CN114432334B - lnc-BIHAA1在制备预防和/或治疗肝纤维化药物中的应用 - Google Patents
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Abstract
本发明公开了长链非编码分子lnc‑BIHAA1在制备预防和/或治疗肝纤维化药物中的应用,属于生物医药材料技术领域。lnc‑BIHAA1在激活的HSC细胞中表达显著上调,通过抑制lnc‑BIHAA1可以显著下调肝纤维化标志物的表达水平,还可以联用其他药物,如马洛替酯,协同防治肝纤维化。lnc‑BIHAA1作为干预治疗肝纤维化的新靶点,具有较好的药物开发和应用前景。
Description
技术领域
本发明涉及生物医药材料技术领域,具体涉及一种长链非编码RNA分子lnc-BIHAA1在制备预防和/或治疗肝纤维化药物中的应用。
背景技术
肝纤维化是一种世界范围内高发病率与高死亡率的疾病,与癌症、艾滋病并称“世纪三大顽疾”。肝纤维化以细胞外基质的累积以及癍痕的形成为特征,目前尚无有效治疗肝纤维化的药物获批上市,唯一可行的治疗方法是消除慢性应激和肝移植。肝纤维化如果不及时治疗,可发展为肝硬化,并伴有一系列致命并发症,如功能性肝功能衰竭、门脉高压、腹水、肝性脑病,甚至肝细胞癌,严重威胁患者的健康和生命。
肝损伤发生时,炎症介质可激活肝组织中的肝星状细胞(hepatic stellatecells,HSC),使其向成纤维细胞分化,同时分泌ECM蛋白和基质金属蛋白酶重塑肝脏组织,促进纤维化的发展。可见,HSC活化是肝纤维化发生发展的中心环节,抑制HSC活化是治疗肝纤维化的关键。因此,亟待开发有效的抑制HSC活化的药物和治疗方法。
发明内容
本发明的目的在于提供一种长链非编码RNA分子lnc-BIHAA1在制备预防和/或治疗肝纤维化药物中的新应用。
第一方面,本发明提供了lnc-BIHAA1在制备预防和/或治疗肝纤维化药物中的应用。
优选地,所述lnc-BIHAA1核苷酸序列如SEQ ID NO.1所示。
第二方面,本发明提供一种防治肝纤维化的药物,所述药物包含干扰lnc-BIHAA1表达的si-lnc-BIHAA1。
在本发明的一些实施例中,所述药物还包含药学上可接受的载体。
在本发明的一些实施例中,所述药物还包含联用药物,所述联用药物包含马洛替酯。
第三方面,本发明还提出了一种在动物模型中发掘和研究肝纤维化治疗机制的方法,该方法包含针对lnc-RNA的作用靶点进行高通量测序和/或基因芯片分析。
优选地,所述lnc-RNA选自lnc-BIHAA1。
与现有技术相比,本发明的有益效果为:
本发明首次提出针对lnc-BIHAA1的干预治疗可作为防治肝纤维化的新策略,发现lnc-BIHAA1在激活的HSC细胞中表达显著上调,抑制lnc-BIHAA1可以显著下调肝纤维化标志物的表达水平。实验结果证实,lnc-BIHAA1是一种调控HSC激活的关键分子,是一种治疗肝纤维化的潜在靶点。lnc-BIHAA1的发现,有利于针对lnc-BIHAA1设计治疗肝纤维化的药物和治疗方法,也有利于在动物模型中发掘和研究肝纤维化的调控机制。
附图说明
图1是实施例3的HSC-T6细胞纤维化标志物的表达水平:其中,A是抑制lnc-BIHAA1后纤维化标志物的qPCR图,B是抑制lnc-BIHAA1后纤维化标志物的WB分析图;
图2是实施例3的HSC-T6细胞纤维化标志物的IHC图:其中左边图是对照组IHC图,右边图是抑制lnc-BIHAA1对HSC-T6细胞纤维化标志物的IHC图;
图3是实施例4检测普通大鼠和肝纤维化大鼠肝脏组织中lnc-BIHAA1的表达水平的qPCR图。
图4是实施例4的肝纤维化大鼠肝组织的病理分析:其中,A是大鼠肝脏组织的HE和Masson染色,B是大鼠肝脏组织中纤维化标志物表达水平的IHC图;
图5是实施例5肝组织纤维化标志物的表达水平图,包括si-NC组、si-lnc-BIHAA1组、马洛替酯组、si-lnc-BIHAA1和马洛替酯联用组。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。本实施例中所使用的材料、试剂等,如无特别说明,为从商业途径得到的试剂和材料。
本实施例中所使用的试剂均为分析纯级试剂,且可从正规渠道商购获得。
实施例1细胞培养、细胞激活
(1)细胞培养
HSC细胞系HSC-T6购买自武汉普诺赛生命科技有限公司。将HSC-T6于高糖(4500mg/L)DMEM培养基中(含10%胎牛血清、100U/mL青霉素和100μg/mL链霉素)培养。
(2)细胞激活
HST-T6可通过在培养基中添加10ng/ml转化生长因子-β1(TGF-β1)孵育24小时进行激活。
实施例2转染构建lnc-BIHAA1抑制的HSC-T6细胞株
在转染前,将HSC-T6(5×105个细胞)接种在六孔板中,将细胞培养基更换为不含血清和抗生素的DMEM。使用Lipofectamine 2000转染试剂(购自Invitrogen)将抑制lnc-BIHAA1的siRNA(si-lnc-BIHAA1)转染到细胞中获得抑制lnc-BIHAA1表达的HSC-T6细胞株。并使用空载体转染无功效siRNA的HSC-T6细胞株作为对照。
各siRNA序列参见表1:
表1 qPCR引物、siRNA序列
实施例3 lnc-BIHAA1对HSC纤维化标志物表达水平影响的实验
1、实验方法
(1)qRT-PCR分析
使用TRIzol试剂(购自Invitrogen)从HSC-T6细胞或肝组织中提取总RNA。使用Prime Script RT试剂盒(购自Takara)合成cDNA。根据制造商的说明,使用Power SYBRGreen PCR Master Mix(购自Bio-Rad)进行基因扩增。在ABI7500实时荧光定量PCR系统上进行qRT-PCR,并使用2-ΔΔCT方法计算相对基因表达。GADPH用作内部对照。各引物序列参见表1。
(2)免疫组化(IHC)分析
标本为细胞爬片时,依次使用4%多聚甲醛固定,0.5%Triton X-100透膜和3%H2O2处理后,在2%山羊血清中封闭;标本为组织石蜡切片时,将切片脱水并置于抗原修复溶液缓冲液(pH 9.0)中,然后在2%正常山羊血清中封闭。然后,标本与MMP2(购自Abcam)、α-SMA(购自Boster)和collagen I(购自Proteintech)等一抗抗体孵育。根据制造商的说明,使用VECTASTIN ABC Elite试剂盒(购自Vector Laboratories)和DAB底物试剂盒(购自Vector Laboratories)处理后检测信号。
(3)Western blot(WB)分析
通过补充有苯甲基磺酰氟的RIPA(购自碧云天)从HSC-T6细胞中提取总蛋白。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离蛋白质,然后转移到PVDF膜上。用5%脱脂奶粉封闭后,将膜与MMP2(1:1000,购自Abcam)、α-SMA(1:1000,购自Boster)、collagen I(1:1000,购自Proteintech)和GAPDH(1:2000,购自Proteintech)等一抗抗体在4℃过夜孵育。第二天,将膜与二抗(1:5000,购自中山生物技术)在室温下孵育1小时。使用增强的化学发光检测系统(GE Healthcare)观察蛋白质。将蛋白质的相对表达标准化为内部对照GAPDH的表达。
2、实验结果
(1)使用qPCR和WB分析对比HSC中纤维化标志物MMP2、α-SMA和collagen I的表达水平,实验结果如图1所示:
图1A-B表明,HSC-T6在激活后,通过对比对照组(TGF-β1+si-NC)和lnc-BIHAA1抑制组(TGF-β1+si-lnc-BIHAA1)各纤维化标志物的表达水平,可以看到干扰lnc-BIHAA1后,HSC-T6的纤维化标志物表达水平普遍下降。
(2)由图2的IHC分析图可知,通过对比对照组(TGF-β1+si-NC)和lnc-BIHAA1抑制组(TGF-β1+si-lnc-BIHAA1)各纤维化标志物的表达水平,可以看到干扰lnc-BIHAA1后,HSC-T6各纤维化标志物染色阳性比例下调,表明HSC-T6的纤维化标志物表达水平普遍下降。
实施例4 lnc-BIHAA1对肝纤维化大鼠影响的实验
选择雄性SD大鼠,体重约130±10g(购自广东省医学实验动物中心)。使用四氯化碳(CCl4;1mL/kg;CCl4:橄榄油,v/v=1:1)在8周内每周两次腹膜内注射诱导肝纤维化。为了研究lnc-BIHAA1对肝纤维化的影响,上述CCl4注射两周后,将干扰lnc-BIHAA1的慢病毒和无功能的对照慢病毒(购自通用生物;6×107TU/只)通过尾静脉注射到大鼠体内。在第11周,收集肝组织通过qPCR分析纤维化标志物的表达水平。
1、大鼠肝脏组织中lnc-BIHAA1的表达水平
使用qPCR检测正常大鼠和肝纤维化大鼠肝脏组织中lnc-BIHAA1的表达水平,实验结果如图3所示。由图3可以看出,在肝纤维化大鼠的肝脏组织中lnc-BIHAA1的表达显著上调,明显高于正常大鼠。
2、大鼠肝组织切片进行IHC分析纤维化标志物的表达水平
对大鼠的肝组织切片分别进行H&E和Masson染色,观察组织形态变化和胶原蛋白含量。检测大鼠组织中纤维化标志物的表达水平,结果如图4所示。H&E染色和Masson染色结果显示,使用慢病毒干扰lnc-BIHAA1的表达后(Hepatic Fibrosis+sh-lnc-BIHAA1),肝脏的病理损伤和纤维化程度有所改善(见图4A)。此外,免疫组化结果显示,使用慢病毒干扰lnc-BIHAA1的表达后(Hepatic Fibrosis+sh-lnc-BIHAA1),肝组织的纤维化标志物的表达显著降低(见图4B)。
实施例5 si-lnc-BIHAA1与马洛替酯(MLT)联用对HSC-T6影响的实验
各组HSC-T6在经过TGF-β1激活后,在添加或不添加10μM MLT(购自selleck)的条件下继续处理48h,然后使用qPCR对比HSC中纤维化标志物MMP2、α-SMA和collagen I的表达水平,实验结果如图5所示。
图5表明:单独马洛替酯处理HSC并无法抑制其纤维化标志物的表达,而在使用si-lnc-BIHAA1下调HSC中lnc-BIHAA1的表达的基础上,添加马洛替酯可进一步提高si-lnc-BIHAA1抑制HSC纤维化的效果,二者在抑制肝纤维化上具有协同作用。
综上可知,使用si-lnc-BIHAA1下调激活的HSC中lnc-BIHAA1的表达可以显著抑制肝纤维化标志物的表达水平,表明lnc-BIHAA1是可以作为治疗肝纤维化的潜在靶点。lnc-BIHAA1的发现,有利于针对lnc-BIHAA1设计治疗肝纤维化的药物和治疗方法,也有利于在动物模型中发掘和研究肝纤维化的调控机制,具有较好的治疗肝纤维化药物开发及应用前景。
尽管已经示出和描述了本说明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本说明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本说明的范围由所附权利要求及其等同物限定。
序列表
<110> 中山大学附属第三医院
<120> lnc-BIHAA1在制备预防和/或治疗肝纤维化药物中的应用
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1775
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggggtcag agcctgcagt accacgtggt ctctgcgctg gcggttcagt accacgtggt 60
ctctgcgctg gtggttcagt accacgtgat ctccgcgctg gtggttcagt aggcttgagc 120
agcactgact actcaccttc tgattttgac ctcccacact ggctcctggg aggatgcgtt 180
acttttcttg agctaggaaa cttgagatgg ctgaattcct tgaccagaag tgcgcccctt 240
cccaatgact cgcaggctcg cagcggggct cggtttcaca cgtcctatga caaaagatac 300
gtcatcaaga ccattaccag cgaggacgtg gcagagatgc acaacattct caagaagtac 360
caccaggtaa cgtcccctta cctctgttga cccttcatgc caaagtgtca tcagggccaa 420
cccttgaact acactcgggc agtgtaacag accacatttg acatgaaggt ctcctccagc 480
ctggggttcc agagtgagtg tggcctgatc cctggccctt aacaagagaa tgcataggct 540
cctatttacg aaatagtcat ctaaattatt taaaataaat ttaatctaat tatgctccta 600
ctgatcccca ttctggttgt aaaaattgct tcgctaaaga tttggcaact ttccagaaga 660
tcccttctgt gccagaagtc agatgttaac cccaagcctg tatctgttta cgctactgag 720
tcccgtgcag ttcccactgt ctggcttttc caggactcac ccgttgccca gttggccttg 780
tagaaaattc tctggtttta gggaattttg tgttggtcac tttctgcccc agttcatccc 840
caaccatggc ggctttgtaa cctcctgggg gaggtgccct cgggagctgc tttctcttcc 900
ctgaaataaa ttccagccta gttttgcatg ctggagcaca gatccacagg tctaactgga 960
tcagtaaaaa agacttctca ctatggagta gcaaggacac caaatgcaga agcaagacag 1020
tcctcagccc agatcccagc cctgtcacct gctgaccccg tggcttctgg attttgagac 1080
ctgtctcctc atttatacaa tggaaagtat ccatacttcc caagtcctgc gaagtcaatg 1140
aggtaacatg tatcaagtcc atagcagagc atctgacatg cactccgtgt gcagcatatg 1200
gaaaagcggt agtactggtg cttgtcatgt aactgaagtt aaacgcctgt cgtgtcacca 1260
taactattaa agtaagtcac cctgagaaat cgtagccttc ttgcacaaat gtcactgctc 1320
atgcagccat tctagtaatt gtcctcaaga gaggtattta tttaaaacac acgtatgcat 1380
gtgtacaata tgcatgtgca caagtgtgta ggtgcatgca cgcgtgcaca cacacacaca 1440
cactcacaca cacacacacg gttgctttca gagtctgtat cagttccctt ggttgtatta 1500
acaaaacaca aactgagtga tttagatagc aaagacattt tgcttgcagt tctagaggct 1560
ttgaatttga gatcaaggaa ccaacatggt agaagatcag agaggactct gcccctgcct 1620
gtgtggttag cgtctcctca cctgggggag acagaaaaaa ctatccttcc tgctaagtgt 1680
aagaatcaca tcagatccac agaaccactt gccacacact ggtgattagg gctttaaatg 1740
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gcgcg 1775
<210> 2
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
cactttctgc cccagttcat 20
<210> 3
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
tcttgcttct gcatttggtg 20
<210> 4
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
accatcggga atgaacgctt 20
<210> 5
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctgtcagcaa tgcctgggta 20
<210> 6
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggagagagca tgaccgatgg 20
<210> 7
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
gggacttctt gaggttgcca 20
<210> 8
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
agctttgatg gcccctatct 20
<210> 9
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggagtgacag gtcccagtgt 20
<210> 10
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 10
gcaagagaga ggccctcag 19
<210> 11
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
tgtgagggag atgctcagtg 20
<210> 12
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
cctgagaaat cgtagcctt 19
<210> 13
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
uucuccgaac gugucacgut t 21
Claims (3)
1.一种防治大鼠肝纤维化的药物,其特征在于,所述药物包含干扰lnc-BIHAA1表达的si-lnc-BIHAA1;所述si-lnc-BIHAA1核苷酸序列如SEQ ID NO.12所示。
2.根据权利要求1所述的药物,其特征在于,还包含药学上可接受的载体。
3.根据权利要求1或2所述的药物,其特征在于,还包含联用药物,所述联用药物为马洛替酯。
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马洛替酯防治大鼠肝纤维化的作用和机制;康毅;王晨晓;罗伟生;黄红;黄旭平;;广西医学(第06期);744-748 * |
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