發(fā)布時(shí)間：2018-01-09 11:18

  本文關(guān)鍵詞：新城疫病毒抑制肝星狀細(xì)胞的活化及其逆轉(zhuǎn)小鼠肝纖維化的研究　出處：《第四軍醫(yī)大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 新城疫病毒 肝纖維化 肝星狀細(xì)胞 肝癌 溶瘤病毒 條件培養(yǎng)基 TGF-β1 CCl_4 共定位

【摘要】： 肝細(xì)胞肝癌(hepatocellular carcinoma,HCC)是肝癌中最常見的組織學(xué)類型。在我國(guó)70%～90%HCC患者伴有肝硬化。在肝癌發(fā)病率呈上升趨勢(shì)的歐美國(guó)家,HCC的發(fā)生幾乎全部源于肝硬化的個(gè)體。而肝硬化嚴(yán)重影響了HCC治療方法的選擇、療效、并發(fā)癥及預(yù)后。目前認(rèn)為,HCC合并肝硬化的發(fā)病機(jī)制基于兩個(gè)方面[1]:1)在損傷因子(如:肝炎病毒、黃曲霉素、代謝病和酒精等)存在下,肝細(xì)胞持續(xù)性受損、變性和壞死所致的炎性反應(yīng)以及細(xì)胞外基質(zhì)(extracellular matrix,ECM)合成過(guò)多,ECM不能被降解、吸收,從而導(dǎo)致在肝內(nèi)大量沉積,形成肝纖維化,最終則引起以纖維組織環(huán)繞增生肝細(xì)胞團(tuán)的硬化結(jié)節(jié),即肝硬化。在這一過(guò)程中,肝星狀細(xì)胞(hepatic stellate cell,HSC)由靜止?fàn)顟B(tài)活化為成肌纖維細(xì)胞,大量分泌ECM,是肝纖維化發(fā)生乃至肝硬化進(jìn)展的主要細(xì)胞學(xué)基礎(chǔ)。2)在肝纖維化炎性環(huán)境中產(chǎn)生的氧自由基等誘變物質(zhì)引起肝細(xì)胞DNA損傷,損傷的肝細(xì)胞應(yīng)答旁分泌細(xì)胞因子的刺激而增殖,這種不斷的損傷、再生誘發(fā)的多基因變異是導(dǎo)致肝細(xì)胞癌變的關(guān)鍵。 HSC活化是肝纖維化發(fā)生的主要原因,肝纖維化是肝癌前病變的重要病理階段。免疫組化研究顯示,HCC合并肝硬化中活化的HSC數(shù)量顯著增高,而肝癌細(xì)胞分泌的有絲分裂因子也具有促進(jìn)HSC活化并增殖的作用[2];活化HSC和癌變細(xì)胞的相互作用會(huì)進(jìn)一步加重肝癌合并肝硬化程度。因此著眼于活化HSC的靶向治療是抑制肝纖維化,改善肝癌狀況的一個(gè)新的研究熱點(diǎn)。目前,抑制肝纖維化的途徑主要集中在:阻斷轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factorβ,TGF-β)信號(hào)通路,以拮抗TGF-β在HSC活化中的作用[3,4];減少細(xì)胞外基質(zhì)ECM的生成,降解組織中過(guò)量沉積的膠原纖維[5,6]。 新城疫病毒(Newcastle disease virus, NDV)是單股負(fù)鏈RNA病毒,屬副粘病毒科。由于多數(shù)RNA病毒具有天然地在腫瘤細(xì)胞選擇性復(fù)制的特點(diǎn),近年來(lái)用這類病毒作為新型運(yùn)載工具在腫瘤基因治療方面引起了人們的關(guān)注。另一方面,NDV作為溶瘤病毒用于腫瘤治療已見臨床報(bào)道。NDV在腫瘤細(xì)胞選擇性復(fù)制的機(jī)制和腫瘤細(xì)胞的干擾素信號(hào)通路缺陷有關(guān)[7]。1976年,McGregor報(bào)道了NDV能夠在活化的T細(xì)胞中復(fù)制,并導(dǎo)致活化T細(xì)胞的死亡[8];Fábián最近又報(bào)道了NDV能夠在轉(zhuǎn)化的細(xì)胞中高效地復(fù)制[9]�；诨罨疕SC是一種非正常的、具有增殖能力的細(xì)胞,我們提出假設(shè):NDV能在這種活化的HSC中復(fù)制,并抑制HSC的活化,進(jìn)而達(dá)到逆轉(zhuǎn)肝纖維化的目的。本論文以抑制活化HSC為出發(fā)點(diǎn),探討NDV在HSC中的復(fù)制率及其對(duì)肝纖維化相關(guān)基因在mRNA和蛋白水平的影響,并通過(guò)建立小鼠肝纖維化模型,研究NDV的體內(nèi)逆轉(zhuǎn)肝纖維化的功能。 第一部分:NDV在人肝癌細(xì)胞條件培養(yǎng)基誘導(dǎo)活化HSC中的復(fù)制 目的:探討人肝癌細(xì)胞FHCC-98條件培養(yǎng)基(conditioned medium, CM)對(duì)人肝星狀細(xì)胞LX-2活化的誘導(dǎo)作用;檢測(cè)NDV在LX-2細(xì)胞中的復(fù)制率。方法:用不同百分含量的FHCC-98 CM刺激LX-2細(xì)胞,MTT法檢測(cè)細(xì)胞的增殖率,TGF-β1為陽(yáng)性對(duì)照。半定量及Real-time定量RT-PCR檢測(cè)40% CM(V/V)或2 ng/ml TGF-β1刺激前后LX-2細(xì)胞中α-平滑肌動(dòng)蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型膠原(collagenⅠ)、金屬蛋白酶組織抑制劑-1(tissue inhibitor of metalloproteinase-1,TIMP-1)和TGF-β1四種活化相關(guān)基因的mRNA表達(dá)。用攜帶增強(qiáng)型綠色熒光蛋白(enhanced greenfluorescent protein, EGFP)的重組NDV(NDFLtag-EGFP)感染不同傳代的LX-2細(xì)胞,熒光顯微鏡下觀察EGFP在細(xì)胞中的表達(dá)率和熒光強(qiáng)度,以反映NDV的復(fù)制率。用流式細(xì)胞術(shù)(flow cytometry, FACS)定量檢測(cè)NDFLtag-EGFP在40% CM或2 ng/ml TGF-β1刺激活化的LX-2細(xì)胞中的復(fù)制。結(jié)果:FHCC-98 CM和TGF-β1均能夠刺激LX-2細(xì)胞的活化。低濃度的CM(10%~40%)更有利于LX-2細(xì)胞的增殖,40% CM刺激時(shí)的細(xì)胞增殖率最大,為25.8%。TGF-β1對(duì)LX-2細(xì)胞增殖的影響呈正相關(guān),且具有劑量依賴性。與無(wú)刺激的對(duì)照組相比,CM或TGF-β1刺激LX-2細(xì)胞活化后,細(xì)胞中α-SMA、collagenⅠ、TIMP-1和TGF-β1四種基因的表達(dá)均有所增加。NDV隨著LX-2細(xì)胞的連續(xù)傳代,其在細(xì)胞中的復(fù)制率也逐漸增加。FACS顯示,LX-2細(xì)胞分別經(jīng)40% CM和2 ng/ml的TGF-β1刺激活化后,NDV在細(xì)胞中的復(fù)制率分別提高了1.76倍和1.52倍。結(jié)論:FHCC-98 CM能誘導(dǎo)LX-2細(xì)胞活化并增殖,NDV在活化LX-2細(xì)胞中的復(fù)制率提高。 第二部分:NDV對(duì)HSC活化的抑制作用 目的:探討NDV在活化HSC中的復(fù)制對(duì)HSC的增殖以及細(xì)胞生物學(xué)功能的影響。方法:用不同滴度的NDV溶瘤株Italien(NDV-Italien)感染經(jīng)CM刺激的LX-2細(xì)胞,MTT法檢測(cè)NDV對(duì)細(xì)胞增殖率的影響。免疫熒光顯微鏡技術(shù)檢測(cè)活化標(biāo)志物α-SMA的表達(dá)變化。實(shí)時(shí)定量RT-PCR檢測(cè)NDV對(duì)四種活化相關(guān)基因α-SMA、collagenⅠ、TIMP-1和TGF-β1 mRNA水平的影響。明膠酶譜法檢測(cè)MMP-2和MMP-9的分泌。結(jié)果:NDV的感染抑制了LX-2細(xì)胞的增殖,細(xì)胞增殖率和病毒滴度呈負(fù)相關(guān)關(guān)系。與未刺激組相比,NDV在CM刺激的LX-2細(xì)胞中的增殖抑制率提高,四種基因的表達(dá)顯著性下調(diào),MMP-2和MMP-9的分泌下調(diào)。結(jié)論:NDV在活化LX-2細(xì)胞中的復(fù)制具有抑制細(xì)胞增殖和HSC活化的作用。 第三部分:NDV對(duì)CCl4誘導(dǎo)的小鼠肝纖維化的逆轉(zhuǎn) 目的:通過(guò)體內(nèi)試驗(yàn)探討NDV對(duì)CCl4誘導(dǎo)的小鼠肝纖維化的抑制作用。方法:體重約20 g的昆明小鼠,每周兩次腹腔注射100μl CCl4/花生油溶液(20%, V/V),對(duì)照組同時(shí)注射100μl生理鹽水(PS),連續(xù)注射8周。最后一次在CCl4注射后3天,尾靜脈注射200μl滴度為1,000 HU(hemagglutination unit)的NDV-Italien 1次或每24 h間隔連續(xù)注射3次。NDV注射24h后處死動(dòng)物,取出肝臟進(jìn)行大體形態(tài)觀察并拍照。肝組織用福爾馬林固定,石蠟包埋,組織切片進(jìn)行常規(guī)HE染色和Masson三色染色。新鮮肝組織用RIPA(non-ionic detergent-containing buffer)裂解液裂解,BCA(bicinchoninic acid)蛋白定量試劑盒測(cè)定總蛋白濃度,取50μg總蛋白上樣進(jìn)行Western blot檢測(cè)α-SMA的表達(dá)。新鮮肝組織進(jìn)行冰凍切片,冷丙酮固定,免疫熒光雙染法檢測(cè)組織中α-SMA和NDV顆粒的表達(dá)和定位。結(jié)果:CCl4誘導(dǎo)8周后,小鼠肝臟出現(xiàn)明顯的纖維化癥狀,可看到肝組織變硬、表面粗糙不平、密集分布大量的白色點(diǎn)狀斑塊。HE染色顯示,纖維化肝臟的組織結(jié)構(gòu)松散,竇周隙增大。Masson三色染色顯示膠原異常沉積。而NDV注射3次后,小鼠肝臟表面的白色斑點(diǎn)顯著減少,膠原沉積降低。Western blot分析表明,α-SMA蛋白水平隨NDV注射次數(shù)增加而降低。免疫熒光雙染顯示,α-SMA和NDV均存在于肝組織的竇周隙,共定位在活化HSC中,而正常小鼠的肝臟組織未見NDV的特異性吸附。結(jié)論:在CCl4誘導(dǎo)的小鼠肝纖維化中,NDV可以選擇性地作用于活化的HSC中,從而抑制肝纖維化。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373;R575.2;R735.7

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 陳鵬;生理節(jié)律與一種新型功能型葡聚糖對(duì)哺乳動(dòng)物肝損傷保護(hù)作用的研究[D];南京理工大學(xué);2011年

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本文編號(hào)：1401189