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肝纤维化(HF)是一种创伤愈合反应,其特征是细胞外基质(ECM)在肝内的过度沉积,导致肝功能丧失和肝脏结构破坏[1]。在正常肝脏中,肝星状细胞(HSC)是位于肝脏窦状隙的DISE腔内,负责储存维生素A。当肝脏受到损伤刺激时,HSC从静止状态转化为具有增殖、类维生素A消失、趋化性、收缩性、纤维生成等特性的肌成纤维细胞(MFs),参与肝纤维化进程[2-4]。
核糖体蛋白S5(RPS5)是核糖体40S小亚基的组成成分,是核糖体生物发生所必需的。越来越多的证据表明,一些核糖体蛋白除了参与蛋白质加工合成之外,还具有许多核糖体外功能[5-6]。例如,RPS5基因对于胚胎干细胞的分化和类胚体的形成至关重要[7];RPS5的β-发夹结构能够与HCV IRES相互作用,并介导HCV的翻译过程[8];RPS5在结肠癌中异常表达,并可能在癌变过程中作为检查点发挥重要作用[9]。此外,RPS5还参与小鼠红白血病细胞分化,其表达下调会影响细胞周期蛋白依赖激酶-2、-4和-6的蛋白水平,并延迟体外红细胞成熟,引起G1/G0细胞周期停滞[10-11]。
本课题组前期研究发现,在肝纤维化过程中,RPS5在静息和激活的HSC内存在差异表达[12-13]。体外实验证实RPS5参与HSC活化,体内研究表明,RPS5基因敲减可加重肝纤维化,RPS5过表达则减轻肝纤维化[13]。但是,我们并没有研究特异性敲减HSC内RPS5的表达对肝纤维化的影响[14-17]。为此,我们构建了GFa2(GFAP启动子)-驱动shRPS5腺病毒,并研究其靶向敲减HSC的RPS5表达对肝纤维化进展的影响。
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高糖DMEM培养基(Gibco);Western及IP细胞裂解液液(Beyotime);BCA蛋白定量试剂盒(Thermo Fisher Scientific);RPS5抗体(Santa Cruz)、ALB、GAPDH抗体(Cell Signaling Technology)、GFAP、α-SMA、胶原蛋白 I抗体(Abcam);山羊(多克隆)抗兔 IgG(H+L)、山羊(多克隆)抗小鼠 IgG(H+L)(LI-COR Biosciences);Odyssey成像系统(LI-COR Biotechnology);SYBR Premix Ex TaqTM PCR 试剂盒(Takara)。
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雄性Sprague-Dawley大鼠,清洁级,重约200 g,购于上海SLAC实验动物有限公司。
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原代HSC和肝细胞从雄性SD大鼠分离,通过蛋白酶-胶原酶消化,单步Nycodenz梯度纯化[13]。分离的HSC和肝细胞在含有10%胎牛血清(FBS)DMEM培养基,5% CO2、37 ℃培养箱中培养。每隔1天更换1次培养基。通过GFAP免疫染色评估,HSC的纯度为90%~95%。本实验于分离后第3天在转分化的HSC上进行。HSC以感染复数(MOI)50感染腺病毒48 h。
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根据AdEasy™ Adenoviral Vector System(AgilentStratagene, USA)的说明制备质粒 pShuttle-GFa2-shRPS5。将GFAP基因启动子(GFa2)、shRPS5序列和必需元件克隆到无启动子穿梭载体pShuttle中,穿梭载体pShuttle-GFa2驱动shRPS5的表达。重组AdGFa2-shRPS5和AdGFa2-shNC腺病毒在293细胞中扩增,通过氯化铯梯度超速离心纯化。使293细胞通过噬斑测定病毒原液的滴度。shRPS5和shNC序列见表1。
表 1 shRPS5和shNC序列
名称 序列 shRPS5 5′-GCTCATGACTGTACGAATTCTCGAGAATTCGTACAGTCAT GAGC-3′ shNC 5′-GCGCGCTTTGTAGGATTCGCTCGAGCGAATCCTACAAA GCGCGC-3′ -
将HSC细胞中的培养基吸弃,用PBS洗涤1次,加入Western及IP细胞裂解液冰上裂解20 min。通过BCA法测量蛋白浓度后将蛋白样品在95 ℃下加热5 min。制胶上样,将20 μg样品进行10% SDS-PAGE电泳,结束后将蛋白质转移到NC膜上。5%脱脂奶粉封闭1 h。分别加入RPS5、ALB、GAPDH、GFAP、α-SMA和I型胶原的单克隆抗体4 ℃孵育过夜,用PBST洗膜3次,每次10 min。山羊(多克隆)抗兔IgG(H+L)或山羊(多克隆)抗小鼠IgG(H+L)室温孵育2 h,PBST洗膜3次,每次10 min。Odyssey红外扫描成像系统对NC膜扫描,观察相关蛋白的表达情况。
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用Trizol试剂盒提取总RNA。用SYBR Premix Ex TaqTM PCR试剂盒,合成cDNA,定量PCR检测基因mRNA表达。以管家基因β-肌动蛋白(β-actin)作为内部对照,并将基因特异性mRNA表达与β-actin表达进行归一化。引物序列见表2。
表 2 实时qPCR引物序列
名称 正向引物序列 反向引物序列 β-actin 5′-CCAT TGAACACGGCATTGTC-3′ 5′-TCATAGATGGGCACAC AGTG-3′ RPS5 5′-AAATGTGCAGGTGTTGACCA-3′ 5′-CACGCTCCTCCTGAAGAATC-3′ α-SMA 5′-CCGAGATCTCACCGACTACC-3′ 5′-TCCA GAGCGACATAGCACAG-3′ collagen I 5′-CCGTGACCTCAAGATG TGCC-3′ 5′-GCTCATACCTTCGCTT CCAA-3′ -
用二甲基亚硝胺(DMN)和胆管结扎术(BDL)的方法建立大鼠肝纤维化模型[13],尾静脉注射腺病毒(4×109pfu)特异性敲减肝内HSC的RPS5水平。
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肝组织切片HE染色分析病理改变情况;羟脯氨酸(羟脯氨酸检测试剂盒)含量测定、切片天狼星红和 Masson染色评价胶原沉积情况;免疫组织化学染色检测α-SMA和RPS5的表达情况。
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数据分析通过SPSS 21统计软件进行,采用t检验分析,实验结果用均数±标准差(
$\bar x $ ±s)表示。与对照组比较,P<0.05 则认为差异具有统计学意义。
Effects of specific knockdown of ribosomal protein S5 in hepatic stellate cells on liver fibrosis
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摘要:
目的 观察特异性敲减肝星状细胞(HSC)内核糖体蛋白S5(RPS5)对大鼠肝纤维化的影响。 方法 构建胶质纤维酸性蛋白(GFAP)启动子驱动的RPS5 shRNA腺病毒,分别用AdGFa2-shRPS5及其对照AdGFa2 shNC转染大鼠原代HSC和肝细胞,通过蛋白印迹法和实时PCR测定RPS5、α-SMA和I型胶原表达情况;采用二甲基亚硝胺(DMN)和胆管结扎术(BDL)的方法建立大鼠肝纤维化模型,尾静脉注射腺病毒特异性敲减肝内HSC的RPS5水平。肝组织切片HE染色分析病理改变情况;羟脯氨酸含量测定、切片天狼星红和 Masson染色评价胶原沉积情况;免疫组织化学染色检测α-SMA和RPS5的表达情况。 结果 AdGFa2-shRPS5能够特异性敲减HSC中的RPS5表达水平,增加α-SMA和I型胶原在体外表达。体内研究结果表明,在两种慢性肝损伤动物模型中,特异性敲减HSC中RPS5表达水平能够促进HSC活化,增加细胞外基质的沉积,促进肝纤维化。 结论 RPS5对HSC激活和肝纤维化发生至关重要,可能是治疗肝纤维化的潜在靶点。 Abstract:Objective To observe the effect of specific knockdown of hepatic stellate cells (HSC) ribosomal protein S5 (RPS5) on liver fibrosis in rats. Methods The glial fibrillary acidic protein (GFAP) promoter-driven RPS5 shRNA adenovirus was established, and AdGFa2-shRPS5 and its control AdGFa2 shNC were used to transfect primary rat HSCs and hepatocytes, respectively. RPS5 was determined by Western-blot and Real Time PCR, α-SMA and type I collagen expression; the rat liver fibrosis model was established by dimethyl nitrosamine (DMN) and bile duct ligation (BDL), and intrahepatic HSC was specifically knocked down by tail vein injection of adenovirus of RPS5 levels. The pathological changes of liver tissue sections were analyzed by HE staining; the content of hydroxyproline, sections of Sirius red and Masson staining were used to evaluate collagen deposition; immunohistochemical staining was used to detect the expression of α-SMA and RPS5. Results AdGFa2-shRPS5 specifically knocked down the expression level of RPS5 in HSC and increased the expression of α-SMA and type I collagen in vitro. The in vivo results showed that in two animal models of chronic liver injury, specific knockdown of RPS5 expression in HSCs promoted HSC activation, increased the deposition of extracellular matrix, and promoted liver fibrosis. Conclusion RPS5 is essential for HSC activation and liver fibrosis, which could be a potential target for the treatment of liver fibrosis. -
表 1 shRPS5和shNC序列
名称 序列 shRPS5 5′-GCTCATGACTGTACGAATTCTCGAGAATTCGTACAGTCAT GAGC-3′ shNC 5′-GCGCGCTTTGTAGGATTCGCTCGAGCGAATCCTACAAA GCGCGC-3′ 表 2 实时qPCR引物序列
名称 正向引物序列 反向引物序列 β-actin 5′-CCAT TGAACACGGCATTGTC-3′ 5′-TCATAGATGGGCACAC AGTG-3′ RPS5 5′-AAATGTGCAGGTGTTGACCA-3′ 5′-CACGCTCCTCCTGAAGAATC-3′ α-SMA 5′-CCGAGATCTCACCGACTACC-3′ 5′-TCCA GAGCGACATAGCACAG-3′ collagen I 5′-CCGTGACCTCAAGATG TGCC-3′ 5′-GCTCATACCTTCGCTT CCAA-3′ -
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