat HCC lung metastatic model offered an experimental platform by which we can study correlation between intratumoral HSC(tHSC) immunology and HCC metastasis.Eventually,isolation and purification of HSC and T lyphocytes paved a way for exploring interaction between tHSC and T cell as well as studying potential mechanism of HSC immunosuppression and invasive and metastasis,thus provided experimental basis for in vivo and intervention study.The aim is to provide new immunotherapy target for HCC metastasis.
Part one Gene different expression between culture-activated hepatic stellate cell and hepatocellular carcinoma cells induction-activated hepatic stellate cell
The main aim of this part was to study whether gene expression profiles are different between two kind of HSC in HCC and normal liver,and provide theoretical basis for follow-up investigation.We compared induced HSC by C5F cell lines conditioned medium with in vitro cultured HSC.Rat HSC were isolated from rat livers by perfusion of collagenase and pronase,followed by centrifugation over Nycodenz gradient.To prepare C5F conditioned medium induce HSC activation. 27100 gene expression was analyzed by cDNA microarray in quiescent, culture-activated and induction-activated HSC,and confirmed by real time polymerase chain reaction and Western blot analysis.Results demonstrated that 1967 genes were differentially expressed in iHSC and aHSC,including novel genes that encode proinflammatory mediators;cell adhesion moleculars;cell surface receptors; transduction molecules;and immunity factors.Some genes including Ccr1,Vcam1, Tagln,Colla1,Xlkd1 and Ccl24 were co-expressed in induction-activated and culture-activated HSC.Some genes including cystatin F,MMP-9,Jun,IgG-2a,Il7r, IAP1 and Igf1 were specifically expressed in induction-activated HSC.HCC cell induction-activated HSCs showed specific gene expression patterns,whereas culture activation only partially reproduced the gene expression changes observed during induced activation,suggesting that HCC cells can induce HSC activation. Induction-activated HSCs play a major role in HCC during invasion and metastasis. The above suggested that induce-activated HSC gene expression patterns are different from culture-activated HSC.Because culture activation does not properly regulate gene expression in HSC,in vivo activation should be considered the gold standard for the study of HSC biology.
Part two Establishment of rat hepatocellular carcinoma lung metastasis model and primary study of intratumoral HSC immunity
The main aim of this part was to study whether T cell apoptosis appeared in hepatocellular carcinoma organization as well as correlation with HCC metastasis.We established rat hepatocellular carcinoma lung metastatic model.First,we injected rat HCC cell line McA-RH7777 1×10~6 into flank subcutaneously in 2 Buffalo rat.When subcutaneous tumor diameter is chalking to 1cm,they were cut into 1mm3 size and implanted rat liver left lobe(n=20).We observed hepatic tumor growth situation by B ultrasonic and MRI and drawn tumor growth curve.Randomly selected 1,2,3 and 4 weeks rats(n=3) and executed.Liver and lung tissue were dyed by HE,and lung metastasis focus amounts were counted.Normal liver and HCC tissue were detected by a-SMA and CD3 immunohistochemical staining or double staining,Tunel and CD3 double staining,then positive cells were counted(cell numbers/mm~2). Relationship in intratumoral HSC(tHSC),T cell apoptosis and lung metastasis was investigated by correlation analysis.Intratumoral HSC and spleen T lymphocytes were cocultured in vitro.Results showed that rat tumor formation rate was 100%,and the tumor in liver was clearly visible to one week by B ultrasonic localization, thereafter growing gradually.Metastatic tumor focus in lung occurred in all rats to four weeks by MRI detection.These are demonstrated by HE staining and pathology. We observed a-SMA expression around and within all HCC,and little in normal liver tissue.A-SMA content gradually increased with prolonged time.In normal liver, 1,2,3 and 4 week HCC,the number of a-SMA positive cells are 2.3±0.8,85±5.8, 151±12,239±14.7 and 321±16.9/mm~2 respectively(P＜0.05).Double CD3 and TUNEL stained firmed T cell apoptosis.The apoptotic T cells in HCC were more frequent than in normal liver and may spatially associated to tHSC.In normal liver,1, 2,3 and 4 week HCC,the number of apoptotic T cell are 1.5±0.5,43±2.4,82±3.1, 99±6.5 and 91±11/mm~2 respectively(P＜0.05).T cell apoptosis directly correlated to tHSC(r=0.711,P＜0.01),and lung metastasis directly correlated to T cell apoptosis in HCC(r=0.561,P＜0.05).To compare with activation T cells and quiescent HSC or quiescent T cells and tHSC cocultivation,T cell apoptosis was more frequently induced in cocultivation of T cell and tHSC(P＜0.05),moreover,tHSC was associated with T lymphocytes through direct contact.The above showed that T lymphocytes by permeating and infiltrating into HCC tissue may be induced apoptosis by tHSC, consequently associated with HCC metastasis.This model has high rates of tumor formation and survival,the activation consistency of HSC around and within HCC, and the histologic form similar to human HSC-T cells patterns,therefore is a reliable animal models that studied HSC immunological function in HCC as well as HCC metastasis correlation.
Part three Experimental research of rat intratumoral HSC inhibit T lymphocyte function
A great deal of activated HSC in HCC tissue are associated with HCC metastasis. But little were known about intratumoral HSC(tHSC) immune response role in HCC. The main aim of this part was to further understand of tHSC immunosuppressive function and mechanism.To study tHSC inhibiting T lymphocyte function in vitro. The main methods are the isolation,culture and purification of HSC from Buffalo rat normal liver and HCC tissue.To detect tHSC surface molecules and gene expression by flow cytometry and fluorescence quantitative RT-PCR.To observe T cell proliferation and cytotoxic activity by ~3H thymine(~3H-TdR) incorporating and releasing experiment.To test IL-2,IL-10 and IFN-γexpression level in T cell by application of ELISA.TUNEL was used as T cell apoptosis detection method.To observe tHSC inhibition to T cell and then the influence of tumor invasion and movement ability by application of Transwell method.The above research results showed that quiescent HSC expressed many important surface molecules such as CD40,CD54,RT1A,RT1D,CD80 and expressed less B7-H1.Expression of CD40, CD54,RT1A,RT1D and CD80 in tHSC were downregulated,but B7-H1 upregulated (P＜0.05).Intratumoral HSC produced many immunosuppression related factors. Addition of the tHSC(but not quiescent) suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation and antigen-specific T cell killing activity in a dose-dependent manner.Addition of tHSC, compared with without addition,T cell apoptosis quantity increased significantly (P＜0.01).Addition of anti-B7-H1 antibodies decreased T cell apoptosis(P＜0.05). Addition of tHSC in activated T cells,compared to without addition,the movement and invasive ability of McA-RH7777 cells were significantly enhanced(P＜0.05). Addition of B7-H1 resistance antibodies,compared to without anti-B7-H1 antibody, weakened tumor cells movement and invasive ability(P＜0.05).Results indicated that high cytokine production by the T cells suggested that the inhibition was probably not a result of suppression of their activation.T-cell division was also found to be normal. The tHSC-induced T cell hyporesponsiveness was associated with enhanced T-cell apoptosis.Intratumoral HSC was associated with markedly enhanced expression of B7-H1.Blockade of B7-H1/PD-1 ligation significantly reduced tHSC immunomodulatory activity,suggesting an important role of B7-H1.In conclusion, the interactions between tHSCs and T cells may contribute to HCC recurrence and metastasis.The above in vitro study provide feasibility basis for contiguous in vivo experiment and interventional investigation.
Conclusions
1 Gene expression profiles of HCC cell induction-activated HSC are different from culture-activated HSC.HCC cell induction-activated HSC showed specific gene expression patterns.This study provided theoretical foundations for investigating biology of HSC in HCC.
2 The established rat HCC lung metastatic models were verified with correlation between intratumoral HSC immunosuppression and HCC metastasis,which provides an experimental platform for further research on relationships between intratumoral HSC immunosuppression and HCC metastasis.
3 Intratumoral HSC inhibit activated T lymphocyte function mainly through promoting T cell apoptosis,but not restraining T cell activation.The intratumoral HSC obviously improved the B7-H1 of expression.Blockade of B7-H1/PD-1 connection decreased intratumoral HSC immunosuppression activity,suggesting B7-H1 important role.Inhibition of T lymphocytes by intratumoral HSC may contribute to HCC recurrence and metastasis.
Novelty
1 To confirm that there are different gene expression patterns between HCC cell induction-activated HSC and culture-activated HSC first time.
2 To prove that Morris HCC model can be used for studying relationships between intratumoral HSC immunosuppression and HCC metastasis first time.
3 Preliminary proof suggest that intratumoral HSC promoted T lymphocytes apoptosis,then may contribute to HCC recurrence and metastasis first time.
Potential merits for clinical application
1 Intratumoral HSC was a potential therapeutic targets inhibiting HCC recurrence and metastasis.
2 This study provided experimental basis for further research the mechanisms of intratumoral HSC immunosuppression and the invasion and metastasis of HCC.
引文
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