CN116970604B - 一种shRNA、慢病毒载体及其构建方法和应用 - Google Patents
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Abstract
本发明提出了一种shRNA、慢病毒载体及其构建方法和应用，所述shRNA的核苷酸序列为：GCAATCAGTTAGCAGACTTGA。慢病毒载体，包括载体质粒，所述载体质粒中包含有上述shRNA序列。本发明提出了慢病毒对TET1酶的敲低作用及其对于认知记忆功能和Wnt信号通路的影响，筛选出了含有特定序列的慢病毒载体对于小鼠海马区TET1酶的敲低作用，以及TET1酶敲低后对于小鼠空间记忆、行为认知和对Wnt通路等方面的影响。
Description
技术领域
本发明属于医疗技术领域，具体涉及一种shRNA、慢病毒载体及其构建方法和应用。
背景技术
阻塞性睡眠呼吸暂停(OSA)是最常见的睡眠呼吸障碍疾病，以慢性间歇性低氧高碳酸血症和睡眠碎片化为核心病理特征，进而诱导全身炎症反应和氧化应激，最终导致全身多系统并发症尤其是神经认知和行为功能异常，严重影响儿童生长发育和远期生活质量。有研究表明OSA与氧化应激的增加有关，其引起的活性氧应激与免疫、炎症、神经发生等多种信号通路有密切的联系。
DNA甲基化及去甲基化修饰参与基因的表达调控，可介导多种生理和病理过程，且二者的动态平衡可以维持遗传表达稳定性，DNA去甲基化酶主要指10-11易位蛋白(ten-eleven-translocation protein，TET)家族，包括TET1、TET2、TET3，是调节DNA甲基化和去甲基化的重要酶类。TET酶是2-氧代戊二酸依赖性双加氧酶家族成员，对神经系统认知相关功能调控起到了重要作用，其介导的DNA羟甲基化过程的动态调节依赖于血氧和氧代谢。TET1酶可促使5mc氧化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine，5hmC)，5hmc在中枢神经系统中含量丰富，而5hmC水平的失调与精神和神经发育障碍的病因有关。TET酶通过调控神经发育相关基因5hmc水平影响神经系统的正常发育和功能。慢病毒载体(lentiviralvictors,LVs)体是一种经过基因改造的病毒载体，在剔除慢病毒致病因子的同时，保留其基因组能整合到宿主基因组的能力，可携带各种外源性基因或改造基因整合至宿主染色体中达到稳定表达的目的。近年来，慢病毒载体在神经科学领域的应用得到了越来越广泛的应用。
发明内容
本发明是为了解决上述技术问题，提出了一种shRNA，以及含有该shRNA的慢病毒载体及其构建方法，并通过该慢病毒载体实现了对TET1酶的敲低，进而在小鼠上试验了该慢病毒载体对于阻塞性睡眠呼吸暂停的治疗作用。
一种shRNA，所述shRNA的核苷酸序列如SEQ ID NO.1所示。
核苷酸序列具体为：GCAATCAGTTAGCAGACTTGA。
一种慢病毒载体，包括载体质粒，所述载体质粒中包含有上述shRNA序列。
可选地，所述载体质粒中还包括筛选标记物的编码核苷酸。
可选地，所述筛选标记物为荧光蛋白。
可选地，所述载体质粒为慢病毒穿梭质粒LV3。
本申请还提出了慢病毒载体的构建方法，包括如下步骤：
步骤一，构建所述载体质粒；
步骤二，将所述载体质粒、慢病毒包装质粒、转染试剂、宿主细胞混合，对宿主细胞进行转染；
步骤三，培养转染后的宿主细胞，得到所述慢病毒载体。
可选地，所述转染试剂为RNAi-Mate。
可选地，所述构建方法还包括：获得所述慢病毒载体后进行筛选，所述筛选包括使用获得的慢病毒载体感染293T细胞，根据荧光蛋白表达情况，筛选出成功感染293T细胞的慢病毒载体。
可选地，所述构建方法还包括：将所述慢病毒载体感染的293T细胞通过RT-PCR检测TET1酶在mRNA水平上的表达情况，筛选出成功敲低TET1酶表达的慢病毒载体。
本申请还提出了一种细胞，所述细胞中包含上述的慢病毒载体。
本申请还提出了上述的shRNA、慢病毒载体、细胞在敲低TET1酶表达和/或降低Wnt信号通路5-羟甲基胞嘧啶中的应用。
本申请还提出了上述shRNA、慢病毒载体、细胞在制备用于治疗阻塞性睡眠呼吸暂停的产品中的应用。所述产品包括药物。
与现有技术相比，本发明具有如下技术效果：
1、本发明提出了一种shRNA序列，其核苷酸序列为：GCAATCAGTTAGCAGACTTGA，测试结果表明，含有该序列的慢病毒载体对TET1酶具有敲低作用。
2、本发明提出了含有上述shRNA序列的慢病毒对TET1酶的敲低作用及其对于认知记忆功能和Wnt信号通路的影响，筛选出了含有特定序列的慢病毒载体对于小鼠海马区TET1酶的敲低作用，以及TET1酶敲低后对于小鼠空间记忆、行为认知和对Wnt通路的影响等方面的影响，并进行了分子生物学验证。
3、本发明的慢病毒的构建方法通过细胞荧光实验精准地检测并筛选到特定序列慢病毒，用RT-PCR对筛选结果进行验证。对小鼠海马区注射慢病毒后对其进行行为学实验，结果同间歇性缺氧组小鼠相比，慢病毒注射组小鼠的空间记忆和认知记忆能力有显著改善。
同时对对照、间歇性缺氧、慢病毒注射干预小鼠海马组织取材进行常规RNA提取,用RT-PCR对筛选结果进行验证。对小鼠海马区注射慢病毒后对其进行行为学实验，结果同间歇性缺氧组小鼠相比，慢病毒注射组小鼠的空间记忆和认知记忆能力有显著改善，且空载体组同缺氧组在行为学和分子生物学验证上无明显差异。同时对对照、间歇性缺氧、慢病毒注射干预和空载体组小鼠海马组织取材进行常规RNA提取，RT-PCR进一步验证了TET1酶的敲低作用。并且发现在间歇性缺氧刺激下TSS区的DNA甲基化及羟甲基化变化情况，筛选到与海马神经发生过程密切相关的Wnt信号通路及下游基因Wnt3a、Ccnd2，并进行RT-PCR验证，结果显示Wnt3a、Ccnd2的5hmC富集水平同间歇性缺氧组较慢病毒注射组显著升高。以上均说明了，一方面，所述的shRNA或含有其的慢病毒载体、细胞可以敲低TET1酶表达和降低Wnt信号通路5-羟甲基胞嘧啶，另一方面，所述的shRNA或含有其的慢病毒载体、细胞可以用于治疗阻塞性睡眠呼吸暂停，改善或治疗阻塞性睡眠呼吸暂停导致的认知功能损伤，如空间记忆和认知记忆能力的改善和治疗。
附图说明
图1为包含shRNA的质粒结构图；
图2为慢病毒载体1转染293T细胞荧光；
图3为RT-PCR验证293T细胞，正常组，慢病毒载体组，空白对照组；
图4为海马组织TET1酶相对表达量验证；
图5为通过巴恩斯迷宫测得的不同组小鼠在训练第5、12天的运动轨迹；
图6为通过巴恩斯迷宫测得的不同组小鼠在训练第1～4天识别目标洞的时间(潜伏期)；
图7为通过巴恩斯迷宫测得的不同组小鼠在训练第5、12天识别目标洞的时间(潜伏期)；
图8为通过Y迷宫测得的不同组小鼠自发交替行为百分比；
图9为通过Y迷宫测得的不同组小鼠进入臂次数；
图10为5-hmc dot blot测试结果；
图11为RT-PCR验证Wnt3a表达量测试结果；
图12为RT-PCR验证Ccnd2表达量测试结果。
各附图中，*表示p<0.05，**表示p<0.01，***表示p<0.001。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。实施例中所用的试验方法如无特殊说明，均为常规方法；所用的材料、试剂等，如无特殊说明，均可从商业途径得到。293T细胞购自中科院细胞库，穿梭质粒LV3(H1/GFP&PURO)和包装质粒(pGag/Pol、pRev、pVSV－G)，RNAi-Mate购自上海吉玛基因。
实施例1
构建慢病毒载体
通过genebank提供TET1(Gene ID:52463)序列，经过分析设计shRNA靶序列，具体序列如下：
由吉玛基因根据特定靶序列合成相应的含shRNA的穿梭质粒LV3(H1/GFP&PURO)，即得到包含shRNA的载体质粒。载体质粒结构如图1所示，其中H1为启动子，GFP为荧光标签，Puro为真核核抗性基因，Amp为原核核抗性基因。
实施例2
1.组装慢病毒载体：
分别在三个无血清DMEM中分别加入含上述shRNA序列(SEQ ID No.1、SEQ IDNo.2、SEQ ID No.3)的穿梭质粒LV3(H1/GFP&PURO)，然后再加入包装质粒(pGag/Pol、pRev、pVSV－G)、RNAi-Mate混匀，加入15cm含293T细胞培养皿中，混匀后37℃、5％CO2培养72小时后收集三种不同的慢病毒浓缩液，并检测滴度。
2.筛选出最适序列的慢病毒载体
筛选过程采用293T细胞转染荧光，RT-PCR验证。具体步骤如下：
取3-8×105个293T细胞，接种于6孔板，过夜培养后，在孔中加入1ul慢病毒和5μg/mL的聚凝胺Polybrene。每孔所加病毒量(μL)＝MOI×感染时的细胞数/病毒滴度×1000,其中293T的MOI值为1。转染24h后更换新鲜培养基，继续培养24～48h,观察荧光表达情况，如图2所示。确定感染成功的慢病毒载体为1号和2号。
最后RT-PCR验证不同序列慢病毒载体的敲低效果。TET1酶敲低效果验证的方法为，收集转染成功的293T细胞。采用RT-PCR技术观察TET1酶在mRNA水平上的表达情况，并与不含shRNA的空载体转染的293T细胞进行对比，TET1酶引物序列如下，SEQ ID No.4、SEQ IDNo.5所示：
| TET1-F | ATTTCCGCATCTGGGAACCTG |
| TET1-R | GGAAGTTGATCTTTGGGGCAAT |
RT-PCR测试结果如图3所示，因此可以最终确定可成功感染293T细胞的最适宜的慢病毒载体为1号，shRNA的核苷酸序列为GCAATCAGTTAGCAGACTTGA。
实施例3
采用实施例2筛选得到的慢病毒载体对间歇性缺氧小鼠进行处理，具体步骤如下：
构建间歇性缺氧小鼠的动物模型：取6w-8wSPF级野生型C57/6J雄性小鼠，间歇性缺氧组于每晚8点开始禁食，次晨8点放入缺氧箱中，严格控制每天8小时(8.00am-4.00pm)慢性间歇性缺氧，保证2分钟内氧浓度下降至5％，1分钟内氧浓度复升到20.9％，并给予小鼠充足的水和食物，温度控制在25℃左右；对照组维持在室内空气中。间歇性缺氧持续4周。
利用众实科技的小动物麻醉定位显微注射系统，将经过4周上述间歇性缺氧造模处理的12周C57BL/6J雄鼠异氟烷麻醉后，固定小鼠剪开头皮后以前后囟连线为纵坐标后囟为正方向建立坐标系，x＝±1.5mm，y＝1.8mm，深度z＝1.5mm对小鼠左右海马各注射1ul慢病毒，注射后缝合头皮，分笼养殖14天。另外设置注射1ul空载体的小鼠的空载体组,不含特异性shRNA序列的穿梭质粒LV3(H1/GFP&PURO)，其方法同组装慢病毒。同时设置正常小鼠和间歇性缺氧造模处理的小鼠作为对照。每组使用小鼠7只。
然后对上述小鼠分别进行行为学实验：
(1)巴恩斯迷宫实验：经过训练，动物学习并记忆目标箱的位置。评估小鼠的空间学习和记忆(包括短期和长期)情况
(2)y迷宫试验：通过自发交替行为评估短期空间工作记忆。
数据统计：采用labmaze动物行为轨迹视频分析系统V3.0收集分析上述实验数据，采用t检验进行统计学分析。
小鼠巴恩斯迷宫实验结果如图5～7所示：图5为通过巴恩斯迷宫测得的不同组小鼠在训练第5、12天的运动轨迹，图6为通过巴恩斯迷宫测得的不同组小鼠在训练第1～4天识别目标洞的时间(潜伏期)。图7为通过巴恩斯迷宫测得的不同组小鼠在训练第5、12天识别目标洞的时间(潜伏期)图7中每组数据从左至右依次为正常组、缺氧组、空载体组、干预组。*表示p<0.05，**表示p<0.01。通过上述实验表明，注射慢病毒对缺氧小鼠干预治疗，可以显著改善小鼠的空间学习和记忆力。
小鼠y迷宫试验结果如图8～9所示，图8为通过Y迷宫测得的不同组小鼠自发交替行为百分比。图9为通过Y迷宫测得的不同组小鼠进入臂次数。实验结果表明，注射慢病毒对缺氧小鼠干预治疗，可显著改善小鼠的短期空间工作记忆。*表示p<0.05，**表示p<0.01。以上数据表明，注射上述慢病毒载体具有治疗阻塞性睡眠呼吸暂停的作用。
对分笼养殖14天后颈椎脱臼法处死小鼠后保留海马组织，常规提取RNA，RT-PCR验证TET1酶在对照、缺氧、干预组以及空载体组中的相对表达。检测结果如图4所示：TET1酶在缺氧处理的小鼠的表达量显著高于对照小鼠。而采用上述慢病毒对缺氧小鼠注射干预后，明显降低了TET1酶表达量，注射空载体则对TET1酶表达量无效。
同时取间歇性缺氧组及对照组小鼠海马样本进行RNA-seq检测，发现基因表达在KEGG通路中表现出显著差异，通过MeDIP-seq、hMeDIP-seq检测DNA甲基化及羟甲基化关键中间产物5mC在间歇性缺氧组中较对照组显著下调，5hmC显著上调；还检测到与海马神经发生过程相关的Wnt信号通路的5hmC水平显著富集，并筛选出该通路中发生显著变化的Wnt3a、Ccnd2基因。
取对照、缺氧及干预组小鼠海马样本进行DNA dot blot检测，如图10所示，发现干预组较缺氧组显著诱导5-hmC下调，同时对其Wnt3a基因和Ccnd2基因进行RT-PCR验证。各基因的RT-PCR引物序列如下:
Wnt3a引物，SEQ ID No.6、SEQ ID No.7所示：
Forward 5’-GCGTGGTGACTGACTGTCTTCTG-3’
Reverse 5’-GTGGTGGGTGGATATAGCAGCATTG-3’
Ccnd2引物，SEQ ID No.8、SEQ ID No.9所示：
Forward 5’-TCGCCCACCTTCCACTCTTCTC-3’
Reverse 5’-GTGCTTCCCTTACCTCCTTCCTTTG-3’
如图11、12所示，经过RT-PCR验证，结果显示干预组较缺氧组显著诱导5-hmC下调。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (9)
1.一种shRNA在制备用于治疗阻塞性睡眠呼吸暂停导致的认知功能损伤的产品中的应用，其特征在于，所述shRNA的核苷酸序列如SEQ ID NO.1所示。
2.一种慢病毒载体在制备用于治疗阻塞性睡眠呼吸暂停导致的认知功能损伤的产品中的应用，所述慢病毒载体包括载体质粒，其特征在于，所述载体质粒中包含权利要求1中所述的shRNA。
3.根据权利要求2所述的应用，其特征在于，所述载体质粒中还包括筛选标记物的编码核苷酸。
4.根据权利要求3所述的应用，其特征在于，所述筛选标记物为荧光蛋白。
5.根据权利要求3所述的应用，其特征在于，所述载体质粒为慢病毒穿梭质粒LV3。
6.权利要求2～5任一项所述的应用，其特征在于，所述慢病毒载体的构建方法包括如下步骤：
步骤一，构建所述载体质粒；
步骤二，将所述载体质粒、慢病毒包装质粒、转染试剂、宿主细胞混合，对宿主细胞进行转染；
步骤三，培养转染后的宿主细胞，得到所述慢病毒载体。
7.根据权利要求6所述的应用，其特征在于，所述构建方法还包括：获得所述慢病毒载体后进行筛选，所述筛选包括使用获得的慢病毒载体感染293T细胞，根据荧光蛋白表达情况，筛选出成功感染293T细胞的慢病毒载体。
8.根据权利要求7所述的应用，其特征在于，所述构建方法还包括：将所述慢病毒载体感染的293T细胞通过RT-PCR检测TET1酶在mRNA水平上的表达情况，筛选出成功敲低TET1酶表达的慢病毒载体。
9.一种细胞在制备用于治疗阻塞性睡眠呼吸暂停导致的认知功能损伤的产品中的应用，其特征在于，所述细胞中包含权利要求2～5任一所述的慢病毒载体。
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| TET1 is a tumour suppressor that inhibits colon cancer growth by derepressing inhibitors of the WNT pathway;Neri et al.;Oncogene;第34卷(第32期);第4168页左栏第1段-4175页左栏第7段 * |
| Tet1-mediated DNA demethylation regulates neuronal cell death induced by oxidative stress;Xin et al.;Sci Rep;第5卷;第1页第1段-第9页左栏第6段 * |
| Xin et al..Tet1-mediated DNA demethylation regulates neuronal cell death induced by oxidative stress.Sci Rep.2015,第5卷第1页第1段-第9页左栏第6段. * |
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