CN113416729A - 一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用 - Google Patents
一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用 Download PDFInfo
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Abstract
本发明属于肝脏疾病技术领域,特别是涉及一组靶向调控甲胎蛋白(AFP)基因的shRNA和cDNA及其应用。本发明提供了一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用。本发明提供的基于该shRNA的重组载体能够沉默甲胎蛋白基因,构建减轻肝损伤、内质网应激和减轻肝细胞坏死的模型。在敲减Afp基因后,会出现小鼠肝损伤及肝内内质网应激加重。在过表达Afp基因后,会出现小鼠肝损伤及肝内内质网应激减轻。表明降低AFP蛋白水平后会加重肝损伤,这说明肝内AFP升高有利于减轻肝损伤,能够应用于制备减轻肝损伤、保护肝脏内质网应激或治疗肝癌的药物。
Description
技术领域
本发明属于肝脏疾病技术领域,特别是涉及一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用。
背景技术
肝脏是体内最大的生物代谢器官,是各种肝炎病毒、多种药物(如抗结核药、化疗药物)、代谢产物(如肠源性内毒素)及多种疾病(如自身免疫性疾病)常侵袭的靶器官,导致肝细胞损伤及肝脏功能异常。因其含有丰富的内质网(ER),在受到刺激时易产生内质网应激(ERS)来促进细胞内环境恢复平衡。ERS可通过影响细胞凋亡及程序性坏死,从而影响肝损伤进程。
现阶段的研究表明,肝损伤会触发肝内甲胎蛋白(AFP)的表达增加,但肝内AFP含量的变化对肝损伤有哪些影响依然不明了。
发明内容
为了解决上述问题,本发明提供了一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用。本发明利用载有本发明提供的靶向敲减甲胎蛋白基因的shRNA的重组载体,实现编码AFP蛋白的基因表达量的敲减,显著降低AFP表达,并加重肝损伤、肝细胞坏死和内质网应激,说明AFP在内质网应激及细胞坏死途径中有保护作用,从而为治疗临床肝脏疾病提供潜在靶点。
为了实现上述目的,本发明提供了如下技术方案:
本发明提供了一种靶向敲减甲胎蛋白基因的shRNA,所述shRNA的核苷酸序列如SEQ ID NO:1所示。
本发明还提供了一种沉默甲胎蛋白基因的重组载体,所述重组载体包括上述shRNA和骨架载体。
优选的,所述骨架载体的类型包括沉默腺相关病毒。
本发明提供了上述shRNA或上述重组载体在构建加重肝损伤的模型中的应用。
本发明还提供了一种甲胎蛋白基因过表达的cDNA,所述cDNA为上述甲胎蛋白基因过表达得到的基因,所述cDNA的核苷酸序列如SEQ ID NO:3所示。
本发明还提供了一种过表达甲胎蛋白基因的重组载体,所述重组载体包括上述的cDNA和骨架载体。
本发明提供了上述cDNA或上述过表达甲胎蛋白基因的重组载体在制备减轻肝损伤的药物中的应用。
本发明提供了上述cDNA或上述过表达甲胎蛋白基因的重组载体在制备保护肝脏内质网应激的药物中的应用。
本发明提供了上述cDNA或上述过表达甲胎蛋白基因的重组载体在制备治疗肝癌的药物中的应用。
本发明提供了一种靶向敲减甲胎蛋白基因的shRNA,所述shRNA的核苷酸序列如SEQ ID NO:1所示。本发明提供的基于该shRNA的重组载体能够沉默甲胎蛋白基因,构建加重肝损伤、加重内质网应激和增加肝细胞坏死的模型。由实施例数据可知,敲减Afp基因后,检测小鼠血清中丙氨酸氨基转移酶(ALT)水平和总胆红素(TBil)水平,均出现了上升现象;肝细胞坏死面积及数量均出现了上升现象;表明降低AFP蛋白水平后会加重肝损伤,说明肝内AFP升高有利于减轻肝损伤,能够应用于制备减轻肝损伤、保护肝脏内质网应激或治疗肝癌的药物。过表达Afp基因,能够有效减轻小鼠肝损伤及肝内内质网应激。
附图说明
图1为本发明提供的重组载体的结构示意图;
图2为对重组载体中插入的shRNA序列进行测序比对鉴定结果;
表1、图3为靶向敲减Afp shRNA小鼠血清中ALT水平变化;
表2、图4为靶向过表达Afp cDNA小鼠血清中ALT水平变化;
表3、图5为靶向敲减Afp shRNA小鼠血清中TBil水平变化;
表4、图6为靶向过表达Afp cDNA小鼠血清中TBil水平变化;
图7为靶向敲减Afp shRNA小鼠肝脏组织的HE染色图;
表6、图8为靶向过表达Afp cDNA小鼠肝脏组织坏死面积的统计结果及HE染色图;
表5、图9为靶向敲减Afp shRNA小鼠肝组织坏死面积的统计结果;
图10为通过免疫印迹法检测靶向敲减Afp shRNA小鼠肝脏中AFP、p-MLKL和caspase-3蛋白的表达;
图11为通过免疫印迹法检测靶向过表达Afp cDNA小鼠肝脏中AFP、p-MLKL和caspase-3蛋白的表达。
具体实施方式
本发明提供了一种靶向敲减甲胎蛋白基因的shRNA,在本发明中,所述shRNA的核苷酸序列如SEQ ID NO:1所示;所述shRNA的靶点的核苷酸序列如SEQ ID NO:2所示。本发明为探究临床肝脏疾病的研究提供了潜在靶点,开拓了肝脏疾病防治药物的新领域。
本发明还提供了一种沉默甲胎蛋白基因的重组载体,所述重组载体包括上述shRNA和骨架载体。在本发明中,所述骨架载体的类型优选包括沉默腺相关病毒,进一步优选为pAAV或携带绿色荧光的腺相关病毒(AAV)载体,在本发明中,所述pAAV骨架载体购自北京合生基因科技有限公司。本发明提供的重组载体具有嗜肝性,能够靶向敲减小鼠肝脏Afp基因,减少AFP表达,能够应用于肝病(包括肝损伤、肝细胞癌)相关的小鼠模型研究中,有利于进一步研究肝内AFP在肝病中的作用。
本发明提供了上述shRNA或上述重组载体在构建加重肝损伤的模型中的应用。在本发明的具体操作中,所述模型的构建方法优选包括以下步骤:将重组载体导入小鼠体内进行预干预,预干预结束后腹腔注射四氯化碳与橄榄油的混合物进行干预;所述导入的次数优选为2次,每次导入的间隔时间优选为36~60h,进一步优选为48h;所述重组载体每次导入小鼠的用量优选为(0.5~1.5)×1010vg/只,进一步优选为1×1010vg/只;所述预干预的时间优选为6周;所述四氯化碳与橄榄油的体积比优选为1:4;所述四氯化碳与橄榄油的混合物的干预时间优选为24h。本发明提供的shRNA及其重组载体,能够应用于肝脏相关疾病的模型研究,为临床肝脏疾病的防治提供新的策略。
本发明还提供了一种甲胎蛋白基因过表达的cDNA,所述cDNA为上述甲胎蛋白基因过表达得到的基因,所述cDNA的核苷酸序列如SEQ ID NO:3所示。
本发明还提供了一种过表达甲胎蛋白基因的重组载体,所述重组载体包括上述的cDNA和骨架载体,即甲胎蛋白重组腺相关病毒血清型8(rAAV8-Afp cDNA)。
本发明提供了上述cDNA或上述过表达甲胎蛋白基因的重组载体在制备减轻肝损伤的药物中的应用。
本发明提供了上述cDNA或上述过表达甲胎蛋白基因的重组载体在制备保护肝脏内质网应激的药物中的应用。
本发明提供了上述cDNA或上述过表达甲胎蛋白基因的重组载体在制备治疗肝癌的药物中的应用。
为了进一步说明本发明,下面结合附图和实施例对本发明提供的一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
1、构建重组载体
(1)构建重组载体:
1)构建沉默甲胎蛋白基因的重组载体,骨架载体为pAAV,骨架信息为pAAV-ITR-hU6-Afp shRNA(mouse,NM_007423)-CAG-EGFP-WPRE-ITR,委托北京合生基因科技有限公司合成,将重组载体命名为rAAV8-Afp shRNA,重组载体结构图谱如图1所示,完成重组载体的构建后,对插入的shRNA序列进行测序比对鉴定结果,鉴定结果如图2所示,由图2可知载体构建成功;
2)构建过表达甲胎蛋白基因的重组载体,骨架载体为pAAV,利用上述方法将骨架载体pAAV与SEQ ID NO:3连接,构建过表达甲胎蛋白基因的重组载体,委托北京合生基因科技有限公司合成,将重组载体命名为rAAV8-Afp cDNA。
(2)构建对照重组载体:
1)构建沉默甲胎蛋白基因的对照重组载体,骨架载体为pAAV,将骨架载体与Control shRNA连接后得到的重组载体命名为rAAV8-control shRNA。Control shRNA的核苷酸序列如SEQ ID NO:4所示;
2)构建过表达甲胎蛋白基因的对照重组载体,骨架载体为pAAV,将重组载体命名为rAAV8-control cDNA。
2、小鼠分组
(1)沉默甲胎蛋白基因实验的分组:
将实验小鼠(BALB/c小鼠,体重为20~25g/只)随机分为4组,每组12只小鼠,并按以下分组给予对应的干预;
1)Control shRNA组:给予rAAV8-control shRNA预干预+橄榄油;
2)Afp shRNA组:给予rAAV8-Afp shRNA预干预+橄榄油;
3)Afp shRNA+四氯化碳(CCl4)组:给予rAAV8-Afp shRNA预干预+CCl4;
4)四氯化碳组:给予rAAV8-control shRNA预干预+CCl4。
(2)过表达甲胎蛋白基因实验的分组:
将上述分组中给予的rAAV8-control shRNA替换为rAAV8-control cDNA,rAAV8-Afp shRNA替换为rAAV8-Afp cDNA,具体分组如下:
1)Control cDNA组:给予rAAV8-control cDNA预干预+橄榄油;
2)Afp cDNA组:给予rAAV8-Afp cDNA预干预+橄榄油;
3)Afp shRNA+四氯化碳(CCl4)组:给予rAAV8-Afp cDNA预干预+CCl4;
4)四氯化碳组:给予rAAV8-control cDNA预干预+CCl4。
3、实验过程
1)进行rAAV8-Afp shRNA/rAAV8-Afp cDNA和rAAV8-control shRNA/rAAV8-control cDNA的转导,具体步骤如下:
通过可视小鼠尾静脉注射固定器固定小鼠,充分固定并暴露小鼠尾静脉,选择尾巴的外侧为常规静脉注射部位,应避开腹侧动脉;注射时针头斜面向上并倾斜30°~45°进针,尽可能由小鼠尾部远端至近端。
75%的酒精消毒尾部静脉注射部位(用纱布热敷局部静脉显露效果更佳),AfpshRNA/Afp cDNA组和Afp shRNA/Afp cDNA+CCl4组,予尾静脉注射rAAV8-Afp shRNA/rAAV8-Afp cDNA(1×1010vg/只),间隔48h重复一次,共2次;Control shRNA/Control cDNA组和CCl4组予尾静脉注射rAAV8-control shRNA/rAAV8-control cDNA(1×1010vg/只),间隔48h重复一次,共2次。
2)当rAAV8-Afp shRNA/rAAV8-Afp cDNA和rAAV8-control shRNA/rAAV8-controlcDNA转导6周后,Afp shRNA/Afp cDNA+CCl4组和CCl4组的小鼠分别予以腹腔注射四氯化碳溶液(四氯化碳与橄榄油以1:4的体积比混合)1mL/kg,Afp shRNA/Afp cDNA组和ControlshRNA/Control cDNA组小鼠分别腹腔注橄榄油0.05mL/10g,分别干预24h。
3)在步骤2)小鼠注射橄榄油或四氯化碳溶液干涉结束后,通过在4L安乐死盒中吸入CO2处死小鼠。小鼠失去知觉后,在维持血液循环的同时收集血液和组织样本进行后续检测。
4、实验结果
1)每只实验小鼠取血1~1.5mL,采用速率法测定血清ALT水平,采用重氮试剂法测定血清TBil水平。测定结果如图3~图6和表1~表4所示。
表1靶向敲减甲胎蛋白小鼠血清ALT水平变化
注:*P<0.05,与Control shRNA组相比;##P<0.01,与CCl4组相比
表2靶向过表达甲胎蛋白小鼠血清ALT水平变化
注:*P<0.05,与Control cDNA组相比;##P<0.01,与CCl4组相比
表3靶向敲减甲胎蛋白小鼠血清TBil水平变化
注:*P<0.05,与Control shRNA组相比;##P<0.01,与CCl4组相比
表4靶向过表达甲胎蛋白小鼠血清TBil水平变化
注:*P<0.05,与Control cDNA组相比;##P<0.01,与CCl4组相比
由表1和图3可知,与CCl4组小鼠相比,Afp shRNA+CCl4组小鼠血清ALT水平显著升高;
由表2和图4可知,与CCl4组小鼠相比,Afp cDNA+CCl4组小鼠血清ALT水平显著降低;
由表3和图5可知,与CCl4组小鼠相比,Afp shRNA+CCl4组小鼠血清TBil水平较对照组明显升高;
由表4和图6可知,与CCl4组小鼠相比,Afp cDNA+CCl4组小鼠血清TBil水平显著降低。
结果表明沉默Afp基因会升高小鼠血清ALT水平和TBil水平并加重肝损伤,过表达Afp基因会降低小鼠血清ALT水平和TBil水平并减轻肝损伤,由此可知,肝内AFP升高有利于减轻肝损伤。
2)取小鼠的肝脏组织0.5cm×0.5cm做病理切片HE染色。借助CaseViewer2.2软件进行观察,选取切片的目的区域进行100倍成像,成像时让组织充满整个视野,保证每张照片的背景光一致,进行坏死面积测量,Afp shRNA/Afp cDNA+CCl4组和CCl4组小鼠肝脏组织的HE染色图如图7和图8所示,肝组织坏死面积的统计结果如表5、表6所示。
表5小鼠肝组织坏死面积百分比(%)
注:##P<0.01,与CCl4组相比
表6小鼠肝组织坏死面积百分比(%)
注:##P<0.01,与CCl4组相比
由表5、图7和图9可知,与CCl4组小鼠相比,Afp shRNA+CCl4组肝细胞坏死亦显著增加(P<0.001);
由表6和图8可知,Afp cDNA+CCl4组肝细胞坏死亦显著下降(P<0.001)。
结果表明靶向敲减Afp的shRNA会显著增加肝细胞坏死;靶向过表达Afp的cDNA肝细胞坏死明显减少,由此可知,肝内AFP升高有利于减轻肝脏细胞坏死。
3)在小鼠肝右叶取出肝组织50mg放入5mL加有50μL PMSF液的免疫沉淀测定裂解缓冲液(成分:Tri-HCL,NACL,NP-40和SDS)中进行匀浆,匀浆结束后用细胞超声破碎仪在4℃下进一步破碎组织至澄清。然后在4℃,12000rpm的条件下离心破碎组织5min,取上清液加入上样缓冲液(5×)(组成:SDS,DTT,溴酚蓝,缓冲盐溶液等,北京索莱宝科技有限公司,货号:P1040)充分混合,体积比为1:4,在100℃的沸水中煮3-5min,待其冷却后放入-20℃冰箱备用。通过免疫印迹法(WesternBlot,WB)检测小鼠肝脏中AFP、p-MLKL和caspase-3蛋白的表达,检测结果如图10和图11所示。
由图10可知,与CCl4组小鼠相比,Afp shRNA+CCl4组显著减少AFP蛋白水平,但是肝内caspase-3和p-MLKL蛋白水平却显著增加(P<0.001);
由图11可知,Afp cDNA+CCl4组则结果相反。现有技术中,casepase-3是细胞凋亡通路的标志性蛋白,p-MLKL的表达可调控细胞程序性坏死。结果表明靶向敲减Afp基因的shRNA会下调AFP蛋白水平,能够加重肝损伤;而靶向过表达Afp基因的cDNA能够保护CCl4诱导的肝损伤,由此可知,肝内AFP升高是肝细胞抗损伤反应的一种表现,AFP有利于减轻肝脏细胞坏死。
由以上实施例可知,本发明公开了小鼠肝脏靶向敲减Afp基因的shRNA及其重组载体、过表达Afp基因的cDNA及其重组载体和在肝损伤的小鼠模型研究中的应用。本发明通过将Afp基因沉默的shRNA导入小鼠体内,实现编码AFP蛋白的基因表达量的敲减,显著降低AFP表达,并加重肝损伤、肝细胞坏死和内质网应激;过表达Afp基因,显著升高AFP表达,并减轻肝损伤、肝细胞坏死和内质网应激,说明AFP在内质网应激及细胞坏死途径中有保护作用,从而为治疗临床肝脏疾病提供潜在靶点。
由上述实验结果可知,本发明提供的过表达基因能够有效上调AFP表达,起到减轻肝损伤的作用。
虽然本发明已以较佳的实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可以做各种改动和修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 遵义医科大学附属医院
<120> 一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用
<160> 4
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gcatccattg caaaggaatt acgaataatt cctttgcaat ggatgc 46
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<213> 人工序列(Artificial Sequence)
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gcatccattg caaaggaatt a 21
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atgaagtgga tcacacccgc ttccctcatc ctcctgctac atttcgctgc gtccaaagca 60
ttgcacgaaa atgagtttgg gatagcttcc tcccagtgcg tgacggagaa gaatgtgctt 120
agcatagcta ccatcacctt tacccagttt gttccggaag ccaccgagga ggaagtgaac 180
aaaatgacta gcgatgtgtt ggctgcaatg aagaaaaact ctggcgatgg gtgtttagaa 240
agccagctat ctgtgtttct ggatgaaatt tgtcatgaga cggaactctc taacaagtat 300
ggactctcag gctgctgcag ccaaagtgga gtggaaagac atcagtgtct gctggcacgc 360
aagaagactg ctccggcctc tgtcccaccc ttccagtttc cagaacctgc cgagagttgc 420
aaagcacatg aagaaaacag ggcagtgttc atgaacaggt tcatctatga agtgtcaagg 480
aggaacccct tcatgtatgc cccagccatt ctgtccttgg ctgctcagta cgacaaggtc 540
gttctggcat gctgcaaagc tgacaacaag gaggagtgct tccagacaaa gagagcatcc 600
attgcaaagg aattaagaga aggaagcatg ttaaatgagc atgtatgttc agtgataaga 660
aaatttggat cccgaaacct ccaggcaaca accattatta agctaagtca aaagttaact 720
gaagcaaatt ttactgagat tcagaagctg gccctggatg tggctcacat ccacgaggag 780
tgttgccaag gaaactcgct ggagtgtctg caggatgggg aaaaagtcat gacatatata 840
tgttctcaac aaaatattct gtcaagcaaa atagcagagt gctgcaaatt acccatgatc 900
caactaggct tctgcataat tcacgcagag aatggcgtca aacctgaagg cttatctcta 960
aatccaagcc agtttttggg agacagaaat tttgcccaat ttcttcagag gaaaaaatca 1020
tgttcatggc aagctttctt catgaatact caagaactca ccccaacctt cctgtctcag 1080
tcattctaag aagctttctt catgaatact caagaactca ccccaacctt cctgtctcag 1140
tcattctaag aattgctaaa acgtaccaga aatattggag aagtgttccc agtctggaaa 1200
tctacctgga tgtcaggaca atctggaaga agaattgcag aaacacatcg aggagagcca 1260
ggcactgtcc aagcaaagct gcgctctcta ccagacctta ggagactaca aattacaaaa 1320
tctgttcctt attggttaca cgaggaaagc ccctcagctg acctcagcag agctgatcga 1380
cctcaccggg aagatggtga gcattgcctc cacgtgctgc cagctcagcg aggagaaatg 1440
gtccggctgt ggtgagggaa tggccgacat tttcattgga catttgtgta taaggaatga 1500
agcaagccct gtgaactctg gtatcagcca ctgctgcaac tcttcgtatt ccaacaggag 1560
gctatgcatc accagttttc tgagggatga aacctatgcc cctcccccat tctctgagga 1620
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<210> 4
<211> 46
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<213> 人工序列(Artificial Sequence)
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aaacgtgaca cgttcggaga acgaattctc cgaacgtgtc acgttt 46
Claims (9)
1.一种靶向敲减甲胎蛋白基因的shRNA,其特征在于,所述shRNA的核苷酸序列如SEQID NO:1所示。
2.一种沉默甲胎蛋白基因的重组载体,其特征在于,所述重组载体包括权利要求1所述的shRNA和骨架载体。
3.根据权利要求2所述的重组载体,其特征在于,所述骨架载体的类型包括沉默腺相关病毒。
4.权利要求1所述的shRNA或权利要求2所述的重组载体在构建加重肝损伤的模型中的应用。
5.一种甲胎蛋白基因过表达的cDNA,其特征在于,所述cDNA为权利要求1所述的甲胎蛋白基因过表达得到的,所述cDNA的核苷酸序列如SEQ ID NO:3所示。
6.一种过表达甲胎蛋白基因的重组载体,其特征在于,所述重组载体包括权利要求5所述的cDNA和骨架载体。
7.权利要求5所述的cDNA或权利要求6所述的重组载体在制备减轻肝损伤的药物中的应用。
8.权利要求5所述的cDNA或权利要求6所述的重组载体在制备保护肝脏内质网应激的药物中的应用。
9.权利要求5所述的cDNA或权利要求6所述的重组载体在制备治疗肝癌的药物中的应用。
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