技术领域：Technical field:
本发明属于生物技术领域，具体地涉及DACT2基因在制备心力衰竭治疗药物中的应用。The invention belongs to the field of biotechnology, and in particular relates to the application of DACT2 gene in the preparation of drugs for treating heart failure.
背景技术：Background technique:
心力衰竭是指由于心脏的收缩功能和(或)舒张功能发生障碍，不能将静脉回心血量充分排出心脏，导致静脉系统血液淤积，动脉系统血液灌注不足，从而引起心脏循环障碍症候群，此种障碍症候群集中表现为肺淤血、腔静脉淤血。心力衰竭并不是一个独立的疾病，而是心脏疾病发展的终末阶段。Heart failure refers to the failure of the systolic function and (or) diastolic function of the heart to fully discharge the venous return blood from the heart, resulting in blood stasis in the venous system and insufficient blood perfusion in the arterial system, resulting in cardiac circulation syndrome. Symptom cluster showed pulmonary congestion, vena cava congestion. Heart failure is not an independent disease, but the end stage of the development of heart disease.
目前用于心力衰竭的诊断方法包括以下几类：(1)心衰标志物：BNP/NT-proBNP，目前心衰的公认客观指标；(2)心肌坏死标志物：肌钙蛋白，检测心肌受损的特异性和敏感性均较高的标示物；(3)超声心动图，可了解心脏的结构和功能；(4)X线检查。The diagnostic methods currently used for heart failure include the following categories: (1) Heart failure markers: BNP/NT-proBNP, currently recognized objective indicators of heart failure; (2) Myocardial necrosis markers: troponin, detection of myocardial damage (3) Echocardiography, which can understand the structure and function of the heart; (4) X-ray examination.
一般的诊断步骤为：根据患者有冠心病、高血压等基础心血管病的病史，运动受限，心功能评级以及超声心动图异常，利钠肽(BNP/NT-proBNP)水平升高等心脏结构或功能异常的客观证据，有收缩性心力衰竭或舒张性心力衰竭的特征，可作出诊断。但是上述诊断方法在心力衰竭发生早期并不适用，因此寻找一种在心力衰竭早期即可判断出疾病存在与否的方法是亟待解决的问题。The general diagnostic steps are: based on the patient's history of underlying cardiovascular diseases such as coronary heart disease and hypertension, exercise restriction, cardiac function rating and echocardiographic abnormalities, elevated levels of natriuretic peptide (BNP/NT-proBNP) and other cardiac structures Or objective evidence of abnormal function, with features of systolic heart failure or diastolic heart failure, can make a diagnosis. However, the above diagnostic methods are not applicable in the early stage of heart failure, so finding a method to judge the presence or absence of the disease in the early stage of heart failure is an urgent problem to be solved.
发明内容：Invention content:
为了弥补现有技术的不足，本发明的目的在于提供DACT2基因在制备心力衰竭治疗药物中的应用。In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide the application of the DACT2 gene in the preparation of drugs for the treatment of heart failure.
为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
本发明提供了DACT2基因和/或其表达产物在制备心力衰竭治疗药物中的应用。The invention provides the application of DACT2 gene and/or its expression product in the preparation of medicine for treating heart failure.
优选，DACT2基因和/或其表达产物的促进剂在制备心力衰竭治疗药物中的应用。Preferably, the application of the promoter of DACT2 gene and/or its expression product in the preparation of medicine for treating heart failure.
优选，所述的DACT2基因和/或其表达产物的促进剂是指能够促进DACT2基因表达或增强DACT2表达功能的试剂。Preferably, the promoter of the DACT2 gene and/or its expression product refers to a reagent that can promote the expression of the DACT2 gene or enhance the expression function of the DACT2.
优选，所述的DACT2基因和/或其表达产物的促进剂是超表达DACT2基因的病毒。Preferably, the promoter of the DACT2 gene and/or its expression product is a virus overexpressing the DACT2 gene.
所述的DACT2基因和/或其表达产物的促进剂是超表达DACT2基因的腺病毒。The promoter of the DACT2 gene and/or its expression product is an adenovirus overexpressing the DACT2 gene.
本发明的第二个目的是提供一种心力衰竭治疗药物，其特征在于，包括能够促进DACT2基因表达或增强DACT2表达功能的试剂作为活性成分。The second object of the present invention is to provide a drug for treating heart failure, which is characterized in that it includes an agent capable of promoting the expression of DACT2 gene or enhancing the expression function of DACT2 as an active ingredient.
本发明的第三个目的是提供DACT2基因在制备抑制α-MHC、ANP、BNP和/或ANF心力衰竭标志物的表达药物中的应用。The third object of the present invention is to provide the application of DACT2 gene in the preparation of drugs for inhibiting the expression of α-MHC, ANP, BNP and/or ANF heart failure markers.
进一步，所述的DACT2基因和/或其表达产物的促进剂是指能够促进DACT2基因表达或增强DACT2表达功能的试剂。Further, the promoter of the DACT2 gene and/or its expression product refers to a reagent that can promote the expression of the DACT2 gene or enhance the expression function of the DACT2.
进一步，所述试剂包括：含有能编码功能性DACT2蛋白的核酸的试剂、DACT2蛋白的激活剂、含有DACT2蛋白质的试剂。Further, the reagents include: reagents containing nucleic acids capable of encoding functional DACT2 proteins, activators of DACT2 proteins, and reagents containing DACT2 proteins.
其中，所述含有能编码功能性DACT2蛋白的核酸的试剂可以是在有利条件下翻译成活性形式的DACT2蛋白的单链核酸(如mRNA)或双链核酸(如DNA)，所述的核酸可以连接在表达载体上或重组到宿主细胞里，只要能编码成活性DACT2蛋白，任何一种DACT2基因的携带方式均可。所述的DACT2蛋白的激活剂是指刺激DACT2蛋白活性、增加DACT2蛋白活性、促进DACT2蛋白活性、增强DACT2蛋白激活、使DACT2蛋白活性敏感化或上调DACT2蛋白活性的试剂，如去甲基化试剂、DACT2启动子和/或增强子特异性的转录激活剂、DACT2蛋白的激动剂(如激活抗体)等。Wherein, the reagent containing a nucleic acid capable of encoding a functional DACT2 protein can be a single-stranded nucleic acid (such as mRNA) or a double-stranded nucleic acid (such as DNA) that is translated into an active form of the DACT2 protein under favorable conditions, and the nucleic acid can be Linked to an expression vector or recombined into a host cell, as long as it can be encoded into an active DACT2 protein, any carrying method of the DACT2 gene is acceptable. The activator of the DACT2 protein refers to a reagent that stimulates the activity of the DACT2 protein, increases the activity of the DACT2 protein, promotes the activity of the DACT2 protein, enhances the activation of the DACT2 protein, sensitizes the activity of the DACT2 protein, or up-regulates the activity of the DACT2 protein, such as demethylation reagents , DACT2 promoter and/or enhancer-specific transcriptional activators, DACT2 protein agonists (such as activating antibodies), etc.
本发明所述携带基因的载体是本领域已知的各种载体，如市售的载体、包括质粒、粘粒、噬菌体、病毒等。The gene-carrying vectors of the present invention are various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like.
在本发明中，术语“宿主细胞”包括原核细胞和真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞和哺乳动物细胞。较佳地，该宿主细胞是真核细胞，如CHO细胞、COS细胞等。In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, such as CHO cells, COS cells and the like.
本发明的药物可以用于补充内源性的DACT2蛋白的缺失或不足，通过提高DACT2蛋白的表达，从而治疗心衰。The medicine of the present invention can be used to supplement the lack or deficiency of endogenous DACT2 protein, and treat heart failure by increasing the expression of DACT2 protein.
进一步，本发明中DACT2蛋白或编码DACT2蛋白的核酸可以通过脂质体给予，所述脂质体的作用是将药物靶向于特定的组织，以及增加药物的半衰期。脂质体包括乳化剂、起泡剂、液态脂质、固态脂质、不溶性单层、磷脂分散剂、表面活性剂等。所述的脂质体中还可以包括能与靶向的细胞中的受体分子结合或其他治疗性或免疫原性组合物。Furthermore, the DACT2 protein or the nucleic acid encoding the DACT2 protein in the present invention can be administered through liposomes, and the function of the liposomes is to target the drug to a specific tissue and increase the half-life of the drug. Liposomes include emulsifiers, foaming agents, liquid lipids, solid lipids, insoluble monolayers, phospholipid dispersants, surfactants, and the like. The liposome may also include other therapeutic or immunogenic compositions capable of binding to receptor molecules in targeted cells.
进一步，本发明的药物还包括药学上可接受的载体，这类载体包括(但并不限于)：稀释剂、赋形剂如水等、填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮；湿润剂如甘油；崩解剂如琼脂、碳酸钙和碳酸氢钠；吸收促进剂季铵化合物；表面活性剂如十六烷醇；吸附载体如高岭土和皂粘土；润滑剂如滑石粉、硬脂酸钙和镁、聚乙二醇等。Further, the medicine of the present invention also includes pharmaceutically acceptable carriers, such carriers include (but not limited to): diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derived alginate, gelatin and polyvinylpyrrolidone; wetting agents such as glycerin; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers quaternary ammonium compounds; surfactants such as cetyl alcohol; and bentonite; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, etc.
本发明的药物导入组织或者细胞的方式可以分为体外或者体内的方式。体外方式包括将含有DACT2基因的药物或者含有DACT2蛋白质的药物导入细胞中，再将细胞移植或回输到体内。体内方式包括直接将含有DACT2基因的药物或者含有DACT2蛋白质的药物注入体内组织中。The method of introducing the drug of the present invention into tissues or cells can be divided into in vitro or in vivo methods. The in vitro method includes introducing the drug containing the DACT2 gene or the drug containing the DACT2 protein into the cells, and then transplanting or reinfusing the cells into the body. The in vivo method includes directly injecting the drug containing the DACT2 gene or the drug containing the DACT2 protein into tissues in the body.
本发明的药物还可与其他治疗心力衰竭的药物联用，多种药物联合使用可以大大提高治疗的成功率。The medicine of the present invention can also be used in combination with other medicines for treating heart failure, and the combined use of multiple medicines can greatly improve the success rate of treatment.
在本发明的上下文中，“DACT2基因”包括人DACT2基因以及人DACT2基因的任何功能等同物的多核苷酸。DACT2基因包括与目前国际公共核酸序列数据库GeneBank中DACT2基因(NC_000006.12)DNA序列具有70％以上同源性，且编码相同功能蛋白质的DNA序列；In the context of the present invention, "DACT2 gene" includes polynucleotides of the human DACT2 gene as well as any functional equivalents of the human DACT2 gene. The DACT2 gene includes a DNA sequence that has more than 70% homology with the DNA sequence of the DACT2 gene (NC_000006.12) in the current international public nucleic acid sequence database GeneBank and encodes the same functional protein;
优选地，DACT2基因的编码序列包括以下任一种DNA分子：Preferably, the coding sequence of the DACT2 gene comprises any of the following DNA molecules:
(1)序列表中SEQ ID NO.1所示的DNA序列；(1) the DNA sequence shown in SEQ ID NO.1 in the sequence listing;
(2)在严格条件下与(1)限定的DNA序列杂交且编码相同功能蛋白质的DNA序列；(2) A DNA sequence that hybridizes to the DNA sequence defined in (1) under stringent conditions and encodes the same functional protein;
(3)与(1)或(2)限定的DNA序列具有70％、优选地，90％以上同源性，且编码相同功能蛋白质的DNA分子。(3) A DNA molecule having 70%, preferably more than 90%, homology with the DNA sequence defined in (1) or (2), and encoding the same functional protein.
在本发明的具体实施方案中，所述DACT2基因的编码序列是SEQ ID NO.1所示的DNA序列。In a specific embodiment of the present invention, the coding sequence of the DACT2 gene is the DNA sequence shown in SEQ ID NO.1.
在本发明的上下文中，DACT2基因表达产物包括人DACT2蛋白以及人DACT2蛋白的部分肽。所述DACT2蛋白的部分肽含有与心里衰竭相关的功能域。In the context of the present invention, DACT2 gene expression products include human DACT2 protein and partial peptides of human DACT2 protein. The partial peptide of the DACT2 protein contains functional domains related to heart failure.
“DACT2蛋白”包括DACT2蛋白以及DACT2蛋白的任何功能等同物。所述功能等同物包括DACT2蛋白保守性变异蛋白质、或其活性片段，或其活性衍生物，等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与人DACT2的DNA杂交的DNA所编码的蛋白质。"DACT2 protein" includes DACT2 protein and any functional equivalents of DACT2 protein. The functional equivalents include DACT2 protein conservative variant proteins, or active fragments thereof, or active derivatives thereof, allelic variants, natural mutants, induced mutants, and those capable of interacting with human DACT2 under high or low stringent conditions. DNA The protein encoded by the DNA that hybridizes.
优选地，DACT2蛋白是具有下列氨基酸序列的蛋白质：Preferably, the DACT2 protein is a protein having the following amino acid sequence:
(1)由序列表中SEQ ID NO.2所示的氨基酸序列组成的蛋白质；(1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;
(2)将SEQ ID NO.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO.2所示的氨基酸序列具有相同功能的由SEQ ID NO.2所示的氨基酸序列衍生的蛋白质。取代、缺失或者添加的氨基酸的个数通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个。(2) The amino acid sequence shown in SEQ ID NO.2 is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as the amino acid sequence shown in SEQ ID NO.2. A protein derived from the amino acid sequence shown in ID NO.2. The number of substituted, deleted or added amino acids is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.
(3)与SEQ ID NO.2所示的氨基酸序列具有至少80％同源性(又称为序列同一性)，优选地，与SEQ ID NO.2所示的氨基酸序列至少约90％至95％的同源性，常为96％、97％、98％、99％同源性的氨基酸序列构成的多肽。(3) having at least 80% homology (also known as sequence identity) with the amino acid sequence shown in SEQ ID NO.2, preferably at least about 90% to 95% with the amino acid sequence shown in SEQ ID NO.2 % homology, usually 96%, 97%, 98%, 99% homology of amino acid sequence composed of polypeptides.
在本发明的具体实施方案中，所述DACT2蛋白是具有SEQ ID NO.2所示的氨基酸序列的蛋白质。In a specific embodiment of the present invention, the DACT2 protein is a protein having the amino acid sequence shown in SEQ ID NO.2.
通常，已知的是，一个蛋白质中一个或多个氨基酸的修饰不会影响蛋白质的功能。本领域技术人员会认可改变单个氨基酸或小百分比的氨基酸或对氨基酸序列的个别添加、缺失、插入、替换是保守修饰，其中蛋白质的改变产生具有相似功能的蛋白质。提供功能相似的氨基酸的保守替换表是本领域公知的。In general, it is known that the modification of one or more amino acids in a protein does not affect the function of the protein. Those skilled in the art will recognize that changes to single amino acids or small percentages of amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications where changes to a protein result in a protein with similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.
通过添加一个氨基酸或多个氨基酸残基修饰的蛋白质的例子是DACT2蛋白的融合蛋白。对于与DACT2蛋白融合的肽或者蛋白质没有限制，只要所得的融合蛋白保留DACT2蛋白的生物学活性即可。An example of a protein modified by adding an amino acid or amino acid residues is a fusion protein of the DACT2 protein. There is no limitation on the peptide or protein fused to the DACT2 protein, as long as the resulting fusion protein retains the biological activity of the DACT2 protein.
本发明的DACT2蛋白也包括对SEQ ID NO.2所示的氨基酸序列的非保守修饰，只要经过修饰的蛋白质仍然能够保留DACT2蛋白的生物学活性即可。在此类修饰蛋白质中突变的氨基酸数目通常是10个或者更少，例如6个或者更少，例如3个或者更少。The DACT2 protein of the present invention also includes non-conservative modifications to the amino acid sequence shown in SEQ ID NO.2, as long as the modified protein can still retain the biological activity of the DACT2 protein. The number of amino acids mutated in such modified proteins is usually 10 or less, such as 6 or less, such as 3 or less.
在本发明的上下文中，“治疗心力衰竭”从疾病的状态变化来分，可以包括疾病的缓解、疾病的完全治愈。In the context of the present invention, "treating heart failure" is divided from the state change of the disease, and may include remission of the disease and complete cure of the disease.
本发明使用体外培养的SD大鼠心肌细胞株H9C2来研究DACT2基因对心力衰竭的治疗作用。本领域技术人员可知，α-MHC、β-MHC、ANP、BNP和ANF为心力衰竭的标志物，因此本发明利用H9C2心肌细胞过表达DACT2来研究DACT2基因与心力衰竭治疗的关系。The invention uses SD rat myocardial cell line H9C2 cultivated in vitro to study the therapeutic effect of DACT2 gene on heart failure. Those skilled in the art know that α-MHC, β-MHC, ANP, BNP and ANF are markers of heart failure, so the present invention uses H9C2 cardiomyocytes to overexpress DACT2 to study the relationship between DACT2 gene and treatment of heart failure.
本发明的优点和有益效果：Advantages and beneficial effects of the present invention:
本发明首次发现了DACT2基因表达与心力衰竭相关，可以将其开发为治疗心力衰竭疾病的潜在治疗药物。The present invention discovers for the first time that DACT2 gene expression is related to heart failure, and it can be developed as a potential therapeutic drug for treating heart failure.
附图说明：Description of drawings:
图1显示利用免疫荧光检测DACT2基因的过表达情况；Figure 1 shows the overexpression of the DACT2 gene detected by immunofluorescence;
图2显示利用RT-PCR检测DACT2基因的过表达情况；Figure 2 shows the overexpression of the DACT2 gene detected by RT-PCR;
图3显示利用WesternBlot检测DACT2蛋白的过表达情况；Figure 3 shows the overexpression of DACT2 protein detected by WesternBlot;
图4显示利用RT-PCR检测过表达DACT2对心肌细胞心衰指标α-MHC和β-MHC表达水平的影响；Figure 4 shows the impact of overexpression of DACT2 on the expression levels of cardiomyocyte heart failure indicators α-MHC and β-MHC detected by RT-PCR;
图5显示利用RT-PCR检测过表达DACT2对心肌细胞心衰指标ANP和BNP表达水平的影响；Figure 5 shows the impact of overexpression of DACT2 on the expression levels of cardiomyocyte heart failure indicators ANP and BNP detected by RT-PCR;
图6显示利用RT-PCR检测过表达DACT2对心肌细胞心衰指标ANF表达水平的影响。Fig. 6 shows the effect of overexpression of DACT2 on the expression level of cardiomyocyte heart failure index ANF detected by RT-PCR.
具体的实施方式：Specific implementation methods:
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(NewYork:ColdSpringHarborLaboratoryPress,1989)中所述的条件，或按照制造厂商所建议的条件。The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the examples are usually in accordance with conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer.
实施例1 DACT2基因过表达Example 1 DACT2 gene overexpression
1、细胞培养1. Cell culture
本研究采用SD大鼠心肌细胞系H9C2。H9C2细胞株，以含抗生素(100U/ml青霉素和100mg/L链霉素)和15％胎牛血清的DMEM(高糖)培养基在37℃、5％CO2的培养箱中培养。2-3天换液1次，使用0.25％胰蛋白酶常规消化传代。In this study, SD rat cardiomyocyte cell line H9C2 was used. H9C2 cell lines were cultivated in an incubator at 37° C. and 5% CO2 with DMEM (high glucose) medium containing antibiotics (100 U/ml penicillin and 100 mg/L streptomycin) and 15% fetal bovine serum. The medium was changed once every 2-3 days, and 0.25% trypsin was used for routine digestion and passage.
2、DACT2基因的过表达2. Overexpression of DACT2 gene
(1)DACT2基因过表达载体的构建(1) Construction of DACT2 gene overexpression vector
过表达DACT2基因(其核苷酸序列如SEQ ID NO.1所示)的Ad5-DACT2-eGFP病毒和Ad5-CON177-eGFP空病毒由上海吉凯基因化学技术有限公司采用Ad-CMV-eGFP载体构建获得。Ad5-DACT2-eGFP virus and Ad5-CON177-eGFP empty virus of overexpression DACT2 gene (its nucleotide sequence is shown in SEQ ID NO.1) adopts Ad-CMV-eGFP vector by Shanghai Jikai Gene Chemical Technology Co., Ltd. Build gets.
(2)转染(2) Transfection
将细胞种于6孔板，培养24h后换含有每个细胞所需病毒multiplicityofinfection(MOI)(过表达DACT2的Ad5-DACT2-eGFP病毒或Ad5-CON177-eGFP空病毒)的新鲜培养基进行感染，18h后，吸去含有病毒的培养基，换新鲜培养基。The cells were planted in a 6-well plate, and after 24 hours of culture, the fresh medium containing the virus multiplicity of infection (MOI) required for each cell (Ad5-DACT2-eGFP virus overexpressing DACT2 or Ad5-CON177-eGFP empty virus) was replaced for infection. After 18 hours, the culture medium containing the virus was aspirated and replaced with fresh culture medium.
(3)免疫荧光检测(3) Immunofluorescence detection
感染48h用荧光显微镜观察细胞的GFP荧光强度。After 48 hours of infection, the GFP fluorescence intensity of the cells was observed with a fluorescence microscope.
(4)RT-PCR检测(4) RT-PCR detection
Ad-DACT2-eGFP感染H9C2细胞48h后使用Trizol法提取RNA，cDNA合成使用PrimeScriptTMRTMasterMix(PerfectRealTime)试剂盒，条件：37℃，15min；85℃，5s；4℃保存。Real-timePCR使用的是TBGreenTMPremixExTaqII(TliRNAseHPlus)试剂盒，检测仪器为LightCycler480荧光定量PCR仪，反应条件为：预变性：95℃，30s；PCR反应：95℃，5s；60℃20s；40个循环；熔解曲线设置：95℃，15s；60℃，1min；95℃，15s。48 hours after Ad-DACT2-eGFP infected H9C2 cells, RNA was extracted using Trizol method, and cDNA was synthesized using PrimeScriptTM RTMasterMix (PerfectRealTime) kit, conditions: 37°C, 15min; 85°C, 5s; 4°C storage. Real-time PCR uses TBGreenTM PremixExTaqII (TliRNAseHPlus) kit, the detection instrument is LightCycler480 fluorescent quantitative PCR instrument, the reaction conditions are: pre-denaturation: 95°C, 30s; PCR reaction: 95°C, 5s; 60°C, 20s; 40 Cycle; melting curve setting: 95°C, 15s; 60°C, 1min; 95°C, 15s.
内参基因选择GAPDH(引物由ThermoFisherScientific公司合成)，空白对照以超纯水代替模板DNA，目标基因的相对表达量采用方法。所使用的相关基因引物序列如下：DACT2上游引物：5’-ACGCAGGGACAGTGGTCTCAT-3’，下游引物：5’-GCAGGAAACAGATGAGCCGG-3’；GAPDH上游引物：5’-ACAGCAACAGGGTGGTGGAC-3’，下游引物：5’-TTTGAGGGTGCAGCGAACTT-3’。上述实验重复3次。GAPDH was selected as an internal reference gene (primers were synthesized by ThermoFisher Scientific Company), the blank control was replaced by ultrapure water for template DNA, and the relative expression of the target gene was obtained by method. The primer sequences of related genes used are as follows: DACT2 upstream primer: 5'-ACGCAGGGACAGTGGTCTCAT-3', downstream primer: 5'-GCAGGAAACAGATGAGCCGG-3'; GAPDH upstream primer: 5'-ACAGCAACAGGGTGGTGGAC-3', downstream primer: 5'- TTTGAGGGTGCAGCGAACTT-3'. The above experiments were repeated 3 times.
(5)Westernblot检测(5) Western blot detection
Ad-DACT2-eGFP感染细胞48h后将培养液吸弃，用预冷的PBS清洗3次，每孔加入100ul含有cocktail的裂解液用细胞刮将细胞刮下，吸至新的EP管，在冰面上裂解细胞30min，4℃离心，13000G10min，吸取上清。BCA法进行蛋白定量。取一定量的蛋白样品，加入6×的LoadingBuffer，100℃加热5min使蛋白变性。取等量蛋白(20μg)进行SDS-PAGE电泳，先以60V的电压至条带出浓缩胶，后以90V恒压跑至溴酚蓝至胶的底沿。用湿转方法将SDS-PAGE胶中蛋白电转移至PVDF膜上，60V恒压的条件下转膜2h。室温下用含5％脱脂奶粉的TBS-T孵育lh以封闭膜上非特异性结合位点，分别加入相应的一抗(用5％脱脂奶粉的TBS-T稀释，DACT2蛋白的一抗)，4℃过夜。次日TBS-T洗膜3次，每次10min。加入相应的辣根过氧化物酶标记的抗鼠或兔IgG二抗(均为1︰5000稀释)，室温孵育1.5h，TBS-T洗膜3次，每次10min。ECL发光液显色，用化学发光成像仪(ChemiDocTouch)曝光。以GAPDH为内参。After Ad-DACT2-eGFP infected cells for 48 hours, the culture medium was discarded, washed 3 times with pre-cooled PBS, and 100ul lysate containing cocktail was added to each well to scrape off the cells with a cell scraper, sucked into a new EP tube, and stored on ice. Cells were lysed on the surface for 30 minutes, centrifuged at 4°C, 13000G for 10 minutes, and the supernatant was aspirated. BCA method for protein quantification. Take a certain amount of protein sample, add 6×LoadingBuffer, heat at 100°C for 5min to denature the protein. Take an equal amount of protein (20 μg) for SDS-PAGE electrophoresis. Firstly, the strips are taken out of the stacking gel with a voltage of 60V, and then run to the bottom edge of the gel with bromophenol blue at a constant voltage of 90V. The protein in the SDS-PAGE gel was transferred to the PVDF membrane by wet transfer method, and transferred to the membrane under the condition of 60V constant voltage for 2h. Incubate for 1 h at room temperature with TBS-T containing 5% skimmed milk powder to block the non-specific binding sites on the membrane, and add the corresponding primary antibody (diluted with 5% skimmed milk powder TBS-T, the primary antibody of DACT2 protein), 4 ℃ overnight. The next day, the membrane was washed 3 times with TBS-T, 10 min each time. Add the corresponding horseradish peroxidase-labeled anti-mouse or rabbit IgG secondary antibody (both diluted 1:5000), incubate at room temperature for 1.5 h, and wash the membrane 3 times with TBS-T, 10 min each time. The ECL luminescent liquid was developed and exposed with a chemiluminescence imager (ChemiDocTouch). GAPDH was used as internal reference.
3、统计学方法3. Statistical methods
实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS19.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。The experiments were repeated 3 times, and the result data were expressed in the form of mean ± standard deviation. SPSS19.0 statistical software was used for statistical analysis, and the difference between the two was tested by t test. It is statistically significant when P<0.05.
4、结果4. Results
如图1所示，Ad-DACT2-eGFP感染H9C2心肌细胞48h后的绿色荧光表达情况。As shown in FIG. 1 , the expression of green fluorescence in H9C2 cardiomyocytes 48 hours after Ad-DACT2-eGFP infection.
如图2所示，RT-PCR检测显示，Ad-DACT2-eGFP感染组(DACT2)与空载组(Control)相比，感染组DACT2的mRNA表达显著升高，且差异具有统计学意义(P<0.05)，提示载体构建、病毒包装、细胞感染、过表达目的基因成功。As shown in Figure 2, RT-PCR detection showed that the mRNA expression of DACT2 in the Ad-DACT2-eGFP infection group (DACT2) was significantly higher than that in the control group (Control), and the difference was statistically significant (P <0.05), indicating that vector construction, virus packaging, cell infection, and overexpression of the target gene were successful.
如图3所示，WesternBlot检测显示，与空载组(Control)相比，Ad-DACT2-eGFP感染组(DACT2)的DACT2的蛋白表达水平明显上调，差异具有统计学意义(P<0.05)。As shown in Figure 3, Western Blot detection showed that the protein expression level of DACT2 in the Ad-DACT2-eGFP infected group (DACT2) was significantly up-regulated compared with the empty group (Control), and the difference was statistically significant (P<0.05).
这些数据提示腺病毒Ad-DACT2-eGFP载体构建、病毒包装、细胞感染成功，并且成功过表达DACT2。These data suggest that the construction of adenovirus Ad-DACT2-eGFP vector, virus packaging, and cell infection were successful, and DACT2 was successfully overexpressed.
实施例2 DACT2基因过表达对心肌细胞心衰指标表达水平的影响Example 2 Effect of overexpression of DACT2 gene on the expression level of heart failure indicators in cardiomyocytes
1、采用RT-PCR检测过表达DACT2基因对H9C2心肌细胞株α-MHC、β-MHC、ANP、BNP及ANF表达水平的影响。1. RT-PCR was used to detect the effect of overexpression of DACT2 gene on the expression levels of α-MHC, β-MHC, ANP, BNP and ANF in H9C2 cardiomyocytes.
2、RT-PCR具体操作步骤同实施例1。2. The specific operation steps of RT-PCR are the same as those in Example 1.
以Ad-DACT2-eGFP感染细胞或Ad5-CON177-eGFP空病毒感染细胞48h后的H9C2心肌细胞作为检测对象。Ad-DACT2-eGFP-infected cells or Ad5-CON177-eGFP empty virus-infected H9C2 cardiomyocytes 48h later were used as detection objects.
内参基因选择GAPDH(引物由ThermoFisherScientific公司合成)，空白对照以超纯水代替模板DNA，目标基因的相对表达量采用方法。所使用的相关基因引物序列如下：α-MHC上游引物：5’-TCCTTTATCGGTATGGAGTCTG-3’，下游引物：5’-TGATCTTGATCTTCATGGTGCT-3’；β-MHC上游引物：5’-CACTCCAGAAGAGAAGAACTCCA-3’，下游引物：5’-ATACTGTTGCCCACTTTGACT-3’；ANP上游引物：5’-AGGAGAAGATGCCGGTAGAAG-3’，下游引物：5’-AGAGCCCTCAGTTTGCTTTTC-3’；BNP上游引物：5’-CAGAAGCTGCTGGAGCTGATAAG-3’，下游引物：5’-TGTAGGGCCTTGGTCCTTTG-3’；ANF上游引物：5’-GGAAGTCAACCCGTCTCA-3’，下游引物：5’-AGCCCTCAGTTTGCTTTT-3’。GAPDH was selected as an internal reference gene (primers were synthesized by ThermoFisher Scientific Company), the blank control was replaced by ultrapure water for template DNA, and the relative expression of the target gene was obtained by method. The primer sequences of related genes used are as follows: α-MHC upstream primer: 5'-TCCTTTATCGGTATGGAGTCTG-3', downstream primer: 5'-TGATCTTGATCTTCATGGTGCT-3'; β-MHC upstream primer: 5'-CACTCCAGAAGAGAAGAACTCCA-3', downstream primer : 5'-ATACTGTTGCCCACTTTGACT-3'; ANP upstream primer: 5'-AGGAGAAGATGCCGGTAGAAG-3', downstream primer: 5'-AGAGCCCTCAGTTTGCTTTTC-3'; BNP upstream primer: 5'-CAGAAGCTGCTGGAGCTGATAAG-3', downstream primer: 5'- TGTAGGGCCTTGGTCCTTTG-3'; ANF upstream primer: 5'-GGAAGTCAACCCGTCTCA-3', downstream primer: 5'-AGCCCCAGTTTGCTTTT-3'.
3、统计学方法3. Statistical methods
实验都是按照重复3次来完成的，结果数据都是以平均值±标准差的方式来表示，采用SPSS19.0统计软件来进行统计分析的，两者之间的差异采用t检验，认为当P<0.05时具有统计学意义。The experiments were repeated 3 times, and the result data were expressed in the form of mean ± standard deviation. SPSS19.0 statistical software was used for statistical analysis, and the difference between the two was tested by t test. It is statistically significant when P<0.05.
4、结果4. Results
如图4所示，RT-PCR检测显示，Ad-DACT2-eGFP感染组(DACT2，下同)与空载组(Control，下同)相比，感染组α-MHC和β-MHC的mRNA表达降低，α-MHC差异具有统计学意义(P<0.05)，但β-MHC差异不具有统计学意义(P>0.05)。As shown in Figure 4, RT-PCR detection showed that the mRNA expression of α-MHC and β-MHC in the Ad-DACT2-eGFP infection group (DACT2, the same below) was compared with the empty load group (Control, the same below). The difference of α-MHC was statistically significant (P<0.05), but the difference of β-MHC was not statistically significant (P>0.05).
如图5所示，RT-PCR检测显示，Ad-DACT2-eGFP感染组与空载组相比，感染组ANP和BNP的mRNA表达均显著降低，差异均具有统计学意义(P<0.05)。As shown in Figure 5, RT-PCR detection showed that the mRNA expressions of ANP and BNP in the Ad-DACT2-eGFP infection group were significantly lower than those in the empty group, and the differences were statistically significant (P<0.05).
如图6所示，RT-PCR检测显示，Ad-DACT2-eGFP感染组与空载组相比，感染组ANFmRNA表达显著降低，差异具有统计学意义(P<0.05)。As shown in Fig. 6, RT-PCR detection showed that the expression of ANF mRNA in the Ad-DACT2-eGFP infection group was significantly lower than that in the empty group, and the difference was statistically significant (P<0.05).
此结果表明，DACT2可能通过抑制α-MHC、ANP、BNP和ANF心力衰竭标志物的表达，对心衰起保护作用，提示Ad-DACT2-eGFP腺病毒可作为心力衰竭疾病的潜在治疗药物。The results indicated that DACT2 may play a protective role in heart failure by inhibiting the expression of α-MHC, ANP, BNP and ANF heart failure markers, suggesting that Ad-DACT2-eGFP adenovirus can be used as a potential therapeutic drug for heart failure.
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。The description of the above embodiments is only for understanding the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.
序列表sequence listing
<110> 中山大学附属第一医院<110> The First Affiliated Hospital of Sun Yat-sen University
<120> DACT2基因在制备心力衰竭治疗药物中的应用<120> Application of DACT2 gene in the preparation of drugs for treating heart failure
<160> 2<160> 2
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 2325<211> 2325
<212> DNA<212>DNA
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 1<400> 1
atgtggacgc cgggcggacc cccggggtcc gcgggctggg accgccgtag gttgggcgcg 60atgtggacgc cgggcggacc cccggggtcc gcgggctggg accgccgtag gttggggcgcg 60
aggttgcgcg cggcgttcgc ggggctgcag gagctgcagg ggctgcgagc cacgcagcag 120aggttgcgcg cggcgttcgc ggggctgcag gagctgcagg ggctgcgagc cacgcagcag 120
gagcgggtac ggggcgccct ggccctgcag cccccgcccg cgcccgccgc gccctgcggc 180gagcgggtac ggggcgccct ggccctgcag cccccgcccg cgcccgccgc gccctgcggc 180
ccccacggcc tccacggccc cgagcagcag ctggaggcgg cgctggccgc gctgcaggag 240ccccacggcc tccacggccc cgagcagcag ctggaggcgg cgctggccgc gctgcaggag 240
cagctgtccc ggctgagaca acaggacatc ggcctgaaga cccacctgga ccagctggac 300cagctgtccc ggctgagaca acaggacatc ggcctgaaga cccacctgga ccagctggac 300
ctgcagatta gcaagctgca gctggatgtg ggcacagcct caggggaggc cctggacagc 360ctgcagatta gcaagctgca gctggatgtg ggcacagcct caggggaggc cctggacagc 360
gacagcaggc ccagctcagg cttttacgag atgagcgacg gtggatcctg ctccctgtcc 420gacagcaggc ccagctcagg cttttacgag atgagcgacg gtggatcctg ctccctgtcc 420
acgtcctgtg cctccgtctg cagtgaccac atctctccct cgctgggcag tttgttgcct 480acgtcctgtg cctccgtctg cagtgaccac atctctccct cgctgggcag tttgttgcct 480
gtggcccagg cccacaaggc caggcccagc atgggggact ggaggccccg gtcggttgat 540gtggcccagg cccacaaggc caggcccagc atgggggact ggaggccccg gtcggttgat 540
gagactactg tgccagcgtg gagaccccag gctaccgagg agggcgccag gcccccaggg 600gagactactg tgccagcgtg gagaccccag gctaccgagg agggcgccag gcccccaggg 600
agcgtggagg atgcaggcca gccgtggggc acattctggc ccaggcctgt gtctacaggt 660agcgtggagg atgcaggcca gccgtggggc attctggc ccaggcctgt gtctacaggt 660
gatcttgaca gagccctgcc ggcggacacg gggctccaga aagccagcgc ggacgccgag 720gatcttgaca gagccctgcc ggcggacacg gggctccaga aagccagcgc ggacgccgag 720
ctcctcgggc tcctctgcca gggggtggat atcccgctgc acgtgccgga ccccaagtat 780ctcctcgggc tccctctgcca gggggtggat atcccgctgc acgtgccgga ccccaagtat 780
cggcaggacc tggtgtccca gggcggcagg gaggtgtacc cgtaccccag ccccctgcac 840cggcaggacc tggtgtccca gggcggcagg gaggtgtacc cgtaccccag ccccctgcac 840
gccgtggctc tacagagccc cctgtttgtc ctgactaagg aaaccccaca gagaggtggc 900gccgtggctc tacagagccc cctgtttgtc ctgactaagg aaaccccaca gagaggtggc 900
ccctcgttcc ctagggagag ccccaggggc cccgcaggtc tgaacaccat ccagactggg 960ccctcgttcc ctagggagag ccccaggggc cccgcaggtc tgaacaccat ccagactgggg 960
ccggtcctcg aggctggccc ggccagggcc agagcttata tcgacaggtt gctgcatctg 1020ccggtcctcg aggctggccc ggccagggcc agagcttata tcgacaggtt gctgcatctg 1020
tggggccggg agaccccagc aaagggtagc gagggagaac agggacctct aaggcatgca 1080tggggccggg agaccccagc aaagggtagc gagggagaac agggacctct aaggcatgca 1080
gcgtccccat ctccacagag gcagggtggc tggagtacag acggtggagg gcgactgctg 1140gcgtccccat ctccacagag gcagggtggc tggagtacag acggtggagg gcgactgctg 1140
gtcttcgccc cagggaggga ggacgaggga gggccagctc agagcagggg tgccggcagg 1200gtcttcgccc cagggaggga ggacgaggga gggccagctc agagcagggg tgccggcagg 1200
ggcgggcccc agcagcaggg atacatgccc cttgagggtc cccagcagtc tggcagcctt 1260ggcgggcccc agcagcaggg acatatgccc cttgagggtc cccagcagtc tggcagcctt 1260
ccagaggagg gctccaagcc ctcaaacagc tgtgtcctca gggagaccat ggtgcaggct 1320ccagaggagg gctccaagcc ctcaaacagc tgtgtcctca gggagaccat ggtgcaggct 1320
tctcccagct caaaggccca gcagacaccc tcagctcagg actatggacg aggcaacatc 1380tctcccagct caaaggccca gcagacaccc tcagctcagg actatggacg aggcaacatc 1380
atatccccat ccaggatgct ggacaagagc ccctcaccgg cctctgggca ctttgcccac 1440atatccccat ccaggatgct ggacaagagc ccctcaccgg cctctgggca ctttgcccac 1440
ccatcctttg ctgccagcct gaaaatgggt ccccccaaga gcaaggctga aaaaatcaag 1500ccatcctttg ctgccagcct gaaaatgggt ccccccaaga gcaaggctga aaaaatcaag 1500
agaagtccca tggacaaggt gctgaggttt gcaaggcagc cgctgcttct actggacagg 1560agaagtccca tggacaaggt gctgaggttt gcaaggcagc cgctgcttct actggacagg 1560
cctgagggag cccatgcagc cccccagcca tccctggagt gggaccctgc ccactggccc 1620cctgaggggag cccatgcagc cccccagcca tccctggagt gggaccctgc ccactggccc 1620
acagggaggg gcgggctcca gcggaggcca gccctggcct gggaggcacc cgggcgctcc 1680acaggggaggg gcggggctcca gcgggaggcca gccctggcct gggaggcacc cgggcgctcc 1680
tgttctgagt ccaccctcta ccccatgcct gtcctcgtcc ccttggcagt ggccccgcag 1740tgttctgagt ccaccctcta ccccatgcct gtcctcgtcc ccttggcagt ggccccgcag 1740
gagagccacc ggacctcagc ccaagccctg ttcccctttg aggcgtcact gctcacctca 1800gagagccacc ggacctcagc ccaagccctg ttcccctttg aggcgtcact gctcacctca 1800
gtggccagga ggaagcatcg ccgctggcag tccaccgtgg agatctcggc ccgggcccgc 1860gtggccagga ggaagcatcg ccgctggcag tccaccgtgg agatctcggc ccgggcccgc 1860
ctggccagct gtcctgagtc taacctgggg ccccccaggc ccgtggccag gagagcaggt 1920ctggccagct gtcctgagtc taacctgggg ccccccaggc ccgtggccag gagagcaggt 1920
ggcccactgg cccggggccg tccctcactg gtccgccagg acgcctacac caggagcgac 1980ggcccactgg cccggggccg tccctcactg gtccgccagg acgcctacac caggagcgac 1980
tcagagccct ccaagcactc ggccgagtgt gacccgcggt tcccgtcagt catcccggag 2040tcagagccct ccaagcactc ggccgagtgt gacccgcggt tcccgtcagt catcccggag 2040
accagcgagg gagagtccag tgaccacacc accaaccgat tcggagaccg tgagtccagc 2100accagcgagg gagagtccag tgaccacacc accaaccgat tcggagaccg tgagtccagc 2100
agcagcgacg aggagggcgg cgcccagagc agggactgtg acctggcact gggctatgtg 2160agcagcgacg aggagggcgg cgcccagagc agggactgtg acctggcact gggctatgtg 2160
gcggccgggc atgcggagct ggcctggacc caggaggccc cggtcagctc ggggccactc 2220gcggccgggc atgcggagct ggcctggacc caggaggccc cggtcagctc ggggccactc 2220
ctgtcccccg tgcccaagct gtgccgtatt aaggcctcca aggccctgaa gaagaagatc 2280ctgtcccccg tgcccaagct gtgccgtatt aaggcctcca aggccctgaa gaagaagatc 2280
cgcaggttcc agccgacggc cctgaaggtc atgaccatgg tgtga 2325cgcaggttcc agccgacggc cctgaaggtc atgaccatgg tgtga 2325
<210> 2<210> 2
<211> 774<211> 774
<212> PRT<212> PRT
<213> 人(Homo sapiens)<213> Human (Homo sapiens)
<400> 2<400> 2
Met Trp Thr Pro Gly Gly Pro Pro Gly Ser Ala Gly Trp Asp Arg ArgMet Trp Thr Pro Gly Gly Pro Pro Gly Ser Ala Gly Trp Asp Arg Arg
1 5 10 151 5 10 15
Arg Leu Gly Ala Arg Leu Arg Ala Ala Phe Ala Gly Leu Gln Glu LeuArg Leu Gly Ala Arg Leu Arg Ala Ala Phe Ala Gly Leu Gln Glu Leu
20 25 30 20 25 30
Gln Gly Leu Arg Ala Thr Gln Gln Glu Arg Val Arg Gly Ala Leu AlaGln Gly Leu Arg Ala Thr Gln Gln Glu Arg Val Arg Gly Ala Leu Ala
35 40 45 35 40 45
Leu Gln Pro Pro Pro Ala Pro Ala Ala Pro Cys Gly Pro His Gly LeuLeu Gln Pro Pro Pro Ala Pro Ala Ala Pro Cys Gly Pro His Gly Leu
50 55 60 50 55 60
His Gly Pro Glu Gln Gln Leu Glu Ala Ala Leu Ala Ala Leu Gln GluHis Gly Pro Glu Gln Gln Leu Glu Ala Ala Leu Ala Ala Leu Gln Glu
65 70 75 8065 70 75 80
Gln Leu Ser Arg Leu Arg Gln Gln Asp Ile Gly Leu Lys Thr His LeuGln Leu Ser Arg Leu Arg Gln Gln Asp Ile Gly Leu Lys Thr His Leu
85 90 95 85 90 95
Asp Gln Leu Asp Leu Gln Ile Ser Lys Leu Gln Leu Asp Val Gly ThrAsp Gln Leu Asp Leu Gln Ile Ser Lys Leu Gln Leu Asp Val Gly Thr
100 105 110 100 105 110
Ala Ser Gly Glu Ala Leu Asp Ser Asp Ser Arg Pro Ser Ser Gly PheAla Ser Gly Glu Ala Leu Asp Ser Asp Ser Arg Pro Ser Ser Gly Phe
115 120 125 115 120 125
Tyr Glu Met Ser Asp Gly Gly Ser Cys Ser Leu Ser Thr Ser Cys AlaTyr Glu Met Ser Asp Gly Gly Ser Cys Ser Leu Ser Thr Ser Cys Ala
130 135 140 130 135 140
Ser Val Cys Ser Asp His Ile Ser Pro Ser Leu Gly Ser Leu Leu ProSer Val Cys Ser Asp His Ile Ser Pro Ser Leu Gly Ser Leu Leu Pro
145 150 155 160145 150 155 160
Val Ala Gln Ala His Lys Ala Arg Pro Ser Met Gly Asp Trp Arg ProVal Ala Gln Ala His Lys Ala Arg Pro Ser Met Gly Asp Trp Arg Pro
165 170 175 165 170 175
Arg Ser Val Asp Glu Thr Thr Val Pro Ala Trp Arg Pro Gln Ala ThrArg Ser Val Asp Glu Thr Thr Val Pro Ala Trp Arg Pro Gln Ala Thr
180 185 190 180 185 190
Glu Glu Gly Ala Arg Pro Pro Gly Ser Val Glu Asp Ala Gly Gln ProGlu Glu Gly Ala Arg Pro Pro Gly Ser Val Glu Asp Ala Gly Gln Pro
195 200 205 195 200 205
Trp Gly Thr Phe Trp Pro Arg Pro Val Ser Thr Gly Asp Leu Asp ArgTrp Gly Thr Phe Trp Pro Arg Pro Val Ser Thr Gly Asp Leu Asp Arg
210 215 220 210 215 220
Ala Leu Pro Ala Asp Thr Gly Leu Gln Lys Ala Ser Ala Asp Ala GluAla Leu Pro Ala Asp Thr Gly Leu Gln Lys Ala Ser Ala Asp Ala Glu
225 230 235 240225 230 235 240
Leu Leu Gly Leu Leu Cys Gln Gly Val Asp Ile Pro Leu His Val ProLeu Leu Gly Leu Leu Cys Gln Gly Val Asp Ile Pro Leu His Val Pro
245 250 255 245 250 255
Asp Pro Lys Tyr Arg Gln Asp Leu Val Ser Gln Gly Gly Arg Glu ValAsp Pro Lys Tyr Arg Gln Asp Leu Val Ser Gln Gly Gly Arg Glu Val
260 265 270 260 265 270
Tyr Pro Tyr Pro Ser Pro Leu His Ala Val Ala Leu Gln Ser Pro LeuTyr Pro Tyr Pro Ser Pro Leu His Ala Val Ala Leu Gln Ser Pro Leu
275 280 285 275 280 285
Phe Val Leu Thr Lys Glu Thr Pro Gln Arg Gly Gly Pro Ser Phe ProPhe Val Leu Thr Lys Glu Thr Pro Gln Arg Gly Gly Pro Ser Phe Pro
290 295 300 290 295 300
Arg Glu Ser Pro Arg Gly Pro Ala Gly Leu Asn Thr Ile Gln Thr GlyArg Glu Ser Pro Arg Gly Pro Ala Gly Leu Asn Thr Ile Gln Thr Gly
305 310 315 320305 310 315 320
Pro Val Leu Glu Ala Gly Pro Ala Arg Ala Arg Ala Tyr Ile Asp ArgPro Val Leu Glu Ala Gly Pro Ala Arg Ala Arg Ala Tyr Ile Asp Arg
325 330 335 325 330 335
Leu Leu His Leu Trp Gly Arg Glu Thr Pro Ala Lys Gly Ser Glu GlyLeu Leu His Leu Trp Gly Arg Glu Thr Pro Ala Lys Gly Ser Glu Gly
340 345 350 340 345 350
Glu Gln Gly Pro Leu Arg His Ala Ala Ser Pro Ser Pro Gln Arg GlnGlu Gln Gly Pro Leu Arg His Ala Ala Ser Pro Ser Pro Gln Arg Gln
355 360 365 355 360 365
Gly Gly Trp Ser Thr Asp Gly Gly Gly Arg Leu Leu Val Phe Ala ProGly Gly Trp Ser Thr Asp Gly Gly Gly Arg Leu Leu Val Phe Ala Pro
370 375 380 370 375 380
Gly Arg Glu Asp Glu Gly Gly Pro Ala Gln Ser Arg Gly Ala Gly ArgGly Arg Glu Asp Glu Gly Gly Pro Ala Gln Ser Arg Gly Ala Gly Arg
385 390 395 400385 390 395 400
Gly Gly Pro Gln Gln Gln Gly Tyr Met Pro Leu Glu Gly Pro Gln GlnGly Gly Pro Gln Gln Gln Gly Tyr Met Pro Leu Glu Gly Pro Gln Gln
405 410 415 405 410 415
Ser Gly Ser Leu Pro Glu Glu Gly Ser Lys Pro Ser Asn Ser Cys ValSer Gly Ser Leu Pro Glu Glu Gly Ser Lys Pro Ser Asn Ser Cys Val
420 425 430 420 425 430
Leu Arg Glu Thr Met Val Gln Ala Ser Pro Ser Ser Lys Ala Gln GlnLeu Arg Glu Thr Met Val Gln Ala Ser Pro Ser Ser Lys Ala Gln Gln
435 440 445 435 440 445
Thr Pro Ser Ala Gln Asp Tyr Gly Arg Gly Asn Ile Ile Ser Pro SerThr Pro Ser Ala Gln Asp Tyr Gly Arg Gly Asn Ile Ile Ser Pro Ser
450 455 460 450 455 460
Arg Met Leu Asp Lys Ser Pro Ser Pro Ala Ser Gly His Phe Ala HisArg Met Leu Asp Lys Ser Pro Ser Pro Ala Ser Gly His Phe Ala His
465 470 475 480465 470 475 480
Pro Ser Phe Ala Ala Ser Leu Lys Met Gly Pro Pro Lys Ser Lys AlaPro Ser Phe Ala Ala Ser Leu Lys Met Gly Pro Pro Lys Ser Lys Ala
485 490 495 485 490 495
Glu Lys Ile Lys Arg Ser Pro Met Asp Lys Val Leu Arg Phe Ala ArgGlu Lys Ile Lys Arg Ser Pro Met Asp Lys Val Leu Arg Phe Ala Arg
500 505 510 500 505 510
Gln Pro Leu Leu Leu Leu Asp Arg Pro Glu Gly Ala His Ala Ala ProGln Pro Leu Leu Leu Leu Asp Arg Pro Glu Gly Ala His Ala Ala Pro
515 520 525 515 520 525
Gln Pro Ser Leu Glu Trp Asp Pro Ala His Trp Pro Thr Gly Arg GlyGln Pro Ser Leu Glu Trp Asp Pro Ala His Trp Pro Thr Gly Arg Gly
530 535 540 530 535 540
Gly Leu Gln Arg Arg Pro Ala Leu Ala Trp Glu Ala Pro Gly Arg SerGly Leu Gln Arg Arg Pro Ala Leu Ala Trp Glu Ala Pro Gly Arg Ser
545 550 555 560545 550 555 560
Cys Ser Glu Ser Thr Leu Tyr Pro Met Pro Val Leu Val Pro Leu AlaCys Ser Glu Ser Thr Leu Tyr Pro Met Pro Val Leu Val Pro Leu Ala
565 570 575 565 570 575
Val Ala Pro Gln Glu Ser His Arg Thr Ser Ala Gln Ala Leu Phe ProVal Ala Pro Gln Glu Ser His Arg Thr Ser Ala Gln Ala Leu Phe Pro
580 585 590 580 585 590
Phe Glu Ala Ser Leu Leu Thr Ser Val Ala Arg Arg Lys His Arg ArgPhe Glu Ala Ser Leu Leu Thr Ser Val Ala Arg Arg Lys His Arg Arg
595 600 605 595 600 605
Trp Gln Ser Thr Val Glu Ile Ser Ala Arg Ala Arg Leu Ala Ser CysTrp Gln Ser Thr Val Glu Ile Ser Ala Arg Ala Arg Leu Ala Ser Cys
610 615 620 610 615 620
Pro Glu Ser Asn Leu Gly Pro Pro Arg Pro Val Ala Arg Arg Ala GlyPro Glu Ser Asn Leu Gly Pro Pro Arg Pro Val Ala Arg Arg Ala Gly
625 630 635 640625 630 635 640
Gly Pro Leu Ala Arg Gly Arg Pro Ser Leu Val Arg Gln Asp Ala TyrGly Pro Leu Ala Arg Gly Arg Pro Ser Leu Val Arg Gln Asp Ala Tyr
645 650 655 645 650 655
Thr Arg Ser Asp Ser Glu Pro Ser Lys His Ser Ala Glu Cys Asp ProThr Arg Ser Asp Ser Glu Pro Ser Lys His Ser Ala Glu Cys Asp Pro
660 665 670 660 665 670
Arg Phe Pro Ser Val Ile Pro Glu Thr Ser Glu Gly Glu Ser Ser AspArg Phe Pro Ser Val Ile Pro Glu Thr Ser Glu Gly Glu Ser Ser Asp
675 680 685 675 680 685
His Thr Thr Asn Arg Phe Gly Asp Arg Glu Ser Ser Ser Ser Asp GluHis Thr Thr Asn Arg Phe Gly Asp Arg Glu Ser Ser Ser Ser Ser Asp Glu
690 695 700 690 695 700
Glu Gly Gly Ala Gln Ser Arg Asp Cys Asp Leu Ala Leu Gly Tyr ValGlu Gly Gly Ala Gln Ser Arg Asp Cys Asp Leu Ala Leu Gly Tyr Val
705 710 715 720705 710 715 720
Ala Ala Gly His Ala Glu Leu Ala Trp Thr Gln Glu Ala Pro Val SerAla Ala Gly His Ala Glu Leu Ala Trp Thr Gln Glu Ala Pro Val Ser
725 730 735 725 730 735
Ser Gly Pro Leu Leu Ser Pro Val Pro Lys Leu Cys Arg Ile Lys AlaSer Gly Pro Leu Leu Ser Pro Val Pro Lys Leu Cys Arg Ile Lys Ala
740 745 750 740 745 750
Ser Lys Ala Leu Lys Lys Lys Ile Arg Arg Phe Gln Pro Thr Ala LeuSer Lys Ala Leu Lys Lys Lys Ile Arg Arg Phe Gln Pro Thr Ala Leu
755 760 765 755 760 765
Lys Val Met Thr Met ValLys Val Met Thr Met Val
770 770
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910430328.XACN110408687A (en) | 2019-05-22 | 2019-05-22 | Application of DACT2 gene in preparation of medicine for treating heart failure |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910430328.XACN110408687A (en) | 2019-05-22 | 2019-05-22 | Application of DACT2 gene in preparation of medicine for treating heart failure |
| Publication Number | Publication Date |
|---|---|
| CN110408687Atrue CN110408687A (en) | 2019-11-05 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910430328.XAPendingCN110408687A (en) | 2019-05-22 | 2019-05-22 | Application of DACT2 gene in preparation of medicine for treating heart failure |
| Country | Link |
|---|---|
| CN (1) | CN110408687A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112156091A (en)* | 2020-09-30 | 2021-01-01 | 中山大学附属第一医院 | Application of hispidulin in preparation of medicine for treating and/or preventing cardiovascular diseases |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1368509A (en)* | 2001-02-08 | 2002-09-11 | 上海市肿瘤研究所 | Human protein with suppression to cancer cell growth and its coding sequence |
| EP1977747A1 (en)* | 2007-04-05 | 2008-10-08 | Max-Delbrück-Centrum | Adaptive cardiac remodeling by beta-catenin downregulation |
| CN105567797A (en)* | 2014-11-11 | 2016-05-11 | 香港中文大学深圳研究院 | PCR primer, method and kit for detecting CpG island methylation of DACT2 gene promoter area |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1368509A (en)* | 2001-02-08 | 2002-09-11 | 上海市肿瘤研究所 | Human protein with suppression to cancer cell growth and its coding sequence |
| EP1977747A1 (en)* | 2007-04-05 | 2008-10-08 | Max-Delbrück-Centrum | Adaptive cardiac remodeling by beta-catenin downregulation |
| CN105567797A (en)* | 2014-11-11 | 2016-05-11 | 香港中文大学深圳研究院 | PCR primer, method and kit for detecting CpG island methylation of DACT2 gene promoter area |
| Title |
|---|
| DANIEL A. FISHER等: "Three Dact gene family membets are expressed during embryonic development and in the adultbrMns of mice", 《DEVELOPMENTAL DYNAMICS》* |
| GEORGIOS KARARLGAS等: "Abstract400：Role of the 17 beta-Estradlol/beta-Catenin Axis in Heart Failure", 《CIRCULATION RESEARCH》* |
| JIAN HOU等: "DACT2 Is a Candidate Tumor Suppressor and PrognosticMarker in Esophageal Squamous Cell Carcinoma", 《AMERICAN ASSOCIATION FOR CANCER RESEARCH》* |
| JIAN WANG等: "Targeted Disruption of Smad4 in Cardiomyocytes Results inCardiac Hypertrophy and Heart Failure", 《CIRCULATION RESEARCH》* |
| 冷秀玉等: "心力衰竭基因治疗的研究进展", 《国外医学.内科学分册》* |
| 张晓梅等: "人Dact2基因真核表达载体的构建及其在结肠癌细胞中的表达", 《胃肠病学和肝病学杂志》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112156091A (en)* | 2020-09-30 | 2021-01-01 | 中山大学附属第一医院 | Application of hispidulin in preparation of medicine for treating and/or preventing cardiovascular diseases |
| Publication | Publication Date | Title |
|---|---|---|
| CN112138159B (en) | Use of lactate dehydrogenase in the treatment of tissue inflammation and fibrosis | |
| JP2003524366A (en) | 64 human secreted proteins | |
| CN110408687A (en) | Application of DACT2 gene in preparation of medicine for treating heart failure | |
| CN105112550B (en) | MTUS1 genes as osteoporosis diagnosis and treatment target | |
| CN104955836A (en) | Variants of the BPIFB4 protein | |
| CN110305950B (en) | Application of DACT2 gene in the preparation of atrial fibrillation diagnosis and treatment products | |
| WO2018191599A1 (en) | Brown fat-selective adipokines | |
| CN111465697B (en) | Pharmaceutical composition for preventing or treating heart failure | |
| CN118027168B (en) | Preparation method and application of MSL recombinant plant protein based on eukaryotic expression | |
| CN105861740B (en) | Purposes of the ABLIM3 gene as cancer of the esophagus diagnosis and treatment marker | |
| CN110946877A (en) | A stem cell biological product for treating liver cirrhosis and its preparation method and application | |
| JP2005538930A (en) | Complex of human FOXC2 protein and FOXC-2 interacting protein | |
| CN111808938A (en) | ATP6V0D2 for early diagnosis or curative effect monitoring of atherosclerosis | |
| CN105506170B (en) | Purposes of the SAV1 gene as fibroid diagnosis and treatment marker | |
| CN113355358B (en) | A kind of preparation method and application of serotype 9 recombinant adeno-associated virus compound agent | |
| US20030148952A1 (en) | Methods and materials for the recruitment of endothelial cells | |
| CN111254200B (en) | New application of Gal3st3 gene | |
| CN115957301A (en) | Cell secretion factor for promoting myocardial infarction repair and application | |
| CN105950752B (en) | Using PEX13 as a gene marker for the diagnosis of cholangiocarcinoma | |
| CN105671158B (en) | Purposes of the FAM63B gene in the product of preparation diagnosis and treatment fibroid | |
| CN105950746B (en) | The diagnosis and treatment target that RASL12 gene is shifted as lung squamous cancer | |
| CN103031293B (en) | Calcitonin-gene-related peptide and AcAPc2 fusion protein and encoding gene thereof | |
| JP2006347979A (en) | Drug for wound treatment and / or promotion of wound healing | |
| CN115944750A (en) | A codable circRNA related to buffalo skeletal muscle development and its application | |
| CN112662768A (en) | Application of circEPHA2 as breast cancer prognosis and tamoxifen drug resistance judgment marker |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | Application publication date:20191105 | |
| RJ01 | Rejection of invention patent application after publication |