大鼠肝星状细胞促进肝癌生长、侵袭及调控免疫微环境的实验研究
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摘要
肝细胞肝癌(hepatocellular carcinoma, HCC)(简称肝癌)是世界上最常见的肿瘤之一,位居世界恶性肿瘤死因第三位,每年至少有610,000死亡病例(其中约半数病例发生在我国)。尽管过去的数十年里肝癌的细胞和分子生物学研究取得很大进展,肝癌的预后仍然很差。手术切除是肿瘤综合治疗中效果最好、使患者获得长期生存最有效的途径,但肝癌根治性切除后5年复发转移率仍高达60%-70%。同时我国肝癌多发生于肝炎(乙型肝炎和丙型肝炎)的基础上,多伴有严重的肝硬化,相当多的原发性肝癌因严重的肝硬化无法手术切除。系统性治疗(包括化疗、分子靶向治疗)是应用广泛、正在深入研究中的主要治疗方法,但治疗数月后肿瘤迅速进展恶化(进一步增殖、转移)成为影响疗效的主要障碍。
以基因组、蛋白组和组织芯片为代表的高通量分子生物学技术的出现和发展,为研究肿瘤发生、进展和转移提供了有力的手段,然而以往对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。肿瘤侵袭与转移是一个多步骤多因素参与的复杂过程,其发生依赖于肿瘤细胞与基质之间的相互作用。间质细胞在肿瘤的发生发展过程中发挥着重要作用,参与调节肿瘤血管生成、结缔组织纤维形成、肿瘤免疫等环节。肝星状细胞(hepatic stellate cell, HSC)也称为Ito细胞,储脂细胞,或窦状隙周(围)细胞,胞浆富含VitA脂滴,HSC约占肝脏细胞总数的13%。HSC位于Diss间隙,具有长的分枝状的细胞突起,通过这些细胞突起包绕肝细胞并与肝窦内皮细胞形成紧密接触,为HSC和肝细胞的相互作用奠定了生理解剖学基础。HSC活化是肝纤维化形成,并最终发展至肝硬化的核心环节。“肝炎-肝纤维化/肝硬化-HCC”被认为是肝癌发生过程中的三部曲。HSC在“炎症-纤维化-肿瘤”这一发生发展过程中充当了重要的介质。近年来研究提示HSC活化在HCC的发生、发展和转移中可能扮演重要角色,通过增强分泌细胞因子、趋化因子、生长因子和ECM蛋白、降低ECM的降解而起作用。而且,在肝癌的间质中伴有活化HSC/MBF(肌成纤维细胞)的浸润,活化的HSC可能通过释放包含生存信号的旁分泌因子促进肝癌形成,机制尚不明。临床证据显示癌周HSC往往和肝癌的早期复发相关。目前为止,普遍认为HSC的基本的非免疫学功能包括维生素A代谢平衡、肝纤维化和调节肝脏血流。更深入的研究发现了HSC的新的功能,HSC具有潜在的免疫学作用。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。
课题组前期实验证实了肝癌相关活化的HSC功能是不同于培养活化的HSC,初步证明瘤内活化HSC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。HCC可诱导HSC活化,而活化的HSC又可促进HCC的发生、发展、浸润和转移,其中活化的HSC对免疫微环境的抑制是可能的作用机制之一,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,有待进一步的研究。
本课题将活化的大鼠HSC与肝癌细胞共培养,首次证实活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,在体外促进肝癌细胞增殖、迁移、侵袭；体内进行HSC与HCC相互作用研究,活化的HSC在大鼠皮下促进肝癌的增殖、侵袭,并伴有免疫微环境的变化；在体外细胞相互作用研究中,HSC促进T细胞凋亡,但未促进DC细胞凋亡；并探索经肝门静脉输注DC细胞改善肝脏局部免疫微环境,为体内肝癌免疫治疗研究、预防肝癌复发转移寻找新的方法和途径。
第一部分大鼠肝星状细胞在体外改变肝癌细胞功能基因表达,促进肝癌细胞增殖、迁移和侵袭
目的：分离并鉴定大鼠肝星状细胞(HSC),在体外活化的HSC可以通过外分泌调控肝癌(HCC)细胞的功能基因表达,促进HCC细胞的增殖、迁移和侵袭。方法：密度梯度离心分离HSC,制备大鼠McA-RH7777肝癌细胞株的条件培养液(CM)培养诱导活化HSC,活化的HSC与McA-RH7777肝癌细胞共培养。应用cDNA微阵列比较了单纯McA-RH7777肝癌细胞、与活化HSC共培养的McA-RH7777肝癌细胞之间28,728基因表达。应用实时RT-PCR和Western blot对芯片结果进行验证。制备活化HSC的CM,通过CCK-8、划痕、Transwell小室等实验,研究活化HSC对肝癌细胞增殖、迁移、侵袭的促进作用。ELISA法检测比较McA-RH7777肝癌细胞、McA-RH7777肝癌细胞+活化HSC共培养两者之间上清中细胞因子的差异。结果：同单纯McA-RH7777肝癌细胞培养相比,和肿瘤诱导活化的HSC(induction-activated HSC, iHSC)共培养的McA-RH7777肝癌细胞多个基因出现显著差异表达,573个基因表达超过2倍上调或下调(其中432个基因为上调,141个基因为下调),包括细胞表面受体、细胞代谢、细胞粘附分子、信号转导通路分子、趋化因子、免疫相关因子等。通过活化HSC旁分泌作用,McA-RH7777肝癌细胞部分基因表达上调,如Tapbp、Ccl2、Cxcll、Cxcl10、Junb、Igf1、Stat1、Irfl、Irf7、Irf9、和Csfl等,部分基因如4-Sep、Cck、Pdgfra、Itgae和Cd36等表达下调。活化HSC在体外通过旁分泌调节促进肝癌细胞增殖、迁移、侵袭。与单纯McA-RH7777肝癌细胞培养相比,McA-RH7777肝癌细胞+活化HSC共培养时,HGF、IL-6、MMP-2、MMP-9等因子升高。结论：活化的HSC与肝癌细胞共培养,活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,促进肝癌细胞增殖、迁移、侵袭,活化的HSC在HCC的生长、侵袭转移中起重要作用。
第二部分大鼠活化肝星状细胞在体内参与改变肿瘤微环境,促进肿瘤生长、侵袭
目的：研究HSC在体内是否参与改变肿瘤免疫微环境,促进肿瘤生长、侵袭。方法：将大鼠肝癌细胞株McA-RH7777种入Buffalo大鼠大腿内侧皮下,约3周后取皮下肿瘤组织,切成1mm3大小植入大鼠肝左叶(n=25),建立Buffalo大鼠肝细胞癌肺转移模型。CT观察肝脏原位移植瘤的生长情况。抽取1、2、3和4周的大鼠(n=5)处死,切取肝癌组织及肺脏。对肝组织和肺组织行HE染色,第1周(早期)及第4周(晚期)肝癌组织行α-SMA、CD3、CD68、S-100免疫组化染色,行阳性细胞计数,对瘤内HSC数和T细胞、巨噬细胞、DC细胞数进行分析比较。皮下成瘤实验：Buffalo大鼠分为2组(每组n=6),对照组皮下注射McA-RH7777细胞(2×106个),实验组皮下注射McA-RH7777细胞(2×106个)+活化HSC(1×106个),比较2组皮下肿瘤的大小、肿瘤免疫微环境。肝内成瘤实验：Buffalo大鼠分为2组(每组n=6),对照组肝左叶内注射McA-RH7777细胞(2×106个),实验组肝左叶内注射McA-RH7777细胞(2×106个)+活化HSC(1×106个),比较2组肝内肿瘤的增殖、肺转移情况。结果：大鼠肝癌原位模型：2周时CT检查可见肿瘤；第4周时CT检出肺转移,5周后检出明显的肺转移病灶,肺转移率100%。大鼠肝癌模型的肝癌组织观察到a-SMA表达,晚期肝癌与早期相比,肿瘤微环境中tHSC (intratumor HSC,瘤内肝星状细胞)、巨噬细胞增加,DC细胞、T细胞减少。皮下成瘤实验：活化HSC促进肝癌皮下肿瘤增殖、侵袭,混合HSC时肿瘤侵犯皮肤,免疫组化提示混合成瘤的肿瘤中存在较多HSC,巨噬细胞增多,DC细胞、T细胞减少。iHSC与肝癌细胞混合后肝内接种没有促进肝癌的增殖、转移。iHSC在体外表现出较强的迁移侵袭能力。结论：大鼠肝癌细胞McA-RH7777的原位肝癌模型中,晚期肝癌组织HSC浸润增加,伴有DC细胞、T淋巴细胞减少,免疫微环境在肿瘤进展过程中发生变化。活化的HSC在体内促进肝癌的增殖、侵袭,并伴有免疫微环境的变化。
第三部分大鼠活化肝星状细胞调控肝癌免疫微环境机制的初步研究
目的：在体外研究HSC对T淋巴细胞、DC细胞的影响,了解HSC对HCC的肿瘤免疫微环境的调控作用及其机制；肝内DC细胞过继免疫改善免疫微环境的作用。方法：使用McA-RH7777肝癌细胞的条件培养基诱导活化新分离的大鼠HSC,分离培养T细胞、DC细胞,iHSC分别与DC细胞、T细胞共培养。LDH释放实验观察iHSC对T细胞的杀伤活性的影响,Tunel法检测T细胞的凋亡,应用Transwell法观察iHSC抑制T细胞对肿瘤侵袭能力的影响,流式细胞分析iHSC对DC细胞诱导凋亡的作用。肝内DC细胞过继免疫实验：Buffalo大鼠分为2组(每组n=6),对照组在肝左叶原位移植瘤组织,干预组经门静脉注射负载肿瘤抗原的DC细胞后再原位移植肝癌,与对照组比较肝内肿瘤的增殖、转移、微环境中免疫细胞。结果：iHSC在体外诱导活化T细胞凋亡,而诱导DC细胞凋亡不明显,iHSC在Transwell小室中诱导T细胞凋亡从而促进肝癌细胞侵袭。经门静脉注射负载肿瘤抗原的DC细胞可以抑制肝癌的增殖和转移,改善免疫微环境。结论：iHSC促进肝癌细胞的侵袭,对HCC的肿瘤免疫微环境的有调控作用,机制可能为促进T细胞凋亡,但未促进DC细胞凋亡。经肝门静脉输注DC细胞可以改善肝脏免疫微环境,抑制肿瘤增殖、转移。
结论
1.活化的HSC与肝癌细胞共培养,活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,促进肝癌细胞增殖、迁移、侵袭,活化的HSC在HCC的生长、侵袭转移中起重要作用。
2.大鼠肝癌细胞McA-RH7777的原位肝癌模型中,晚期肝癌组织HSC浸润增加,伴有DC细胞、T淋巴细胞减少,免疫微环境在肿瘤进展过程中发生变化。活化的HSC在体内促进肝癌的增殖、侵袭,并伴有免疫微环境的变化。
3.肿瘤诱导活化的HSC促进肝癌细胞的侵袭,对HCC的肿瘤免疫微环境的有调控作用,机制可能为促进T细胞凋亡,但未促进DC细胞凋亡；经肝门静脉输注DC细胞可以改善肝脏免疫微环境,抑制肿瘤增殖、转移。创新点
1.首次证实活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,促进肝癌细胞增殖、迁移、侵袭,参与其中的基因和因子是多个的、非单一独特的。
2.首次证实肿瘤诱导活化的HSC对HCC的免疫微环境的调控作用,未促进DC细胞凋亡；经肝门静脉输注DC细胞可以改善肝脏免疫微环境,抑制肿瘤增殖、转移。潜在应用价值
1.肝癌微环境中的活化HSC是反应肝癌生物学特性的指标之一,为深入研究瘤内HSC在肝癌免疫微环境中的免疫抑制机制提供实验基础。
2.HSC是抑制肝癌复发转移的潜在治疗靶点,对于伴有肝硬化背景的肝癌针对HSC的治疗意义更加重大。
Hepatocellular carcinoma (HCC) is one of the most common cancer in the world, and the third leading cause of cancer-related death worldwide, accounting for at least610,000deaths per year, nearly half of them in China. Despite tremendous achievements of cellular and molecular biology in HCC have been made during past decades, the prognosis of HCC remains dismal. Although surgery achieves the best outcome in well-selected candidates, the5-year recurrence rate remains as high as60-70%after HCC resection. At the same time, about80%of HCC cases are associated with chronic hepatitis (type B or C hepatitis) and cirrhosis in China, and most of them lost the chance to accept the currative operation. Although the systematic therapies including chemotherapy and targeted therapy were investigated deeply and applied widespread in the patients with late stage, the tumors always progress after several months treatment remains a major obstacle for more therapeutic efficacy.
The novel high throughput analysis technologies, such as genomics, protemics and tissue microarray, greatly facilitate the studies on the progression and metastasis of tumor. However, none of the findings has turned into a breakthrough in the prevention and treatment for HCC. The vast majority of previous studies focused solely on malignant cells themselves, and ignored the important role of stromal cells in the malignant transformation. The invason and metastasis of HCC are complicated procedures with multi-step and multi-factor, and also depened on the interaction between malignant cells and stromal cells. 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 initiation and development of cancer. HSC, also referred as Ito cells, lipocytes, or perisinusoidal cells, comprise13%of the total number of liver cells. They localized in Disse and characterized by their long processes by which they closely embrace hepatocytes and contact with sinusoidal endothelial. The activation of hepatic stellate cells (HSC) is recognized as a central event in the development of hepatic fibrosis and lastly, cirrhosis. A three-step process of "inflammation-fibrosis/cirrhosis-HCC" is believed to be involved in hepatocarcinogenesis, the activation of hepatic stellate cells (HSCs) may serve as an important mediator in the process of inflammation-fibrosis-carcinoma axis, by increasing secretion of cytokines, chemokines, growth factors, and extracellular matrix proteins and decreasing degradation of extracellular matrix. Moreover, in HCC the stroma is infiltrated by activated HSC/myofibroblasts. Activated HSCs release paracrine factors that may promote hepatocarcinogenesis through unknown mechanisms including release of survival signals. Clinical evidences disclosed that peritumoral HSCs were related to more early HCC recurrences.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. Recently, studies suggested that it is possible that HSC play a great role in the progression of HCC, even in tumor metastasis, but the related mechanism remains unclear.
Our research group confirmed that: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. Intratumoral HSC inhibit activated T lymphocyte function mainly through promoting T cell apoptosis, and then promote tumor cells and movement ability in vitro.
In this study, we take induction-activated HSCs (iHSC) and McA-RH-7777hepatoma cells to co-culture, and confirm that iHSC alter expression genes and promote the growth and invation of McA-RH-7777hepatoma cells in vitro through the mechanism of paracrine secretion. iHSCs also enhance growth and invation of the subcutaneouly hepatoma in rat with the alteration of immune microenviroment. Subsequently, we investigate the interaction between iHSCs and T lyphocytes or DCs in vitro to explore potenial mechanism of HSC immunosuppression in HCC microenviroment, the results show iHSCs induce T cell apoptosis, but not promote DCs apoptosis. Eventually, we attempt to improve the immune microenviroment in liver by infusion DCs through portal vein to control the development and metastasis of HCC, and provided experimental basis and intervention study in vivo. The aim is to explore the novel immunotherapy for reccurence and metastsis of HCC
Part1Rat hepatic stellate cells alter the gene expression profiles and promote the growth, migration, invation of hepatocellular carcinoma cells in vitro
The aim of this part was to investigate the induction-activated HSCs (iHSC) with tumor-CM alter the gene expression profiles and promote the growth, migration, of hepatocellular carcinoma cells in vitro through the mechanisms including release of paracrine factors. Rat HSCs were isolated from rat livers by perfusion of collagenase and pronase, followed by centrifugation over percoll density gradient. We prepared rat McA-RH7777hepatoma cells conditioned medium (CM) and induced HSCs activation in vitro. The iHSCs were collected and co-cultured with McA-RH7777cells in transwell systems. We compared the gene expression profiles of co-cultured McA-RH7777cells with the control McA-RH7777cells.28,728gene expression was analyzed by cDNA microarray, and confirmed by real time polymerase chain reaction and Western blot analysis. We prepared iHSC CM, and investigated wether iHSC CM promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through the experiments, such as CCK-8(cell count kit-8), scratch repair, transwell invation. The cytokines of supernant in the co-cultured McA-RH7777cells and the control McA-RH7777cells were assayed by ELISA and compared.
Results demonstrated that573genes were differentially expressed in the co-cultured McA-RH7777cells and control McA-RH7777cells with more than2-fold up/down-regulated (among them,432genes were up-regulated and141genes were down-regulated), including cell surface receptors, metabolic process, cell adhesion moleculars, signal transduction molecules, chemokines and immunity factors. By the release of paracrine factors from iHSC, some genes in the co-cultured McA-RH7777cells were up regulated such as Tapbp, Ccl2, Cxcll, Cxcl10, Junb, Igfl, Statl, Irfl, Irf7, Irf9and Csfl, and some genes including4-Sep, Cck, Pdgfra, Itgae, Cd36were specifically down-regulated. iHSC also promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through release of paracrine factors. Compared with the control McA-RH7777cells, some cytokines of supernant were increased in the co-cultured McA-RH7777cells, such as HGF, IL-6, MMP-2, MMP-9. In conclusion, iHSCs can alter the gene expression profiles and promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through the release of paracrine factors. The above suggested that iHSC play a major role in HCC during growth, and metastasis.
Part2Rat hepatic stellate cells promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment in vivo
The aim of this part was to investigate whether HSCs promoted the growth, invasion of hepatocellular carcinoma, involved the evolution of tumor immune microenviroment in vivo. McA-RH7777hepatoma cells (3×106cells) were injected into flank subcutaneously in Buffalo rats. After3weeks, the long diameter of subcutaneous tumor was about2cm, the tumor tissures were cut into1mm3size and implanted into rat liver left lobe (n=25). Hepatic tumor growth was observed by CT scan.
At the time of1,2,3,4weeks after implantation, the rats (n=5) were randomly selected to sacrifice, liver and lung tissue were dyed by HE. Early stage (1week after implantation) and late stage (4week after implantation) hepatoma tissue were detected by a-SMA, CD3, CD68, and S-100immunohistochemical staining. Then positive cells were counted and compared in the early and late stage hepatoma tissue. Experiment of hypodermic tumorigenicity:Buffalo rats were divided into2groups (n=6), and injected subcutaneously with a cell suspension of0.2ml containing either:(A, control group)2×106McA-RH7777cells,(B, exprimental group) a mixture of2×106McA-RH7777cells and1×106induction-activated HSC. The tumor growth and immune microenviroment were detected and compared in the2groups. Experiment of tumorigenicity in liver:Buffalo rats were divided into2groups (n=6), and injected into each rat liver left lobe with a cell suspension of0.2ml containing either:(A, control group)2×106McA-RH7777cells,(B, exprimental group) a mixture of2×106McA-RH7777cells and1×106induction-activated HSC. The tumor growth and lung metastasis were detected and compared in the2groups. Results showed that rat tumor formation rate was100%. The tumors in liver were clearly visible at2week by CT scan, and the lung metastases were detected at4weeks and obviuosly at5weeks by CT scan. The rate of lung metastasis formation was also100%. We observed a-SMA expression within all HCC. Compared with the early stage HCC, the a-SMA content (intratumal HSC, tHSC) and macrophages increased while the dendritic cells (DCs) and T lymphocytes decreased in the late stage HCC. The induction-activated HSCs promote growth and invasion of hypodermic hepatoma in Bufallo rats with more HSCs, macrophages and less DCs, T lymphocytes in the tumor immune microenviroment. The mixture of McA-RH7777cells and induction-activated HSC innoculation in liver didn't promote the gowth or metastasis of hepatoma, compared to the simple McA-RH7777cells. The above showed that the rat orthotope hepatoma model has high rates of tumor formation and lung metastasis. During the progression of hepatoma, the immune microenviroment changes and evolves with infiltrated tHSC increase and DCs, T lymphocytes decrease. Rat HSC promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment in vivo.
Part3Preliminary experimental research on the mechanism by which rat activated hepatic stellate cells regulated the immunity microenviroment in hepatocellular carcinoma
Activated HSC in HCC tissue are associated with immunosuppressive microenviroment and early recurrence. But little were known about HSC immune interaction with immunocytes in HCC. The aim of this part was to investigate iHSC immunosuppressive function in immune microenviroment and mechanism, and whether iHSCs effected T lymphocytes or DCs in vitro.
We prepared rat McA-RH7777hepatoma cells conditioned medium (CM) and induced HSCs activation in vitro. T lymphocytes and DCs were isolated, purified and cultured respectively. The iHSCs were collected and co-cultured with actived-T lymphocytes (aT) or DCs respectively. To assay iHSC inhibition to aT cytotoxic activity by LDH releasing experiment. TUNEL was used to detect T cell apoptosis induced by iHSC. To observe iHSC inhibition to aT and then the influence of tumor invasion ability by application of Transwell method. Flow cytometry was used to detect DC apoptosis induced by iHSC. Experiment of adoptive immunity of DCs in liver:Buffalo rats were divided into2groups (n=6), the rats in control group were implanted into each rat liver left lobe with a1mm3size tumor tissue, the rats in intervention group were infused with DCs loaded with tumor antigen through hepatic vein and then implanted with tumor tissue. The tumor growth, lung metastasis and immunocytes in microenviroment were detected and compared in the2groups. Results showed that activated T cell apoptosis quantity increased significantly by iHSC induction in vitro while DC apoptosis quantity didn't increase. Addition of iHSC induced activated T cells apoptosis, compared to without addition, the invasive ability of McA-RH7777hepatoma cells were significantly enhanced (P<0.05) in Transwell systems. Compared to the control group, tumor weight and lung metastases were decreased and local immune state were improved (P<0.05) in the intervention group which accepted adoptive immunity of DCs. In summary, iHSC promotes HCC invasion and regulates immune microenviroment through the mechanism of which iHSC induced activated T cell apoptosis, not DC apoptosis.Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of hepatoma, and improves local immune state in vivo. The above in vivo study provide feasibility basis for therapy of HCC.
Conclusions
1. Induction activated HSCs can alter the gene expression profiles and promote the growth, migration, invasion of hepatocellular carcinoma cells in vitro through the release of paracrine factors. The above suggested that iHSC play a major role in HCC during growth, and metastasis.
2. The rat orthotope hepatoma model has high rates of tumor formation and lung metastasis. During the progression of hepatoma, the immune microenviroment changes and evolves with infiltrated tHSC increase and DCs, T lymphocytes decrease. Rat HSC promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment. in vivo.
3. Induction activated HSC promotes HCC invasion and regulates immune microenviroment through the mechanism of which iHSC induced activated T cell apoptosis, not DC apoptosis.Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of hepatoma, and improves local immune state in vivo. The above in vivo study provide feasibility basis for therapy of HCC.
Novelty of this study
1For the first time, we confirm that induction activated HSCs can alter the gene expression profiles and promote the growth, migration, invasion of hepatocellular carcinoma cells through the release of paracrine factors.
2To confirm that induction activated HSC regulates HCC immune microenviroment, and iHSC does not enhance DC apoptosis. Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of HCC, and improves local immune state in vivo.
The potential application of this study
1Induction ctivated HSC in HCC microenviroment was a potential biomark of HCC characteristics. This study provided experimental basis for further research the mechanisms of intratumoral HSC immunosuppression in HCC immune microenviroment.
2HSC was a potential treatment target inhibiting HCC recurrence and metastasis, especially for HCC associated with fibrosis/cirrhosis.
以基因组、蛋白组和组织芯片为代表的高通量分子生物学技术的出现和发展,为研究肿瘤发生、进展和转移提供了有力的手段,然而以往对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。肿瘤侵袭与转移是一个多步骤多因素参与的复杂过程,其发生依赖于肿瘤细胞与基质之间的相互作用。间质细胞在肿瘤的发生发展过程中发挥着重要作用,参与调节肿瘤血管生成、结缔组织纤维形成、肿瘤免疫等环节。肝星状细胞(hepatic stellate cell, HSC)也称为Ito细胞,储脂细胞,或窦状隙周(围)细胞,胞浆富含VitA脂滴,HSC约占肝脏细胞总数的13%。HSC位于Diss间隙,具有长的分枝状的细胞突起,通过这些细胞突起包绕肝细胞并与肝窦内皮细胞形成紧密接触,为HSC和肝细胞的相互作用奠定了生理解剖学基础。HSC活化是肝纤维化形成,并最终发展至肝硬化的核心环节。“肝炎-肝纤维化/肝硬化-HCC”被认为是肝癌发生过程中的三部曲。HSC在“炎症-纤维化-肿瘤”这一发生发展过程中充当了重要的介质。近年来研究提示HSC活化在HCC的发生、发展和转移中可能扮演重要角色,通过增强分泌细胞因子、趋化因子、生长因子和ECM蛋白、降低ECM的降解而起作用。而且,在肝癌的间质中伴有活化HSC/MBF(肌成纤维细胞)的浸润,活化的HSC可能通过释放包含生存信号的旁分泌因子促进肝癌形成,机制尚不明。临床证据显示癌周HSC往往和肝癌的早期复发相关。目前为止,普遍认为HSC的基本的非免疫学功能包括维生素A代谢平衡、肝纤维化和调节肝脏血流。更深入的研究发现了HSC的新的功能,HSC具有潜在的免疫学作用。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。
课题组前期实验证实了肝癌相关活化的HSC功能是不同于培养活化的HSC,初步证明瘤内活化HSC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。HCC可诱导HSC活化,而活化的HSC又可促进HCC的发生、发展、浸润和转移,其中活化的HSC对免疫微环境的抑制是可能的作用机制之一,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,有待进一步的研究。
本课题将活化的大鼠HSC与肝癌细胞共培养,首次证实活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,在体外促进肝癌细胞增殖、迁移、侵袭；体内进行HSC与HCC相互作用研究,活化的HSC在大鼠皮下促进肝癌的增殖、侵袭,并伴有免疫微环境的变化；在体外细胞相互作用研究中,HSC促进T细胞凋亡,但未促进DC细胞凋亡；并探索经肝门静脉输注DC细胞改善肝脏局部免疫微环境,为体内肝癌免疫治疗研究、预防肝癌复发转移寻找新的方法和途径。
第一部分大鼠肝星状细胞在体外改变肝癌细胞功能基因表达,促进肝癌细胞增殖、迁移和侵袭
目的：分离并鉴定大鼠肝星状细胞(HSC),在体外活化的HSC可以通过外分泌调控肝癌(HCC)细胞的功能基因表达,促进HCC细胞的增殖、迁移和侵袭。方法：密度梯度离心分离HSC,制备大鼠McA-RH7777肝癌细胞株的条件培养液(CM)培养诱导活化HSC,活化的HSC与McA-RH7777肝癌细胞共培养。应用cDNA微阵列比较了单纯McA-RH7777肝癌细胞、与活化HSC共培养的McA-RH7777肝癌细胞之间28,728基因表达。应用实时RT-PCR和Western blot对芯片结果进行验证。制备活化HSC的CM,通过CCK-8、划痕、Transwell小室等实验,研究活化HSC对肝癌细胞增殖、迁移、侵袭的促进作用。ELISA法检测比较McA-RH7777肝癌细胞、McA-RH7777肝癌细胞+活化HSC共培养两者之间上清中细胞因子的差异。结果：同单纯McA-RH7777肝癌细胞培养相比,和肿瘤诱导活化的HSC(induction-activated HSC, iHSC)共培养的McA-RH7777肝癌细胞多个基因出现显著差异表达,573个基因表达超过2倍上调或下调(其中432个基因为上调,141个基因为下调),包括细胞表面受体、细胞代谢、细胞粘附分子、信号转导通路分子、趋化因子、免疫相关因子等。通过活化HSC旁分泌作用,McA-RH7777肝癌细胞部分基因表达上调,如Tapbp、Ccl2、Cxcll、Cxcl10、Junb、Igf1、Stat1、Irfl、Irf7、Irf9、和Csfl等,部分基因如4-Sep、Cck、Pdgfra、Itgae和Cd36等表达下调。活化HSC在体外通过旁分泌调节促进肝癌细胞增殖、迁移、侵袭。与单纯McA-RH7777肝癌细胞培养相比,McA-RH7777肝癌细胞+活化HSC共培养时,HGF、IL-6、MMP-2、MMP-9等因子升高。结论：活化的HSC与肝癌细胞共培养,活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,促进肝癌细胞增殖、迁移、侵袭,活化的HSC在HCC的生长、侵袭转移中起重要作用。
第二部分大鼠活化肝星状细胞在体内参与改变肿瘤微环境,促进肿瘤生长、侵袭
目的：研究HSC在体内是否参与改变肿瘤免疫微环境,促进肿瘤生长、侵袭。方法：将大鼠肝癌细胞株McA-RH7777种入Buffalo大鼠大腿内侧皮下,约3周后取皮下肿瘤组织,切成1mm3大小植入大鼠肝左叶(n=25),建立Buffalo大鼠肝细胞癌肺转移模型。CT观察肝脏原位移植瘤的生长情况。抽取1、2、3和4周的大鼠(n=5)处死,切取肝癌组织及肺脏。对肝组织和肺组织行HE染色,第1周(早期)及第4周(晚期)肝癌组织行α-SMA、CD3、CD68、S-100免疫组化染色,行阳性细胞计数,对瘤内HSC数和T细胞、巨噬细胞、DC细胞数进行分析比较。皮下成瘤实验：Buffalo大鼠分为2组(每组n=6),对照组皮下注射McA-RH7777细胞(2×106个),实验组皮下注射McA-RH7777细胞(2×106个)+活化HSC(1×106个),比较2组皮下肿瘤的大小、肿瘤免疫微环境。肝内成瘤实验：Buffalo大鼠分为2组(每组n=6),对照组肝左叶内注射McA-RH7777细胞(2×106个),实验组肝左叶内注射McA-RH7777细胞(2×106个)+活化HSC(1×106个),比较2组肝内肿瘤的增殖、肺转移情况。结果：大鼠肝癌原位模型：2周时CT检查可见肿瘤；第4周时CT检出肺转移,5周后检出明显的肺转移病灶,肺转移率100%。大鼠肝癌模型的肝癌组织观察到a-SMA表达,晚期肝癌与早期相比,肿瘤微环境中tHSC (intratumor HSC,瘤内肝星状细胞)、巨噬细胞增加,DC细胞、T细胞减少。皮下成瘤实验：活化HSC促进肝癌皮下肿瘤增殖、侵袭,混合HSC时肿瘤侵犯皮肤,免疫组化提示混合成瘤的肿瘤中存在较多HSC,巨噬细胞增多,DC细胞、T细胞减少。iHSC与肝癌细胞混合后肝内接种没有促进肝癌的增殖、转移。iHSC在体外表现出较强的迁移侵袭能力。结论：大鼠肝癌细胞McA-RH7777的原位肝癌模型中,晚期肝癌组织HSC浸润增加,伴有DC细胞、T淋巴细胞减少,免疫微环境在肿瘤进展过程中发生变化。活化的HSC在体内促进肝癌的增殖、侵袭,并伴有免疫微环境的变化。
第三部分大鼠活化肝星状细胞调控肝癌免疫微环境机制的初步研究
目的：在体外研究HSC对T淋巴细胞、DC细胞的影响,了解HSC对HCC的肿瘤免疫微环境的调控作用及其机制；肝内DC细胞过继免疫改善免疫微环境的作用。方法：使用McA-RH7777肝癌细胞的条件培养基诱导活化新分离的大鼠HSC,分离培养T细胞、DC细胞,iHSC分别与DC细胞、T细胞共培养。LDH释放实验观察iHSC对T细胞的杀伤活性的影响,Tunel法检测T细胞的凋亡,应用Transwell法观察iHSC抑制T细胞对肿瘤侵袭能力的影响,流式细胞分析iHSC对DC细胞诱导凋亡的作用。肝内DC细胞过继免疫实验：Buffalo大鼠分为2组(每组n=6),对照组在肝左叶原位移植瘤组织,干预组经门静脉注射负载肿瘤抗原的DC细胞后再原位移植肝癌,与对照组比较肝内肿瘤的增殖、转移、微环境中免疫细胞。结果：iHSC在体外诱导活化T细胞凋亡,而诱导DC细胞凋亡不明显,iHSC在Transwell小室中诱导T细胞凋亡从而促进肝癌细胞侵袭。经门静脉注射负载肿瘤抗原的DC细胞可以抑制肝癌的增殖和转移,改善免疫微环境。结论：iHSC促进肝癌细胞的侵袭,对HCC的肿瘤免疫微环境的有调控作用,机制可能为促进T细胞凋亡,但未促进DC细胞凋亡。经肝门静脉输注DC细胞可以改善肝脏免疫微环境,抑制肿瘤增殖、转移。
结论
1.活化的HSC与肝癌细胞共培养,活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,促进肝癌细胞增殖、迁移、侵袭,活化的HSC在HCC的生长、侵袭转移中起重要作用。
2.大鼠肝癌细胞McA-RH7777的原位肝癌模型中,晚期肝癌组织HSC浸润增加,伴有DC细胞、T淋巴细胞减少,免疫微环境在肿瘤进展过程中发生变化。活化的HSC在体内促进肝癌的增殖、侵袭,并伴有免疫微环境的变化。
3.肿瘤诱导活化的HSC促进肝癌细胞的侵袭,对HCC的肿瘤免疫微环境的有调控作用,机制可能为促进T细胞凋亡,但未促进DC细胞凋亡；经肝门静脉输注DC细胞可以改善肝脏免疫微环境,抑制肿瘤增殖、转移。创新点
1.首次证实活化HSC通过旁分泌改变McA-RH7777肝癌细胞多个基因的表达,促进肝癌细胞增殖、迁移、侵袭,参与其中的基因和因子是多个的、非单一独特的。
2.首次证实肿瘤诱导活化的HSC对HCC的免疫微环境的调控作用,未促进DC细胞凋亡；经肝门静脉输注DC细胞可以改善肝脏免疫微环境,抑制肿瘤增殖、转移。潜在应用价值
1.肝癌微环境中的活化HSC是反应肝癌生物学特性的指标之一,为深入研究瘤内HSC在肝癌免疫微环境中的免疫抑制机制提供实验基础。
2.HSC是抑制肝癌复发转移的潜在治疗靶点,对于伴有肝硬化背景的肝癌针对HSC的治疗意义更加重大。
Hepatocellular carcinoma (HCC) is one of the most common cancer in the world, and the third leading cause of cancer-related death worldwide, accounting for at least610,000deaths per year, nearly half of them in China. Despite tremendous achievements of cellular and molecular biology in HCC have been made during past decades, the prognosis of HCC remains dismal. Although surgery achieves the best outcome in well-selected candidates, the5-year recurrence rate remains as high as60-70%after HCC resection. At the same time, about80%of HCC cases are associated with chronic hepatitis (type B or C hepatitis) and cirrhosis in China, and most of them lost the chance to accept the currative operation. Although the systematic therapies including chemotherapy and targeted therapy were investigated deeply and applied widespread in the patients with late stage, the tumors always progress after several months treatment remains a major obstacle for more therapeutic efficacy.
The novel high throughput analysis technologies, such as genomics, protemics and tissue microarray, greatly facilitate the studies on the progression and metastasis of tumor. However, none of the findings has turned into a breakthrough in the prevention and treatment for HCC. The vast majority of previous studies focused solely on malignant cells themselves, and ignored the important role of stromal cells in the malignant transformation. The invason and metastasis of HCC are complicated procedures with multi-step and multi-factor, and also depened on the interaction between malignant cells and stromal cells. 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 initiation and development of cancer. HSC, also referred as Ito cells, lipocytes, or perisinusoidal cells, comprise13%of the total number of liver cells. They localized in Disse and characterized by their long processes by which they closely embrace hepatocytes and contact with sinusoidal endothelial. The activation of hepatic stellate cells (HSC) is recognized as a central event in the development of hepatic fibrosis and lastly, cirrhosis. A three-step process of "inflammation-fibrosis/cirrhosis-HCC" is believed to be involved in hepatocarcinogenesis, the activation of hepatic stellate cells (HSCs) may serve as an important mediator in the process of inflammation-fibrosis-carcinoma axis, by increasing secretion of cytokines, chemokines, growth factors, and extracellular matrix proteins and decreasing degradation of extracellular matrix. Moreover, in HCC the stroma is infiltrated by activated HSC/myofibroblasts. Activated HSCs release paracrine factors that may promote hepatocarcinogenesis through unknown mechanisms including release of survival signals. Clinical evidences disclosed that peritumoral HSCs were related to more early HCC recurrences.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. Recently, studies suggested that it is possible that HSC play a great role in the progression of HCC, even in tumor metastasis, but the related mechanism remains unclear.
Our research group confirmed that: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. Intratumoral HSC inhibit activated T lymphocyte function mainly through promoting T cell apoptosis, and then promote tumor cells and movement ability in vitro.
In this study, we take induction-activated HSCs (iHSC) and McA-RH-7777hepatoma cells to co-culture, and confirm that iHSC alter expression genes and promote the growth and invation of McA-RH-7777hepatoma cells in vitro through the mechanism of paracrine secretion. iHSCs also enhance growth and invation of the subcutaneouly hepatoma in rat with the alteration of immune microenviroment. Subsequently, we investigate the interaction between iHSCs and T lyphocytes or DCs in vitro to explore potenial mechanism of HSC immunosuppression in HCC microenviroment, the results show iHSCs induce T cell apoptosis, but not promote DCs apoptosis. Eventually, we attempt to improve the immune microenviroment in liver by infusion DCs through portal vein to control the development and metastasis of HCC, and provided experimental basis and intervention study in vivo. The aim is to explore the novel immunotherapy for reccurence and metastsis of HCC
Part1Rat hepatic stellate cells alter the gene expression profiles and promote the growth, migration, invation of hepatocellular carcinoma cells in vitro
The aim of this part was to investigate the induction-activated HSCs (iHSC) with tumor-CM alter the gene expression profiles and promote the growth, migration, of hepatocellular carcinoma cells in vitro through the mechanisms including release of paracrine factors. Rat HSCs were isolated from rat livers by perfusion of collagenase and pronase, followed by centrifugation over percoll density gradient. We prepared rat McA-RH7777hepatoma cells conditioned medium (CM) and induced HSCs activation in vitro. The iHSCs were collected and co-cultured with McA-RH7777cells in transwell systems. We compared the gene expression profiles of co-cultured McA-RH7777cells with the control McA-RH7777cells.28,728gene expression was analyzed by cDNA microarray, and confirmed by real time polymerase chain reaction and Western blot analysis. We prepared iHSC CM, and investigated wether iHSC CM promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through the experiments, such as CCK-8(cell count kit-8), scratch repair, transwell invation. The cytokines of supernant in the co-cultured McA-RH7777cells and the control McA-RH7777cells were assayed by ELISA and compared.
Results demonstrated that573genes were differentially expressed in the co-cultured McA-RH7777cells and control McA-RH7777cells with more than2-fold up/down-regulated (among them,432genes were up-regulated and141genes were down-regulated), including cell surface receptors, metabolic process, cell adhesion moleculars, signal transduction molecules, chemokines and immunity factors. By the release of paracrine factors from iHSC, some genes in the co-cultured McA-RH7777cells were up regulated such as Tapbp, Ccl2, Cxcll, Cxcl10, Junb, Igfl, Statl, Irfl, Irf7, Irf9and Csfl, and some genes including4-Sep, Cck, Pdgfra, Itgae, Cd36were specifically down-regulated. iHSC also promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through release of paracrine factors. Compared with the control McA-RH7777cells, some cytokines of supernant were increased in the co-cultured McA-RH7777cells, such as HGF, IL-6, MMP-2, MMP-9. In conclusion, iHSCs can alter the gene expression profiles and promote the growth, migration, invation of hepatocellular carcinoma cells in vitro through the release of paracrine factors. The above suggested that iHSC play a major role in HCC during growth, and metastasis.
Part2Rat hepatic stellate cells promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment in vivo
The aim of this part was to investigate whether HSCs promoted the growth, invasion of hepatocellular carcinoma, involved the evolution of tumor immune microenviroment in vivo. McA-RH7777hepatoma cells (3×106cells) were injected into flank subcutaneously in Buffalo rats. After3weeks, the long diameter of subcutaneous tumor was about2cm, the tumor tissures were cut into1mm3size and implanted into rat liver left lobe (n=25). Hepatic tumor growth was observed by CT scan.
At the time of1,2,3,4weeks after implantation, the rats (n=5) were randomly selected to sacrifice, liver and lung tissue were dyed by HE. Early stage (1week after implantation) and late stage (4week after implantation) hepatoma tissue were detected by a-SMA, CD3, CD68, and S-100immunohistochemical staining. Then positive cells were counted and compared in the early and late stage hepatoma tissue. Experiment of hypodermic tumorigenicity:Buffalo rats were divided into2groups (n=6), and injected subcutaneously with a cell suspension of0.2ml containing either:(A, control group)2×106McA-RH7777cells,(B, exprimental group) a mixture of2×106McA-RH7777cells and1×106induction-activated HSC. The tumor growth and immune microenviroment were detected and compared in the2groups. Experiment of tumorigenicity in liver:Buffalo rats were divided into2groups (n=6), and injected into each rat liver left lobe with a cell suspension of0.2ml containing either:(A, control group)2×106McA-RH7777cells,(B, exprimental group) a mixture of2×106McA-RH7777cells and1×106induction-activated HSC. The tumor growth and lung metastasis were detected and compared in the2groups. Results showed that rat tumor formation rate was100%. The tumors in liver were clearly visible at2week by CT scan, and the lung metastases were detected at4weeks and obviuosly at5weeks by CT scan. The rate of lung metastasis formation was also100%. We observed a-SMA expression within all HCC. Compared with the early stage HCC, the a-SMA content (intratumal HSC, tHSC) and macrophages increased while the dendritic cells (DCs) and T lymphocytes decreased in the late stage HCC. The induction-activated HSCs promote growth and invasion of hypodermic hepatoma in Bufallo rats with more HSCs, macrophages and less DCs, T lymphocytes in the tumor immune microenviroment. The mixture of McA-RH7777cells and induction-activated HSC innoculation in liver didn't promote the gowth or metastasis of hepatoma, compared to the simple McA-RH7777cells. The above showed that the rat orthotope hepatoma model has high rates of tumor formation and lung metastasis. During the progression of hepatoma, the immune microenviroment changes and evolves with infiltrated tHSC increase and DCs, T lymphocytes decrease. Rat HSC promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment in vivo.
Part3Preliminary experimental research on the mechanism by which rat activated hepatic stellate cells regulated the immunity microenviroment in hepatocellular carcinoma
Activated HSC in HCC tissue are associated with immunosuppressive microenviroment and early recurrence. But little were known about HSC immune interaction with immunocytes in HCC. The aim of this part was to investigate iHSC immunosuppressive function in immune microenviroment and mechanism, and whether iHSCs effected T lymphocytes or DCs in vitro.
We prepared rat McA-RH7777hepatoma cells conditioned medium (CM) and induced HSCs activation in vitro. T lymphocytes and DCs were isolated, purified and cultured respectively. The iHSCs were collected and co-cultured with actived-T lymphocytes (aT) or DCs respectively. To assay iHSC inhibition to aT cytotoxic activity by LDH releasing experiment. TUNEL was used to detect T cell apoptosis induced by iHSC. To observe iHSC inhibition to aT and then the influence of tumor invasion ability by application of Transwell method. Flow cytometry was used to detect DC apoptosis induced by iHSC. Experiment of adoptive immunity of DCs in liver:Buffalo rats were divided into2groups (n=6), the rats in control group were implanted into each rat liver left lobe with a1mm3size tumor tissue, the rats in intervention group were infused with DCs loaded with tumor antigen through hepatic vein and then implanted with tumor tissue. The tumor growth, lung metastasis and immunocytes in microenviroment were detected and compared in the2groups. Results showed that activated T cell apoptosis quantity increased significantly by iHSC induction in vitro while DC apoptosis quantity didn't increase. Addition of iHSC induced activated T cells apoptosis, compared to without addition, the invasive ability of McA-RH7777hepatoma cells were significantly enhanced (P<0.05) in Transwell systems. Compared to the control group, tumor weight and lung metastases were decreased and local immune state were improved (P<0.05) in the intervention group which accepted adoptive immunity of DCs. In summary, iHSC promotes HCC invasion and regulates immune microenviroment through the mechanism of which iHSC induced activated T cell apoptosis, not DC apoptosis.Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of hepatoma, and improves local immune state in vivo. The above in vivo study provide feasibility basis for therapy of HCC.
Conclusions
1. Induction activated HSCs can alter the gene expression profiles and promote the growth, migration, invasion of hepatocellular carcinoma cells in vitro through the release of paracrine factors. The above suggested that iHSC play a major role in HCC during growth, and metastasis.
2. The rat orthotope hepatoma model has high rates of tumor formation and lung metastasis. During the progression of hepatoma, the immune microenviroment changes and evolves with infiltrated tHSC increase and DCs, T lymphocytes decrease. Rat HSC promote growth and invasion of hepatocellular carcinoma and alter the tumor immune microenviroment. in vivo.
3. Induction activated HSC promotes HCC invasion and regulates immune microenviroment through the mechanism of which iHSC induced activated T cell apoptosis, not DC apoptosis.Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of hepatoma, and improves local immune state in vivo. The above in vivo study provide feasibility basis for therapy of HCC.
Novelty of this study
1For the first time, we confirm that induction activated HSCs can alter the gene expression profiles and promote the growth, migration, invasion of hepatocellular carcinoma cells through the release of paracrine factors.
2To confirm that induction activated HSC regulates HCC immune microenviroment, and iHSC does not enhance DC apoptosis. Adoptive immunity of DCs through hepatic vein could inhibit proliferation and metastasis of HCC, and improves local immune state in vivo.
The potential application of this study
1Induction ctivated HSC in HCC microenviroment was a potential biomark of HCC characteristics. This study provided experimental basis for further research the mechanisms of intratumoral HSC immunosuppression in HCC immune microenviroment.
2HSC was a potential treatment target inhibiting HCC recurrence and metastasis, especially for HCC associated with fibrosis/cirrhosis.
引文
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[14]Ooi LL, Bay BH, Ng RT. An animal model for the study of hepatic stellate cells and hepatocellular carcinoma interaction. Ann Acad Med Singapore,1999,28(1):95-98.
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[17]王伟,官阳,杨木兰,等.活化肝星状细胞与肝细胞肝癌发生及转移的相关性.临床与实验病理学杂志,2007,23(4)：492-494.
[18]吕丽,王莉芬,王丽丽,等.肝星状细胞、肝细胞生长因子与肝癌相关性研究.肝脏,2005,10(3)：213-214.
[19]Chan Y, Lily MA, Wu PC, et al. Myofibroblasts in hepatitis B related cirrhosis and hepatoellular carcinoma. J Clin Pathol,1992,45(5):446-448.
[20]Ooi LP, Crawford DH, Gotley DC, et al. Evidence that myofibroblast-like cells are the cellular source of capsular collagen in hepatocelluar carcinoma. J Hepatol, 1997,26(4):798-807.
[21]Bridle KR, Crawford DH, Powell LW, et al. Role of myofibroblasts in tumor encapsulation of hepatocelluar carcinoma in haemochromatosis. Liver,2001,21(2): 96-104.
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