CN109652542B - Gsα基因在制备抗腹主动脉瘤药物中的应用 - Google Patents
Gsα基因在制备抗腹主动脉瘤药物中的应用 Download PDFInfo
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Abstract
本发明涉及Gsα基因在制备抗腹主动脉瘤药物中的应用。本发明通过构建平滑肌特异性敲除Gsα的基因敲除小鼠，公开了Gsα基因在调节平滑肌表型转化、维持平滑肌收缩功能中的重要作用；同时利用皮下泵入AngII来构建小鼠腹主动脉瘤模型，发现Gsα基因敲除小鼠能够显著增大腹主动脉瘤发病率；另外，本发明还制备了Gsα基因的慢病毒，通过小鼠尾静脉注射Gsα基因的慢病毒，能够显著减小小鼠腹主动脉瘤发病率。本发明以Gsα基因为作用靶标，制备或筛选抗腹主动脉瘤的药物，该药物在腹主动脉瘤的治疗中具有重要意义。
Description
技术领域
本发明涉及Gsα基因在制备抗腹主动脉瘤药物中的应用，属于医药技术领域。
背景技术
腹主动脉瘤是指腹主动脉局部呈瘤样扩张，管腔直径超过正常值50％的一种病理状态，它是具有潜在致死危险的疾病，一旦瘤体破裂，死亡率超过80％。在美国，腹主动脉瘤的年发病率约为20-40人/10万人，每年约有15000人死于腹主动脉瘤，占疾病死因的第13位，在我国腹主动脉瘤的发病率也呈逐年上升的趋势，65岁以上男性超声检出腹主动脉瘤的发病率高达5％。腹主动脉瘤管腔瘤样扩张常伴随腹主动脉管壁慢性炎症浸润、平滑肌细胞层变薄、弹力纤维结构破坏和细胞外基质合成/降解失衡。腹主动脉瘤的发病机制尚未清楚，目前认为与平滑肌细胞表型转化、炎症反应、氧化应激和家族遗传等诸多因素相关，临床上一直未找到防治腹主动脉瘤的有效药物。因此，进一步阐明腹主动脉瘤的发生机制并寻找有效的干预措施是当前心血管病领域中一个亟需解决的重大课题。
平滑肌细胞是腹主动脉中层最主要的组成部分，而收缩能力是血管平滑肌细胞最重要的功能。正常的血管平滑肌细胞表现为收缩表型，主要表达收缩蛋白如α-SMA、SM22、SMMHC、Calponin等。当发生腹主动脉瘤时，血管平滑肌细胞由收缩型向合成型转化，表现为收缩型蛋白表达降低，而合成型蛋白如Vimentin、Osteopontin表达升高，同时分泌MMP2/MMP9、Collagen及Fibronectin等蛋白，导致腹主动脉管壁僵硬，收缩能力下降。因此，平滑肌细胞表型转化是腹主动脉瘤发生的重要机制。
G蛋白即鸟嘌呤核苷酸结合蛋白，它是一个G蛋白家族，现已发现的G蛋白有20余种，所有类型的G蛋白都是由3个不同的亚基组成的，分别为α、β、γ亚基，不同G蛋白结构和功能的差异主要在α亚基上。对效应器(如PLC，AC等)起激活作用的G蛋白称为激活型G蛋白(Gs蛋白)，而其α亚基则称为Gsα。Gsα主要通过调节AC(腺苷酸环化酶)的活性来调节靶细胞中cAMP(环磷酸腺苷)的水平，进而影响PKA信号通路的下游事件。
条件性基因敲除是指在特定的组织细胞或者细胞发育的特定阶段敲除某一特定基因的实验技术。条件性基因敲除一般采用Cre/loxP重组系统，Cre重组酶是一种位点特异性重组酶，能介导两个loxP位点之间的特异性重组，使loxP位点间的基因序列被删除或重组。Cre重组酶介导两个loxP位点间的重组是一个动态、可逆的过程，可以分为三种情况：1)如果两个loxP位点位于一条DNA链上，且方向相同，Cre重组酶能有效切除两个loxP位点间的序列；2)如果两个loxP位点位于一条DNA链上，但方向相反，Cre重组酶能导致两个loxP位点间的序列倒位；3)如果两个loxP位点分别位于两条不同的DNA链或染色体上，Cre重组酶能介导两条DNA链的交换或染色体易位。条件性基因敲除对于在特定的组织细胞和/或特定的时间研究特定基因的功能，以及更好地建立人类疾病的动物模型都具有十分重要的意义。
发明内容
针对现有技术的不足，本发明提供Gsα基因在制备抗腹主动脉瘤药物中的应用。
本发明的技术方案如下：
Gsα基因在制备抗腹主动脉瘤药物中的应用，所述Gsα基因的核苷酸序列如SEQ IDNo.1所示。
根据本发明优选的，所述Gsα基因在制备抗腹主动脉瘤药物中的应用，包括以下两个方面：
(1)将Gsα基因作为作用靶标应用于抗腹主动脉瘤药物的制备；
(2)将Gsα基因作为作用靶标应用于抗腹主动脉瘤药物的筛选。
根据本发明优选的，所述Gsα基因作为作用靶标应用于抗腹主动脉瘤药物的制备是指：将Gsα基因作为药物或制剂的作用靶标，来研制针对腹主动脉瘤的药物或制剂，从而提高平滑肌细胞Gsα基因的表达水平。
根据本发明优选的，所述Gsα基因作为作用靶标应用于抗腹主动脉瘤药物的筛选是指：将Gsα基因作为药物或制剂的作用靶标，对药物或者制剂进行筛选，以找到可以促进平滑肌细胞Gsα基因表达的药物或制剂作为抗腹主动脉瘤的备选药物或制剂。
根据本发明优选的，所述抗腹主动脉瘤药物包括但不限于：核酸分子、碳水化合物、脂类、小分子化学药、抗体药、多肽、蛋白质或慢病毒。
进一步优选的，所述核酸分子包括但不限于：反义寡合苷酸、双链RNA或者短发夹RNA。
进一步优选的，所述慢病毒，其有效成分含有如SEQ ID No.1所示的核苷酸序列，是经过病毒包装而成，通过表达Gsα基因来降低腹主动脉瘤的发生。
本发明的有益效果：
本发明通过构建平滑肌特异性敲除Gsα的基因敲除小鼠，公开了Gsα基因在调节平滑肌表型转化、维持平滑肌收缩功能中的重要作用；同时利用皮下泵入AngII来构建小鼠腹主动脉瘤模型，发现Gsα基因敲除小鼠能够显著增大腹主动脉瘤发病率，从而为制备或筛选促进平滑肌细胞Gsα基因表达的药物或制剂作为抗腹主动脉瘤的备选药物或制剂提供了理论依据。
本发明制备了Gsα基因的慢病毒(Lenti-Gsα)，通过小鼠尾静脉注射Gsα基因的慢病毒(Lenti-Gsα)，能够显著减小小鼠腹主动脉瘤发病率。本发明以Gsα基因为作用靶标，制备或筛选抗腹主动脉瘤的药物，该药物在腹主动脉瘤的治疗中具有重要意义。
附图说明
图1为条件性敲除Gsα基因的策略流程图；
图2为GsαSMKO小鼠主动脉平滑肌细胞中Gsα蛋白的表达情况；
图中，Control代表对照鼠，GsαSMKO代表基因敲除鼠，GAPDH为甘油醛-3-磷酸脱氢酶，为内参蛋白；
图3为GsαSMKO小鼠主动脉平滑肌细胞中cAMP活性的变化情况；
图中，Control代表对照鼠，GsαSMKO代表基因敲除鼠；
图4为小鼠腹主动脉瘤的成瘤情况；
图5为小鼠尾静脉注射慢病毒Lenti-Gsα后腹主动脉瘤的成瘤情况。
具体实施方式
下面结合实施例进一步阐述本发明，实施例仅用于说明本发明，而非限制本发明的保护范围。
实施例中涉及药品及试剂，若无特殊说明，均为普通市售产品；实施例中涉及实验操作，若无特殊说明，均按照本领域常规操作进行。
实施例1小鼠腹主动脉瘤模型的构建
将Gsαflox/flox小鼠与平滑肌特异表达Cre重组酶的小鼠SM22-CreERT2交配，得到Gsαflox/flox/Cre+小鼠，连续五天向Gsαflox/flox/Cre+小鼠腹腔注射1毫克他莫西芬药物(Sigma公司)，刺激Cre重组酶表达，从而将Gsαflox/flox/Cre+小鼠中loxP位点之间的序列重组掉，即将Gsαflox/flox/Cre+小鼠平滑肌内的Gsα基因敲除掉，抑制了Gsα基因的功能，从而得到平滑肌特异性敲除Gsα基因的小鼠，该小鼠用GsαSMKO表示。图1为条件性敲除Gsα基因的策略。
腹腔注射他莫西芬药物后第16天，将GsαSMKO小鼠的主动脉取出，同时取对照鼠(未注射他莫西芬药物的Gsαflox/flox/Cre+小鼠)的主动脉，分别剥去主动脉外膜，加蛋白裂解液研磨样品，各取40微克蛋白上样做Western blot验证，用Gsα蛋白的抗体检测，结果如图2所示，发现GsαSMKO小鼠的主动脉中大部分Gsα基因被去除，无法表达Gsα蛋白，从而为后续的研究提供了很好的动物模型。
cAMP是Gsα下游最为重要的第二信使，cAMP活性高低可以反应Gsα基因的敲除情况。腹腔注射他莫西芬药物后第16天，将GsαSMKO小鼠和对照鼠(未注射他莫西芬药物的Gsαflox/flox/Cre+小鼠)的主动脉取出，显微镜下去除主动脉外膜组织，加0.1M的HCl研磨，离心后取上清，使用Elisa试剂盒检测GsαSMKO小鼠和对照鼠的主动脉中cAMP活性。结果如图3所示，发现GsαSMKO小鼠主动脉平滑肌中cAMP活性明显降低。
将GsαSMKO小鼠回交至ApoE-/-背景8代以上，使之非常接近ApoE-/-背景，以减少个体之间的差异，从而得到较为稳定的同时敲除Gsα基因和ApoE基因的双敲小鼠，双敲小鼠用ApoE-/-/GsαSMKO表示，同窝不带Cre重组酶基因的ApoE-/-/Gsαflox/flox小鼠作为对照。
对10周龄的对照小鼠ApoE-/-/Gsαflox/flox和双敲小鼠ApoE-/-/GsαSMKO背部皮下植入Alzet微量泵，分别按照1000ng/Kg/min的速度泵入AngII，连续泵入28天，即可得到小鼠腹主动脉瘤模型。
实施例2平滑肌Gsα基因在小鼠腹主动脉瘤中的作用
将实施例1得到的双敲小鼠ApoE-/-/GsαSMKO和对照小鼠不带Cre重组酶基因的ApoE-/-/Gsαflox/flox分为四组，每组至少6只，在10周龄时进行处理，四组的小鼠及处理情况如下：
第一组：对照小鼠ApoE-/-/Gsαflox/flox，泵入生理盐水；
第二组：对照小鼠ApoE-/-/Gsαflox/flox，泵入AngII；
第三组：双敲小鼠ApoE-/-/GsαSMKO，泵入生理盐水；
第四组：双敲小鼠ApoE-/-/GsαSMKO，泵入AngII；
泵入的操作流程为：在小鼠背部皮下植入Alzet微量泵，分别按照1000ng/Kg/min的速度泵入AngII，或者分别泵入等量生理盐水，连续泵入28天。
28天后安乐死小鼠，取小鼠主动脉，小鼠腹主动脉瘤的成瘤情况如图4所示，发现敲除Gsα基因后能够显著增大腹主动脉瘤发病率。
实施例3针对Gsα基因的慢病毒的制备
1、pFUGW-Gsα载体的构建：
1)将pFUGW质粒(Addgene购买，#14883)经内切酶HindIII和KpnI双酶切，双酶切产物进行琼脂糖凝胶电泳，切胶回收；
2)以平滑肌细胞cDNA为模板，经PCR扩增，纯化，制得Gsα基因片段；
PCR扩增的特异引物核苷酸序列如下：
正向引物：5’-AAGCTTATGGGCTGCCTCGGGAACAGTA-3’；
反向引物：5’-GGTACCTTAGAGCAGCTCGTACTGACGAA-3’；
Gsα基因的核苷酸序列如SEQ ID No.1所示；
3)将步骤2)制得的Gsα基因片段经内切酶HindIII和KpnI双酶切，制得Gsα基因双酶切产物；
4)将步骤3)Gsα基因双酶切产物进行琼脂糖凝胶电泳，切胶并回收含1185bp大小的DNA片段，纯化，得到纯化后的Gsα基因双酶切产物；
5)将步骤1)回收后的pFUGW双酶切产物和步骤4)纯化后的Gsα基因双酶切产物进行连接，制得连接产物；
6)将步骤5)制得的连接产物转化大肠杆菌，经抗性筛选和测序验证后，提取阳性质粒pFUGW-Gsα。
2、Gsα基因的慢病毒(Lenti-Gsα)的包装制备
将上述得到的阳性质粒pFUGW-Gsα和辅助质粒psPAX2(Addgene购买，#12260)、pMD2.G(Addgene购买，#12259)，按照4：3：1的质量比，通过转染试剂Ultra-PEI，共转入293T细胞，转染后3天收集病毒，经超离心纯化，同时测定病毒滴度，制得表达Gsα的慢病毒Lenti-Gsα。
实施例4Gsα基因的慢病毒对腹主动脉瘤的作用
将实施例1得到的双敲小鼠ApoE-/-/GsαSMKO分为两组，每组至少6只，在10周龄时进行处理，分别尾静脉注射慢病毒Lenti-Gsα及对照无关慢病毒Lenti-LacZ(山东维真生物科技有限公司)，两组的小鼠及处理情况如下：
实验组：ApoE-/-/GsαSMKO小鼠，注射Lenti-Gsα；
对照组：ApoE-/-/GsαSMKO小鼠，注射Lenti-LacZ；
两组小鼠均在背部皮下植入Alzet微量泵，分别按照1000ng/Kg/min的速度泵入AngII，连续泵入28天。28天后安乐死小鼠，取小鼠主动脉，小鼠腹主动脉瘤的成瘤情况如图5所示，小鼠尾静脉注射慢病毒Lenti-Gsα后能够显著减小腹主动脉瘤。
以上结果证实了Gsα基因在维持血管收缩功能中的关键作用，平滑肌敲除Gsα基因能够增大患腹主动脉瘤的几率，而通过给Gsα基因敲除小鼠尾静脉注射Gsα基因慢病毒的方法加以治疗，能够减小腹主动脉瘤的患病率。
SEQUENCE LISTING
<110> 山东大学
<120> Gsα基因在制备抗腹主动脉瘤药物中的应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1185
<212> DNA
<213> Homo sapiens
<400> 1
atgggctgcc tcgggaacag taagaccgag gaccagcgca acgaggagaa ggcgcagcgt 60
gaggccaaca aaaagatcga gaagcagctg cagaaggaca agcaggtcta ccgggccacg 120
caccgcctgc tgctgctggg tgctggagaa tctggtaaaa gcaccattgt gaagcagatg 180
aggatcctgc atgttaatgg gtttaatgga gagggcggcg aagaggaccc gcaggctgca 240
aggagcaaca gcgatggtga gaaggcaacc aaagtgcagg acatcaaaaa caacctgaaa 300
gaggcgattg aaaccattgt ggccgccatg agcaacctgg tgccccccgt ggagctggcc 360
aaccccgaga accagttcag agtggactac atcctgagtg tgatgaacgt gcctgacttt 420
gacttccctc ccgaattcta tgagcatgcc aaggctctgt gggaggatga aggagtgcgt 480
gcctgctacg aacgctccaa cgagtaccag ctgattgact gtgcccagta cttcctggac 540
aagatcgacg tgatcaagca ggctgactat gtgccgagcg atcaggacct gcttcgctgc 600
cgtgtcctga cttctggaat ctttgagacc aagttccagg tggacaaagt caacttccac 660
atgtttgacg tgggtggcca gcgcgatgaa cgccgcaagt ggatccagtg cttcaacgat 720
gtgactgcca tcatcttcgt ggtggccagc agcagctaca acatggtcat ccgggaggac 780
aaccagacca accgcctgca ggaggctctg aacctcttca agagcatctg gaacaacaga 840
tggctgcgca ccatctctgt gatcctgttc ctcaacaagc aagatctgct cgctgagaaa 900
gtccttgctg ggaaatcgaa gattgaggac tactttccag aatttgctcg ctacactact 960
cctgaggatg ctactcccga gcccggagag gacccacgcg tgacccgggc caagtacttc 1020
attcgagatg agtttctgag gatcagcact gccagtggag atgggcgtca ctactgctac 1080
cctcatttca cctgcgctgt ggacactgag aacatccgcc gtgtgttcaa cgactgccgt 1140
gacatcattc agcgcatgca ccttcgtcag tacgagctgc tctaa 1185
Claims (3)
1.Gsα基因在制备抗腹主动脉瘤药物中的应用，所述Gsα基因的核苷酸序列如SEQ IDNo.1所示。
2.如权利要求1所述的应用，其特征在于，所述抗腹主动脉瘤药物为慢病毒。
3.如权利要求2所述的应用，其特征在于，所述慢病毒，其有效成分含有如SEQ ID No.1所示的核苷酸序列，是经过病毒包装而成，通过表达Gsα基因来降低腹主动脉瘤的发生。
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| Heterotrimeric G stimulatory protein α subunit is required for intestinal smooth muscle contraction in mice;qin x等;《gastrpemterology》;20170430;第152卷;1114-1125，手稿版共18 * |
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