CN1142272C - 携带人肝细胞生长因子基因的重组腺病毒的用途 - Google Patents
携带人肝细胞生长因子基因的重组腺病毒的用途 Download PDFInfo
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Abstract
本发明涉及生物医学领域，具体地说是涉及一种携带人肝细胞生长因子基因的重组腺病毒及其在纤维性疾病防治中的应用。将人肝细胞生长因子全长编码区cDNA插入到复制缺陷型腺病毒载体GT4050上，获得一载导人肝细胞生长因子基因的重组腺病毒。该重组腺病毒尾静脉注射入大鼠体内，对大鼠肝纤维化有治疗作用。该重组腺病毒涂抹于创口表面有预防疤痕的功效。
Description
技术领域
本发明涉及生物医学领域，具体地说是涉及一种携带人肝细胞生长因子基因基因的重组腺病毒在制备纤维性疾病防治药物中的应用。
背景技术
纤维性疾病如肝纤维化、肺纤维化、肾纤维化及疤痕的形成等，是严重影响人类生命健康的疾病之一。肝纤维化是慢性肝病最主要的病理特征之一，也是慢性病毒性肝炎、代谢紊乱或慢性酒精中毒等疾病进一步发展的主要原因之一。目前国内对肝纤维化的治疗还仅仅停留在延缓和阻断纤维化过程方面，而对于已经形成的肝纤维化，尚无有效手段能够逆转纤维化过程，达到治疗肝纤维化的效果。国外最近虽有用裸露质粒治疗肝纤维化的文献报道(1)，但由于存在需要量大、多次反复用药等缺点而难以在临床广泛应用。应用重组腺病毒治疗肝纤维化国内外尚未见报道。
疤痕的形成是创伤愈合的产物和象征。然而过度的疤痕增生则是一种病态表现，是创伤外科的一种重要并发症，可影响外观，影响机体功能，疤痕晚期还可发生癌变。目前人们对疤痕增生后的治疗主要靠手术、加压、冷冻等方法，均不是理想的手段。所以对疤痕的预防有着极其重要的意义。目前国内外尚无用生物工程外用药物预防疤痕的报道。
鉴于细胞因子在纤维性疾病中发挥的重要作用，目前有关细胞因子与纤维形成和控制之间的研究倍受人们的关注。肝细胞生长因子(hepatocytegrowth factor，HGF)最初被认为是一种肝细胞有丝分裂原，后来发现其对多种组织细胞的分裂、扩散和形态发生起重要的调解作用。尤其在肝脏的发育和再生中，HGF起着至关重要的作用。作为一种间质细胞来源的细胞因子，HGF能够明显地抑制转化生长因子(TGFβ)的产生和增强胶原酶的活性，而对成纤维细胞的增殖则无刺激活性，因而能够有效降低组织中TGFβ的水平、降解胶原纤维，具有防治纤维性疾病的作用。
重组腺病毒因其可介导目的基因高效地转染各种细胞的特性而倍受瞩目，已作为重要的基因载体系统迅速发展且用于基因治疗的临床实验。由于通过静脉输入的腺病毒大多数集中在肝脏，因而成为以肝脏为靶器官的基因的理想载体。伤口局部应用重组腺病毒，其转染效率很高，优于基因枪方法的转染效率。
综上所述，纤维性疾病的防治尚无有效的手段，目前仍是医学中的一大难题。
发明内容
本发明的目的在于公开一种携带人肝细胞生长因子基因的重组腺病毒在临床上可以制成一定滴度的注射液、涂抹液或喷雾剂等，用于纤维性疾病防治的药物。
本发明的具体实施方案如下：
1、将从人胎肝cDNA文库分离得到的人肝细胞生长因子编码区cDNA克隆到pBLUSCRIPT载体的BamH I和Sal I多克隆位点上(pBLUSCRIPT-HGF)：
将HGFcDNA由pBLUSCRIPT-HGF的Apa I和Not I位点切出，克隆至pXCJL1-CMV/PA载体的Hind III和Not I位点上，该载体含有CMV启动子和polyA信号，以及腺病毒基因组左端序列，从而获得携带人肝细胞生长因子基因表达盒以及腺病毒基因组左端序列的穿梭质粒(pXCJL1-CMV/PA-HGF)。见图1。
2、将穿梭质粒pXCJL1-CMV/PA-HGF和含E1、E3及包装信号区缺失的复制缺陷型5型腺病毒基因组的质粒GT4050共转染293细胞(以腺病毒E1基因转化的人胚肾细胞系)，通过在细胞内发生同源重组获得携带人肝细胞生长因子基因的重组腺病毒。该重组腺病毒的结构为：
5’ITR CMV/HGF/polyA Ad3328-3’ITR
见图2。
3、将该重组腺病毒通过大鼠尾静脉注射入用DMN(二硝基亚硝酸)法建立的大鼠致死性肝纤维化模型，通过组织病理学方法观察到注射该重组腺病毒组(治疗组)肝细胞空泡变性程度轻、数量较少，纤维组织薄且短(图3(d)，(e))而对照组显示明显的纤维增生和多而严重的肝细胞空泡变性，并可见假小叶(图3(a)，(b)，(c))。从而证实该重组腺病毒对大鼠肝纤维化有治疗作用。
4、用新西兰大白兔通过显微外科的方法建立兔耳疤痕模型，在手术同时将该重组腺病毒涂抹于创口表面(预防组)，结果表明预防组较对照组创面愈合快，炎性反应轻，疤痕的形成小(图4(a)，(b))；HE染色显示预防组较对照组表皮角化轻、真皮层厚度小，纤维组织薄(图5(a)，(b))。从而证实该重组腺病毒对疤痕形成有一定的预防作用。
本发明提供一种携带人肝细胞生长因子基因的重组腺病毒，该重组腺病毒可以以液体形式被配制成注射液、涂抹液(用于体表)等用于防治纤维性疾病。纤维性疾病的类型可以是肝纤维化，也可以是疤痕，也可以是身体其他组织器官的纤维化。实施例将通过描述携带人肝细胞生长因子基因的重组腺病毒对肝纤维化和皮肤疤痕的基因治疗来进一步阐述本发明。
应用本发明提供的重组腺病毒防治纤维性疾病是从纤维形成机制入手，利用腺病毒高效转染及嗜肝性特点，向患者体内导入人肝细胞生长因子基因，通过抑制TGFβ的产生和增强基质降解酶如胶原酶的活性，来降低组织中TGFβ的水平和降解胶原纤维，达到防治纤维性疾病的目的。因此应用本发明提供的重组腺病毒防治纤维性疾病比以往的防治手段更具有科学性，疗效更明确，更彻底，而且能够逆转纤维化过程。若本发明得以应用，将为目前尚无有效防治手段的纤维性疾病的临床治疗提供一种有效的防治手段。
附图说明
结合附图进一步说明本发明：
图1：携带人肝细胞生长因子基因及含腺病毒组左端序列的穿梭质粒的构建示意图。
图2：载导人肝细胞生长因子基因的重组腺病毒构建示意图。
图3：肝细胞生长因子治疗肝纤维化的组织病理学切片
(a)(H.E.染色，100×)(b)(H.E.染色，100×)
(c)(H.E.染色，100×)(d)(H.E.染色，100×)
(e)(H.E.染色，100×)
图4：肝细胞生长因子预防疤痕形成的大体外观图
(a)对照组 (b)预防组
图5：肝细胞生长因子预防疤痕作用的组织切片
(a)(H.E.染色，100×)对照组
(b)(H.E.染色，100×)预防组
参考文献：
(1)Takahiro Ueki，Yasufumi Kaneda，Hiroko Tsutsui，et al.Hepatocytegrowth factor gene therapy of liver cirrhosis in rats.Nature Medicie，1999；5(2)：226-230
具体实施方式
实施例一 重组腺病毒的构建、扩增及纯化一、重组腺病毒的构建
携带人肝细胞生长因子基因的穿梭质粒(pXCJL1-CMV/HGF/polyA)的构建过程如图1所示。将pXCJL1-CMV/HGF/polyA与携带腺病毒基因组DNA的重组质粒GT4050共转染293细胞，二者在细胞内发生同源重组，形成携带人肝细胞生长因子基因的重组腺病毒(命名为Ad-HGF)，其结构示意图如图2所示。二、重组腺病毒的制备与纯化
将293细胞接种于650ml细胞培养瓶中，待细胞生长至90％汇合状态时，按10个空斑形成单位(pfu)/细胞的量加入病毒，36-48小时后，细胞出现完全病变效应(CPE)，收集细胞并冻存于-80℃，累计30-40瓶时一次纯化。三、将出现细胞病变效应的293细胞于-80℃和37℃间反复冻融三次，2500转/分钟离心10分钟，收集上清；制备氯化铯密度梯度：依次将用无菌PBS配制的0.5ml 1.5g/ml氯化铯溶液、2.5ml 1.35g/ml氯化铯溶液和2.5ml1.25g/ml氯化铯溶液加入超速离心管中；将6ml待纯化病毒上清加于各管氯化铯梯度液之上；35000转/分钟10℃离心1小时，于1.35g/ml氯化铯溶液和1.25g/ml氯化铯溶液之间出现白色雾状病毒带，收集病毒带并与1.35g/ml氯化铯溶液混合，35000转/分钟10℃离心18小时，吸出病毒带，以1-2倍的Hanks液稀释病毒，以Hanks液为透析液4℃透析病毒，每2小时更换一次透析液，共换液5次；取出纯化的病毒液，加终浓度为10％的无菌甘油，分装后-80℃保存。空斑实验确定其滴度为8.6×1010空斑形成单位/毫升(pfu/ml)。
实施例二 重组腺病毒介导人肝细胞生长因子基因
对大鼠肝纤维化治疗的评价一、大鼠肝纤维化模型的制备
Wistar雄性大鼠30只，体重200-250克。随机抽取26只大鼠制备纤维化模型：将1克二硝基亚硝胺(DMN)溶于100毫升无菌生理盐水中，使终浓度达1％。大鼠皮下给药，每次1ml/kg体重，每周两次，连续4周；另外4只大鼠作为正常组，除不给药外，其余条件均与模型组相同。末次给药后10天，随机取模型组和正常组各两只大鼠，取肝组织做HE切片染色，观察肝纤维化大鼠肝脏的病理形态变化；肝脏病理学检查：模型组大鼠的肝脏明显肿大，边缘钝，质韧；在光镜下可见肝细胞空泡坏死，有较多的中性粒细胞和淋巴细胞浸润，汇管区成纤维细胞增多，纤维组织增生，有些还有出血性病变。正常组大鼠则无上述病变，证明肝纤维化模型制成。二、重组腺病毒对肝纤维化的治疗作用
取12只模型组大鼠，随机分为治疗和对照两组，通过尾静脉注给治疗组大鼠100μl8.6×107pfu/μl的Ad-HGF病毒，而给对照组大鼠注射300μl2.8×107pfu/μl的Ad-GFP病毒(Leibowitz G Beattie GM，Kafri T.et al.Gene transferto human pancreatic endocrine cells using viral vectors.Diabetes.1999Apr；48(4)：745-53.)，次日重复给药一次。给药40天后，取大鼠肝组织做HE染色，观察两组大鼠肝脏的病理形态。治疗组大鼠的肝细胞空泡变性程度较轻，数量较少，未见有中性粒细胞浸润，仅见少量的淋巴细胞，更没有出血性病变；汇管区成纤维细胞数量较少，纤维组织层次较薄且延伸范围较短；有的大鼠肝标本，细胞变性程度较轻没有炎性细胞的浸润，汇管区看不到明显的纤维增生，达到了基本治愈的效果。而对照组大鼠肝组织病理形态与治疗前相比无明显改变。如图3所示。
实施例三 重组腺病毒介导人肝细胞生长因子基因
对疤痕形成的预防作用一、疤痕动物模型
新西兰雌性大白兔，体重2.5-3.5kg。耳腹侧无血管处剃毛消毒后，一环钻以显微外科方法切除全层皮肤直至软骨表面，圆形切口的直径为0.6cm，每侧4-6个。二、效果评价
切除皮肤的同时将重组腺病毒涂抹于伤口表面，每次8ul(8.6×107pfu/μl)(Ad-HGF组)，每天一次，连续3天；对照组伤口则涂抹同剂量Ad-GFP。术后第22天将创面与邻近无创伤处皮肤一并切下，做石蜡切片，行HE染色。结果表明涂抹Ad-HGF组较涂Ad-GFP组创面愈合快，炎性反应轻，疤痕的形成小，如图4所示；HE染色显示涂Ad-HGF组较涂Ad-GFP表皮角化轻、真皮层厚度小，纤维组织薄。如图5所示。
Claims (5)
1、携带人肝细胞生长因子基因的重组腺病毒在制备纤维性疾病防治药物中的应用，该重组腺病毒为携带人肝细胞生长因子基因的E1、E3缺失的复制缺陷型5型腺病毒。
2、根据权利要求1所述的应用，其特征在于所述药物剂型为注射液、涂抹液或喷雾剂。
3、根据权利要求1或2所述的应用，其特征在于纤维性疾病为肝纤维化，通过静脉注射注射液给药。
4、根据权利要求1或2所述的应用，其特征在于纤维性疾病为疤痕，通过创口表面涂或喷涂抹液给药。
5、根据权利要求1或2所述的应用，其特征在于纤维性疾病为肺纤维化。
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| US20030103941A1 (en) * | 2001-10-09 | 2003-06-05 | Crombleholme Timothy M. | Materials and methods for preventing or reducing scar formation |
| KR100562824B1 (ko) * | 2002-03-20 | 2006-03-23 | 주식회사 바이로메드 | 유전자 발현효율이 높으며 간세포 성장인자의 두 가지이형체를 동시에 발현하는 하이브리드 간세포 성장인자유전자 |
| CN100441226C (zh) * | 2002-04-26 | 2008-12-10 | 中国人民解放军军事医学科学院放射医学研究所 | 用于加速创伤修复及防治并发症的方法 |
| EP1547618B1 (en) * | 2002-10-02 | 2010-05-26 | AnGes MG, Inc. | Drug for auditory dysfunction |
| WO2008011216A2 (en) | 2006-05-16 | 2008-01-24 | Pro-Pharmaceuticals, Inc. | Galactose-pronged polysaccharides in a formulation for antifibrotic therapies |
| CN101864452B (zh) * | 2009-04-16 | 2012-01-11 | 中国人民解放军军事医学科学院军事兽医研究所 | 一种双特异抗肿瘤重组腺病毒及其构建方法和应用 |
| ES2869884T3 (es) * | 2011-09-16 | 2021-10-26 | Galectin Therapeutics Inc | Composiciones de galacto-ramnogalacturonato para el tratamiento de la esteatohepatitis no alcohólica y la enfermedad de hígado graso no alcohólico |
| MX2014014979A (es) | 2012-06-06 | 2015-06-17 | Galectin Therapeutics Inc | Composiciones de galacto-ramnogalacturonato para el tratamiento de las enfermedades asociadas con la sintasa de oxido nitrico inducible elevada. |
| CN103446188B (zh) * | 2013-09-02 | 2017-01-25 | 中国人民解放军第三军医大学第一附属医院 | Tradd基因过表达慢病毒在抑制皮肤疤痕形成中的应用 |
| CN114392361B (zh) * | 2022-01-23 | 2023-06-02 | 重庆医科大学附属儿童医院 | 一种羧甲基壳聚糖-腺病毒混合物及其应用 |
| CN115161289B (zh) * | 2022-03-14 | 2023-12-05 | 东南大学 | 一种用于炎症性疾病治疗的重组腺相关病毒及其构建方法和应用 |
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