摘要
肝细胞肝癌(hepatocellular carcinoma,HCC)是肝癌中最常见的组织学类型。在我国70%～90%HCC患者伴有肝硬化。在肝癌发病率呈上升趋势的欧美国家,HCC的发生几乎全部源于肝硬化的个体。而肝硬化严重影响了HCC治疗方法的选择、疗效、并发症及预后。目前认为,HCC合并肝硬化的发病机制基于两个方面[1]:1)在损伤因子(如:肝炎病毒、黄曲霉素、代谢病和酒精等)存在下,肝细胞持续性受损、变性和坏死所致的炎性反应以及细胞外基质(extracellular matrix,ECM)合成过多,ECM不能被降解、吸收,从而导致在肝内大量沉积,形成肝纤维化,最终则引起以纤维组织环绕增生肝细胞团的硬化结节,即肝硬化。在这一过程中,肝星状细胞(hepatic stellate cell,HSC)由静止状态活化为成肌纤维细胞,大量分泌ECM,是肝纤维化发生乃至肝硬化进展的主要细胞学基础。2)在肝纤维化炎性环境中产生的氧自由基等诱变物质引起肝细胞DNA损伤,损伤的肝细胞应答旁分泌细胞因子的刺激而增殖,这种不断的损伤、再生诱发的多基因变异是导致肝细胞癌变的关键。
HSC活化是肝纤维化发生的主要原因,肝纤维化是肝癌前病变的重要病理阶段。免疫组化研究显示,HCC合并肝硬化中活化的HSC数量显著增高,而肝癌细胞分泌的有丝分裂因子也具有促进HSC活化并增殖的作用[2];活化HSC和癌变细胞的相互作用会进一步加重肝癌合并肝硬化程度。因此着眼于活化HSC的靶向治疗是抑制肝纤维化,改善肝癌状况的一个新的研究热点。目前,抑制肝纤维化的途径主要集中在:阻断转化生长因子β(transforming growth factorβ,TGF-β)信号通路,以拮抗TGF-β在HSC活化中的作用[3,4];减少细胞外基质ECM的生成,降解组织中过量沉积的胶原纤维[5,6]。
新城疫病毒(Newcastle disease virus, NDV)是单股负链RNA病毒,属副粘病毒科。由于多数RNA病毒具有天然地在肿瘤细胞选择性复制的特点,近年来用这类病毒作为新型运载工具在肿瘤基因治疗方面引起了人们的关注。另一方面,NDV作为溶瘤病毒用于肿瘤治疗已见临床报道。NDV在肿瘤细胞选择性复制的机制和肿瘤细胞的干扰素信号通路缺陷有关[7]。1976年,McGregor报道了NDV能够在活化的T细胞中复制,并导致活化T细胞的死亡[8];Fábián最近又报道了NDV能够在转化的细胞中高效地复制[9]。基于活化HSC是一种非正常的、具有增殖能力的细胞,我们提出假设:NDV能在这种活化的HSC中复制,并抑制HSC的活化,进而达到逆转肝纤维化的目的。本论文以抑制活化HSC为出发点,探讨NDV在HSC中的复制率及其对肝纤维化相关基因在mRNA和蛋白水平的影响,并通过建立小鼠肝纤维化模型,研究NDV的体内逆转肝纤维化的功能。
第一部分:NDV在人肝癌细胞条件培养基诱导活化HSC中的复制
目的:探讨人肝癌细胞FHCC-98条件培养基(conditioned medium, CM)对人肝星状细胞LX-2活化的诱导作用;检测NDV在LX-2细胞中的复制率。方法:用不同百分含量的FHCC-98 CM刺激LX-2细胞,MTT法检测细胞的增殖率,TGF-β1为阳性对照。半定量及Real-time定量RT-PCR检测40% CM(V/V)或2 ng/ml TGF-β1刺激前后LX-2细胞中α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原(collagenⅠ)、金属蛋白酶组织抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)和TGF-β1四种活化相关基因的mRNA表达。用携带增强型绿色荧光蛋白(enhanced greenfluorescent protein, EGFP)的重组NDV(NDFLtag-EGFP)感染不同传代的LX-2细胞,荧光显微镜下观察EGFP在细胞中的表达率和荧光强度,以反映NDV的复制率。用流式细胞术(flow cytometry, FACS)定量检测NDFLtag-EGFP在40% CM或2 ng/ml TGF-β1刺激活化的LX-2细胞中的复制。结果:FHCC-98 CM和TGF-β1均能够刺激LX-2细胞的活化。低浓度的CM(10%~40%)更有利于LX-2细胞的增殖,40% CM刺激时的细胞增殖率最大,为25.8%。TGF-β1对LX-2细胞增殖的影响呈正相关,且具有剂量依赖性。与无刺激的对照组相比,CM或TGF-β1刺激LX-2细胞活化后,细胞中α-SMA、collagenⅠ、TIMP-1和TGF-β1四种基因的表达均有所增加。NDV随着LX-2细胞的连续传代,其在细胞中的复制率也逐渐增加。FACS显示,LX-2细胞分别经40% CM和2 ng/ml的TGF-β1刺激活化后,NDV在细胞中的复制率分别提高了1.76倍和1.52倍。结论:FHCC-98 CM能诱导LX-2细胞活化并增殖,NDV在活化LX-2细胞中的复制率提高。
第二部分:NDV对HSC活化的抑制作用
目的:探讨NDV在活化HSC中的复制对HSC的增殖以及细胞生物学功能的影响。方法:用不同滴度的NDV溶瘤株Italien(NDV-Italien)感染经CM刺激的LX-2细胞,MTT法检测NDV对细胞增殖率的影响。免疫荧光显微镜技术检测活化标志物α-SMA的表达变化。实时定量RT-PCR检测NDV对四种活化相关基因α-SMA、collagenⅠ、TIMP-1和TGF-β1 mRNA水平的影响。明胶酶谱法检测MMP-2和MMP-9的分泌。结果:NDV的感染抑制了LX-2细胞的增殖,细胞增殖率和病毒滴度呈负相关关系。与未刺激组相比,NDV在CM刺激的LX-2细胞中的增殖抑制率提高,四种基因的表达显著性下调,MMP-2和MMP-9的分泌下调。结论:NDV在活化LX-2细胞中的复制具有抑制细胞增殖和HSC活化的作用。
第三部分:NDV对CCl4诱导的小鼠肝纤维化的逆转
目的:通过体内试验探讨NDV对CCl4诱导的小鼠肝纤维化的抑制作用。方法:体重约20 g的昆明小鼠,每周两次腹腔注射100μl CCl4/花生油溶液(20%, V/V),对照组同时注射100μl生理盐水(PS),连续注射8周。最后一次在CCl4注射后3天,尾静脉注射200μl滴度为1,000 HU(hemagglutination unit)的NDV-Italien 1次或每24 h间隔连续注射3次。NDV注射24h后处死动物,取出肝脏进行大体形态观察并拍照。肝组织用福尔马林固定,石蜡包埋,组织切片进行常规HE染色和Masson三色染色。新鲜肝组织用RIPA(non-ionic detergent-containing buffer)裂解液裂解,BCA(bicinchoninic acid)蛋白定量试剂盒测定总蛋白浓度,取50μg总蛋白上样进行Western blot检测α-SMA的表达。新鲜肝组织进行冰冻切片,冷丙酮固定,免疫荧光双染法检测组织中α-SMA和NDV颗粒的表达和定位。结果:CCl4诱导8周后,小鼠肝脏出现明显的纤维化症状,可看到肝组织变硬、表面粗糙不平、密集分布大量的白色点状斑块。HE染色显示,纤维化肝脏的组织结构松散,窦周隙增大。Masson三色染色显示胶原异常沉积。而NDV注射3次后,小鼠肝脏表面的白色斑点显著减少,胶原沉积降低。Western blot分析表明,α-SMA蛋白水平随NDV注射次数增加而降低。免疫荧光双染显示,α-SMA和NDV均存在于肝组织的窦周隙,共定位在活化HSC中,而正常小鼠的肝脏组织未见NDV的特异性吸附。结论:在CCl4诱导的小鼠肝纤维化中,NDV可以选择性地作用于活化的HSC中,从而抑制肝纤维化。
Hepatocellular carcinoma (HCC) is the most common histological type of liver cancer. About seventy to ninety percent of HCC patients are accompanied with liver cirrhosis in China. In Western countries which have a increasing occurrence tendency of HCC in recent years, the liver carcinogenesis is almost derived from the liver cirrhosis, influencing the therapeutic choice, therapeutic effect, complications, and prognosis for the treatment of HCC. The mechanisms of HCC development in cirrhosis are based on two aspects: 1) under the exist of some detriment factors, such as hepatitis virus, flavacin, metabolism disease, and alcohol, hepatocytes are damaged persistently, resulting in liver cell degeneration and necrosis followed by excess synthesis of extracellular matrix (ECM) induced by inflammatory reaction. ECM cannot be degraded and deposited in hepatic tissue, which contributs to the liver fibrogenesis finally cirrhosis characterized by nodular regeneration which is surrounded by new formed fibrous tissue. Hepatic stellate cells (HSC) transform from quiescent to activated state and transdifferentiates into myofibroblasts which secrete excessive ECM, playing an important role in the development of hepatic fibrosis and cirrhosis. 2) Reactive oxygen species are generated under the inflammatory circumstance which then induces DNA damage in hepatocytes. The damaged hepatocytes are proliferated in response to the cytokines secreted via paracrine. The alternation between damage and regeneration induces polygenic mutations initiating carcinogenesis. Liver fibrogenesis caused by HSC activation which is an important pathologic stage during the development of HCC. Immunohistochemistry shows that a number of activated HSCs exits in hepatic tissue from HCC complicating cirrhosis. While mitogenic factors secreted by HCC cells promote the activation and proliferation of HSC. The interaction between activated HSC and malignant cells aggravate the HCC development. Targeted therapy focusing on the activated HSC is noval strategy for repression of liver fibrosis. There are two methods to repress the activation of HSC: blocking transforming growth factorβ(TGF-β) signal pathway to antagonize the function of TGF-βon activation of HSC; decreasing the synthesis of ECM and degrading the excessively deposited collagen in liver tissue.
Newcastle disease virus (NDV) contains nonsegmented single-stranded negative-sense RNA and is a member of the family Paramyxoviridae. Because most RNA virus have the characteristics of selective replication in tumor cells, NDV is interesting to be a new viral carrier in tumor gene therapy in recent years. In the other hand, NDV is applied clinically in tumor therapy due to the oncolytic properties. The mechanism of the NDV replication in tumor cells is based on the defective interferon signal pathway in these tansformed cells. In 1976, McGregor reported NDV replicated in activated T cells and induced the cell death. Recently, Fábián confirmed that NDV can replicate in abnormal transformed cells effectively. Activated HSC is also a kind of“abnormal”and is capable of proliferation, we presumed that NDV can replicate in these cells followed by repression of the activation of cells and reversion of hepatic fibrosis. In this dissertation my intention is focusing on the repression of HSC activation by an oncolytic strain NDV-Italien, investigation of the NDV replication in activated HSC and its effects on decrease of the levels of mRNA and protein in those genes which are related to hepatic fibrosis. We evaluated the repression of liver fibrosis by NDV in vivo through mouse hepatic fibrosis model.
PartⅠNDV replication in the activated HSC induced with human hepatocellular carcinoma conditioned medium
Aim: To explore the function of conditioned medium (CM) from human HCC cell line FHCC-98 on the activation of human hepatic stellate cell line LX-2 and to investigate the replication efficiency of NDV in LX-2 cells. Methods: LX-2 cells were stimulated with different concentrated CM from FHCC-98 cells and the proliferation of LX-2 cells after stimulation was detected by MTT assay in which TGF-β1 was used as positive control to evaluate the role of CM. Semi-quantitative and Real-time quantitative RT-PCR was used to measure the changes of mRNA levels ofα-SMA, collagenⅠ, TIMP-1, and TGF-β1 which are related with HSC activation in LX-2 cells after stimulation by 40% CM or TGF-β1. After infected by NDFLtag-EGFP, a recombinant NDV carring enhanced green fluorescent protein (EGFP), virus replication in LX-2 cells allows the expression of EGFP which can be observed under fluroscent microscopy and quantitated by flow cytometry (FACS). Results: Either FHCC-98 CM or TGF-β1 can stimulate the activation of LX-2 cells. Low concentrations of CM are beneficial to the cell proliferation than the high ones. The maximum proliferation rate is 25.8% with 40% CM stimulation. TGF-β1 induced the proliferation of LX-2 cells in a dose-dependent way. After stimulated by 40% CM or TGF-β1, the mRNA expression levels ofα-SMA, collagenⅠ, TIMP-1, and TGF-β1 were up-regulated comparing with the unstimulated control group. The efficiency of NDV replication was increased in the consecutive passages of LX-2 cells, FACS analysis showed that the efficiency of NDV replication in 40% CM and TGF-β1 stimulated cells were enhanced to 1.76 and 1.52 folds compared with the controls. Conclusion: CM from FHCC-98 cells induced the proliferation and activation of LX-2 cells, the replication efficiency of NDV was increased in this activated LX-2 cells.
PartⅡRepression of HSC activation by NDV
Aim: To explore the influence of NDV replication in LX-2 cells on the cell viability and behavior of cellular biology. Methods: CM-stimulated LX-2 cells were infected by different titres of oncolytic NDV-Italien, the proliferation rate of LX-2 cells was detected by MTT assay. Immunofluorescent technique was used to detect the expression ofα-SMA in LX-2 cells. Real-time quantitative RT-PCR was used to evaluate the expression ofα-SMA, collagenⅠ, TIMP-1, and TGF-β1 in the activated LX-2 cells after NDV infection. Secretion of MMPs in LX-2 cells was determined by gelatin zymography. Results: NDV infection in LX-2 cells repressed the cell proliferation in a dose-dependent manner. Compared with control, the growth inhibition rate of activated LX-2 cells was increased. The mRNA expression of four genes,α-SMA, collagenⅠ, TIMP-1, and TGF-β1 in activated LX-2 cells were down-regulated and the secretion of MMP-2 and MMP-9 was decreased after NDV infection. Conclusion: The replication of NDV in activated LX-2 cells repressed the cell proliferation and attenuated the activation of LX-2 cells.
PartⅢReversion of hepatic fibrosis by NDV in mice induced by CCl4
Aim: To explore the influence of NDV treatment on the reversion of hepatic fibrosis in mice induced by CCl4 in vivo. Methods: Kunming mice (weight about 20 g) were injected intraperitoneally (i.p.) 100μl carbon tetrachloride (CCl4)/peanut oil solution (20%, V/V) twice a week, the mice were given 100μl physiological saline (PS) replacing CCl4 as PS group for 8 weeks. Each mouse was treated with 1,000 hemagglutination unit (HU) NDV-Italien in 200μl volume via tail vein for one time or 3 times with 24 hour-interval after the final infusion of CCl4. The mice were sacrificed at 24 h-post injection of NDV and liver tissues were excised for grossly examination with naked eye and taking photograph. Tissue samples were fixed in 10% formalin, then embedded in paraffin, the sections were stained by HE and masson’s trichrome methods. Fresh tissue samples were lysed in RIPA lysis buffers and protein contents was determined by BCA assay. Fifty microgram total protein for each sample was subjected to 10% SDS-PAGE electrophoresis and detected the expression ofα-SMA by western blot analysis. Cryostat sections of liver tissues were fixed with cold acetone and followed by observision of NDV virions andα-SMA expression under fluorescent microscopy with double staining technique. Results: After 8 weeks of CCl4 induction, the mouse liver showed fibrosis symptom severely. Hepatic tissues become hard, coarse and there were large amounts of white plaques on liver surface. HE staining showed that tissue structure in fibrosis liver was loose and perisinusoidal space is dilated. Masson’s trichrome staining revealed that collagen in fibrosis tissue was deposited abnormally. After three times injection of NDV, white plaques on liver surface were dispelled dramatically and deposition of collagen in liver tissue was suppressed. Immunofluorescence microscope showed thatα-SMA and NDV were detected in the perisinusoidal spaces and co-located in activated HSCs, while no specific adsorption was visualized in liver tissues of normal control mice. Conclusion: In CCl4-induced mouse hepatic fibrosis, NDV repressed the activation of HSC and reversed fibrosis of liver through its selective replication in the activated HSC.
引文
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