CN106755090A - 一种以凋亡信号调节激酶1n端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法 - Google Patents
一种以凋亡信号调节激酶1n端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法 Download PDFInfo
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Abstract
本发明公开了凋亡信号调节激酶1（ASK1）的用途，以凋亡信号调节激酶1 N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物。本发明还公开了以凋亡信号调节激酶1 N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物的方法。本发明首次公开了ASK1的N端二聚化对于ASK1的磷酸化激活以及ASK1‑JNK信号通路具有重要意义，对于肝脏细胞脂肪变性具有重要的调控作用，可促进肝脏脂肪变性的发展。以ASK1的N端二聚化为靶点，通过检测候选物质是否抑制ASK1的N端二聚化，可以筛选新型预防、缓解和/或治疗脂肪性肝炎的药物。
Description
技术领域
本发明属于生物技术领域，具体地说，本发明涉及一种以凋亡信号调节激酶1(ASK1,Apoptosis signal-regulated kinase 1)N端二聚化为靶点的筛选模型的建立，并将筛选得到的ASK1N端二聚化抑制剂应用于制备预防、缓解和/或治疗脂肪性肝炎疾病的药物中。
背景技术
凋亡信号调节激酶1(ASK1,Apoptosis signal-regulated kinase 1)是有丝分裂原激活的蛋白激酶激酶激酶(MAPKKK)家族成员之一。ASK1蛋白包含1375个氨基酸残基，分子量约为160kDa[1]。ASK1的蛋白结构分为5部分，其中N端的46-277号氨基酸为硫氧还蛋白(Trx)结合区，297-324号氨基酸为N端卷曲螺旋结构域，384-655号氨基酸为TRAF结合区，687-945号氨基酸为ASK1的激酶结构域，而1239-1295号氨基酸则为C端卷曲螺旋结构域[2]。在正常状态下，ASK1通过C端的卷曲螺旋结构域进行同源寡聚反应，形成分子量约为1500-2000kDa的高分子量复合体，同时Trx结合在ASK1的N端的Trx结合区，形成ASK1信号小体。Trx的特异性结合可抑制TRAF2、TRAF6与ASK1结合，从而抑制ASK1的活性。当受到如TNF-α、LPS、内质网压力、氧化压力等刺激时，Trx迅速被激活并与ASK1分离，TRAF2、TRAF6被募集并结合到ASK1的TRAF结合区上，ASK1的激酶结构域被磷酸化，使ASK1激活，诱导SEKI2-JNK及MKK3/MKK6-p38信号级联反应[3]。ASK1对于多种疾病均具有重要的调控作用，其可促进AngⅡ诱导的心肌肥厚、心脏重构、间质纤维化以及冠状动脉重构等[4]；可促进脑缺血再灌注后神经元-小神经胶质细胞的死亡，从而恶化脑卒中疾病的发展[5]；在缺血再灌注诱导的急性肾损伤模型中，ASK1被显著激活[6]。基于ASK1的激活机制以及其对多种疾病的调控作用，目前已有多种研究致力于研发可调控ASK1信号级联反应的药物，以达到治疗疾病的目的。
肝脏是机体的一个主要的调控糖代谢以及脂肪代谢的组织器官，而以肝细胞脂质聚集以及肝脏脂肪变性为病理表现的脂肪性肝炎，由于其在世界范围内的高发病率，已经成为人类的一个主要的健康问题。其可进一步损害消化系统功能、降低人体免疫力、减弱解毒功能、影响激素代谢等，严重影响了人们的身体健康和生活质量，也给社会带来沉重负担。因此，如何有效治疗脂肪性肝炎，如何高效的筛选能有效治疗脂肪性肝炎这一疾病的药物成为现阶段亟待解决的问题。有研究表明，在高脂饮食诱导的Ⅱ型糖尿病以及肝脏脂肪变性模型中，ASK1起到了明显的促进作用[7]。但ASK1的作用机制还有必要进行进一步的研究，从而找到肝脏脂肪变性调节的良好靶点。
参考文献
[1]Ichijo H,Nishida E,Irie K,et al.Induction of apoptosis by ASK1,amammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways[J].Science,1997,275(5296):90-94.
[2]Fujino G,Noguchi T,Matsuzawa A,et al.Thioredoxin and TRAF familyproteins regulate reactive oxygen species-dependent activation of ASK1through reciprocal modulation of the N-terminal homophilic interaction ofASK1[J].Molecular and cellular biology,2007,27(23):8152-8163.
[3]T.Noguchi,K.Takeda,A.Matsuzawa,K.Saegusa,H.Nakano,J.Gohda,J.Inoue,H.Ichijo,Recruitment of tumor necrosis factor receptorassociated factorfamily proteins to apoptosis signal-regulating kinase 1 signalosome isessential for oxidative stress-induced cell death,J.Biol.Chem.280(2005)37033–37040.
[4]Izumiya Y,Kim S,Izumi Y,et al.Apoptosis signal-regulating kinase 1plays a pivotal role in angiotensin II–induced cardiac hypertrophy andremodeling[J].Circulation Research,2003,93(9):874-883.
[5]Shi Y,Pu H,Hu X,et al.Aberrant Activation of ASK1 MediatesProinflammatory and Neurotoxic Microglial Responses After Cerebral Ischemia/Reperfusion[J].Stroke,2016,47(Suppl 1):A147-A147.
[6]Terada Y,Inoshita S,Kuwana H,et al.Important role of apoptosissignal-regulating kinase 1 in ischemic acute kidney injury[J].Biochemical andbiophysical research communications,2007,364(4):1043-1049.
[7]Yamamoto E,Dong Y F,Kataoka K,et al.Olmesartan preventscardiovascular injury and hepatic steatosis in obesity and diabetes,accompanied by apoptosis signal regulating kinase-1inhibition[J].Hypertension,2008,52(3):573-580.
发明内容
本发明的目的是克服现有技术的缺陷，提供一种凋亡信号调节激酶1的用途，以凋亡信号调节激酶1N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物，所述的药物能够抑制凋亡信号调节激酶1N端二聚化。
在本发明的第二方面，提供一种以凋亡信号调节激酶1N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物的方法，包括如下步骤：
(a)将含有凋亡信号调节激酶1N端的体系和候选物质进行接触；所述的含有凋亡信号调节激酶1N端的体系是含有凋亡信号调节激酶1N端的细胞，或是含含有凋亡信号调节激酶1N端的溶液；
(b)观察候选物质对于凋亡信号调节激酶1N端二聚化的影响；
其中，若所述候选物质可抑制凋亡信号调节激酶1N端二聚化，则表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
在另一优选例中，包括如下步骤：
步骤(a)包括：在测试组中，将候选物质加入到含有凋亡信号调节激酶1N端的体系中；和/或
步骤(b)包括：检测测试组的体系中凋亡信号调节激酶1N端的二聚化作用，并与对照组比较，其中所述的对照组是不添加所述候选物质的含有凋亡信号调节激酶1N端的体系；
其中，如果测试组中检测结果显示凋亡信号调节激酶1N端二聚化受抑制，就表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
在另一优选例中，
步骤(a)包括：
(1)选用CheckMateTM哺乳动物双杂交系统，分别将编码凋亡信号调节激酶1N端1-678aa的DNA连接至编码DNA结合结构域的pBIND载体以及转录激活结构域的pACT载体中，并将两种载体转染至动物细胞中，构建凋亡信号调节激酶1N端二聚化哺乳动物双杂交筛选系统；若凋亡信号调节激酶1的N端正常二聚化，则当上述细胞继续转染入pG5luc载体后，萤火虫荧光素酶报告基因高水平表达；若凋亡信号调节激酶1的N端二聚化被抑制，则pG5luc载体上的萤火虫荧光素酶报告基因不表达；通过Dual-Luciferase双荧光素酶报告基因检测系统可分析凋亡信号调节激酶1的N端二聚化情况；
其中，编码凋亡信号调节激酶1N端1-678aa的氨基酸序列如SEQ NO：1所示，DNA序列如SEQ NO：2所示；
(2)将候选多肽的核苷酸片段分别连接至psi-Flag载体中，将候选多肽质粒psi-flag-Peptides与pG5luc质粒同时转染上述已构建的用于凋亡信号调节激酶1N端二聚化筛选的动物细胞；
步骤(b)包括：转染成功的细胞在培养24小时后检测萤火虫荧光素酶以及Renilla荧光素酶的RLU，并计算二者比值，根据得到的比值来比较不同样品间多肽抑制作用的程度。
在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T。
在另一优选例中，
步骤(a)包括：
(1)分别将权利要求5所述的编码凋亡信号调节激酶1N端1-678氨基酸的DNA连接至pcDNA5-HA以及pcDNA5-Myc质粒中，构建HA-ASK1N和Myc-ASK1N质粒，并分别或同时转染动物细胞；
(2)将候选多肽的DNA分别连接至psi-Flag载体中，将psi-flag-Peptides质粒分别转染至同时含有HA-ASK1N和Myc-ASK1N质粒的动物细胞中；
步骤(b)包括：转染成功的细胞在培养24h后通过免疫共沉淀及Western blot实验检测免疫沉淀反应后的蛋白溶液中HA-ASK1N和Myc-ASK1N的含量；
免疫共沉淀采用anti-HA将目标蛋白与protein A/G琼脂糖珠相连，Western blot采用anti-Myc作为一抗，如果同时转染HA-ASK1N、Myc-ASK1N和某一候选物质的psi-flag-Peptide的动物细胞组与仅转染HA-ASK1N或Myc-ASK1N的对照组相同，未检测到明显Myc蛋白条带，则该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T。
在另一优选例中，还包括对获得的潜在物质进行进一步的细胞实验和/或动物试验，以选出抑制凋亡信号调节激酶1N端二聚化的物质，用于预防、缓解和/或治疗脂肪性肝炎。
本发明利用腺相关病毒AAV8这种肝脏靶向的基因治疗载体，介导筛选到的潜在物质多肽S1在食蟹猕猴肝脏组织中过表达，通过高脂饮食诱导的肥胖模型(diet inducedobesity，DIO)研究多肽S1的功能，结果发现与AAV8-GFP对照组食蟹猕猴相比，AAV8-S1组猕猴体重、BMI指标无显著差异。通过血脂含量测定以及反应肝功能的酶(ALT、AST)活性测定结果表明AAV8-S1组猕猴血清中甘油三脂和低密度胆固醇含量显著降低，高密度胆固醇含量增加，ALT酶活性显著降低；肝脏组织病理染色结果表明AAV8-S1组猕猴肝脏组织中脂质蓄积明显减少，肝脏脂肪变性显著减轻。这表明在猕猴中，多肽S1同样能够抑制脂肪肝的发生。
在本发明的第三方面，提供一种凋亡信号调节激酶1的用途，用于制备抑制肝脏脂肪变性的组合物。
在本发明的第四方面，提供一种体外调节肝脏细胞脂肪变性的方法，所述方法包括：调节细胞内凋亡信号调节激酶1N端二聚化。
本发明的主要优点在于：
本发明人通过对ASK1在高脂饮食诱导的Ⅱ型糖尿病以及肝脏脂肪变性模型中起到的明显的促进作用的机制研究，首次发现ASK1的N端二聚化对于ASK1的磷酸化激活以及ASK1-JNK信号通路具有重要意义，对于肝脏细胞脂肪变性具有重要的调控作用，可促进肝脏脂肪变性的发展。以ASK1的N端二聚化为靶点，通过检测候选物质是否抑制ASK1的N端二聚化，可以筛选新型预防、缓解和/或治疗脂肪性肝炎的药物。
附图说明
图1是萤火虫荧光素酶与Renilla荧光素酶RLU的比值结果；
图2是转染不同质粒的HEK-293T细胞的裂解液以及裂解液经免疫共沉淀后得到的蛋白溶液的Western blot检测结果；
图3是转染不同多肽质粒的L02细胞经PA刺激1h后细胞内ASK1-JNK1信号通路的Western blot检测结果；
图4是转染不同多肽质粒的L02细胞经PA刺激12h后油红O染色图及细胞内甘油三酯含量定量检测结果。
图5是腺相关病毒AAV8介导的食蟹猕猴肝脏中多肽S1过表达效率检测结果
a为生理盐水对照组、AAV8-GFP对照组和AAV8-S1组食蟹猕猴肝脏组织中病毒转染效率结果；b为AAV8-GFP对照组和AAV8-S1组在门静脉注射病毒载体后食蟹猕猴肝脏组织S1多肽表达情况检测结果。
图6是AAV8-GFP和AAV8-S1组食蟹猕猴体重以及BMI指标结果图
a为体重结果图，b为BMI指标结果图。
图7是AAV8-GFP和AAV8-S1组食蟹猕猴血脂含量以及肝功能测定结果图
a为血清甘油三酯含量测定结果图，b为血清总胆固醇含量测定结果图，c为血清高密度胆固醇含量测定结果图，d为血清低密度胆固醇含量测定结果图，e为谷丙转氨酶(ALT)含量测定结果图，f为谷草转氨酶(AST)含量测定结果图(*：p＜0.05vs AAV8-GFP组)。
图8是AAV8-GFP和AAV8-S1组食蟹猕猴肝脏组织HE和油红O染色结果图。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。
实验方法
(1)细胞培养
HEK-293T细胞来源于人胚胎肾脏细胞，培养于DMEM高糖培养基(含10％FBS，1％青霉素-链霉素)中；L02细胞来源于人正常肝细胞，培养于DMEM高糖培养基(含10％FBS，1％青霉素-链霉素)中，保证培养箱内的空气湿度，5％的CO2含量，37℃培养。
(2)载体构建
1)哺乳动物双杂交检测载体pACT-ASK1N和pBIND-ASK1N的构建
利用CheckMateTM哺乳动物双杂交系统(Promega,E2440)，构建ASK1N二聚化检测载体：
①利用上游引物1：5’-CGGGATCCGGATGAGCACGGAGGCGGACGA-3’，下游引物1：5’-TGATGTCATTCTGGTGCTCCTCGCCCTCGC-3’，从ASK1(NCBI：BC088829)基因cDNA中PCR扩增得到ASK1N1片段；
②利用上游引物2：5’-GGAGCACCAGAATGACATCAGGAAAGCTCG-3’，下游引物2：5’-GCTCTAGATCAATCATATTCATAGTCATACTCCAGC-3’，从ASK1(NCBI：BC088829)基因cDNA中PCR扩增得到ASK1N2片段；
③利用上游引物1：5’-CGGGATCCGGATGAGCACGGAGGCGGACGA-3’，下游引物2：5’-GCTCTAGATCAATCATATTCATAGTCATACTCCAGC-3’，从ASK1N1和ASK1N2混合模板中，搭桥PCR扩增得到ASK1N片段；
④pACT载体和pBIND载体分别利用BamH I+Xba I酶切后与扩增得到的目标基因连接，得到pACT-ASK1N和pBIND-ASK1N载体；
2)用于免疫共沉淀验证目标多肽对ASK1N端二聚化的抑制作用的HA-ASK1N和Myc-ASK1N载体的构建
HA-ASK1N载体的构建：利用上游引物：5’-CGGGATCCATGAGCACGGAGGCGGACGA-3’，下游引物：5’-TGCGGCCGCTCAATCATATTCATAGTCATACTCCAGC-3’，扩增ASK1(NCBI：BC088829)基因N端序列，将扩增得到的产物以及pcDNA5-HA载体用限制性内切酶BamHI(NEB，R0136L)和NotI(NEB，R0189L)酶切连接，得到HA-ASK1N载体。
Myc-ASK1N载体的构建：利用上游引物：5’-GAAGATCTATGAGCACGGAGGCGGACGA-3’，下游引物：5’-CATGCCATGGTCAATCATATTCATAGTCATACTCCAGC-3’，扩增ASK1(NCBI：BC088829)基因N端序列，将扩增得到的产物以及pcDNA5-Myc载体用限制性内切酶BgIII(NEB，R0144L)和NcoI(NEB，R0193L)酶切连接，得到Myc-ASK1N载体。
3)构建候选多肽质粒
所用多肽氨基酸序列如下：
Peptide1：
LHNGRSKEQRLKEQLGAQQEPVKKSIQESEAFLPQSIPEERYKMKSKPLGICLIIDCI
Peptide2：
MSAEVIHQVEEALDTDEKEMLLFLCRDVAIDVVPPNVRDLLDILRERGKLSVGDLAEL
Peptide3：
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW
Peptide4：
GVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
①利用上游引物：5’-CGGGATCCCTCCATAATGGGAGAAG-3’，下游引物：5’-GCTCTAGAAATGCAATCGATTATC-3’，从cFLIP(NCBI：NM_003879.5)基因cDNA中PCR扩增得到Peptide1片段；
②利用上游引物：5’-CGGGATCCATGTCTGCTGAAGTC-3’，下游引物：5’-GCTCTAGATCACAGTTCAGCCAAGTC-3’，从cFLIP(NCBI：NM_003879.5)基因cDNA中PCR扩增得到Peptide2片段；
③利用上游引物：5’-CGGGATCCATGGTGAGCAAGGGCG-3’，下游引物：5’-GCTCTAGATCACCAGGGCACGGGCAG-3’，从GFP(NCBI：KX130867.1)基因DNA中PCR扩增得到Peptide3片段；
④利用上游引物：5’-CGGGATCCGGCGTGCAGTGCTTC-3’，下游引物：5’-GCTCTAGATCACTCGATGCGGTTCAC-3’，从GFP(NCBI：KX130867.1)基因DNA中PCR扩增得到Peptide4片段；
⑤psi-Flag载体分别利用BamH I+Xba I酶酶切后与扩增得到的目标基因连接，得到psi-flag-peptides(Flag-peptides)载体。
(3)构建ASK1N二聚化哺乳动物双杂交筛选系统
①将上述构建得到的ASK1N哺乳动物双杂交检测载体在HEK-293T细胞中包装病毒72h，收集细胞培养液，用于感染；
②慢病毒感染前18-24小时，将HEK-293T贴壁细胞铺到6孔板中。使细胞在慢病毒感染时的数量为2×105/孔左右；
③第二天，用含有6μg/ml polybrene的2ml新鲜培养基替换原培养基，加入适量病毒悬液；
④继续培养48小时后，用新鲜培养基替换含有病毒的培养基，转染待筛选多肽质粒。
(4)目标多肽质粒瞬时转染
①按照以下步骤配置转染溶液：
a取0.5μg psi-Flag-Peptides+0.5μg pG5luc质粒加至200μl无血清的DMEM培养基中，涡旋震荡混匀；
b轻微涡旋震荡混匀TurboFect转染试剂，加入2μl至a的培养基中，立即吹打混匀，室温静置20min；
②将上述转染溶液加入至铺有稳定表达ASK1N二聚化哺乳动物双杂交筛选系统的24孔培养皿中，摇晃混匀，37℃培养24h。
(5)Luciferase检测荧光
①裂解细胞：将培养了一段时间后的目标细胞吸尽细胞培养液，直接加入报告基因细胞裂解液；充分裂解后，10,000-15,000g离心3-5min，取上清液用于测定；
②溶解萤火虫荧光素酶检测试剂和Renilla荧光素酶检测缓冲液，并达到室温，将Renilla荧光素酶检测底物(100X)置于冰浴或冰盒上备用；
③按照每个样品需100μl的量，取适量Renilla荧光素酶检测缓冲液，按照1:100加入Renilla荧光素酶检测底物(100X)配制成Renilla荧光素酶检测工作液；
④开启荧光测定仪，将测定间隔设为2s，测定时间设为10s；
⑤每个样品测定时，取样品20-100μl，加入100μ升萤火虫荧光素酶检测试剂，用枪打匀混匀后测定RLU(relative light unit)，以报告基因细胞裂解液为空白对照；
⑥完成测定萤火虫荧光素酶步骤后，加入100μl Renilla荧光素酶检测工作液，用枪打匀混匀后测定RLU(relative light unit)；
⑦在以Renilla荧光素酶为内参的情况下，用萤火虫荧光素酶测定得到的RLU值除以Renilla荧光素酶测定得到的RLU值。根据得到的比值来比较不同样品间多肽抑制作用的程度。
(6)免疫共沉淀
①将上述构建的HA-ASK1N和Myc-ASK1N质粒，分别或同时转染HEK-293T细胞。之后将psi-flag-Peptides质粒分别转染至同时含有HA-ASK1N和Myc-ASK1N质粒的HEK-293T细胞中。
②转染24小时后收获细胞，加入适量细胞裂解缓冲液(含蛋白酶抑制剂)，冰上裂解30min，细胞裂解液于4摄氏度，用最大转速离心30min后取上清液；
③取少量裂解液以备Western blot分析，剩余裂解液加1μg anti-HA抗体(Sigma，#H6908)，4℃缓慢摇晃孵育过夜；
④取10μl protein A/G琼脂糖珠(11719394001and 11719386001,Roche)，用适量裂解缓冲液洗3次，每次3,000rpm离心3min；
⑤将预处理过的10μl protein A/G琼脂糖珠加入到和抗体孵育过夜的细胞裂解液中4℃缓慢摇晃孵育2-4h，使抗体与protein A/G琼脂糖珠偶连；
⑥免疫沉淀反应后，在4℃以3,000rpm速度离心3min，将琼脂糖珠离心至管底；将上清小心吸去，琼脂糖珠用1ml裂解缓冲液洗3-4次；最后加入15μl的2×SDS上样缓冲液，沸水煮5分钟；
⑦SDS-PAGE,Western blot分析。
(7)Western blot
1)蛋白质提取
细胞加入裂解液，运用BCA Protein Assay Kit定量收集蛋白样品。
2)上样与电泳
①将配制好的凝胶板架在电泳槽中，加入电泳内液和电泳外液，每个电泳槽需200ml电泳内液，电泳外液需要灌满电泳槽体积的2/3。
②把蛋白样品上样到SDS-PAGE胶加样孔内，点样完成后开始电泳。
3)转膜
①按要求配制转膜液于4℃预冷。
②按8cm×5.9cm裁剪好PVDF膜并在一角剪个缺口作为膜的左上角，使用前在甲醇中浸泡15秒后放入转膜液中备用。
③将夹板左右摊开，负极向右。黑色的一边为负极，白色为正极，两边各铺上2张海绵和5张滤纸(海绵和滤纸预先用转移缓冲液浸湿)。
④取出凝胶板中的凝胶，去除多余部分，用转膜液洗涤凝胶，将凝胶平铺在负极的滤纸上，赶走气泡，将PVDF膜覆盖其上，剪缺口的地方应与大Marker的一角对齐，赶走气泡后覆盖上左边的滤纸及海绵(不能有气泡)，夹上夹板。
⑤将夹板放入转膜槽中，转膜槽的负极(黑色面)应与夹板的负极(黑色面)放在一起，灌满转膜液以淹没凝胶。
⑥转膜槽接通电源，电压设为250V，电流设为0.2A。开始电泳转移，起始电压应大于100V，如果低于100V，可适当调高电流至所需电压，转移1.5小时。
⑦转移结束后，取出PVDF膜。
4)封闭
把蛋白膜放置到预先准备好的TBS中，洗去膜上的转膜液。蛋白膜放入封闭液中，在摇床上缓慢摇动，室温封闭1-4小时。
5)一抗孵育
①用TBST洗涤蛋白膜3次，每次5分钟。
②封口机将薄膜封入杂交袋中，加上一抗，封口，尽可能不留空气。
③将杂交袋放入4℃摇床中，过夜。
6)二抗孵育
①将薄膜取出用TBST洗涤3次，每次5分钟，回收一抗。
②将膜放入对应的加有二抗的二抗稀释液中，避光孵育1小时。
7)蛋白检测
孵育后用TBST洗涤3次，每次5min。利用Bio-Rad Chemi Doc XRS+凝胶成像系统检测目的条带。
【实施例1】筛选可抑制ASK1的N端二聚化的多肽
稳定表达ASK1N二聚化检测质粒的HEK-293T分为5组，分别标记为A、B、C、D、E，其中A组在目标多肽瞬时转染时只添加pG5luc质粒而不添加psi-flag-Peptide质粒，B、C、D、E组分别用psi-flag-Peptide1、2、3、4质粒与pG5luc质粒转染HEK-293T细胞，即5组分别为：
A:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+pG5luc
B:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide1+pG5luc
C:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide2+pG5luc
D:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide3+pG5luc
E:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide4+pG5luc
添加转染溶液后，细胞于37℃培养24h，之后收集细胞，进行Luciferase荧光检测。
荧光检测结果如图1所示，A组不添加psi-flag-Peptide质粒，ASK1的N端正常二聚化，萤火虫荧光素酶RLU值除以Renilla荧光素酶RLU值约为1.9；当添加psi-flag-Peptide1质粒时(B组)，萤火虫荧光素酶RLU值除以Renilla荧光素酶RLU值相比于无多肽组显著降低，即Peptide1抑制了ASK1的N端二聚化；而当分别添加psi-flag-Peptide 2/3/4(C、D、E组)时，比值结果相比于A组无显著变化，即Peptide2、3、4不影响ASK1的N端二聚化。
【实施例2】免疫共沉淀验证多肽对ASK1的N端二聚化的抑制作用
HEK-293T细胞分为7组，编号为A、B、C、D、E、F、G，A组以及B组细胞分别只转染HA-ASK1N或Myc-ASK1N质粒，C、D、E、F、G组细胞同时转染HA-ASK1N和Myc-ASK1N两种质粒，48小时后筛选阳性细胞。所得细胞如下：
A组：HEK-293T细胞(HA-ASK1N)
B组：HEK-293T细胞(Myc-ASK1N)
C、D、E、F、G组：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)
之后进行目标多肽质粒瞬时转染，A、B、C三组细胞中只加入同等量的不含目标多肽质粒的转染溶液，D、E、F、G组细胞分别加入含psi-flag-Peptide1/psi-flag-Peptide2/psi-flag-Peptide3/psi-flag-Peptide4质粒的转染溶液，即7组分别为：
A：HEK-293T细胞(HA-ASK1N)
B：HEK-293T细胞(Myc-ASK1N)
C：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)
D：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide1
E：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide2
F：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide3
G：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide4
转染24小时后收获细胞，进行免疫共沉淀分析，验证目标多肽对ASK1的N端二聚化的影响。Western blot分析分析中所用一抗信息：HA(Sigam，#H6908)，Myc(MBL，#M192-3)，Flag(Sigma，#F3165)，所需二抗信息：HRP AffiniPure Goat Anti-Rabbit IgG(H+L)(Jackson，#111-035-003)，Biotin AffiniPure Goat Anti-Mouse IgG(H+L)(Abbkine，A21210)。
在免疫共沉淀过程中，protein A/G琼脂糖珠通过HA抗体与细胞裂解液中的HA-ASK1N相结合，若ASK1的N端正常二聚化，则免疫沉淀反应后得到的上样蛋白溶液中含有HA-ASK1N以及Myc-ASK1N，Western blot分析结果可见HA-ASK1N和Myc-ASK1N两条条带；若ASK1的N端二聚化被抑制，则Myc-ASK1N不会被检测到。
Western blotting分析结果如图2所示，细胞裂解液的Western blotting分析显示在B-G组细胞中，Myc-ASK1N均正常表达，且表达量一致；在D-G组细胞中，不同多肽表达含量一致。免疫共沉淀后Western blotting结果表明A组可见清晰HA-ASK1N条带；B组无明显条带；C组可清晰检测到两条条带；E、F、G组的检测结果与C组相同，即ASK1的N端二聚化不被抑制；而在D组中，当含有Peptide1时，Myc-ASK1N的检测条带明显变弱，HA-ASK1N条带强弱不变，这一结果进一步验证了Peptide1可抑制ASK1的N端二聚化。
【实施例3】抑制ASK1的N端二聚化可抑制ASK1-JNK1信号通路
通过Western blot分析来检测ASK1的N端二聚化被抑制后对细胞内JNK1信号通路的影响。所需一抗信息：p-ASK1(Cell Signaling Technology，#3765)，ASK1(GeneTex，#GTX107921)，p-MKK7(Aviva Systems Biology，#OAAF05547)，MKK7(Cell SignalingTechnology，#4172)，p-JNK1(NOVUS，#NB100-82009)，JNK1(Abcam，#ab199380)，Flag(Sigma，#F3165)；所需二抗信息：HRP AffiniPure Goat Anti-Rabbit IgG(H+L)(Jackson,#111-035-003),Biotin AffiniPure Goat Anti-Mouse IgG(H+L)(Abbkine,A21210)。
L02细胞分5组于培养皿中培养，编号为A、B、C、D、E，37℃培养至细胞密度为70％，A组细胞转染psi-flag质粒作为对照，B、C、D、E组分别转染psi-Flag-Peptides1、2、3、4，转染24h后，各组的一部分细胞培养基中加棕榈酸酯(PA)刺激1h，同时各组的另一部分细胞培养基中加入BSA处理1h作为对照。收集细胞，Western blot分析细胞内ASK1-JNK1信号通路中涉及的各蛋白表达含量变化。结果如图3所示，其中Flag蛋白表达含量在各组中无明显变化，说明B、C、D、E 4组在多肽质粒转染后多肽表达量基本一致。与其他组相比，仅B组p-ASK1、p-MKK7、p-JNK1蛋白含量显著降低，即ASK1的N端二聚化被抑制后，会抑制MKK7、JNK1的磷酸化，抑制ASK1-JNK1信号通路。
【实施例4】抑制ASK1的N端二聚化可促进细胞脂质代谢，抑制脂肪性肝炎。
L02细胞分5组于培养皿中培养，编号为A、B、C、D、E，A组细胞转染psi-flag质粒作为对照，B、C、D、E组分别转染psi-flag-Peptides1、2、3、4。转染24h后，各组一部分细胞的培养基中加入PA处理12h，同时另一部分用BSA处理12h作为对照在(Ctl)。之后进行油红O染色，并用甘油三酯(Triglyceride)分析试剂盒(比色法)(Cayman，10010303)检测细胞内甘油三酯的含量，以A组BSA对照组细胞甘油三酯检测值为1，计算其余各组检测结果的相对值，得到各组细胞甘油三酯相对含量。油红O染色步骤如下：
(1)样品组和对照组用1X PBS洗涤2次，加入300μl 3％多聚甲醛固定20min；
(2)加入1x PBS洗涤2次后，加入60％异丙醇漂洗10s；
(3)加入1x PBS洗涤2次，通风橱吹干；
(4)每孔500μl加入油红O染色1h；
(5)加入1x PBS洗涤2次，60％异丙醇进行分选，再加入1x PBS洗2次；镜检，拍照；
结果如图4所示，A为油红O染色图，可见A组大量细胞呈红色，表明PA刺激12h后，细胞内出现明显的脂质沉积；C、D、E组细胞染色结果与A组相似，均呈现大面积红色；而在B组中，当细胞内表达多肽1时，经PA刺激12h后，被油红O染红的细胞较少，且细胞内着色面积较小。B为细胞内相对甘油三酯含量测定结果，经BSA处理的对照组细胞内甘油三酯含量均较低，且5组细胞甘油三酯含量无显著性差异，而经PA处理12h后，相比于各组的对照组，细胞内甘油三酯含量显著增加，但转染Flag-Peptide1的B组细胞内甘油三酯含量的增加程度显著低于其他组。上述结果表明Peptide1可通过抑制ASK1的N端二聚化抑制肝细胞脂肪变性，ASK1的N端二聚化对筛选治疗脂肪性肝炎的药物具有重要意义，其可作为一个新的治疗靶点，应用于脂肪性肝炎疾病的治疗中。
【实施例5】构建多肽S1过表达腺相关病毒载体系统(AAV8-S1/AAV8-GFP)
本实施例所述的多肽S1为实施例1-4筛选出来的Peptide1。
AAV8为一种肝脏靶向的基因治疗载体，本发明中使用AAV8介导S1多肽在食蟹猴肝脏中的过表达，以研究多肽S1过表达对非酒精性脂肪肝的影响。
(1)用引物PX458-HindIII-F(CCCAAGCTTGGTACCACTAGTGTCGACgaattcGGCAGTGGAGAGGG)和PX458-BgLII-R(GGAAGATCTTTACTTGTACAGCTCGTCCATGCC)从质粒PX458(Addgene，48138)中扩增T2A-EGFP片段，连接至使用HindIII(NEB，R0104L)和BgLII(NEB，R0144L)双酶切的AAV载体pAAV-MCS中，构建pAAV-T2A-EGFP载体。为了便于以后使用，再合成MCS-Oligo1-SacI(CtctagactcgagaccggtCTTAAGGCTAGCGATATCGGATCCAAGCTTGGTAC)和MCS-Oligo2-KpnI(CAAGCTTGGATCCGATATCGCTAGCCTTAAGACCGGTCTCGAGTCTAGAGAGCT)，通过变性，融合之后连入经SacI(NEB，R0156L)和KpnI(NEB，R0142L)酶切的上述pAAV-T2A-EGFP载体中，构建pAAV-MCS-T2A-EGFP载体。
(2)利用上游引物：5’-GCTCTAGAgccaccATGCTCCATAATGGGAGAAG-3’，下游引物：5’-CGGGATCCCTTGTCATCGTCGTCCTTGTAATCAATGCAATCGATTATC-3’，从cFLIP(NCBI：NM_003879.5)基因cDNA中PCR扩增得到S1肽段的编码序列；将S1肽段的编码序列用XbaI(NEB，R0145L)和BamHI(NEB，R0136L)双酶切，然后克隆到经同样酶切的pAAV-MCS-T2A-EGFP载体中，得到pAAV-S1-T2A-EGFP载体，此载体中S1片段与EGFP在同一读码框内，在表达S1肽段的同时表达EGFP，便于切片观察。
(3)将三质粒转染系统(pAAV-S1-T2A-EGFP，pAAV-Helper和pAAV-2/8)用PEI(Polysciences cat#24765)共转染至AAV293细胞中，转染72h后收集细胞，超声裂解后去除细胞碎片(AAV8-GFP对照组三质粒转染系统为：pAAV-MCS-T2A-EGFP，pAAV-Helper和pAAV-2/8)。
(4)用氯化铯梯度离心的方法纯化病毒液，用1x PBS+5％Sorbitol-in Slide-A-Lyzer dialysis cassettes透析纯化的病毒液，去除氯化铯，获得腺相关病毒AAV8-S1和AAV8-GFP。
(5)荧光定量PCR检测病毒滴度，具体步骤如下：
a.取10μL纯化后的病毒液，加入100μL DNase裂解液(10mM Tris·Cl,pH 7.5、10mM MgCl2、2mM CaCl2、50U/ml DNase I)，37℃孵育1小时。
b.加入0.5M的EDTA，混合均匀后70℃孵育10分钟，加入终浓度为50μg/ml的蛋白酶K，50摄氏度过夜孵育。
c.在99℃下孵育10分钟，彻底灭活蛋白酶K。加入灭菌双蒸水至1ml，取出2μL补水至400μL用作荧光定量PCR的模板。
d.按照9.1×1011个1000bp的双链DNA分子计算，将pAAV-S1-T2A-EGFP/pAAV-MCS-T2A-EGFP质粒稀释至5×101至5×108八个浓度梯度，制成标准品。
e.分别取2μL样品和标准品，用GFP特异性的引物Forward 5’-AGCAGCACGACTTCTTCAAGTCC；和Reverse 5’-TGTAGTTGTACTCCAGCTTGTGC做荧光定量PCR。根据标准品指定标准曲线，计算出病毒中的载体拷贝数(病毒滴度)。
【实施例6】腺相关病毒(AAV8-S1)介导的肝脏S1多肽过表达食蟹猕猴模型的构建
本实施例所述的多肽S1为实施例1-4筛选出来的Peptide1。
腺相关病毒载体通过门静脉注射至食蟹猕猴体内，在肝脏中过表达多肽S1。
食蟹猕猴高脂饲料(购自北京华阜康生物科技有限公司，#5043，热量百分比：蛋白质:17.86％；碳水化合物:58.8％；脂肪:22.34％)喂养2天后，随机分为三组(每组6-8只，分别注射AAV8-S1、AAV8-GFP、生理盐水)。门静脉注射需要开腹手术，术前禁食10-14小时，禁水6小时。术前先肌肉注射阿托品0.05mg/kg(减少术中腺体的分泌)，约10分钟后肌肉注射舒眠宁Ⅱ0.1mL/kg。待实验猴麻醉后称量体重，备皮，将猕猴以仰卧位固定于手术台上，连接心电监护仪，监测血氧饱和度、血压、心率，用碘酒消毒手术区域3遍。手术及助手洗手消毒穿手术衣，给实验猴消毒铺巾，准备手术。沿腹中线分层划开皮肤，打开腹腔，找到门静脉后缓慢将含有AAV8-S1病毒载体(滴度5.88E+08Tu/mL，血清型AAV2/8)1mL的缓冲液注射入门静脉内，注射完成后用无菌小纱布块按压止血，检查无出血情况后准备缝合。同时静脉匀速滴注青霉素400万单位，预防感染。分层关闭腹腔，穿好腹带防止手术后切口张力过大和减少腹腔内出血(AAV8-GFP、生理盐水组分别注射同等体积的含同等量AAV8-GFP病毒以及同等体积的生理盐水，其方法与AAV8-S1组相同)。高脂饲养20周后，穿刺活检术取出组织通过免疫荧光以及Western blot检测腺相关病毒介导的多肽S1的过表达。
食蟹猕猴通过高脂饲料#5043饲养30周，并每天供应150g时令水果。所有动物均自由饮水。
【实施例7】食蟹猕猴肝脏过表达多肽S1抑制高脂饲养引起的脂肪肝发生
本实施例所述的多肽S1为实施例1-4筛选出来的Peptide1。
所有实验猴每两周检测生理指标，包括体重，体温，呼吸，心率，血压，腰围，臀围，坐高等指标。每4周进行腹部超声检测。第0周及30周时采集血液5mL分离血清后进行空腹肝脏脂质(甘油三酯、总胆固醇、高密度脂蛋白、低密度脂蛋白)及肝功能(ALT、AST)检测。通过穿刺活检术取出组织通过GFP荧光检测以及Western blot检测腺相关病毒介导的多肽S1的过表达。
1、荧光检测以及肝脏组织Western blot分析
制作肝脏组织冰冻切片，在荧光显微镜下观察GFP的绿色荧光强度，以确定腺相关病毒的感染效率；肝脏组织Western blot分析检测肝脏组织中多肽S1的表达情况。
(1)肝脏组织冰冻切片
将活体穿刺取出的组织置于并动机中包埋，包埋完成后使用冰冻切片机切片(切片厚度5μm)，用载玻片接近组织片，将其粘贴于载玻片上。冰冻切片置于荧光显微镜下观察绿色荧光强度。
(2)Western blot检测多肽S1表达
1)蛋白质提取
①于-80℃取出肝脏组织样本，放入干冰中。每个EP管放入3-4颗钢珠，放入干冰中预冷。用眼科剪剪下所需样本放入对应的EP管，称重并记录每个样本的重量。
②裂解液中加入PMSF，混匀，加入相应量的裂解液到样品中，迅速摇匀。
③于-80℃预冷研磨仪适配器中研磨样品，研磨参数设置为30Hz/秒，90秒。
④研磨结束后，冰上放置10分钟，取出钢珠。
⑤超声裂解仪裂解样本5KHz/次，每次1秒，间隔1秒，重复10次。超声完成后冰上放置10分钟。
⑥样本放入4℃预冷的离心机中离心，4℃，12000转/分钟离心30min。
⑦吸取上清转移到新的EP管中，4℃，14000转/分钟离心10min。
⑧吸取上清转移到新的EP管中继续离心，4℃，14000转/分钟离心5min。准确吸取清液并利用BCA Protein Assay Kit(PierecTM，23225)进行蛋白定量。
2)配置SDS-PAGE凝胶并进行上样与电泳
①将配制好的凝胶板架在电泳槽中，加入电泳内液和电泳外液。每个电泳槽需200ml电泳内液。电泳外液需要灌满电泳槽体积的2/3。
②把蛋白样品上样到SDS-PAGE胶加样孔内，点样完成后开始电泳。
3)转膜
①按要求配制转膜液于4℃预冷。
②按8cm×5.9cm裁剪好PVDF膜并在一角剪个缺口作为膜的左上角，使用前在甲醇中浸泡15秒后放入转膜液中备用。
③将夹板左右摊开，负极向右。黑色的一边为负极，白色为正极，两边各铺上2张海绵和5张滤纸(海绵和滤纸预先用转移缓冲液浸湿)。
④取出凝胶板中的凝胶，去除多余部分，用转膜液洗涤凝胶，将凝胶平铺在负极的滤纸上，赶走气泡，将PVDF膜覆盖其上，剪缺口的地方应与大Marker的一角对齐，赶走气泡后覆盖上左边的滤纸及海绵(不能有气泡)，夹上夹板。
⑤将夹板放入转膜槽中，转膜槽的负极(黑色面)应与夹板的负极(黑色面)放在一起，灌满转膜液以淹没凝胶。
⑥转膜槽接通电源，电压设为250V，电流设为0.2A。开始电泳转移，起始电压应大于100V，如果低于100V，可适当调高电流至所需电压，转移1.5小时。
⑦转移结束后，取出PVDF膜。
4)封闭
把蛋白膜放置到预先准备好的TBS中，洗去膜上的转膜液。蛋白膜放入封闭液中，在摇床上缓慢摇动，室温封闭1-4小时。
5)一抗(anti-Flag(Sigma，#F3165))孵育
①用TBST洗涤蛋白膜3次，每次5分钟。
②封口机将薄膜封入杂交袋中，加上一抗，封口，尽可能不留空气。
③将杂交袋放入4℃摇床中，过夜。
6)二抗(Biotin AffiniPure Goat Anti-Mouse IgG(H+L)(Abbkine，A21210))孵育
①将薄膜取出用TBST洗涤3次，每次5分钟，回收一抗。
②将膜放入对应的加有二抗的二抗稀释液中，避光孵育1小时。
7)蛋白检测
孵育后用TBST洗涤3次，每次5min。利用Bio-Rad Chemi Doc XRS+凝胶成像系统检测目的条带。
2、生理生化指标检测
(1)生理指标检测
禁食10-14小时，禁水6小时，盐酸氯胺酮10mg/kg肌肉注射麻醉，检测以上生理指标并记录相关实验数据。
(2)生化指标检测
实验前禁食10-14小时，禁水6小时，盐酸氯胺酮10mg/kg肌肉注射麻醉，静脉采血后收集血液5mL，分离学清后送公司检测(武汉迪安)脂质及肝功能情况。
3、肝脏组织病理染色相关实验
制作冰冻、石蜡切片，并进行HE和油红O染色。
(1)肝脏脱水，透明，浸蜡
活体穿刺取出的肝脏组织标本置于甲醛固定液中固定，取固定好的部分肝叶组织于标记的包埋框内，在小流量流水冲洗30分钟以上。按照以下流程在机器上设置以下程序，①脱水：75％酒精(45分钟)→75％酒精(45分钟)→85％酒精(45分钟)→85％酒精(45分钟)→95％酒精(45分钟)→95％酒精(45分钟)→无水酒精(1小时)→无水酒精(1小时)；②透明：二甲苯(1小时)→二甲苯(1小时)；③浸腊(65℃)：石蜡(1小时)→石蜡(1小时)。待组织冲洗完毕后，将包含组织的包埋框装进机器篮筐内，启动上述程序。上述程序完成后，取出组织包埋框送病理室包埋组织，同时清洗机器备用。
(2)肝脏组织切片
使用切片机切片(切片厚度5μm)。
(3)肝脏组织苏木精-伊红(HE)染色
将肝脏组织石蜡切片放入65℃烘箱(30分钟)→二甲苯中(5分钟×3次)→100％酒精(1分钟)→90％酒精(1分钟)→70％酒精(1分钟)→蒸馏水洗→苏木素(5分钟)→自来水洗去切片上的浮色→1％盐酸酒精(1至3秒)→自来水洗数下→Scott促蓝液(碳酸氢钠0.35g，硫酸镁2g，蒸馏水100mL)(1分钟)→自来水洗数下→伊红(1分钟)→蒸馏水洗去切片上的浮色→70％酒精一下→90％酒精一下→100％酒精(30秒×3次)→二甲苯(2分钟×3次)→在二甲苯未干时封片，拍照。
(4)肝脏组织油红O染色
①将冰冻肝脏组织切片在通风橱中风干30分钟，4％多聚甲醛固定10分钟。置于双蒸水中稍洗10分钟，以除去组织表明的多聚甲醛。
②以60％异丙醇处理1分钟。
③用油红O(公司sigma，货号O0625，浓度0.5克/100mL 100％异丙醇)染色30分钟。
④之后以60％异丙醇漂洗1分钟×3次，直至背景干净。
⑤用Mayer’s苏木素染液(5滴)淡染细胞核。
⑥水漂洗，稀碳酸锂水溶液中促蓝，充分水洗，水洗至细胞核蓝化。
⑦用甘油明胶封片，拍照。
4、检测结果
荧光检测结果如图5a，AAV8-GFP以及AAV8-S1组猕猴肝脏组织在荧光显微镜下均可见明显绿色荧光，且两组之间荧光强度无显著差异，说明AAV8病毒载体在两组猕猴肝脏组织中转染率相同。肝脏组织Western blot检测S1多肽表达结果如图5b所示，可见在AAV8-GFP中未见Western blot条带，即未检测到S1表达，而在AAV8-S1组中可见明显条带，即AAV8-S1门静脉注射组猕猴肝脏中S1多肽表达显著。
猕猴体重及BMI测定结果如图6所示。AAV8-GFP以及AAV8-S1组食蟹猕猴从实验开始直至高脂饲养30周，两组动物体重均无显著差异(图6a)，且其BMI结果也无明显差异(图6b)。
血脂以及ASL、AST检测结果如图7所示。在高脂饲料饲养30周后，AAV8-S1组猕猴血清甘油三酯含量显著低于AAV8-GFP对照组(图7a)；两组动物血清总胆固醇含量无明显差异(图7b)，但AAV8-S1组猕猴血清高密度胆固醇含量高于AAV8-GFP组(图7c)，低密度胆固醇含量低于AAV8-GFP组(图7d)。两组猕猴AST酶含量无显著差异但AAV8-S1组ALT酶含量显著低于AAV8-GFP组(图7e、f)。这些结果表明，多肽S1抑制了高脂饮食引起的肝功能的恶化以及脂肪肝的发生。
肝脏组织病理染色结果如图8所示，高脂饲料饲养30周后的AAV8-GFP组猕猴肝脏切片经HE染色后可见明显空泡化且融合连成片状，肝脏细胞形态被破坏，AAV8-S1组空泡化较AAV8-GFP组明显减轻(图8上)；油红O染色结果可见AAV8-GFP组猕猴的肝门静脉周围呈大面积红色，提示有大量脂质沉积，而AAV8-S1组红色面积显著减少，脂质沉积量降低(图8下)。病理染色结果表明多肽S1过表达显著减轻了食蟹猕猴由高脂饮食导致的肝脏脂肪变性以及肝脏脂质沉积。多肽S1可抑制食蟹猕猴脂肪肝疾病的发生。
上述结果显示，多肽S1的过表达可显著减轻HFD诱导下发生的脂肪肝病变。多肽S1对改善脂肪肝具有显著的作用。多肽S1可有望成为一种治疗脂肪肝的新型药物。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 武汉大学
<120> 一种以凋亡信号调节激酶1
N端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法
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Thr Leu His Phe Gly Lys Leu Asp Phe Gly Glu Thr Thr Val Leu Asp
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cagcgagtag ataatatcga agtcttgaca gcagatattg tcataaatct gttactttcc 960
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ccaacctttg atttggcctc ccatcaccat gtgaagtttc attatgcatt tgcactgaat 1080
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gcagctggac accagtttga atcttccttt gagctccgga aagttggggt gaagctaagt 1380
agtcttcttg gtaaaaaggg aaacttggaa aaactccaga gctactggga agttggattt 1440
tttctggggg ccagcgtcct agccaatgac cacatgagag tcattcaagc atctgaaaag 1500
ctttttaaac tgaagacacc agcatggtac ctcaagtcta ttgtagagac aattttaata 1560
tataagcatt ttgtgaaact gaccacagaa cagcctgtgg ccaagcaaga acttgtggac 1620
ttttggatgg atttcctggt cgaggccaca aagacagatg ttactgtggt taggtttcca 1680
gtattaatat tagaaccaac caaaatctat caaccttctt atttgtctat caacaatgaa 1740
gttgaggaaa agacaatctc tatttggcac gtgcttcctg atgacaagaa aggtatacat 1800
gagtggaatt ttagtgcctc ttctgtcagg ggagtgagta tttctaaatt tgaagaaaga 1860
tgctgctttc tttatgtgct tcacaattct gatgatttcc aaatctattt ctgtacagaa 1920
cttcattgta aaaagttttt tgagatggtg aacaccatta ccgaagagaa ggggagaagc 1980
acagaggaag gagactgtga aagtgacttg ctggagtatg actatgaata tgat 2034
<210> 3
<211> 30
<212> DNA
<213> 人工序列
<400> 3
cgggatccgg atgagcacgg aggcggacga 30
<210> 4
<211> 30
<212> DNA
<213> 人工序列
<400> 4
tgatgtcatt ctggtgctcc tcgccctcgc 30
<210> 5
<211> 30
<212> DNA
<213> 人工序列
<400> 5
ggagcaccag aatgacatca ggaaagctcg 30
<210> 6
<211> 36
<212> DNA
<213> 人工序列
<400> 6
gctctagatc aatcatattc atagtcatac tccagc 36
<210> 7
<211> 30
<212> DNA
<213> 人工序列
<400> 7
cgggatccgg atgagcacgg aggcggacga 30
<210> 8
<211> 36
<212> DNA
<213> 人工序列
<400> 8
gctctagatc aatcatattc atagtcatac tccagc 36
<210> 9
<211> 28
<212> DNA
<213> 人工序列
<400> 9
cgggatccat gagcacggag gcggacga 28
<210> 10
<211> 37
<212> DNA
<213> 人工序列
<400> 10
tgcggccgct caatcatatt catagtcata ctccagc 37
<210> 11
<211> 28
<212> DNA
<213> 人工序列
<400> 11
gaagatctat gagcacggag gcggacga 28
<210> 12
<211> 38
<212> DNA
<213> 人工序列
<400> 12
catgccatgg tcaatcatat tcatagtcat actccagc 38
<210> 13
<211> 58
<212> PRT
<213> 人类
<400> 13
Leu His Asn Gly Arg Ser Lys Glu Gln Arg Leu Lys Glu Gln Leu Gly
1 5 10 15
Ala Gln Gln Glu Pro Val Lys Lys Ser Ile Gln Glu Ser Glu Ala Phe
20 25 30
Leu Pro Gln Ser Ile Pro Glu Glu Arg Tyr Lys Met Lys Ser Lys Pro
35 40 45
Leu Gly Ile Cys Leu Ile Ile Asp Cys Ile
50 55
<210> 14
<211> 58
<212> PRT
<213> 人类
<400> 14
Met Ser Ala Glu Val Ile His Gln Val Glu Glu Ala Leu Asp Thr Asp
1 5 10 15
Glu Lys Glu Met Leu Leu Phe Leu Cys Arg Asp Val Ala Ile Asp Val
20 25 30
Val Pro Pro Asn Val Arg Asp Leu Leu Asp Ile Leu Arg Glu Arg Gly
35 40 45
Lys Leu Ser Val Gly Asp Leu Ala Glu Leu
50 55
<210> 15
<211> 58
<212> PRT
<213> 人类
<400> 15
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
50 55
<210> 16
<211> 58
<212> PRT
<213> 人类
<400> 16
Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp
1 5 10 15
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
20 25 30
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
35 40 45
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
50 55
<210> 17
<211> 25
<212> DNA
<213> 人工序列
<400> 17
cgggatccct ccataatggg agaag 25
<210> 18
<211> 24
<212> DNA
<213> 人工序列
<400> 18
gctctagaaa tgcaatcgat tatc 24
<210> 19
<211> 23
<212> DNA
<213> 人工序列
<400> 19
cgggatccat gtctgctgaa gtc 23
<210> 20
<211> 26
<212> DNA
<213> 人工序列
<400> 20
gctctagatc acagttcagc caagtc 26
<210> 21
<211> 24
<212> DNA
<213> 人工序列
<400> 21
cgggatccat ggtgagcaag ggcg 24
<210> 22
<211> 26
<212> DNA
<213> 人工序列
<400> 22
gctctagatc accagggcac gggcag 26
<210> 23
<211> 23
<212> DNA
<213> 人工序列
<400> 23
cgggatccgg cgtgcagtgc ttc 23
<210> 24
<211> 26
<212> DNA
<213> 人工序列
<400> 24
gctctagatc actcgatgcg gttcac 26
Claims (13)
1.一种凋亡信号调节激酶1的用途，其特征在于，以凋亡信号调节激酶1 N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物。
2.如权利要求1所述的用途，其特征在于，所述的药物能够抑制凋亡信号调节激酶1 N端二聚化。
3.一种以凋亡信号调节激酶1 N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物的方法，其特征在于，所述方法包括步骤：
（a）将含有凋亡信号调节激酶1 N端的体系和候选物质进行接触；所述的含有凋亡信号调节激酶1 N端的体系是含有凋亡信号调节激酶1 N端的细胞，或是含含有凋亡信号调节激酶1 N端的溶液；
（b）观察候选物质对于凋亡信号调节激酶1 N端二聚化的影响；
其中，若所述候选物质可抑制凋亡信号调节激酶1 N端二聚化，则表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
4.如权利要求3所述的方法，其特征在于，
步骤（a）包括：在测试组中，将候选物质加入到含有凋亡信号调节激酶1 N端的体系中；和/或
步骤（b）包括：检测测试组的体系中凋亡信号调节激酶1 N端的二聚化作用，并与对照组比较，其中所述的对照组是不添加所述候选物质的含有凋亡信号调节激酶1 N端的体系；
其中，如果测试组中检测结果显示凋亡信号调节激酶1 N端二聚化受抑制，就表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
5.如权利要求3所述的方法，其特征在于，
步骤（a）包括：
（1）选用CheckMate™ 哺乳动物双杂交系统，分别将编码凋亡信号调节激酶1N端1-678号氨基酸的DNA连接至编码DNA结合结构域的pBIND载体以及转录激活结构域的pACT载体中，并将两种载体转染至动物细胞中，构建凋亡信号调节激酶1 N端二聚化哺乳动物双杂交筛选系统；若凋亡信号调节激酶1的N端正常二聚化，则当上述细胞继续转染入pG5luc载体后，萤火虫荧光素酶报告基因高水平表达；若凋亡信号调节激酶1的N端二聚化被抑制，则pG5luc载体上的萤火虫荧光素酶报告基因不表达；通过Dual-Luciferase双荧光素酶报告基因检测系统可分析凋亡信号调节激酶1的N端二聚化情况；
其中，编码凋亡信号调节激酶1N端1-678号氨基酸的氨基酸序列如SEQ NO：1所示，核苷酸序列如SEQ NO：2所示；
（2）将候选短肽的核苷酸片段分别连接至psi-Flag载体中，将候选短肽质粒psi-flag-Peptides与pG5luc质粒同时转染上述已构建的用于凋亡信号调节激酶1 N端二聚化筛选的动物细胞；
步骤（b）包括：转染成功的细胞在培养24小时后检测萤火虫荧光素酶以及Renilla荧光素酶的RLU，并计算二者比值，根据得到的比值来比较不同样品间短肽抑制作用的程度。
6.如权利要求5所述的方法，其特征在于，步骤（a）所用动物细胞选自HEK-293T、L02、Hela、 Huh7、 Hepg2、A549、3T3、MEFs、H9C2。
7.如权利要求6所述的方法，其特征在于，步骤（a）所用动物细胞选自HEK-293T。
8.如权利要求3所述的方法，其特征在于，
步骤（a）包括：
（1）分别将权利要求5所述的编码凋亡信号调节激酶1 N端1-678氨基酸的DNA连接至pcDNA5-HA以及pcDNA5-Myc质粒中，构建HA-ASK1N和Myc-ASK1N质粒，并分别或同时转染动物细胞；
（2）将候选短肽的DNA分别连接至psi-Flag载体中，将psi-flag-Peptides质粒分别转染至同时含有HA-ASK1N和Myc-ASK1N质粒的动物细胞中；
步骤（b）包括：转染成功的细胞在培养24h后通过免疫共沉淀及Western blot实验检测免疫沉淀反应后的蛋白溶液中HA-ASK1N和Myc-ASK1N的含量；
免疫共沉淀采用anti-HA将目标蛋白与protein A/G 琼脂糖珠相连，Western blot采用anti-Myc作为一抗，如果同时转染HA-ASK1N、Myc-ASK1N和某一候选物质的psi-flag-Peptide的动物细胞组与仅转染HA-ASK1N或Myc-ASK1N的对照组相同，未检测到明显Myc蛋白条带，则该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
9.如权利要求8所述的方法，其特征在于，步骤（a）所用动物细胞选自HEK-293T、L02、Hela、 Huh7、 Hepg2、A549、3T3、MEFs、H9C2。
10.如权利要求9所述的方法，其特征在于，步骤（a）所用动物细胞选自HEK-293T。
11.如权利要求3-10所述的方法，其特征在于，所述的方法还包括步骤：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以选出抑制凋亡信号调节激酶1 N端二聚化的物质，用于预防、缓解和/或治疗脂肪性肝炎。
12.一种凋亡信号调节激酶1的用途，其特征在于，用于制备抑制肝脏脂肪变性的组合物。
13.一种体外调节肝脏细胞脂肪变性的方法，其特征在于，所述方法包括：调节细胞内凋亡信号调节激酶1N端二聚化。
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| PCT/CN2017/081936 WO2018094964A1 (zh) | 2016-11-25 | 2017-04-25 | 一种以凋亡信号调节激酶1n端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法 |
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| CN115429879A (zh) * | 2021-06-01 | 2022-12-06 | 中国人民解放军海军军医大学第三附属医院 | 靶向抑制gata3在促进肝脏再生及改善肝损伤中的应用 |
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| CN1164236A (zh) * | 1994-08-24 | 1997-11-05 | 哈佛大学校长及研究员协会 | 鉴别对治疗自身免疫疾病有效的化合物的方法 |
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| CN115429879A (zh) * | 2021-06-01 | 2022-12-06 | 中国人民解放军海军军医大学第三附属医院 | 靶向抑制gata3在促进肝脏再生及改善肝损伤中的应用 |
| CN115429879B (zh) * | 2021-06-01 | 2024-02-13 | 中国人民解放军海军军医大学第三附属医院 | 靶向抑制gata3在促进肝脏再生及改善肝损伤中的应用 |
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