CN116751849A - LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用 - Google Patents
LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用 Download PDFInfo
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Abstract
本发明提供了LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，所述LncRNA MSTRG16457.2的核苷酸序列如SEQ ID NO:1所示，所述产品通过检测LncRNA MSTRG16457.2的表达水平诊断主动脉夹层。本发明通过构建大鼠主动脉夹层模型，在主动脉夹层模型的LncRNA中筛选发现了LncRNA MSTRG16457.2的新用途，并进一步研究了LncRNA MSTRG16457.2表达对主动脉夹层的影响，为主动脉夹层的快速诊断和治疗提供了新的可行性方案。
Description
技术领域
本发明属于生物医药技术领域，涉及LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用。
背景技术
主动脉夹层(aortic dissection，AD)指主动脉腔内的血液通过主动脉内膜的破口进入主动脉壁中膜而形成的血肿，并沿主动脉长轴方向扩展，造成主动脉真假两腔分离的一种病理改变，因通常呈继发瘤样改变，过去此种情况被称为主动脉夹层动脉瘤(aorticdissecting aneurysm)，现多改称为主动脉夹层血肿(aortic dissectin ghematoma)，或主动脉夹层分离，简称主动脉夹层。
主动脉夹层临床特点为急性起病，突发剧烈疼痛、休克和血肿压迫相应的主动脉分支血管时出现的脏器缺血症状。主动脉夹层是心血管疾病的灾难性危重急症，如不及时诊治，48小时内死亡率可高达50％，故早期诊断和治疗非常必要。
发明内容
本发明的目的在于提供LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，为临床上快速诊断和治疗主动脉夹层提供一种新的可行性方案。
为实现上述目的，本发明采用的技术方案为：
LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，所述LncRNA MSTRG16457.2的核苷酸序列如SEQ ID NO:1所示。
优选地，所述产品通过检测LncRNA MSTRG16457.2的表达水平诊断主动脉夹层。
更优选地，诊断主动脉夹层的产品包括试剂或试剂盒。
更优选地，LncRNA MSTRG16457.2在主动脉夹层的生物学样品中的表达水平升高。
优选地，所述产品通过干扰LncRNA MSTRG16457.2的表达水平抑制主动脉夹层的形成。
更优选地，治疗主动脉夹层的产品包括表达LncRNA MSTRG16457.2的腺病毒载体。
本发明的有益效果在于：
本发明通过构建大鼠主动脉夹层模型，在主动脉夹层模型的LncRNA中筛选发现了LncRNA MSTRG16457.2的新用途，并进一步研究了LncRNA MSTRG16457.2表达对主动脉夹层的影响，为主动脉夹层的快速诊断和治疗提供了新的可行性方案。
附图说明
图1为3个LncRNA的PCR检测结果。
图2为本发明造模过程中的部分照片。
图3为qPCR检测各组大鼠主动脉组织中MSTRG16457.2表达情况(*P＜0.05VSControl)。
图4为载体pAdEasy-U6-CMV-EGFP的图谱。
图5为PCR检测各组中LncRNA MSTRG16457.2的表达水平。*p<0.05，与对照组相比。
图6为HE和MASSON染色观察主动脉夹层的病理情况。
具体实施方式
为了更清楚地说明本发明，下面结合实施例并对照附图对本发明作进一步详细说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例
本发明实现的主要流程包括：
一、LncRNA MSTRG16457.2的筛选
根据高通量分析结果筛选出3个LncRNA：MSTRG7090.4(SEQ ID NO:3)、MSTRG16456.1(SEQ ID NO:2)和MSTRG16457.2(SEQ ID NO:1)，如图1所示(图中1，2，3对应SD大鼠的编号)，PCR结果显示与对照组(Control)相比，模型组(Model)中MSTRG16457.2的表达水平均显著上升，差异具有统计学意义；因此选择LncRNA MSTRG16457.2进行研究。
二、LncRNA MSTRG16457.2在主动脉夹层的表达情况
1.动物材料
30只SD大鼠，雄性，3周龄，SPF级，购于湖南斯莱克景达实验动物有限公司，许可证号SCXK(湘)2019-0004
2.试剂：β-氨基丙腈(批号：C12511686，厂家：上海麦克林生化科技有限公司)、血管紧张素Ⅱ(批号：4474-91-3，厂家：上海源叶生物科技有限公司)
3.实验过程：
饮用水中添加β-氨基丙腈(0.12％)，后续每三天额外增加0.02％氨基丙腈和每天每只两次颈部皮下注射血管紧张素(0.4mg/ml)0.3ml，持续21天。
qPCR检测各组大鼠主动脉组织中MSTRG16457.2表达情况，结果见图3，PCR结果显示与对照组(Control)相比，模型组(Model)中MSTRG16457.2的表达水平均显著上升，差异具有统计学意义。
以上实验结果表明，检测LncRNA MSTRG16457.2的表达水平的试剂或试剂盒可用于诊断主动脉夹层，LncRNA MSTRG16457.2在主动脉夹层的生物学样品中的表达水平升高。三、LncRNA MSTRG16457.2腺病毒载体构建和病毒包装
(一)腺病毒载体的制备
主要流程：
1.选择载体pAdEasy-U6-CMV-EGFP，载体图谱如图4所示，并进行设计目的片段PCR引物；
扩增条件：
2.根据如下表中顺序依次加入每种试剂，轻轻吸打混匀，置于37℃水浴锅中反应1-2h；酶切结束之后进行琼脂糖凝胶电泳，回收目的片段；
载体酶切体系如下：
3.干扰片段与载体连接：
连接反应体系(20μL)如下：
以上连接液在22℃连接1-2h，或者16℃连接过夜。
4.转化感受态DH5a或者stbl3，菌液涂板，培养12-16h；
5.挑选单克隆行进菌落验证；
6.选择菌落验证正确的阳性克隆进行测序；
7.测序正确的克隆样品进行质粒抽提。
(二)腺病毒包装
实验流程：
采用Adeasy腺病毒系统进行病毒包装过程。质粒载体进行高纯度无内毒素抽提后，采用LipofiterTM转染试剂将质粒转染293A细胞。需要说明的是，腺病毒载体构建和病毒包装流程为本领域较为成熟的技术，可通过常规技术操作实现，在此不做具体描述。
四、LncRNA MSTRG16457.2对主动脉夹层的影响
饮用水中添加β-氨基丙腈(0.12％)，后续每三天额外增加0.02％氨基丙腈和每天每只两次颈部皮下注射血管紧张素(0.4mg/ml)0.3ml。总共作用21天。作为AD组。
腺病毒干预：尾静脉注射腺病毒10μl。以不作任何处理的正常大鼠作为对照组(Control)。
取样：造模结束后第4天，处死各组大鼠。
PCR检测各组中LncRNA MSTRG16457.2的表达水平，结果如图5所示，与对照组(Control)和空载组(NC)相比，干扰组中LncRNA MSTRG16457.2的表达水平显著下降，差异具有统计学意义(P<0.05)。
病理观察结果见图6，结果显示，对照组动脉结构正常，中内膜结构完整，Masson染色可看出弹力纤维及肌层完好无破裂。模型组可见内皮损伤撕裂，且血管壁中形成假腔，弹力纤维断裂甚至消失，肌层破坏严重，胶原纤维增生，血管弹性降低。干扰LncRNAMSTRG16457.2会得到改善，说明干扰LncRNA MSTRG16457.2表达能够显著抑制主动脉夹层的形成。
以上实验结果表明，可通过干扰LncRNA MSTRG16457.2的表达水平抑制主动脉夹层的形成从而达到治疗主动脉夹层的目的。
显然，本发明的上述实施例仅仅是为更清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，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他不同形式的变化或变动，这里无法对所有的实施方法予以穷举，凡是属于本发明的技术方案所引申出的显而易见的变化或变动仍处于本发明的保护范围之列。
Claims (6)
1.LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，所述LncRNAMSTRG16457.2的核苷酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，其特征在于，所述产品通过检测LncRNA MSTRG16457.2的表达水平诊断主动脉夹层。
3.根据权利要求2所述的LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，其特征在于，诊断主动脉夹层的产品包括试剂或试剂盒。
4.根据权利要求2或3所述的LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，其特征在于，LncRNA MSTRG16457.2在主动脉夹层的生物学样品中的表达水平升高。
5.根据权利要求1所述的LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，其特征在于，所述产品通过干扰LncRNA MSTRG16457.2的表达水平抑制主动脉夹层的形成。
6.根据权利要求5所述的LncRNA MSTRG16457.2在制备诊断或治疗主动脉夹层的产品中的应用，其特征在于，治疗主动脉夹层的产品包括表达LncRNA MSTRG16457.2的腺病毒载体。
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