新城疫病毒抑制肝星状细胞的活化及其逆转小鼠肝纤维化的研究

发布时间：2018-01-09 11:18

本文关键词：新城疫病毒抑制肝星状细胞的活化及其逆转小鼠肝纤维化的研究　出处：《第四军医大学》2008年博士论文　论文类型：学位论文

【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。
[Abstract]:Hepatocellular carcinoma (hepatocellular, carcinoma, HCC) is the most common histological type of liver cancer in China. 70% ~ 90%HCC in patients with cirrhosis. The incidence of liver cancer is on the rise of the United States and Europe, individual HCC occur almost exclusively derived from the liver cirrhosis. The curative effect and the serious influence of HCC treatment method selection., complications and prognosis. At present, the pathogenesis of liver cirrhosis complicated with HCC [1] based on two aspects: 1) in damage factors (such as hepatitis, aflatoxin, metabolic disease and alcohol) the presence of persistent liver cell damage, inflammatory reaction and extracellular matrix caused by degeneration and necrosis (extracellular matrix, ECM) synthesis over ECM cannot be degraded, absorbed, resulting in a large number of deposition in the liver, hepatic fibrosis, and ultimately caused the sclerotic nodules, with fibrous tissue around the liver cell proliferation group is a in cirrhosis. In the process of hepatic stellate cells (hepatic stellate cell, HSC) from static state to myofibroblast activation, secretion of ECM,.2 is the main cellular basis of the progression of liver fibrosis and cirrhosis) oxygen free radical induced substance produced in liver fibrosis, inflammatory environment caused by damage to the liver cells of liver DNA. The cellular response to injury paracrine cytokine stimulation and proliferation, this constant injury, multi gene mutation induced regeneration is a key cause of liver cancer.
HSC activation is a major cause of liver fibrosis, liver fibrosis is an important pathological stage of hepatic precancerous lesion. Immunohistochemical study showed that the number of HSC activated HCC was significantly higher in patients with cirrhosis, and liver cancer cells secrete mitogenic factors can promote the activation of HSC and effect of [2] proliferation and activation of HSC and interaction; malignant cells aggravate liver cancer with cirrhosis. It focuses on the activation of HSC targeted therapy is to inhibit liver fibrosis, a new research hotspot to improve liver cancer. At present, inhibition of hepatic fibrosis mainly in: inhibition of transforming growth factor beta (transforming beta growth factor, TGF- beta) signaling pathway. The antagonist of TGF- beta in the role of HSC in the activation of [3,4]; reduce the formation of extracellular matrix ECM, collagen deposition in tissues of [5,6]. excessive degradation
Newcastle disease virus (Newcastle disease, virus, NDV) is a single stranded RNA virus, belonging to Paramyxoviridae. Because most RNA virus has the natural characteristics of selective replication in tumor cells, in recent years, with the virus as a new carrier in gene therapy of tumor has aroused people's attention. On the other hand NDV, as an oncolytic virus for cancer treatment has been reported in clinical.NDV mechanism of tumor selective replication and tumor cell interferon signaling pathway related to defects in [7].1976, McGregor reported NDV in activated T cells of complex system, and leads to the activation of T cells, Bi F death [8]; n recently reported NDV in transformed cells efficiently based on replication of [9]. activated HSC is a non normal, with the proliferation of cells, we hypothesized that NDV can replicate in the activation of HSC and inhibition of HSC activation, In order to reverse liver fibrosis. The inhibition of activated HSC as the starting point of NDV in HSC replication rate and its effect on liver fibrosis related genes in mRNA and protein levels, and through the establishment of a mouse model of hepatic fibrosis in vivo, reverse liver fiber research of NDV function.
Part one: replication of NDV in activated HSC induced by human hepatoma cell conditioned medium
Objective: To investigate the human hepatoma FHCC-98 cells conditioned medium (conditioned medium CM) to induce human hepatic stellate cells activated by LX-2; detection of NDV replication in LX-2 cells. Methods: using different percentages of FHCC-98 CM stimulation of LX-2 cells, the proliferation rate of cells was detected by MTT, TGF- beta 1 as a positive control. Semi quantitative Real-time and quantitative RT-PCR detection 40% CM (V/V) or ng/ml TGF- 1 beta 2 before and after stimulation of LX-2 cells in alpha smooth muscle actin (-smooth muscle alpha actin, alpha -SMA), collagen type I (collagen I), tissue inhibitor of metalloproteinase -1 (tissue inhibitor of metalloproteinase-1, TIMP-1) and the expression of TGF- 1 beta four activation related gene mRNA. By carrying enhanced green fluorescent protein (enhanced greenfluorescent, protein, EGFP) of the recombinant NDV (NDFLtag-EGFP) infection in different passages of LX-2 cells, the observation of EGFP in cells under fluorescent microscope The expression rate and intensity, to reflect the NDV replication rate. Using flow cytometry (flow cytometry FACS) quantitative detection of NDFLtag-EGFP in 40% CM or 2 ng/ml TGF- beta 1 stimulated LX-2 cells in duplicate. Results: FHCC-98 CM and TGF- beta 1 were able to stimulate the activation of LX-2 cells at low concentrations. CM (10%~40%) is more conducive to the proliferation of LX-2 cells in 40% CM stimulated cell proliferation rate, as the effect of 25.8%.TGF- beta 1 on the proliferation of LX-2 cells was positively correlated with dose dependent. Compared with the control group without stimulation, CM or TGF- beta 1 stimulates LX-2 cell activation, cell alpha -SMA. Collagen I showed the expression of TIMP-1 and TGF- four beta 1 gene.NDV increases with continuous passage of LX-2 cells, the replication in the cell rate is gradually increased.FACS, LX-2 cells were treated with 40% CM and 2 ng/ml TGF- 1 beta stimulation after activation of NDV replication in cell division rate Do not increase 1.76 and 1.52 times. Conclusion: FHCC-98 CM can induce LX-2 cells to activate and proliferate, and the replication rate of NDV in activated LX-2 cells is increased.
The second part: the inhibitory effect of NDV on the activation of HSC
Objective: To investigate the effect of NDV in the activation of HSC replication on HSC proliferation and cell biological function. Methods: NDV oncolytic strain Italien different titers (NDV-Italien) infection after CM stimulation of LX-2 cells, affect the detection of NDV MTT method for detection of cell proliferation rate. Immunofluorescence microscopy expression of activation markers alpha changes -SMA. Real time quantitative RT-PCR detection of NDV four activation related gene of alpha -SMA, collagen 1, TIMP-1 and TGF- 1 beta mRNA level. Gelatinase spectrum method to detect MMP-2 and MMP-9. Results: NDV infection inhibited the proliferation of LX-2 cells, there was a negative correlation between the cell proliferation rate and virus titer. Compared with the untreated group, the proliferation of NDV in CM stimulated LX-2 cells inhibition rate increased, significantly reduced expression of four genes, inhibiting MMP-2 secretion and MMP-9. Conclusion: NDV can inhibit cell proliferation in LX-2 cell activation in replication The effect of colonization and HSC activation.
The third part: the reversal of NDV induced liver fibrosis in mice induced by CCl4
Objective: To investigate the in vivo inhibitory effect of NDV on CCl4 induced liver fibrosis in mice. Methods: weight of about 20 g Kunming mice two times a week, intraperitoneal injection of 100 L CCl4/ peanut oil solution (20%, V/V), the control group were injected with 100 L saline (PS), continuous injection for 8 weeks at last in the CCl4 3 days after injection, intravenous injection of 200 L titer was 1000 HU (hemagglutination unit) NDV-Italien 3 times or 1 times every 24 h interval of continuous injection of.NDV injection of 24h after the death of animal, remove the liver for the observation of general morphology and pictures. With Ma Lin Foer fixed paraffin embedded liver tissue., tissue sections were stained by HE and Masson staining. Fresh liver tissue with RIPA (non-ionic detergent-containing buffer) lysis, BCA (bicinchoninic acid) to determine the total protein concentration assay kit, 50 g total protein sample was Western blot. To detect the expression of alpha -SMA. Fresh liver tissues were frozen and fixed in cold acetone, the expression and localization of immunofluorescence double staining assay in tissue alpha -SMA and NDV particles. Results: CCl4 after 8 weeks of induction, the mouse liver fibrosis appeared obvious symptoms, can be seen in liver tissue of hard, rough surface, dense distribution a large number of white plaques on.HE staining showed that the loose structure of liver fibrosis, perisinusoidal space increases.Masson's trichrome staining showed abnormal deposition of collagen and NDV. After 3 times of injection, mice liver surface white spots significantly reduced, low.Western blot analysis showed that the reduction of collagen deposition, -SMA protein level decreased with the increase in the number of NDV injection. Double immunofluorescence staining showed that -SMA and NDV exist in liver perisinusoidal space, CO localization in the activation of HSC, and the specific adsorption of normal mice liver tissues of NDV. Conclusion: in CCl4 induced small In rat liver fibrosis, NDV can selectively act in the activated HSC and inhibit liver fibrosis.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R373;R575.2;R735.7

【引证文献】