慢病毒介导的星形胶质细胞升高基因-1表达下调对肝星状细胞生物学行为的影响
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摘要
肝纤维化(hepatic fibrosis)是由于各种慢性肝病反复损伤导致的以细胞外基质(extracellular matrix, ECM)过度沉积为特征的病理生理过程，其病因主要包括病毒感染、非酒精或酒精性脂肪性肝炎、免疫损伤和胆汁淤积等。包括肝星状细胞(hepatic stellate cells, HSCs)在内的不同来源的肝脏肌成纤维细胞是此病理生理过程的主要驱动因素。
静态的HSCs富含维生素A，位于肝细胞及窦内皮细胞之间的狄氏间隙。肝脏受到损伤时，细胞内维生素A脂滴消失，静态HSCs转化为肌成纤维样细胞。这一转化过程对肝纤维化的发生、发展至关重要，称作静态HSCs的“激活”。活化的HSCs被赋予了多种能力，包括增殖速度加快、ECM分泌增加、迁移能力提高等。
目前，肝纤维化尚缺乏有效治疗手段。肝纤维化逆转需要纤维瘢痕降解消退，此基于胶原分泌细胞数量减少、能力弱化。已有研究证实活化的HSCs是最主要的促纤维化因素，因此，清除活化的HSCs、抑制其增殖、诱导凋亡、抑制其胶原分泌及迁移能力，是逆转肝纤维化的关键治疗靶点和重要策略。
星型胶质细胞升高基因-1(astrocyte elevated gene-1,AEG-1)，也被称作异粘蛋白(metadherin, MTDH)，于2002年在人类免疫缺陷病毒(human immunodeficiency virus, HIV)及肿瘤坏死因子-α (tumor necrosisfactor, TNF-α)感染的星型胶质细胞中首次被发现。在过去的十余年间，对AEG-1的研究多集中在与肿瘤的关系上，包括乳腺癌、恶性胶质瘤、成神经细胞瘤及肝癌等。研究认为，AEG-1能够促进肿瘤的发生、发展，与肿瘤的预后密切相关，被认为是一种癌基因。例如，AEG-1在肝细胞癌中高表达，且表达量与癌症分期相关，表达越高则预后较差。近年来，随着国内外学者研究的不断深入，发现AEG-1的功能并非只是局限在肿瘤范围，在非肿瘤的生理、病理过程中也发挥着重要作用，如生长发育、炎症、神经退行性变等。这也促进人们对此基因功能谱的认识及内在机制的研究日益深入。
AEG-1可通过多条信号转导通路发挥作用，最重要的有磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase，PI3K)/Akt、核因子-κB (nuclearfactor-κB, NF-κB)、细胞外信号调节激酶(extracellular regulated kinase,ERK)等通路，而这些通路在肝纤维化发生、发展中均发挥重要作用。研究证明，在多种细胞中过表达AEG-1可促进细胞的增殖及侵袭、迁移能力，抑制其凋亡。更重要的是，上调AEG-1能促进细胞的炎症反应及血管新生能力，而这些也是肝纤维化形成的关键因素。但迄今为止，AEG-1与肝纤维化及HSCs之间的关系尚无报道。
本课题旨在研究AEG-1在肝纤维化形成及活化HSCs中的表达，应用慢病毒介导的RNA干扰(RNAinterference, RNAi)技术下调AEG-1表达后，观察其对HSCs细胞活化、凋亡和迁移等生物学行为的影响，并进一步研究导致此些影响所涉及的信号转导通路。本学位论文由以下四部分组成：
第一部分：星形胶质细胞升高基因-1在肝纤维化大鼠肝组织及活化HSCs中的表达
目的：研究AEG-1在大鼠纤维化肝脏及促纤维化因子刺激后HSCs中的表达。
方法：应用胆总管结扎(bile duct ligation，BDL)及二甲基亚硝胺(dimethyl-nitrosamine，DMN)腹腔注射两种方法建立大鼠肝纤维化模型。BDL模型组结扎胆总管，假手术组只分离胆总管不结扎，均于术后2wk取材。DMN模型组腹腔注射1%DMN，每周连续3d，对照组腹腔注射生理盐水，均于4wk后取材。肝组织石蜡切片经HE与天狼星红染色(Sirius Red)检测肝脏病理变化。免疫组织化学法(immunohistochemistry,IHC)观察AEG-1的表达及定位。Western blot及RT-real time PCR检测AEG-1蛋白及mRNA的表达。采用原位灌流、梯度密度离心技术分离大鼠原代HSCs，倒置荧光显微镜观察刚分离静止的大鼠原代HSCs，应用免疫细胞化学染色技术检测α-SMA表达以鉴定活化的大鼠原代HSCs。应用细菌脂多糖(lipopolysaccharide, LPS:0μg/ml,0.5μg/ml,1.5μg/ml)及转化生长因子-β (transforming growth factor beta, TGF-β:0ng/ml,5ng/ml,10ng/ml)刺激HSC-T624h，应用LPS (1.5μg/ml)或TGF-β (10ng/ml)刺激HSC-T6或原代大鼠HSCs0h,24h或48h，Western blot检测干预前后AEG-1蛋白的表达变化。
结果：①HE与天狼星红染色结果证明BDL和DMN诱导的大鼠肝纤维化模型成功建立。BDL诱导的纤维化模型可见肝脏结构紊乱，大量胶原沉积，胆管明显扩张和增生；DMN诱导的肝纤维化模型除肝脏结构紊乱外，尚可见明显的出血性坏死、肝窦充血及大量炎细胞浸润。②免疫组织化学法染色显示，BDL及DMN诱导的肝纤维化大鼠肝脏细胞AEG-1表达明显升高，主要定位于细胞质，而对照组基本无表达。③Western blot及RT-real time-PCR检测显示，BDL及DMN模型组较对照组AEG-1均明显升高(P<0.05)。④TGF-β (0ng/ml,5ng/ml,10ng/ml)刺激HSC-T624h，5ng/ml刺激组AEG-1蛋白水平无明显升高，10ng/ml组AEG-1蛋白水平明显升高。应用10ng/ml TGF-β刺激HSC-T624h及48h，结果显示，48h刺激组较24h组AEG-1蛋白水平进一步升高(P<0.05)。⑤LPS (0μg/ml,0.5μg/ml,1.5μg/ml)刺激HSC-T624h，0.5μg/ml刺激组AEG-1蛋白水平无明显升高，1.5μg/ml组AEG-1蛋白水平明显升高。应用1.5μg/ml LPS刺激HSC-T624h及48h，结果显示，48h刺激组较24h组AEG-1蛋白水平进一步升高(P<0.05)。⑥采用肝脏原位灌注链酶和胶原酶，以Nycodenz为介质，密度梯度离心一步法成功分离出大鼠原代HSCs。细胞得率为1.0×107至1.5×107/只，细胞存活率和纯度均大于90％。在倒置显微镜下观察刚分离的HSCs呈圆形，胞浆中富含脂滴，在波长328nm的荧光显微镜下观察，刚分离的原代HSCs呈自发蓝色荧光。培养至14d，大鼠HSCs呈活化状态，脂滴消失，变成梭形肌成纤维样细胞。⑦原代HSCs性质鉴定。刚分离的HSCs免疫细胞化学染色α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)表达阴性；原代培养14d后α-SMA阳性染色率达100％。⑧应用10ng/mlTGF-β及1.5μg/ml LPS刺激原代大鼠HSCs24h及48h，刺激24h组AEG-1蛋白表达无明显升高，刺激48h组AEG-1蛋白表达则明显升高(P<0.05)。
结论：AEG-1在大鼠BDL及DMN诱导的肝纤维化组织中明显升高。TGF-β及LPS刺激HSC-T6及原代大鼠HSCs可使AEG-1表达升高，且呈时间、剂量依赖性。
第二部分：星形胶质细胞升高基因-1表达下调对肝星状细胞活化的影响
目的：构建慢病毒介导的短发卡RNA (short hairpin RNA, shRNA)下调AEG-1的表达，观察其对HSC-T6细胞增殖、凋亡及胶原合成的影响。
方法：设计并合成可抑制AEG-1表达的shRNA，根据转染率摸索出适合靶细胞的感染复数(multiplicity of infection, MOI)，转染进入HSC-T6细胞中。利用western blot和RT-real-time PCR方法检测AEG-1蛋白和mRNA表达情况，筛选出一条抑制效率最高的shRNA。成功下调HSC-T6的AEG-1表达后，CCK-8(Cell Counting Kit-8)检测细胞增殖；流式细胞学分析细胞周期变化；western blot和RT-real time PCR方法检测Ⅰ型胶原及α-SMA水平的变化。
结果：①通过预实验摸索出MOI值为20。转染进入细胞后，根据蛋白及mRNA检测结果，所设计的3条shRNA质粒片段均可显著抑制AEG-1蛋白及基因的表达，蛋白干扰的效果分别达到75.22%、60.21%及64.36%，mRNA表达分别下降85.52%、69.96%及71.10%，与空白对照组相比均有显著性差异(P<0.05)。②下调AEG-1表达促进了HSC-T6细胞增殖，培养24h，Lenti-shAEG-1组(1.13±0.13)较Lenti-shCon组(1.57±0.22)明显下降(P<0.05)。培养48h，Lenti-shAEG-1组(1.49±0.19)较Lenti-shCon组(2.12±0.24)进一步下降(P<0.01)。Lenti-shCon组与uninfected组相比均无明显变化。③流式细胞学检测下调AEG-1表达可影响HSC-T6的细胞周期。 Lenti-shAEG-1组(48.67±4.35%)较Lenti-shCon组(29.70±3.08%) G0/G1期细胞比例上升(P<0.05)，而G2/M期细胞比例下降(4.90±1.71vs15.07±3.37, P<0.05)，表明细胞被阻滞在G0/G1期。④下调AEG-1后，HSC-T6的α-SMA的蛋白(0.31±0.11vs0.78±0.12, P<0.05)及mRNA (0.57±0.27vs1.14±0.23, P<0.05)水平较阴性对照组分别下降了60.32%及41.34%。⑤Ⅰ型胶原蛋白(0.36±0.12vs0.73±0.23, P<0.05)及mRNA (0.57±0.11vs0.99±0.11, P<0.05)水平较阴性对照组分别下降了50.72%及42.54%。而两者的阴性对照组较未感染组无明显变化。
结论：成功构建靶向AEG-1的shRNA并下调AEG-1表达。下调AEG-1表达可抑制HSC-T6的活化。
第三部分：星形胶质细胞升高基因-1表达下调诱导肝星状细胞凋亡、抑制迁移
目的：观察shRNA下调AEG-1表达后对HSC-T6细胞的凋亡和迁移的影响。
方法：下调HSC-T6的AEG-1表达后，膜联蛋白(AnnexinV)/藻红蛋白(Phycoerythrin，PE)联合标记流式细胞术检测HSCs凋亡率；末段脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末段标记(terminaldeoxynucleotidy transferrase UTP-nick end labeling，TUNEL)检测HSC-T6凋亡；扫描电镜观察细胞形态；RT-Real time PCR测定caspase-3mRNA的表达；划痕实验及Transwell小室检测细胞迁移。
结果：①下调AEG-1后，HSC-T6的TUNEL染色阳性细胞率较对照组提高了1.93倍(P<0.05)。②流式细胞技术检测显示，下调AEG-1表达后，Lenti-shAEG-1组细胞凋亡率(27.12±5.72%)较Lenti-shCon组(8.88±3.20%)及uninfected (7.97±2.52%)组明显升高(P<0.01)。③AEG-1shRNA干扰HSC-T6细胞后，与阴性对照组相比，细胞体积缩小，细胞表面微绒毛缩短，伪足缩短或消失。④下调AEG-1升高了Caspase-3的mRNA表达，Lenti-shAEG-1组较Lenti-shCon组升高了1.86倍(P<0.05)。⑤划痕愈合实验：划痕后培养24h，Lenti-shAEG-1组划痕愈合率(0.41±0.06)较Lenti-shCon组(0.56±0.05)下降26.8%(P<0.05)。培养48h后，划痕愈合率Lenti-shAEG-1组(0.59±0.13)较Lenti-shCon组(0.88±0.12)下降33.7%(P<0.05)。⑥Transwell assay实验：Tanswell小室中接种细胞后，细胞培养箱培养24h，结果显示，Lenti-shAEG-1组细胞迁移数(18.20±3.44)较Lenti-shCon组(46.60±4.85, P<0.05)降低了71.21%。
结论：下调AEG-1表达后，可以促进HSC-T6的凋亡，抑制其迁移。
第四部分：星形胶质细胞升高基因-1调控肝星状细胞生物学行为的信号转导机制
目的：研究慢病毒介导shRNA下调AEG-1表达对HSC-T6细胞内PI3K/Akt、ERK及P38MAPK信号转导通路的调节作用。
方法：慢病毒介导的shRNA下调HSC-T6的AEG-1表达后，Westernblot检测PI3K/Akt及ERK、P38MAPK磷酸化及总蛋白活化表达情况。
结果：①下调AEG-1表达可以抑制PI3K、Akt水平，而对总蛋白无明显影响。ShAEG-1组与shCON组比较，p-PI3K/PI3K、p-AktThr308/Akt和p-AktSer473/Akt明显下调(P<0.05)。②下调AEG-1表达可以抑制ERK、P38MAPK水平，而对总蛋白无明显影响。ShAEG-1组与shCON组比较，p-ERK/ERK和p-P38/P38明显下调(P<0.05)。
结论：下调AEG-1表达对HSC-T6生物学行为的调控可能是通过抑制PI3K/Akt及ERK、P38的磷酸化实现的。
Hepatic fibrosis, characterized by accumulation of excessiveextracellular matrix (ECM), is a wound-healing response to all chronic liverdiseases including viral infection, non-alcoholic and alcoholic steatohepatitis,immune injury, and others. This process is driven by a heterogeneouspopulation of hepatic myofibroblasts, which mainly derive from hepaticstellate cells (HSCs).
During liver injury of any etiology, quiescent HSCs, which are vitaminA–rich cells residing between hepatocytes and sinusoidal endothelial cells inthe subendothelial space of Disse, lost vitamin A droplets and convert tomyofibroblast-like cells. This orchestrated response to liver Injury, named as“activation”, is the most important event of hepatic fibrosis. These activatedcells are capable to elevate deposition of ECM components, increaseproliferation and enhance migration. Although many potential anti-fibrotictargets have been fixed，there are no effective treatment of hepatic fibrosis upto now. Regression of liver fibrosis is associated with resorption of fibrousscar and the disappearance of collagen-producing myofibroblasts. It isbelieved that HSCs are the major collagen-producing cells of all fibrogenicmyofibroblasts. Clearance of activated HSCs is a key mechanism forachieving resolution of fibrosis and is linked with regression of fibrotictissue. Suppression of activated HSCs proliferation, or induction of apoptosisis considered as a useful strategy for antifibrotic therapy.
Astrocyte elevated gene-1(AEG-1), also called metadherin (MTDH)and lysine-rich CEACAM1coisolated (LYRIC), was initially identified as aHIV-1and tumor necrosis factor-α-inducible gene in primary human fetalastrocytes. Over the past decade, AEG-1emerged as a positive regulator oftumorigenesis and a valuable prognostic marker of a diverse array of cancers, such as breast cancer, malignant glioma, neuroblastoma and liver cancer.AEG-1expression was utilized in stratification of hepatocelluar carcinoma(HCC) and high level of AEG-1is poportional to poor prognosis. Recently,studies increasingly focus on functions of AEG-1beyond cancer, such asdevelopment, inflammation and neurodegeneration.
AEG-1is reported to be a downstream target of Ha-ras and participatesin diverse signaling pathways and interacts with phosphatidylinositol3-linase (PI3K)/Akt, nuclear factor-κB and extracellular signal-regulatedkinase (ERK), which are molecules also involved in liver fibrogenesis.Forced overexpression of AEG-1is demonstrated to increase proliferation,inhibit apoptosis and promote invasion and migration in different types ofcells. It is also noteworthy that up-regulation of AEG-1is associated withenhanced angiogenesis and inflammation, which are key factors of hepaticfibrosis formation. However, the relationship between AEG-1and hepaticfibrogenesis is not known.
This study was performed to evaluate the function of AEG-1in hepaticfibrogenesis. Lentiviral delivery of shRNA was used to obtain stablesilencing of AEG-1in the rat HSC-T6cell line, and meanwhile, the effects ofAEG-1on cell proliferation, apoptosis, migration andphenotype wereexamined, as well as the possibly associated molecular mechanisms. Theexperiments contain four parts, shown as follows：
Part1: The expression of AEG-1in fibrotic rats and HSCs.
Objective: To explore the expression of AEG-1in fibrotic rats inducedby common bile duct ligated (BDL) or dimethylnitrosamine (DMN) andactivated HSCs stimulated by transforming growth factor-β (TGF-β) orlipopolysaccharide (LPS).
Methods: hepatic fibrosis was induced by BDL and intraperitonealinjections of DMN．For the bile duct ligation (BDL) model of liver fibrosis,rats were laparotomized after anesthetized. The common bile duct wasdouble ligated and sectioned before the abdomen closed. The sham operationwas performed similarly without BDL. Rats were sacrificed2weeks after operation. For the DMN model of liver fibrosis, rats were injectedintraperitoneally with DMN, which was diluted1:100in0.15M NaCl, orwith vehicle (NaCl) at a dose of1μL/100g of body weight. The injectionswere given on the first three consecutive days of each week over a period4weeks. Livers were fixed in4%paraformaldehyde, embedded in paraffin andsectioned. Sections were used for hematoxylin and eosin (H&E), Sirius redstaining and immunohistochemistry following standard procedures.Furthermore，the expressions of AEG-1in above-mentioned groups wereexamined by RT-real time PCR and western blot. Primary rat HSCs wereisolated using pronase/collagenase perfusion digestion followed by densitygradient centrifugation. The quiescent HSCs immediately after plating weretested by fluorescence microscope to evaluate the purity of the cultures andα-SMA monoclonal antibody was used to identify the activated primaryHSCs by immunocytochemistry. To investigate the expression of AEG-1inactivated HSCs, we incubated cells with TGF-β and LPS of variousconcentrations (TGF-β:0ng/ml,5ng/ml,10ng/ml; LPS:0μg/ml,0.5μg/ml,1.5μg/ml) and timespans (0h,24h,48h), followed by western blot.
Results:①To study the potential role of AEG-1in hepatic fibrosis, weused two well-established models, BDL and DMN, to induce liver fibrosis.As examined by H&E and Sirius red staining, the livers of BDL rats showedsever distortion of architecture, proliferating bile ductules and collagendeposition. Compared to the characters above, the liver of DMN ratsexhibited more hemorrhagic necrosis, sinusoidal congestion andinflammatory cells. These data confirmed that both fibrotic models weresuccessfully established.②AEG-1expression in fibrotic liver wassignificant increased, while little was detected in liver treated with sham orsaline detected by immunohistochemistry. AEG-1was localizedpredominantly in the cytoplasmic region.③Up-regulation of AEG-1infibrotic liver was also confirmed by RT-real time PCR analysis and westernblot (P<0.05).④The expression of AEG-1in HSC-T6stimulated by TGF-βwas up-regulated in a dose-and time-dependent manner.⑤The expression of AEG-1in HSC-T6stimulated by LPS was up-regulated in a dose-andtime-dependent manner.⑥Primary rat HSCs were isolated successfully bysequential digestion of the liver with pronase and eollagenase, followed bysingle step density gradient centrifugation with Nycodenz. Cell viability andcell purity were greater than90％, with a yield ranging from1.2×107to2.0×107HSCs/rat. The primary HSC immediately after plating is round andin rich of lipid droplet under the inverted microscope, while HSCs show blueunder the ultraviolet light (L=328nm). After14days culture，activated HSCslose retinoid and become fusiform myofibroblast-like cell.⑦To indentify theprimary HSCs identification α-SMA staining was performed at day l(quiescent, α-SMA negative cells)and day14(activated，α-SMA positivecells) by immunocytochemistry．⑧The expressions of AEG-1in primary ratHSCs stimulated by TGF-β (10ng/ml) or LPS (1.5μg/ml) for48h wereup-regulated.
Conclusions: AEG-1expression in fibrotic liver was significantincreased. TGF-β, as well as LPS, induced AEG-1expression of HSCs in adose-and time-dependent manner.
Part2: Knockdown of AEG-1inhibits activation of HSC-T6cells
Objective: To investigate the influences of down-regulation of AEG-1on HSC-T6activation.
Methods: Three shRNA oligonucleotide duplexes targeting rat AEG-1sequence were synthesized and cloned into a lentivirus-based vector carryingthe green fluorescent protein (GFP) gene by GeneChem. Cells wereharvested after infection. Protein and mRNA were detected to determineAEG-1knockdown efficiency and screen for the most efficient shRNAwhich was then used for subsequent experiments. cell count kit-8was usedin cell proliferation assay. The cell cycle was analyzed by flow cytometer.The expression of collagen Ⅰ and α-SMA were examined by RT-real timePCR and western blot after knockdown of AEG-1.
Results:①AEG-1expression in HSC-T6cells infected with sh-AEG-1lentivirus was significantly decreased at both mRNA and protein levels as compared to control cells. The protein interference efficiencies of threeshRNAs were75.22%,60.21%and64.36%respectively. The mRNAinterference efficiencies of three shRNAs were85.52%,69.96%and71.10%respectively.②Knockdown of AEG-1markedly suppressed cell viability ofHSC-T6cells examined by CCK-8.③Cell cycle analysis showed thatLv-shAEG-1infection led to an increase of cells population in G0/G1phase,but a corresponding decrease in G2/M phase, indicating that knockdown ofAEG-1induced cell cycle arrest in the G0/G1phase.④Gene abolishment ofAEG-1by shRNA interference significantly reduced the expression ofcollagen Ⅰ in both protein and mRNA levels (P<0.05).⑤Gene abolishmentof AEG-1by shRNA interference significantly reduced the expression ofα-SMAin both protein and mRNAlevels (P<0.05).
Conclusion: Knockdown of AEG-1inhibits the activation of HSC-T6cells
Part3: Knockdown of AEG-1induces apoptosis and inhibits migrationof HSC-T6
Objectives: To investigate the influences of knockdown of AEG-1oncell apoptosis and migration of HSC-T6.
Methods: AEG-1was knockdown by Lenti-shAEG-1in HSC-T6. Theextent of apoptosis was quantified and visualized by Annexin V-PE/7AADstaining. The TUNEL technique using the In Situ Cell Death Detection kitPOD was performed to measure nuclear DNA fragmentation. Morphologicalexamination of HSC-T6was performed by scanning electron microscopy. Toverify the role of AEG-1on HSC-T6migration, wound-healing assay andtranswell insert chambers assay were performed.
Results:①Knockdown of AEG-1by Lv-shAEG-1infection increasedTUNEL-positive cells distribution at10.43%, as compared to5.01%and5.43%in uninfected and LV-shCon infected cells, respectively.②A flowcytometric analysis also demonstrated that as compared to the LV-shConinfected cells, knockdown of AEG-1in the cells infected with Lv-shAEG-1significantly induced cell apoptosis (7.97±2.52%vs27.12±5.72%, P<0.05). ③Analysis of mRNA showed enhanced Caspase-3expression inLv-shAEG-1infected cells compared with LV-shCon cells.④Knockdown ofAEG-1reduced HSCs cell volume, shortened microvilli on cell surface andvanished dendritic pseudopodia.⑤In the wound-healing assay, the distancemoved by a wounded cell significantly decreased in Lv-shAEG-1group at24h (P<0.05) and even pronounced at48h (P<0.05) after treatment.⑥Inthe transwell insert chambers assay, the number of Lv-shAEG-1infectedcells (18.20±3.44) which penetrated the chamber membrane wassignificantly less than the uninfected cells (58.60±4.82; P<0.05) andLV-shCon infected cells (46.60±4.85; P <0.05).
Conclusion: Knockdown of AEG-1induces apoptosis and reduces cellmigration capacity of HSC-T6
Part4: The mechanism of intracellular signal transduction thatcontribute to the impacts of AEG-1on HSC-T6biologicalbehaviours
Objectives: To explore the regulation of AEG-1knockdown onPI3K/Akt, ERK and P38MAPK signalling pathways in HSC-T6．
Methods: AEG-1was knockdown by Lenti-shAEG-1in HSC-T6. Thephosphorylated and totle protein expressions of PI3K/Akt, ERK and P38MAPK were examined by Western blot.
Results:①Knockdown of AEG-1in HSC-T6cells inhibitedphosphorylation of PI3-K and Akt (P<0.05), while total expression of PI3-Kand Akt were unchanged.②Knockdown of AEG-1in HSC-T6cellsinhibited phosphorylation of ERK and P38MAPK (P<0.05), while totalexpression of ERK and P38MAPK were unchanged.
Conclusion: Knockdown of AEG-1changed HSC-T6biologicalbehaviours probably via down-regulation of phosphorylations of PI3-K, Akt,ERK and P38MAPK signaling pathways
静态的HSCs富含维生素A，位于肝细胞及窦内皮细胞之间的狄氏间隙。肝脏受到损伤时，细胞内维生素A脂滴消失，静态HSCs转化为肌成纤维样细胞。这一转化过程对肝纤维化的发生、发展至关重要，称作静态HSCs的“激活”。活化的HSCs被赋予了多种能力，包括增殖速度加快、ECM分泌增加、迁移能力提高等。
目前，肝纤维化尚缺乏有效治疗手段。肝纤维化逆转需要纤维瘢痕降解消退，此基于胶原分泌细胞数量减少、能力弱化。已有研究证实活化的HSCs是最主要的促纤维化因素，因此，清除活化的HSCs、抑制其增殖、诱导凋亡、抑制其胶原分泌及迁移能力，是逆转肝纤维化的关键治疗靶点和重要策略。
星型胶质细胞升高基因-1(astrocyte elevated gene-1,AEG-1)，也被称作异粘蛋白(metadherin, MTDH)，于2002年在人类免疫缺陷病毒(human immunodeficiency virus, HIV)及肿瘤坏死因子-α (tumor necrosisfactor, TNF-α)感染的星型胶质细胞中首次被发现。在过去的十余年间，对AEG-1的研究多集中在与肿瘤的关系上，包括乳腺癌、恶性胶质瘤、成神经细胞瘤及肝癌等。研究认为，AEG-1能够促进肿瘤的发生、发展，与肿瘤的预后密切相关，被认为是一种癌基因。例如，AEG-1在肝细胞癌中高表达，且表达量与癌症分期相关，表达越高则预后较差。近年来，随着国内外学者研究的不断深入，发现AEG-1的功能并非只是局限在肿瘤范围，在非肿瘤的生理、病理过程中也发挥着重要作用，如生长发育、炎症、神经退行性变等。这也促进人们对此基因功能谱的认识及内在机制的研究日益深入。
AEG-1可通过多条信号转导通路发挥作用，最重要的有磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase，PI3K)/Akt、核因子-κB (nuclearfactor-κB, NF-κB)、细胞外信号调节激酶(extracellular regulated kinase,ERK)等通路，而这些通路在肝纤维化发生、发展中均发挥重要作用。研究证明，在多种细胞中过表达AEG-1可促进细胞的增殖及侵袭、迁移能力，抑制其凋亡。更重要的是，上调AEG-1能促进细胞的炎症反应及血管新生能力，而这些也是肝纤维化形成的关键因素。但迄今为止，AEG-1与肝纤维化及HSCs之间的关系尚无报道。
本课题旨在研究AEG-1在肝纤维化形成及活化HSCs中的表达，应用慢病毒介导的RNA干扰(RNAinterference, RNAi)技术下调AEG-1表达后，观察其对HSCs细胞活化、凋亡和迁移等生物学行为的影响，并进一步研究导致此些影响所涉及的信号转导通路。本学位论文由以下四部分组成：
第一部分：星形胶质细胞升高基因-1在肝纤维化大鼠肝组织及活化HSCs中的表达
目的：研究AEG-1在大鼠纤维化肝脏及促纤维化因子刺激后HSCs中的表达。
方法：应用胆总管结扎(bile duct ligation，BDL)及二甲基亚硝胺(dimethyl-nitrosamine，DMN)腹腔注射两种方法建立大鼠肝纤维化模型。BDL模型组结扎胆总管，假手术组只分离胆总管不结扎，均于术后2wk取材。DMN模型组腹腔注射1%DMN，每周连续3d，对照组腹腔注射生理盐水，均于4wk后取材。肝组织石蜡切片经HE与天狼星红染色(Sirius Red)检测肝脏病理变化。免疫组织化学法(immunohistochemistry,IHC)观察AEG-1的表达及定位。Western blot及RT-real time PCR检测AEG-1蛋白及mRNA的表达。采用原位灌流、梯度密度离心技术分离大鼠原代HSCs，倒置荧光显微镜观察刚分离静止的大鼠原代HSCs，应用免疫细胞化学染色技术检测α-SMA表达以鉴定活化的大鼠原代HSCs。应用细菌脂多糖(lipopolysaccharide, LPS:0μg/ml,0.5μg/ml,1.5μg/ml)及转化生长因子-β (transforming growth factor beta, TGF-β:0ng/ml,5ng/ml,10ng/ml)刺激HSC-T624h，应用LPS (1.5μg/ml)或TGF-β (10ng/ml)刺激HSC-T6或原代大鼠HSCs0h,24h或48h，Western blot检测干预前后AEG-1蛋白的表达变化。
结果：①HE与天狼星红染色结果证明BDL和DMN诱导的大鼠肝纤维化模型成功建立。BDL诱导的纤维化模型可见肝脏结构紊乱，大量胶原沉积，胆管明显扩张和增生；DMN诱导的肝纤维化模型除肝脏结构紊乱外，尚可见明显的出血性坏死、肝窦充血及大量炎细胞浸润。②免疫组织化学法染色显示，BDL及DMN诱导的肝纤维化大鼠肝脏细胞AEG-1表达明显升高，主要定位于细胞质，而对照组基本无表达。③Western blot及RT-real time-PCR检测显示，BDL及DMN模型组较对照组AEG-1均明显升高(P<0.05)。④TGF-β (0ng/ml,5ng/ml,10ng/ml)刺激HSC-T624h，5ng/ml刺激组AEG-1蛋白水平无明显升高，10ng/ml组AEG-1蛋白水平明显升高。应用10ng/ml TGF-β刺激HSC-T624h及48h，结果显示，48h刺激组较24h组AEG-1蛋白水平进一步升高(P<0.05)。⑤LPS (0μg/ml,0.5μg/ml,1.5μg/ml)刺激HSC-T624h，0.5μg/ml刺激组AEG-1蛋白水平无明显升高，1.5μg/ml组AEG-1蛋白水平明显升高。应用1.5μg/ml LPS刺激HSC-T624h及48h，结果显示，48h刺激组较24h组AEG-1蛋白水平进一步升高(P<0.05)。⑥采用肝脏原位灌注链酶和胶原酶，以Nycodenz为介质，密度梯度离心一步法成功分离出大鼠原代HSCs。细胞得率为1.0×107至1.5×107/只，细胞存活率和纯度均大于90％。在倒置显微镜下观察刚分离的HSCs呈圆形，胞浆中富含脂滴，在波长328nm的荧光显微镜下观察，刚分离的原代HSCs呈自发蓝色荧光。培养至14d，大鼠HSCs呈活化状态，脂滴消失，变成梭形肌成纤维样细胞。⑦原代HSCs性质鉴定。刚分离的HSCs免疫细胞化学染色α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)表达阴性；原代培养14d后α-SMA阳性染色率达100％。⑧应用10ng/mlTGF-β及1.5μg/ml LPS刺激原代大鼠HSCs24h及48h，刺激24h组AEG-1蛋白表达无明显升高，刺激48h组AEG-1蛋白表达则明显升高(P<0.05)。
结论：AEG-1在大鼠BDL及DMN诱导的肝纤维化组织中明显升高。TGF-β及LPS刺激HSC-T6及原代大鼠HSCs可使AEG-1表达升高，且呈时间、剂量依赖性。
第二部分：星形胶质细胞升高基因-1表达下调对肝星状细胞活化的影响
目的：构建慢病毒介导的短发卡RNA (short hairpin RNA, shRNA)下调AEG-1的表达，观察其对HSC-T6细胞增殖、凋亡及胶原合成的影响。
方法：设计并合成可抑制AEG-1表达的shRNA，根据转染率摸索出适合靶细胞的感染复数(multiplicity of infection, MOI)，转染进入HSC-T6细胞中。利用western blot和RT-real-time PCR方法检测AEG-1蛋白和mRNA表达情况，筛选出一条抑制效率最高的shRNA。成功下调HSC-T6的AEG-1表达后，CCK-8(Cell Counting Kit-8)检测细胞增殖；流式细胞学分析细胞周期变化；western blot和RT-real time PCR方法检测Ⅰ型胶原及α-SMA水平的变化。
结果：①通过预实验摸索出MOI值为20。转染进入细胞后，根据蛋白及mRNA检测结果，所设计的3条shRNA质粒片段均可显著抑制AEG-1蛋白及基因的表达，蛋白干扰的效果分别达到75.22%、60.21%及64.36%，mRNA表达分别下降85.52%、69.96%及71.10%，与空白对照组相比均有显著性差异(P<0.05)。②下调AEG-1表达促进了HSC-T6细胞增殖，培养24h，Lenti-shAEG-1组(1.13±0.13)较Lenti-shCon组(1.57±0.22)明显下降(P<0.05)。培养48h，Lenti-shAEG-1组(1.49±0.19)较Lenti-shCon组(2.12±0.24)进一步下降(P<0.01)。Lenti-shCon组与uninfected组相比均无明显变化。③流式细胞学检测下调AEG-1表达可影响HSC-T6的细胞周期。 Lenti-shAEG-1组(48.67±4.35%)较Lenti-shCon组(29.70±3.08%) G0/G1期细胞比例上升(P<0.05)，而G2/M期细胞比例下降(4.90±1.71vs15.07±3.37, P<0.05)，表明细胞被阻滞在G0/G1期。④下调AEG-1后，HSC-T6的α-SMA的蛋白(0.31±0.11vs0.78±0.12, P<0.05)及mRNA (0.57±0.27vs1.14±0.23, P<0.05)水平较阴性对照组分别下降了60.32%及41.34%。⑤Ⅰ型胶原蛋白(0.36±0.12vs0.73±0.23, P<0.05)及mRNA (0.57±0.11vs0.99±0.11, P<0.05)水平较阴性对照组分别下降了50.72%及42.54%。而两者的阴性对照组较未感染组无明显变化。
结论：成功构建靶向AEG-1的shRNA并下调AEG-1表达。下调AEG-1表达可抑制HSC-T6的活化。
第三部分：星形胶质细胞升高基因-1表达下调诱导肝星状细胞凋亡、抑制迁移
目的：观察shRNA下调AEG-1表达后对HSC-T6细胞的凋亡和迁移的影响。
方法：下调HSC-T6的AEG-1表达后，膜联蛋白(AnnexinV)/藻红蛋白(Phycoerythrin，PE)联合标记流式细胞术检测HSCs凋亡率；末段脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末段标记(terminaldeoxynucleotidy transferrase UTP-nick end labeling，TUNEL)检测HSC-T6凋亡；扫描电镜观察细胞形态；RT-Real time PCR测定caspase-3mRNA的表达；划痕实验及Transwell小室检测细胞迁移。
结果：①下调AEG-1后，HSC-T6的TUNEL染色阳性细胞率较对照组提高了1.93倍(P<0.05)。②流式细胞技术检测显示，下调AEG-1表达后，Lenti-shAEG-1组细胞凋亡率(27.12±5.72%)较Lenti-shCon组(8.88±3.20%)及uninfected (7.97±2.52%)组明显升高(P<0.01)。③AEG-1shRNA干扰HSC-T6细胞后，与阴性对照组相比，细胞体积缩小，细胞表面微绒毛缩短，伪足缩短或消失。④下调AEG-1升高了Caspase-3的mRNA表达，Lenti-shAEG-1组较Lenti-shCon组升高了1.86倍(P<0.05)。⑤划痕愈合实验：划痕后培养24h，Lenti-shAEG-1组划痕愈合率(0.41±0.06)较Lenti-shCon组(0.56±0.05)下降26.8%(P<0.05)。培养48h后，划痕愈合率Lenti-shAEG-1组(0.59±0.13)较Lenti-shCon组(0.88±0.12)下降33.7%(P<0.05)。⑥Transwell assay实验：Tanswell小室中接种细胞后，细胞培养箱培养24h，结果显示，Lenti-shAEG-1组细胞迁移数(18.20±3.44)较Lenti-shCon组(46.60±4.85, P<0.05)降低了71.21%。
结论：下调AEG-1表达后，可以促进HSC-T6的凋亡，抑制其迁移。
第四部分：星形胶质细胞升高基因-1调控肝星状细胞生物学行为的信号转导机制
目的：研究慢病毒介导shRNA下调AEG-1表达对HSC-T6细胞内PI3K/Akt、ERK及P38MAPK信号转导通路的调节作用。
方法：慢病毒介导的shRNA下调HSC-T6的AEG-1表达后，Westernblot检测PI3K/Akt及ERK、P38MAPK磷酸化及总蛋白活化表达情况。
结果：①下调AEG-1表达可以抑制PI3K、Akt水平，而对总蛋白无明显影响。ShAEG-1组与shCON组比较，p-PI3K/PI3K、p-AktThr308/Akt和p-AktSer473/Akt明显下调(P<0.05)。②下调AEG-1表达可以抑制ERK、P38MAPK水平，而对总蛋白无明显影响。ShAEG-1组与shCON组比较，p-ERK/ERK和p-P38/P38明显下调(P<0.05)。
结论：下调AEG-1表达对HSC-T6生物学行为的调控可能是通过抑制PI3K/Akt及ERK、P38的磷酸化实现的。
Hepatic fibrosis, characterized by accumulation of excessiveextracellular matrix (ECM), is a wound-healing response to all chronic liverdiseases including viral infection, non-alcoholic and alcoholic steatohepatitis,immune injury, and others. This process is driven by a heterogeneouspopulation of hepatic myofibroblasts, which mainly derive from hepaticstellate cells (HSCs).
During liver injury of any etiology, quiescent HSCs, which are vitaminA–rich cells residing between hepatocytes and sinusoidal endothelial cells inthe subendothelial space of Disse, lost vitamin A droplets and convert tomyofibroblast-like cells. This orchestrated response to liver Injury, named as“activation”, is the most important event of hepatic fibrosis. These activatedcells are capable to elevate deposition of ECM components, increaseproliferation and enhance migration. Although many potential anti-fibrotictargets have been fixed，there are no effective treatment of hepatic fibrosis upto now. Regression of liver fibrosis is associated with resorption of fibrousscar and the disappearance of collagen-producing myofibroblasts. It isbelieved that HSCs are the major collagen-producing cells of all fibrogenicmyofibroblasts. Clearance of activated HSCs is a key mechanism forachieving resolution of fibrosis and is linked with regression of fibrotictissue. Suppression of activated HSCs proliferation, or induction of apoptosisis considered as a useful strategy for antifibrotic therapy.
Astrocyte elevated gene-1(AEG-1), also called metadherin (MTDH)and lysine-rich CEACAM1coisolated (LYRIC), was initially identified as aHIV-1and tumor necrosis factor-α-inducible gene in primary human fetalastrocytes. Over the past decade, AEG-1emerged as a positive regulator oftumorigenesis and a valuable prognostic marker of a diverse array of cancers, such as breast cancer, malignant glioma, neuroblastoma and liver cancer.AEG-1expression was utilized in stratification of hepatocelluar carcinoma(HCC) and high level of AEG-1is poportional to poor prognosis. Recently,studies increasingly focus on functions of AEG-1beyond cancer, such asdevelopment, inflammation and neurodegeneration.
AEG-1is reported to be a downstream target of Ha-ras and participatesin diverse signaling pathways and interacts with phosphatidylinositol3-linase (PI3K)/Akt, nuclear factor-κB and extracellular signal-regulatedkinase (ERK), which are molecules also involved in liver fibrogenesis.Forced overexpression of AEG-1is demonstrated to increase proliferation,inhibit apoptosis and promote invasion and migration in different types ofcells. It is also noteworthy that up-regulation of AEG-1is associated withenhanced angiogenesis and inflammation, which are key factors of hepaticfibrosis formation. However, the relationship between AEG-1and hepaticfibrogenesis is not known.
This study was performed to evaluate the function of AEG-1in hepaticfibrogenesis. Lentiviral delivery of shRNA was used to obtain stablesilencing of AEG-1in the rat HSC-T6cell line, and meanwhile, the effects ofAEG-1on cell proliferation, apoptosis, migration andphenotype wereexamined, as well as the possibly associated molecular mechanisms. Theexperiments contain four parts, shown as follows：
Part1: The expression of AEG-1in fibrotic rats and HSCs.
Objective: To explore the expression of AEG-1in fibrotic rats inducedby common bile duct ligated (BDL) or dimethylnitrosamine (DMN) andactivated HSCs stimulated by transforming growth factor-β (TGF-β) orlipopolysaccharide (LPS).
Methods: hepatic fibrosis was induced by BDL and intraperitonealinjections of DMN．For the bile duct ligation (BDL) model of liver fibrosis,rats were laparotomized after anesthetized. The common bile duct wasdouble ligated and sectioned before the abdomen closed. The sham operationwas performed similarly without BDL. Rats were sacrificed2weeks after operation. For the DMN model of liver fibrosis, rats were injectedintraperitoneally with DMN, which was diluted1:100in0.15M NaCl, orwith vehicle (NaCl) at a dose of1μL/100g of body weight. The injectionswere given on the first three consecutive days of each week over a period4weeks. Livers were fixed in4%paraformaldehyde, embedded in paraffin andsectioned. Sections were used for hematoxylin and eosin (H&E), Sirius redstaining and immunohistochemistry following standard procedures.Furthermore，the expressions of AEG-1in above-mentioned groups wereexamined by RT-real time PCR and western blot. Primary rat HSCs wereisolated using pronase/collagenase perfusion digestion followed by densitygradient centrifugation. The quiescent HSCs immediately after plating weretested by fluorescence microscope to evaluate the purity of the cultures andα-SMA monoclonal antibody was used to identify the activated primaryHSCs by immunocytochemistry. To investigate the expression of AEG-1inactivated HSCs, we incubated cells with TGF-β and LPS of variousconcentrations (TGF-β:0ng/ml,5ng/ml,10ng/ml; LPS:0μg/ml,0.5μg/ml,1.5μg/ml) and timespans (0h,24h,48h), followed by western blot.
Results:①To study the potential role of AEG-1in hepatic fibrosis, weused two well-established models, BDL and DMN, to induce liver fibrosis.As examined by H&E and Sirius red staining, the livers of BDL rats showedsever distortion of architecture, proliferating bile ductules and collagendeposition. Compared to the characters above, the liver of DMN ratsexhibited more hemorrhagic necrosis, sinusoidal congestion andinflammatory cells. These data confirmed that both fibrotic models weresuccessfully established.②AEG-1expression in fibrotic liver wassignificant increased, while little was detected in liver treated with sham orsaline detected by immunohistochemistry. AEG-1was localizedpredominantly in the cytoplasmic region.③Up-regulation of AEG-1infibrotic liver was also confirmed by RT-real time PCR analysis and westernblot (P<0.05).④The expression of AEG-1in HSC-T6stimulated by TGF-βwas up-regulated in a dose-and time-dependent manner.⑤The expression of AEG-1in HSC-T6stimulated by LPS was up-regulated in a dose-andtime-dependent manner.⑥Primary rat HSCs were isolated successfully bysequential digestion of the liver with pronase and eollagenase, followed bysingle step density gradient centrifugation with Nycodenz. Cell viability andcell purity were greater than90％, with a yield ranging from1.2×107to2.0×107HSCs/rat. The primary HSC immediately after plating is round andin rich of lipid droplet under the inverted microscope, while HSCs show blueunder the ultraviolet light (L=328nm). After14days culture，activated HSCslose retinoid and become fusiform myofibroblast-like cell.⑦To indentify theprimary HSCs identification α-SMA staining was performed at day l(quiescent, α-SMA negative cells)and day14(activated，α-SMA positivecells) by immunocytochemistry．⑧The expressions of AEG-1in primary ratHSCs stimulated by TGF-β (10ng/ml) or LPS (1.5μg/ml) for48h wereup-regulated.
Conclusions: AEG-1expression in fibrotic liver was significantincreased. TGF-β, as well as LPS, induced AEG-1expression of HSCs in adose-and time-dependent manner.
Part2: Knockdown of AEG-1inhibits activation of HSC-T6cells
Objective: To investigate the influences of down-regulation of AEG-1on HSC-T6activation.
Methods: Three shRNA oligonucleotide duplexes targeting rat AEG-1sequence were synthesized and cloned into a lentivirus-based vector carryingthe green fluorescent protein (GFP) gene by GeneChem. Cells wereharvested after infection. Protein and mRNA were detected to determineAEG-1knockdown efficiency and screen for the most efficient shRNAwhich was then used for subsequent experiments. cell count kit-8was usedin cell proliferation assay. The cell cycle was analyzed by flow cytometer.The expression of collagen Ⅰ and α-SMA were examined by RT-real timePCR and western blot after knockdown of AEG-1.
Results:①AEG-1expression in HSC-T6cells infected with sh-AEG-1lentivirus was significantly decreased at both mRNA and protein levels as compared to control cells. The protein interference efficiencies of threeshRNAs were75.22%,60.21%and64.36%respectively. The mRNAinterference efficiencies of three shRNAs were85.52%,69.96%and71.10%respectively.②Knockdown of AEG-1markedly suppressed cell viability ofHSC-T6cells examined by CCK-8.③Cell cycle analysis showed thatLv-shAEG-1infection led to an increase of cells population in G0/G1phase,but a corresponding decrease in G2/M phase, indicating that knockdown ofAEG-1induced cell cycle arrest in the G0/G1phase.④Gene abolishment ofAEG-1by shRNA interference significantly reduced the expression ofcollagen Ⅰ in both protein and mRNA levels (P<0.05).⑤Gene abolishmentof AEG-1by shRNA interference significantly reduced the expression ofα-SMAin both protein and mRNAlevels (P<0.05).
Conclusion: Knockdown of AEG-1inhibits the activation of HSC-T6cells
Part3: Knockdown of AEG-1induces apoptosis and inhibits migrationof HSC-T6
Objectives: To investigate the influences of knockdown of AEG-1oncell apoptosis and migration of HSC-T6.
Methods: AEG-1was knockdown by Lenti-shAEG-1in HSC-T6. Theextent of apoptosis was quantified and visualized by Annexin V-PE/7AADstaining. The TUNEL technique using the In Situ Cell Death Detection kitPOD was performed to measure nuclear DNA fragmentation. Morphologicalexamination of HSC-T6was performed by scanning electron microscopy. Toverify the role of AEG-1on HSC-T6migration, wound-healing assay andtranswell insert chambers assay were performed.
Results:①Knockdown of AEG-1by Lv-shAEG-1infection increasedTUNEL-positive cells distribution at10.43%, as compared to5.01%and5.43%in uninfected and LV-shCon infected cells, respectively.②A flowcytometric analysis also demonstrated that as compared to the LV-shConinfected cells, knockdown of AEG-1in the cells infected with Lv-shAEG-1significantly induced cell apoptosis (7.97±2.52%vs27.12±5.72%, P<0.05). ③Analysis of mRNA showed enhanced Caspase-3expression inLv-shAEG-1infected cells compared with LV-shCon cells.④Knockdown ofAEG-1reduced HSCs cell volume, shortened microvilli on cell surface andvanished dendritic pseudopodia.⑤In the wound-healing assay, the distancemoved by a wounded cell significantly decreased in Lv-shAEG-1group at24h (P<0.05) and even pronounced at48h (P<0.05) after treatment.⑥Inthe transwell insert chambers assay, the number of Lv-shAEG-1infectedcells (18.20±3.44) which penetrated the chamber membrane wassignificantly less than the uninfected cells (58.60±4.82; P<0.05) andLV-shCon infected cells (46.60±4.85; P <0.05).
Conclusion: Knockdown of AEG-1induces apoptosis and reduces cellmigration capacity of HSC-T6
Part4: The mechanism of intracellular signal transduction thatcontribute to the impacts of AEG-1on HSC-T6biologicalbehaviours
Objectives: To explore the regulation of AEG-1knockdown onPI3K/Akt, ERK and P38MAPK signalling pathways in HSC-T6．
Methods: AEG-1was knockdown by Lenti-shAEG-1in HSC-T6. Thephosphorylated and totle protein expressions of PI3K/Akt, ERK and P38MAPK were examined by Western blot.
Results:①Knockdown of AEG-1in HSC-T6cells inhibitedphosphorylation of PI3-K and Akt (P<0.05), while total expression of PI3-Kand Akt were unchanged.②Knockdown of AEG-1in HSC-T6cellsinhibited phosphorylation of ERK and P38MAPK (P<0.05), while totalexpression of ERK and P38MAPK were unchanged.
Conclusion: Knockdown of AEG-1changed HSC-T6biologicalbehaviours probably via down-regulation of phosphorylations of PI3-K, Akt,ERK and P38MAPK signaling pathways
引文
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