CN107080849A - 肝损伤治疗的靶点和药物 - Google Patents
肝损伤治疗的靶点和药物 Download PDFInfo
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Abstract
本发明公开了一种肝损伤治疗的靶点和药物，所述药物能够用于调控肝星状细胞活性以预防或治疗化学性肝损伤导致的肝纤维化的物质。本发明所述的GGPPS可以通过特异性调控肝星状细胞活性调节化学性肝损伤导致的肝纤维化，可以作为特异靶向治疗肝纤维化的新靶点。本发明所述药物能够靶向性、特异性的治疗肝纤维化。
Description
技术领域
本发明属于靶向药物领域，涉及一种肝损伤治疗的药物，具体而言，涉及治疗肝损伤的 GGPPS靶点，以及通过该靶点治疗肝损伤的药物。
背景技术
我国是慢性肝病的高发地区。据统计，我国目前约有1.2亿肝病患者，另外每年近100万 新发病人群。每年因肝硬化、肝癌死亡人数则为40多万人。近年来，卫生部公布的我国疾病 发生率数字表明，肝病发病率仍高居榜首。由于肝病预后差，相当一部分慢性肝病可转为肝 硬化和肝癌。因此慢性肝病对人类的健康危害极大，如何防治一直是我国传染病工作的重中 之重。
肝脏作为机体的主要代谢器官，起着去氧化、储存肝糖、分泌性蛋白质的合成等等作用。 肝脏是口服药物、酒精和其它肠道吸收的异物的最初代谢场所，极易受到化学物质诱导的损 伤。诸如急性和慢性肝炎、肝中毒、肝细胞坏死、急性脂肪病变、胆汁淤积、肝硬化和肝癌 等均可不同程度的对肝脏造成肝损伤。损伤程度有轻微的肝结构和功能变化如急性肝衰竭、 肝硬化，到肝癌不。肝损伤后会诱导肝脏发生一系列修复反应，肝纤维增生就是其中极为重 要的一个环节。肝纤维化(Hepatic fibrosis,HF)的特点是细胞外基质的产生和分解平衡被打 破，造成胶原和其它细胞外基质在肝内过度沉积。肝纤维化是各种慢性肝病共有的病理基础 和必经阶段，是影响慢性肝病的重要环节。如不加控制，可逆的肝纤维化可进而发展成为不 可逆的肝硬化，而且绝大多数还会导致肝癌形成。肝星状细胞(Hepatic stellate cell,HSC) 是肝非实质细胞的一种，存在于肝血窦内皮细胞与肝细胞间的Disse间隙，占肝脏中细胞比 例的15％。HSC的活化、增殖是肝纤维化发生的中心环节，因此，对HSC活化从而导致肝 纤维化发生机制的研究在临床诊断治疗纤维化方面具有重要的意义。当肝脏受损时，HSC被 激活，失去了对维生素A和脂质储存的特性转变成活化形式，即从静止的肝星状细胞转化为 肌成纤维样细胞。HSC的活化过程受到转化生长因子β(Transforming growth factorβ，TGF-β) 和血小板源生长因子(Platelet-derivedgrowth factor,PDGF)两个细胞因子的调控，在这两个细 胞因子促进下活化的HSC表达α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)、合成细胞外 基质(Extracellularmatrix，ECM)，最终会导致肝的纤维化。因此，在过去的一二十年中， 对HSC的研究成为研究肝纤维化的主导方向。由于肝纤维化是各种慢性肝病最后走向肝硬化 甚至于肝癌的必由之路，因而在此阶段抗肝纤维化治疗，可以阻断甚至逆转肝纤维化的发生， 从而防止肝硬化和肝癌的产生，对慢性肝病的防治具有重要的意义。
香叶基香叶基二磷酸合成酶(geranylgeranyl diphosphate,GGPP)是甲羟戊酸代谢途径中 的关键蛋白，在小G蛋白香叶基修饰中发挥重要作用，促进小G蛋白激活。香叶基化是重要 的蛋白质翻译后修饰之一，一般发生在蛋白质C末端保守的半胱氨酸。发生香叶基化的蛋白 质包括Ras和Ras相关的小G蛋白如Rho、Rab、Rac，以及三聚体G蛋白的γ亚基，这些受调控 的蛋白质都是信号传递通路中重要的分子开关调节蛋白。已有研究报道过GGPP在逆转他汀 类药物引起的肝损伤中发挥关键作用。当用他汀类药物抑制HMG-CoA还原酶活性时，可抑 制肝细胞活性，引起细胞死亡。然而，这种抑制作用可以被甲羟戊酸或者GGPP所逆转。GGPPS 可以催化法尼基焦磷酸(Fanesyl pyrophosphate，FPP)与异戊烯焦磷酸(Isopenteny pryophosphate，IPP)的缩合反应，产生由20个碳组成的香叶基焦磷酸，促进下游蛋白发生 香叶基化修饰。在甲羟戊酸代谢途径中，GGPPS是催化GGPP和FPP合成的分支点酶。 GGPPS这一催化酶的功能已经在许多疾病中被揭示。近年研究发现GGPPS在香烟烟雾导致 肺部炎症的信号通路中发挥作用。小鼠睾丸支持细胞中GGPPS的特异性敲除引起不育症，心 脏特异性敲除GGPPS导致了心肌细胞肥厚性增生。
发明内容
解决的技术问题：为了克服现有技术的缺陷，获得一个特异靶向治疗肝纤维化的新靶点， 本发明提供了一种肝损伤治疗的靶点和药物。
技术方案：用于调控肝星状细胞活性以预防或治疗化学性肝损伤导致的肝纤维化的物 质。
优选的，所述物质是香叶基二磷酸合成酶抑制剂。
进一步的，所述抑制剂是香叶基二磷酸合成酶特异性的干扰质粒。
进一步的，所述干扰质粒的小干扰RNA的编码序列为：SEQ ID NO:1～2。序列如下：
SEQ ID NO:1 5'-CCAGAUUAGAGAUGAUUAUTT-3'；
SEQ ID NO:2 5'-AUAAUCAUCUCUAAUCUGGTT-3'。
优选的，所述干扰质粒由维生素A偶联的脂质体包裹。
优选的，所述干扰质粒靶向干扰肝星状细胞的GGPPS。
优选的，所述维生素A脂质体包裹的干扰质粒靶向干扰肝星状细胞的GGPPS，抑制肝星 状细胞的活化。
优选的，所述维生素A脂质体包裹的干扰质粒靶向干扰肝星状细胞的GGPPS，抑制CCl4 化学损伤诱导的肝纤维化。
上述任一所述物质在制备预防或治疗化学性肝损伤导致的肝纤维化的药物或药物组合物 中的应用。
有益效果：肝星状细胞的活化、增殖是肝纤维化发生的中心环节。当肝脏受到损伤刺激 后，肝星状细胞可以经历从静止的肝星状细胞到具有增殖性、成纤维性和收缩性的肌成纤维 样细胞(MF)的转化过程。激活后的肝星形细胞可自分泌TGF、PDGF、ET等细胞因子使活 化得以持续并合成大量细胞外基质，使得肝脏发生纤维化。
本发明所述药物具有以下优点：针对肝星状细胞，并有高度的特异性；对肝实质细胞及 其他非实质细胞的毒性小；应能有效逆转进展性肝纤维化，而不是仅仅阻止新的胶原的沉积。 此外，本发明充分利用了肝星形细胞是维生素A的存储细胞的特性，实现了将小干扰RNA 靶向到肝星状细胞而不影响肝实质细胞和其它的非实质细胞。由于GGPPS在肝星状细胞活化 过程中起着重要作用，利用小干扰RNA特异的降低肝星状细胞中GGPPS的表达，能够抑制 肝星状细胞的活化，并从根本上抑制了胶原的产生，有效的控制肝纤维化的发展。
相比较而言，目前科研领域虽然利用转基因手段敲除肝星状细胞中某些基因可以控制肝 纤维化，但转基因不可能作为抗纤维化治疗的手段，仅适用于基础研究。此外，一些药物被 报道可以作用到肝星状细胞，但是鉴于其靶向性差，安全性差和副作用大，也未能成功的用 于肝纤维化治疗。综上所述，本发明在肝纤维化治疗上具有靶向性强、安全性高、作用明显 以及副作用小的特点，可能作为理想的纤维化治疗手段。
附图说明
图1是GGPPS mRNA水平在肝硬化病人样本中变化图；A为定量PCR的方法检测肝硬化病人病理组织中GGPPS表达图；B为定量PCR的方法检测肝硬化相关基因COL1A1的表 达图；C为定量PCR的方法检测肝硬化相关基因CTGF的表达图；*，与正常肝脏比p﹤0.05； D为免疫蛋白印记检测肝硬化病人病理组织中GGPPS以及肝硬化相关基因α-SMA的表达 图；
图2是GGPPS mRNA水平在CCl4诱导的肝损伤中变化图；对GGPPS肝脏敲除小鼠和对照小鼠腹腔注射CCl4，搜集不同时间肝脏样品提取RNA，荧光定量PCR检测肝硬化相关 基因COL1A1(A)以及GGPPS(B)的表达。**，与0小时相比p﹤0.01；
图3是CCl4化学物质诱导的小鼠肝纤维化中GGPPS特异的高表达于HSC；(A)免疫荧光染色检测α-SMA(绿)和GGPPS(红)在CCl4诱导的慢性小鼠肝纤维化中的表达(放 大倍数：×200)；(B)密度梯度离心分离肝纤维化小鼠和对照小鼠的HSC，Western blotting 检测GGPPS的表达；
图4是VA-Liposome-siRNAggpps-FAM靶向HSC；(A)荧光检测冰冻切片中α-SMA 的表达以及FAM的分布(放大倍数：×200)；(B)Real-time PCR验证HSC中GGPPS的 干扰；
图5是靶向干扰HSC中GGPPS表达可以抑制HSC活化和肝纤维化；(A)Westernblotting 检测GGPPS、α-SMA和TGF-β1的表达；(B)Real-time PCR检测小鼠肝脏中HSC活化标 志物和纤维化标志物的表达。*，与olive相比p＜0.05，**，与olive相比p＜0.01；##，与NC相比p＜0.01)；(C)天狼猩红染色测定肝纤维化的代表图片(放大倍数：×200)。右 图为天狼猩红染色的统计结果，**，p＜0.01；
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明 精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。 若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 GGPPS在肝硬化及CCl4诱导的急性肝损伤中表达的检测
实验中使用的是8周龄的雄性B6小鼠。急性损伤模型中，小鼠腹腔注射每克体重1uL 的CCl4(CCl4与橄榄油1:3混合)。慢性肝损伤模型中，小鼠根据体重每周腹腔注射2次1uL /g的CCl4(CCl4与橄榄油1:3混合)，连续注射12周。
首先，对搜集的病人肝硬化样品和对照样品以及CCl4诱导的小鼠急性肝损伤样品进行 RNA提取。具体方法如下；取肝脏组织约20mg于1mL Trizol，组织匀浆器匀浆至无块状后， 加入200μL氯仿剧烈震荡，室温静置5min后4℃12 000rpm离心15min。转移上层水相 至新的eppendorf管，加入等体积异丙醇后轻柔混匀，-20℃静置30min。4℃12 000rpm离心10min，弃上清，沉淀用1mL预冷的75％的乙醇洗涤，RNA最终溶于150uL DEPC水。55℃ 水浴充分溶解后测RNA浓度。
接下来将RNA反转录为反转录为cDNA。该试验采用Takara公司的PrimeScriptTMRT reagent试剂盒进行。每10uL反应体系中加入5×PrimerScript Buffer 4μL，RNA 800ng余下 部分用ddH2O补足20μL。然后将加好的反转录体系置于PCR仪中，37℃反应30min，然后85℃5s。反转录结束后，CDNA用ddH2O稀释5倍后实时荧光定量PCR检测GGPPS、CTGF和COL1A1的表达。事实荧光定量PCR中使用的引物序列如下：
此外，我们还在蛋白水平检测了GGPPS与纤维化相关因子α-SMA和TGF-β的表达。取肝组织30mg，加入300μL组织裂解缓冲液(其中含20mM Tris-HCl PH7.5；4mM EDTA； 2％SDS；1.0mmol/L Na3VO4；2.0mmol/L NaF；100.0μg/mL PMSF；1.0μg/mL cocktail)， 冰上匀浆后，4℃，12 000rpm离心15min，收集上清夜于另一新eppendorf管中，进行Western 印迹，具体方法如下：测定浓度后蛋白样品加入相应量的6×蛋白上样缓冲液，混匀后，99℃ 煮样5min，使蛋白变性。将处理好的蛋白样品按50μg/孔进行SDS-PAGE胶电泳分离，按 常规方法转印至PVDF膜上，一抗GGPPS(1:200稀释)、TGF-β(1：100稀释)、α-SMA (1：1000稀释)或β-Actin(1:1000稀释)4℃孵育过夜，PBST洗膜，每次10min，共3次。 二抗使用辣根过氧化物酶偶联IgG(1:20000稀释)室温孵育1h，再次PBST洗膜，每次5 分钟，共6次。之后暗室中配置ECL-plus显色液，混匀后滴加在平整放置的PVDF膜上，反 应40sec，随即将PVDF膜置于保鲜膜中固定在曝光盒内，上层覆盖感光胶片，根据荧光强 度选择不同的曝光时间，进行曝光。
实时荧光定量PCR和免疫蛋白印迹结果显示GGPPS在硬化样品中显著增加,具体结果如 图1和图2所示。
实施例2免疫荧光检测CCl4诱导的肝纤维化中GGPPS和α-SMA的分布
肝脏石蜡切片的制作:肝脏组织样生理盐水洗后，4％多聚甲醛固定过夜。50％乙醇1h→75％乙醇1h→90％乙醇1h→100％乙醇1h梯度脱水，二甲苯透明15min×2times，60℃ 浸蜡≥3h，石蜡包埋并切片。
1)5uM厚的石蜡切片脱蜡复水：二甲苯Ⅰ30min→二甲苯Ⅱ10min→100％乙醇3 min→95％乙醇3min→85％乙醇3min→70％乙醇3min→50％乙醇3min→H2O 5min×2
2)抗原修复：柠檬酸钠缓冲液微波修复，高火3min，中高2.5min，低火7min，自然冷却至室温。
3)打孔：含0.5％Tween-20和0.5％TritionX-100的PBS室温处理15min。
4)封闭：正常山羊血清室温封闭1h。
5)Ki67抗体用山羊血清按1：200稀释，滴于玻片上，4℃过夜。
6)PBS×3times，每次5min。
7)荧光二抗用山羊血清1：200稀释，室温1h。
8)PBS×3times，每次5min。
9)DAPI(1μg/mL终浓度)室温染15min。
10)PBS×3times，每次5min。
11)90％甘油封片
12)倒置荧光显微镜观察拍照。
通过免疫荧光共染GGPPS和α-SMA我们发现在小鼠肝纤维化的早期，GGPPS特异的高表达与HSC细胞,具体结果如图3所示。
实施例3原代HSC的分离
小鼠腹腔注射10％水合氯醛麻醉，75％乙醇消毒后固定在鼠板上。打开腹腔，暴露下腔 静脉和肝门静脉。针头从下腔静脉插入，确定针头插入血管后剪开肝门静脉。灌注液1以 10mL/min速度灌注7min后更换预热的灌注液2，以同样速度灌注10min。将消化好的肝脏取 出放入20mL含10％胎牛血清的DMEM培养基终止消化。用镊子将肝脏被摸撕开，晃散，然后滴管吹打，70uM筛网过滤后，4℃，50×g离心2min。上清4℃，2000×g离心10min后， 用2mL含胎牛血清的DMEM悬浮，铺在含33％percoll分离液的离心管中，2000×g离心10min。 吸出HSC细胞，用滴管吸取纯净的星形细胞。在星形细胞中加入培养液使总体积约20ml， 1000×g离心10min，以去除percoll。分离出来的原代HSC用于提取蛋白和RNA。
实施例4 VA-Liposome-siRNAggpps-FAM混合物的制备
1)脂质体准备：用ddH2O将冻干的脂质体配置成1mM(DC-16-4)母液，边加ddH2O 边震荡。
2)VA-coupled liposomes的准备：25℃下，在1.5mL eppendorf管中将200nmol维生 素A(DMSO配制)通过vertex混合到100nmol DC-16-4中。
3)VA-coupled liposomes carrying siRNAggpps(VA-lip-siRNAggpps)准备：将siRNAggpps溶液(580pmol/ml in ddH2O)加入到the VA-coupled liposome溶液中，25℃。其中siRNA和DC-16-4的比例为1:11.5(mol/mol)，siRNA和liposome的比例(wt/wt)为 1:1。
4)混合液用micropartition system(VIVASPIN 2concentrator 30000MWCO PES，VIVASCIENCE)去除掉未与脂质体结合的vitamin A和siRNA。micropartition system(VIVASPIN 2concentrator 30000MWCO PES，VIVASCIENCE)。1500g 25℃离心5min， 共3次。用PBS洗脱一次，并配制成需要的比例。
CCl4诱导12周，将此复合物以0.75mg/kg浓度通过尾静脉注射到小鼠体内，连续6次注射后检测小鼠肝纤维化的程度以及相关基因的表达。
实施例5肝脏冰冻切片的制备及免疫荧光染色
肝脏组织4％PFA室温固定2h，转移至30％蔗糖4℃脱水过夜，OCT包埋后-20℃切片(8μM)。切片置于-80℃保存。
FAM荧光与α-SMA共染按以下步骤进行：
1)玻片室温干燥，PBS洗5min×2；
2)含0.5％Tween-20和0.5％TritionX-100的PBS室温处理15min；
3)山羊血清室温封闭1h；
4)α-SMA抗体4℃孵育过夜；
5)PBS×3times，每次5min；
6)荧光二抗用山羊血清1：200稀释，室温1h；
7)PBS×3times，每次5min。
8)DAPI(1μg/mL终浓度)室温染15min。
9)PBS×3times，每次5min。
10)90％甘油封片
11)倒置荧光显微镜观察拍照
α-SMA染色显示CCl4诱导的慢性肝损伤模型建模成功。携带FAM的小干扰RNA分布在α-SMA染色阳性的区域，说明利用维生素A将包裹GGPPS siRNA的脂质体靶向到HSC 的行性,具体结果如图4所示。
实施例6天狼猩红染色
石蜡切片进行常规的脱蜡复水；天青石液染5-10min；蒸馏水洗3次；天狼星红饱和苦味 酸浓染15-30min；无水乙醇直接分化和脱水；二甲苯透明；中性树胶封固。天狼星红染色结 果显示，与对照组相比，HSC中GGPPS特异敲降的小鼠肝脏中胶原的积累明显减少具体结 果如图5所示。
SEQUENCE LISTING
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Claims (9)
1.用于调控肝星状细胞活性以预防或治疗化学性肝损伤导致的肝纤维化的物质。
2.根据权利要求1所述的物质，其特征在于，所述物质是香叶基二磷酸合成酶抑制剂。
3.根据权利要求2所述的物质，其特征在于，所述抑制剂是香叶基二磷酸合成酶特异性的干扰质粒。
4.根据权利要求3所述的物质，其特征在于，所述干扰质粒的小干扰RNA的编码序列为：SEQ ID NO:1～2。
5.根据权利要求3或4所述的物质，其特征在于，所述干扰质粒由维生素A偶联的脂质体包裹。
6.根据权利要求5所述的物质，其特征在于，所述干扰质粒靶向干扰肝星状细胞的GGPPS。
7.根据权利要求6所述的物质，其特征在于，所述维生素A脂质体包裹的干扰质粒靶向干扰肝星状细胞的GGPPS，抑制肝星状细胞的活化。
8.根据权利要求6所述的物质，其特征在于，所述维生素A脂质体包裹的干扰质粒靶向干扰肝星状细胞的GGPPS，抑制CCl4化学损伤诱导的肝纤维化。
9.权利要求1～4任一所述的物质在制备预防或治疗化学性肝损伤导致的肝纤维化的药物或药物组合物中的应用。
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