TW201818959A - Use of FBP aldolase in preparation of drug activating AMPK - Google Patents

Use of FBP aldolase in preparation of drug activating AMPK Download PDF

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TW201818959A
TW201818959A TW106138391A TW106138391A TW201818959A TW 201818959 A TW201818959 A TW 201818959A TW 106138391 A TW106138391 A TW 106138391A TW 106138391 A TW106138391 A TW 106138391A TW 201818959 A TW201818959 A TW 201818959A
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aldolase
drug
inhibits
ampk
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林聖彩
張宸崧
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廈門大學
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Abstract

Disclosed is the use of FBP aldolase in the preparation of a drug activating AMPK. Also disclosed are the uses of FBP aldolase in the preparation of a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, a drug for preventing and/or treating diabetes, a drug for preventing and/or treating tumours, a drug for preventing and/or treating Parkinson's disease, a drug for preventing and/or treating Alzheimer's disease, or a drug for prolonging the lifespan of an organism. The use of FBP aldolase as a target to develop drugs to activate AMPK overcomes the difficulties of directly using AMPK as a drug target in the prior art and has good application prospects.

Description

FBP　ALDOLASE在製備啟動AMPK的藥物中的用途Use of FBP ALDOLASE in preparing AMPK-initiating medicine

本發明屬於生物醫藥領域，涉及FBP aldolase在製備啟動AMPK的藥物中的用途。本發明還涉及在製備抑制膽固醇合成的藥物、降低脂肪酸合成的藥物、預防及/或治療糖尿病的藥物、預防及/或治療腫瘤的藥物、預防及/或治療帕金森氏症的藥物、預防及/或治療阿茲海默症的藥物或者延長哺乳動物壽命的藥物中的用途。The invention belongs to the field of biomedicine and relates to the use of FBP aldolase in the preparation of a medicament for starting AMPK. The present invention also relates to preparation of a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, a drug for preventing and / or treating diabetes, a drug for preventing and / or treating tumors, a drug for preventing and / or treating Parkinson's disease, prevention and And / or use in a drug for treating Alzheimer's disease or a drug for extending the life of a mammal.

5’- 腺苷一磷酸活化的蛋白激酶（AMPK）是調節細胞和機體能量平衡的重要分子。AMPK由3 個不同的亞基所組成，各個亞基分別具有數個同功型：α亞基（α1或α2）；β亞基（β1或β2）；及γ亞基（γ1、γ2 或γ3）；總共有12種可能的雜三聚體的同功型，每種同功型均有其特異性分佈的組織類型，各種同功型共同組成了廣泛分佈在機體內的所有組織中的AMPK複合體。另外，AMPK的12種雜三聚體的功能是類似的，或者說目前並未見其不同功能的報導，其差別僅僅在組織特異性分佈上，任意一種同功型的AMPK啟動都能夠引起同樣的下游蛋白的啟動。5'-adenosine monophosphate-activated protein kinase (AMPK) is an important molecule that regulates the energy balance of cells and the body. AMPK consists of 3 different subunits, each of which has several isotypes: α subunit (α1 or α2); β subunit (β1 or β2); and γ subunit (γ1, γ2, or γ3) ); There are a total of 12 possible heterotrimeric isoforms, each isotype has its specific distribution of tissue types, and various isoforms together form AMPK that is widely distributed in all tissues in the body Complex. In addition, the functions of the 12 heterotrimers of AMPK are similar, or no different functions have been reported at present. The difference is only in the tissue-specific distribution. The activation of any kind of AMPK can cause the same Of downstream proteins.

傳統的觀點認為，AMPK的啟動是由其別構啟動因數——AMP/ADP及其類似物介導的。AMP/ADP能夠結合AMPK的γ亞基，引起全酶的結構變化，使其更易被其上游激酶磷酸化其α亞基上的蘇氨酸172位位點（p-AMPKα）從而被啟動。除了γ亞基引起的別構啟動，AMPK的β亞基也能被糖原等代謝物結合並被其調節活力。The traditional view is that the activation of AMPK is mediated by its allosteric activation factor, AMP / ADP and its analogs. AMP / ADP can bind to the γ subunit of AMPK, causing structural changes of the whole enzyme, making it more likely to be activated by its upstream kinase to phosphorylate the threonine 172 site (p-AMPKα) on its α subunit and thus be activated. In addition to the allosteric activation caused by the gamma subunit, the beta subunit of AMPK can also be bound by metabolites such as glycogen and regulated by it.

活化的AMPK 能夠磷酸化多種底物，經典的AMPK底物包括3-羥基-3-甲基戊二醯輔酶A（HMG-CoA）還原酶1 和乙醯輔酶A 羧化酶（ACC）2 ，其分別抑制膽固醇生物合成及降低脂肪酸合成，導致脂質合成代謝途徑被抑制，脂類分解途徑被加強，而這一功能與抑制糖尿病的形成和發展有重要的聯繫3 。AMPK還能夠促進葡萄糖轉運子4（GLUT4）向細胞膜上的轉移，後者能夠顯著促進血液中葡萄糖的吸收和同化，降低血糖，該作用與胰島素通路相平行，因而在具有胰島素抵抗的糖尿病病患的治療中具有重要的作用4-5 。上述AMPK對於脂質和蛋白質合成的抑制作用能夠顯著抑制腫瘤細胞的生長，從而抑制腫瘤（例如黑色素瘤、胰腺癌、卵巢癌或乳腺癌）的發生和發展6 。此外，AMPK能夠通過PGC1和SIRT等分子，上調體內NAD+ /NADH的比例，該功能被認為和長壽密切相關7-9 。線上蟲（C .elegans ）、果蠅和小鼠中都已經證明了啟動AMPK能夠顯著延長這些有機體的壽命10-12 。此外，AMPK還是自噬作用重要的調節因數。例如，AMPK能夠通過啟動ULK1（Unc-51 Like Autophagy Activating Kinase 1），顯著地促進巨自噬作用（macroautophagy）的發生，巨自噬作用是生命體維持能量和物質代謝平衡的基本過程之一，從生理功能的角度來看，它已被證明和糖尿病、腫瘤等重大疾病的發生密切相關13-14 。特別地，AMPK的這一功能還和脂肪組織的棕化密切相關15-16 ，後者被認為是緩解糖尿病，重塑健康脂肪組織的重要過程，啟動的AMPK也確實通過促進脂肪棕化抑制了糖尿病病情的發展17 。AMPK還能夠通過啟動MFF（mitochondrial fission factor），促進線粒體自噬作用（mitophagy）18 ，而後者的失調和帕金森氏症、阿茲海默症等重要的神經系統疾病有著重要的聯繫19 。由於AMPK 對碳水化合物、脂肪和膽固醇代謝及生物合成具有多功能性的作用，所以AMPK是治療重大疾病最吸引人的藥物靶標之一。近二十年來，學術界已經運用了多種篩選方法，以AMPK作為靶標得到了大量AMPK的啟動劑，並展開了大量研究。Activated AMPK can phosphorylate a variety of substrates. Classic AMPK substrates include 3-hydroxy-3-methylglutaridine coenzyme A (HMG-CoA) reductase 1 and acetamidine coenzyme A carboxylase (ACC) 2 , It inhibits cholesterol biosynthesis and decreases fatty acid synthesis, respectively, leading to the inhibition of lipid anabolic pathways and enhanced lipid breakdown pathways, and this function is important to inhibit the formation and development of diabetes 3 . AMPK can also promote the transfer of glucose transporter 4 (GLUT4) to the cell membrane, which can significantly promote the absorption and assimilation of glucose in the blood and reduce blood sugar. This effect is parallel to the insulin pathway, and therefore in patients with diabetes with insulin resistance Has an important role in treatment 4-5 . The aforementioned inhibitory effect of AMPK on lipid and protein synthesis can significantly inhibit the growth of tumor cells, thereby inhibiting the occurrence and development of tumors (such as melanoma, pancreatic cancer, ovarian cancer or breast cancer) 6 . In addition, AMPK can up-regulate the ratio of NAD + / NADH in the body through molecules such as PGC1 and SIRT. This function is considered to be closely related to longevity 7-9 . C. elegans , Drosophila and mice have been shown to activate AMPK to significantly extend the lifespan of these organisms by 10-12 . In addition, AMPK is an important regulator of autophagy. For example, AMPK can significantly promote the occurrence of macroautophagy through the activation of ULK1 (Unc-51 Like Autophagy Activating Kinase 1), which is one of the basic processes for a living body to maintain energy and material metabolism balance. From the perspective of physiological functions, it has been shown to be closely related to the occurrence of major diseases such as diabetes and tumors 13-14 . In particular, this function of AMPK is also closely related to the browning of adipose tissue 15-16 , which is considered to be an important process for relieving diabetes and remodeling healthy adipose tissue. The initiated AMPK does indeed inhibit diabetes by promoting fat browning Development of the condition 17 . AMPK can also promote mitochondrial fission factor (MFF) to promote mitophagy 18 , which is imbalanced with important neurological diseases such as Parkinson's disease and Alzheimer's disease 19 . Because AMPK has multifunctional effects on carbohydrate, fat and cholesterol metabolism and biosynthesis, AMPK is one of the most attractive drug targets for the treatment of major diseases. In the past two decades, a variety of screening methods have been used in the academic community, and a large number of AMPK initiators have been obtained with AMPK as a target, and a lot of research has been carried out.

然而，這些研究的結果表明，以AMPK作為藥物的直接靶標有諸多的缺陷，例如藥效不足或特異性較低。目前發現的最為成熟的AMPK啟動劑大多應用於糖尿病的治療領域。以廣泛應用的AMPK啟動劑二甲雙胍為例，該藥物對機體副作用極小，通過啟動AMPK能夠顯著降低血糖和脂肪肝水準，從而緩解糖尿病病情20 ，因而在眾多AMPK啟動劑中具有很大的優勢。但是，由於二甲雙胍分子對細胞膜的通透性較差，需要相應的轉運蛋白將其轉運進細胞中，而這些轉運蛋白僅僅分佈於少數幾個組織如肝臟中，從而極大地限制了該藥物藥效的行使和AMPK功能的發揮21 。除二甲雙胍之外，目前在臨床實驗中走得最遠的藥物是A-769662，該化合物能夠直接結合β亞基從而別構啟動AMPK22 。與二甲雙胍相比，A-769662具有良好的細胞膜通透性，能夠進入大部分組織中，並具有良好的持續性，能夠長時間發揮作用23 。然而，A-769662不適合口服23 ，這大大限制了其應用範圍。更為麻煩的是，關於A-769662的特異性，近年來已經有報導表明，其還具有複數除了AMPK之外的靶點24 。另外，據本發明人所知，除此之外的其它藥物均未進入臨床實驗階段22 。However, the results of these studies indicate that there are many shortcomings in using AMPK as a direct target for drugs, such as insufficient efficacy or low specificity. The most mature AMPK initiators found so far are mostly used in the treatment of diabetes. AMPK agent to start Metformin is widely used as an example, the drug with minimal side effects on the body, can significantly reduce the level of blood sugar and fatty liver by activating AMPK, thus alleviating diabetes condition 20, which has a great advantage in many AMPK start agent. However, due to the poor permeability of metformin molecules to cell membranes, corresponding transporters are required to transport them into cells, and these transporters are only distributed in a few tissues such as the liver, which greatly limits the efficacy of the drug. Exercise and play of AMPK functions 21 . In addition to metformin, the drug that has gone the farthest in clinical trials is A-769662, which can directly bind to the β subunit to allosterically activate AMPK 22 . Compared with metformin, A-769662 has a good cell membrane permeability, can enter most tissues, and has good continuity, it can play a role in 23 long. However, A-769662 is not suitable for oral administration 23, which greatly limits its applications. More trouble is, with regard to the specific A-769662, has been reported in recent years showed that in addition to also having a plurality of AMPK targets 24. Further, according to the present inventors' knowledge, none of the other drugs in addition to 22 to enter clinical trials.

醛縮酶（fructose-1,6-bisphosphate aldolase，簡稱為FBP aldolase，本發明中亦簡稱為aldolase）包括醛縮酶A、醛縮酶B以及醛縮酶C，是糖代謝中的重要的代謝酶，在糖酵解途徑中，它催化六碳的果糖-1,6-二磷酸（FBP）產生三碳的甘油醛-3-磷酸（G3P）和磷酸二羥丙酮（DHAP），後者進一步經過多次酶促反應產生丙酮酸；同時，在糖異生途徑中，它能夠催化這一反應的逆過程。Aldolase所催化的這一過程是不能被其它代謝酶補償的。目前，已知的aldolase的功能還僅僅侷限在其代謝酶本身的性質上。目前已經報導，aldolase的某些突變體可能與果糖不耐受有關，但具體機制還不明確。值得一提的是，在腫瘤組織中，Aldolase的表達水準明顯升高，這可能促進了瓦氏效應（Warburg effect）和腫瘤細胞的發展，而在腫瘤細胞中敲低aldolase會直接引起腫瘤細胞生長的停止25 。Fructose-1,6-bisphosphate aldolase (referred to as FBP aldolase, also referred to as aldolase in the present invention) includes aldolase A, aldolase B and aldolase C, which are important metabolisms in sugar metabolism. An enzyme, in the glycolytic pathway, it catalyzes six carbon fructose-1,6-diphosphate (FBP) to produce three carbon glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), the latter further Multiple enzymatic reactions produce pyruvate; at the same time, in the gluconeogenesis pathway, it can catalyze the reverse process of this reaction. This process catalyzed by Aldolase cannot be compensated by other metabolic enzymes. At present, the function of known aldolase is limited to the nature of its metabolic enzymes. It has been reported that some mutants of aldolase may be related to fructose intolerance, but the specific mechanism is not clear. It is worth mentioning that the expression level of Aldolase is significantly increased in tumor tissues, which may promote the Warburg effect and the development of tumor cells, and knocking down aldolase in tumor cells will directly cause tumor cell growth. Stop 25 .

早在1970年以前，研究人員就設計了眾多不能被aldolase催化的果糖-1,6-二磷酸的類似物，在體外實驗中起到競爭果糖-1,6-二磷酸和aldolase結合的作用來達到抑制aldolase的效果。然而，這些抑制劑都不能透過細胞膜、進入細胞發揮作用，其應用也只停留在體外的生化實驗水準上。在生理水準上，目前唯一報導的aldolase的抑制劑是TDZD-8（報導於2016年，CAS #327036-89-5，使用MDA-MB-231細胞作為生理模型）26 。但是，早先該抑制劑是作為細胞內的另一種激酶——GSK3的經典抑制劑被廣泛應用，也就是說，該抑制劑有明確的非aldolase的靶標，且其對於aldolase的IC50 和對於GSK3的IC50 是接近的。除此之外，本發明人初步的實驗結果表明，TDZD-8無法在MEF細胞中起到抑制aldolase的作用，這至少說明該抑制劑對aldolase的抑制不具有普遍性。As early as 1970, researchers have designed many analogs of fructose-1,6-diphosphate that cannot be catalyzed by aldolase. They compete for the combination of fructose-1,6-diphosphate and aldolase in in vitro experiments. To achieve the effect of inhibiting aldolase. However, none of these inhibitors can penetrate the cell membrane and enter the cells to play a role, and their application is only at the level of in vitro biochemical experiments. At physiological level, the only reported inhibitor of aldolase is TDZD-8 (reported in 2016, CAS # 327036-89-5, using MDA-MB-231 cells as a physiological model) 26 . However, earlier this inhibitor was widely used as another classic intracellular GSK3 inhibitor, that is, the inhibitor has a clear non-aldolase target, and its IC 50 for aldolase and for GSK3 The IC 50 is close. In addition, the preliminary experimental results of the inventors show that TDZD-8 cannot play a role in inhibiting aldolase in MEF cells, which at least indicates that the inhibitor does not have universal inhibition of aldolase.

目前，亟需開發新的啟動AMPK的藥物或者技術手段。At present, there is an urgent need to develop new drugs or technical means to start AMPK.

本發明人經過深入的研究和創造性的勞動，發現了一新的AMPK的調節因數——醛縮酶（aldolase），該因數能夠直接調節AMPK的啟動，並將在將來作為一調節AMPK的重要靶點被進一步研究與應用；並且本發明人還驚奇地發現，下調Aldolase基因表達水準，或者抑制Aldolase，能夠顯著地啟動AMPK，具有應用於低AMPK水準相關疾病的潛力。由此提供了下述發明：After intensive research and creative labor, the inventor discovered a new regulation factor of AMPK-aldolase, which can directly regulate the activation of AMPK, and will be an important target for regulating AMPK in the future. The point has been further studied and applied; and the inventors have also surprisingly found that down-regulating the level of Aldolase gene expression, or inhibiting Aldolase, can significantly activate AMPK and has the potential to apply to diseases associated with low AMPK levels. The following inventions are thus provided:

本發明的一方面涉及選自如下的（1）－（6）項中的任一項在製備啟動AMPK的藥物或者在製備篩選啟動AMPK的藥物的模型中的用途： （1） Aldolase； （2） 編碼Aldolase的多核苷酸； （3） 核酸構建體，其含有用於完全敲除或者部分敲除Aldolase基因的多核苷酸；較佳地，該多核苷酸為siRNA例如shRNA，或者為用於CRISPR/Cas9系統的guide RNA； （4） 宿主細胞，其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除；較佳地，其含有第（3）項所述的核酸構建體； （5） 抑制或阻斷Aldolase活性的藥物； （6） 抑制或降低Aldolase基因表達水準的藥物。One aspect of the present invention relates to the use of any one selected from the following items (1) to (6) in the preparation of a drug that initiates AMPK or in the model for the screening of a drug that initiates AMPK: (1) Aldolase; (2) ) A polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or Guide RNA of the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially deleted; preferably, it contains the nucleic acid construct according to item (3); ( 5) Drugs that inhibit or block Aldolase activity; (6) Drugs that inhibit or decrease Aldolase gene expression level.

在本發明的一實施方案中，所述的用途，其中，該抑制或阻斷Aldolase活性的藥物為抗Aldolase的抗體或TDZD-8；較佳地，該抗體為單克隆抗體。In one embodiment of the present invention, the use, wherein the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.

在本發明的一實施方案中，所述的用途，其中，該抑制或降低Aldolase基因表達水準的藥物選自siRNA例如shRNA，以及用於CRISPR-Cas9 系統的guide RNA。In one embodiment of the present invention, the use, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.

本發明涉及Aldolase作為藥物靶點在製備啟動AMPK的藥物中的用途。The present invention relates to the use of Aldolase as a drug target in the preparation of a AMPK-initiating medicament.

本發明的實施例2中，通過抑制aldolase來啟動AMPK。具體而言，利用慢病毒介導shRNA感染方法，抑制ALDOA-C（即ALDOA、ALDOB和ALDOC）基因的表達，然後通過對AMPK磷酸化的檢測發現AMPK被啟動。其中，術語“shRNA”是指小髮夾結構RNA（Short Hairpin RNA，簡稱“shRNA”）是一種短髮夾結構的RNA序列，可以經由質粒表達而來，並干擾靶基因表達。In Example 2 of the present invention, AMPK is activated by inhibiting aldolase. Specifically, the lentivirus-mediated shRNA infection method was used to suppress the expression of ALDOA-C (ie, ALDOA, ALDOB, and ALDOC) genes, and then AMPK was found to be activated by detection of AMPK phosphorylation. The term "shRNA" refers to a short hairpin RNA (Short Hairpin RNA), which is a short hairpin RNA sequence that can be expressed through a plasmid and interferes with the expression of a target gene.

在本發明的一實施方案中，該shRNA靶向ALDOA、ALDOB和ALDOC。在本發明的一實施方案中，該shRNA包括： 選自SEQ ID NO: 7和SEQ ID NO: 8所示序列中的至少一條， 選自SEQ ID NO: 9和SEQ ID NO: 10所示序列中的至少一條，以及 選自SEQ ID NO: 11和SEQ ID NO: 12所示序列中的至少一條。In one embodiment of the invention, the shRNA targets ALDOA, ALDOB, and ALDOC. In one embodiment of the present invention, the shRNA includes: at least one selected from the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, and selected from the sequences shown by SEQ ID NO: 9 and SEQ ID NO: 10 And at least one selected from the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12.

在本發明的一實施方案中，該模型可以是哺乳動物細胞（例如人或小鼠的細胞）或者哺乳動物（例如人或小鼠）。如果該待測藥物能夠抑制或降低模型中的Aldolase基因表達水準，或者抑制或阻斷模型中的Aldolase活性水準，則可以作為候選藥物。In one embodiment of the invention, the model may be a mammalian cell (such as a human or mouse cell) or a mammal (such as a human or mouse). If the test drug can inhibit or reduce the level of Aldolase gene expression in the model, or inhibit or block the level of Aldolase activity in the model, it can be a candidate drug.

本發明的另一方面涉及選自如下的（1）至（6）項中的任一項在製備抑制膽固醇合成的藥物、降低脂肪酸合成的藥物、抗肥胖（例如預防肥胖或者減肥）的藥物、預防及/或治療糖尿病的藥物、預防及/或治療腫瘤的藥物、預防及/或治療帕金森氏症的藥物、預防及/或治療阿茲海默症的藥物、抗衰老的藥物或者用於延長哺乳動物壽命的藥物中的用途： （1） Aldolase； （2） 編碼Aldolase的多核苷酸； （3） 核酸構建體，其含有用於完全敲除或者部分敲除Aldolase基因的多核苷酸；較佳地，該多核苷酸為siRNA例如shRNA，或者為用於CRISPR/Cas9系統的guide RNA； （4） 宿主細胞，其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除；較佳地，其含有第（3）項所述的核酸構建體； （5） 抑制或阻斷Aldolase活性的藥物； （6） 抑制或降低Aldolase基因表達水準的藥物。Another aspect of the present invention relates to a drug selected from any one of the following items (1) to (6) for preparing a drug that inhibits cholesterol synthesis, a drug that reduces fatty acid synthesis, an anti-obesity (such as preventing obesity or losing weight), Drugs for the prevention and / or treatment of diabetes, drugs for the prevention and / or treatment of tumors, drugs for the prevention and / or treatment of Parkinson's disease, drugs for the prevention and / or treatment of Alzheimer's disease, anti-aging drugs or for Uses in medicines to extend the life of mammals: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; Preferably, the polynucleotide is an siRNA such as shRNA, or guide RNA used in the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially deleted; preferably It contains the nucleic acid construct according to item (3); (5) a drug that inhibits or blocks Aldolase activity; (6) inhibits or reduces the level of Aldolase gene expression Drug.

較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer and breast cancer.

在本發明的一實施方案中，所述的用途，其中，該抑制或阻斷Aldolase活性的藥物為抗Aldolase的抗體或TDZD-8；較佳地，該抗體為單克隆抗體。In one embodiment of the present invention, the use, wherein the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.

在本發明的一實施方案中，所述的用途，其中，該抑制或降低Aldolase基因表達水準的藥物選自siRNA例如shRNA，以及用於CRISPR-Cas9 系統的guide RNA。In one embodiment of the present invention, the use, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.

在本發明的一實施方案中，該shRNA靶向ALDOA、ALDOB和ALDOC。在本發明的一實施方案中，該shRNA包括： 選自SEQ ID NO: 7和SEQ ID NO: 8所示序列中的至少一條， 選自SEQ ID NO: 9和SEQ ID NO: 10所示序列中的至少一條，以及 選自SEQ ID NO: 11和SEQ ID NO: 12所示序列中的至少一條。In one embodiment of the invention, the shRNA targets ALDOA, ALDOB, and ALDOC. In one embodiment of the present invention, the shRNA includes: at least one selected from the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, and selected from the sequences shown by SEQ ID NO: 9 and SEQ ID NO: 10 And at least one selected from the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12.

本發明的再一方面涉及一種在體內或體外啟動AMPK的方法，包括抑制Aldolase活性或者下調Aldolase基因表達水準的步驟，例如，包括抑制有需求的受試者中或者細胞中的Aldolase的活性或者下調Aldolase基因的表達水準的步驟。Yet another aspect of the present invention relates to a method for initiating AMPK in vivo or in vitro, comprising the steps of inhibiting Aldolase activity or down-regulating Aldolase gene expression level, for example, including inhibiting Aldolase activity or down-regulating in a subject in need or in a cell Aldolase gene expression level steps.

本發明的再一方面涉及一種篩選選自如下的藥物的方法，包括加入待測藥物，以及檢測Aldolase活性或者檢測Aldolase基因表達水準的步驟： 啟動AMPK的藥物、抑制膽固醇合成的藥物、降低脂肪酸合成的藥物、抗肥胖的藥物、預防及/或治療糖尿病的藥物、預防及/或治療腫瘤的藥物、預防及/或治療帕金森氏症的藥物、預防及/或治療阿茲海默症的藥物、抗衰老的藥物或者用於延長哺乳動物壽命的藥物。較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。Yet another aspect of the present invention relates to a method for screening a drug selected from the group consisting of adding a test drug, and detecting Aldolase activity or detecting an Aldolase gene expression level: a drug that activates AMPK, a drug that inhibits cholesterol synthesis, and reduces fatty acid synthesis Drugs, anti-obesity drugs, drugs for preventing and / or treating diabetes, drugs for preventing and / or treating tumors, drugs for preventing and / or treating Parkinson's disease, drugs for preventing and / or treating Alzheimer's disease , Anti-aging drugs or drugs used to extend the life of mammals. Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer and breast cancer.

如果該待測藥物能夠抑制或降低Aldolase基因表達水準，或者抑制或阻斷Aldolase活性水準，則可以作為候選藥物。例如： 在本發明的一實施方案中，將待測藥物加入到分離的哺乳動物例如人或小鼠的細胞中，以不加待測藥物的細胞作為對照。If the drug under test can inhibit or reduce the level of Aldolase gene expression, or inhibit or block the level of Aldolase activity, it can be a candidate drug. For example: In one embodiment of the present invention, the test drug is added to the cells of an isolated mammal such as a human or a mouse, and cells without the test drug are used as a control.

在本發明的一實施方案中，將待測藥物施與哺乳動物例如人或小鼠，觀察或者檢測目標症狀或者指標是否有改善。In one embodiment of the present invention, a test drug is administered to a mammal, such as a human or a mouse, to observe or detect whether the target symptom or index is improved.

本發明的再一方面涉及一種重組載體，其含有下調Aldolase基因表達水準的siRNA例如shRNA，或者用於CRISPR-Cas9 系統的guide RNA；較佳地，該重組載體為重組慢病毒載體。Another aspect of the present invention relates to a recombinant vector, which contains an siRNA such as shRNA that down-regulates the expression level of the Aldolase gene, or a guide RNA used in the CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector.

在本發明的一實施方案中，該shRNA靶向ALDOA、ALDOB和ALDOC。在本發明的一實施方案中，該shRNA包括： 選自SEQ ID NO: 7和SEQ ID NO: 8所示序列中的至少一條， 選自SEQ ID NO: 9和SEQ ID NO: 10所示序列中的至少一條，以及 選自SEQ ID NO: 11和SEQ ID NO: 12所示序列中的至少一條。In one embodiment of the invention, the shRNA targets ALDOA, ALDOB, and ALDOC. In one embodiment of the present invention, the shRNA includes: at least one selected from the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, and selected from the sequences shown by SEQ ID NO: 9 and SEQ ID NO: 10 And at least one selected from the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12.

本發明的再一方面涉及一種宿主細胞，其含有本發明的重組載體，或者其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除。Still another aspect of the present invention relates to a host cell containing the recombinant vector of the present invention, or a polynucleotide encoding Aldolase therein is completely or partially deleted.

本發明的再一方面涉及一種藥物組合物，其包含本發明的重組載體或者本發明的宿主細胞，可選地，其還包含藥學上可接受的輔料。Yet another aspect of the present invention relates to a pharmaceutical composition comprising a recombinant vector of the present invention or a host cell of the present invention, optionally, further comprising a pharmaceutically acceptable excipient.

在本發明的一實施方案中，所述的藥物組合物，其用於啟動AMPK、抑制膽固醇合成、降低脂肪酸合成、抗肥胖、預防及/或治療糖尿病、預防及/或治療腫瘤、預防及/或治療帕金森氏症、預防及/或治療阿茲海默症、抗衰老或者用於延長哺乳動物壽命。較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。In one embodiment of the present invention, the pharmaceutical composition is used to start AMPK, inhibit cholesterol synthesis, reduce fatty acid synthesis, anti-obesity, prevent and / or treat diabetes, prevent and / or treat tumors, prevent and / Or treat Parkinson's disease, prevent and / or treat Alzheimer's disease, anti-aging or use to extend the life of mammals. Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer and breast cancer.

本發明還涉及一種治療及/或預防高膽固醇症、糖尿病、腫瘤、帕金森氏症或阿茲海默症的方法或者一種抗肥胖（例如預防肥胖或者減肥）、抗衰老或延長哺乳動物壽命的方法，包括抑制受試者的Aldolase活性或者下調受試者的Aldolase基因表達水準的步驟；例如包括給予受試者有效量的本發明的宿主細胞或者組合物的步驟；例如包括給予有需求的受試者以有效量的抑制或阻斷Aldolase活性的藥物或者抑制或降低Aldolase基因表達水準的藥物的步驟； 較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。The invention also relates to a method of treating and / or preventing hypercholesterolemia, diabetes, tumors, Parkinson's disease or Alzheimer's disease or an anti-obesity (such as preventing obesity or losing weight), anti-aging or prolonging the life of mammals A method comprising the steps of inhibiting the Aldolase activity of a subject or down-regulating the level of Aldolase gene expression in the subject; for example, the step of administering to the subject an effective amount of a host cell or composition of the present invention; The step of the tester using an effective amount of a drug that inhibits or blocks Aldolase activity or a drug that inhibits or reduces the level of Aldolase gene expression; preferably, the tumor is any one selected from melanoma, pancreatic cancer, ovarian cancer, and breast cancer One or more.

較佳地，該抑制或阻斷Aldolase活性的藥物為抗Aldolase的抗體或TDZD-8；較佳地，該抗體為單克隆抗體。Preferably, the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody.

較佳地，該抑制或降低Aldolase基因表達水準的藥物選自siRNA例如shRNA，以及用於CRISPR-Cas9 系統的guide RNA。Preferably, the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA used in the CRISPR-Cas9 system.

抑制受試者的Aldolase活性的水準或者下調受試者的Aldolase基因表達的水準，取決於許多因素，例如所治療病況的嚴重程度，患者或動物的性別、年齡、體重及個體反應，以及待治療患者的病況和既往病史來選定。本領域通常的做法是，從低於為得到所需治療效果及/或預防效果而要求的水準開始，逐漸增加劑量，直到得到所需的效果。The level of inhibition of the subject's Aldolase activity or the level of down-regulation of the subject's Aldolase gene expression depends on many factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, and the treatment The patient's condition and past medical history are selected. It is common practice in the art to start with a dose that is lower than required to obtain the desired therapeutic and / or preventive effect, and gradually increase the dose until the desired effect is obtained.

本發明還涉及選自如下的（1）－（6）項中的任一項，其用於啟動AMPK或用於製備啟動AMPK的藥物或者用於製備篩選啟動AMPK的藥物的模型： （1） Aldolase； （2） 編碼Aldolase的多核苷酸； （3） 核酸構建體，其含有用於完全敲除或者部分敲除Aldolase基因的多核苷酸；較佳地，該多核苷酸為siRNA例如shRNA，或者為用於CRISPR/Cas9系統的guide RNA； （4） 宿主細胞，其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除；較佳地，其含有第（3）項所述的核酸構建體； （5） 抑制或阻斷Aldolase活性的藥物； （6） 抑制或降低Aldolase基因表達水準的藥物。The present invention also relates to any one selected from the following items (1) to (6), which is used for starting AMPK or for preparing a drug for starting AMPK or for preparing a model for screening a drug for starting AMPK: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, Or guide RNA for the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially deleted; preferably, it contains the nucleic acid described in item (3) Constructs; (5) drugs that inhibit or block Aldolase activity; (6) drugs that inhibit or reduce the level of Aldolase gene expression.

本發明還涉及選自如下的（1）－（6）項中的任一項，其用於製備抑制膽固醇合成的藥物、降低脂肪酸合成的藥物、抗肥胖的藥物、預防及/或治療糖尿病的藥物、預防及/或治療腫瘤的藥物、預防及/或治療帕金森氏症的藥物、預防及/或治療阿茲海默症的藥物、抗衰老的藥物或者用於延長哺乳動物壽命的藥物： （1） Aldolase； （2） 編碼Aldolase的多核苷酸； （3） 核酸構建體，其含有用於完全敲除或者部分敲除Aldolase基因的多核苷酸；較佳地，該多核苷酸為siRNA例如shRNA，或者為用於CRISPR/Cas9系統的guide RNA； （4） 宿主細胞，其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除；較佳地，其含有第（3）項所述的核酸構建體； （5） 抑制或阻斷Aldolase活性的藥物； （6） 抑制或降低Aldolase基因表達水準的藥物。The present invention also relates to any one selected from the following items (1) to (6), which is used for preparing a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, an anti-obesity drug, and preventing and / or treating diabetes Drugs, drugs for the prevention and / or treatment of tumors, drugs for the prevention and / or treatment of Parkinson's disease, drugs for the prevention and / or treatment of Alzheimer's disease, anti-aging drugs or drugs for extending the life of mammals: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA For example, shRNA, or guide RNA used in the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially knocked out; preferably, it contains the enzyme of item (3) (5) a drug that inhibits or blocks Aldolase activity; (6) a drug that inhibits or reduces Aldolase gene expression level.

較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer and breast cancer.

在本發明中，醛縮酶（fructose-1,6-bisphosphate aldolase，簡稱為FBP aldolase）在本發明中亦簡稱為aldolase，包括3個異構體ALDOA、ALDOB和ALDOC。當提及Aldolase或Aldolase的氨基酸序列時，其包括Aldolase的蛋白全長，還包括其融合蛋白。然而，本領域技術人員理解，在Aldolase的氨基酸序列中，可天然產生或人工引入突變或變異（包括但不限於置換，缺失及/或添加），而不影響其生物學功能。在本發明的一實施方案中，Aldolase為人Aldolase。較佳地，Aldolase為選自ALDOA、ALDOB和ALDOC中的任意一種、兩種或三種。當Aldolase為ALDOA、ALDOB和ALDOC三者時，亦表示為“ALDOA-C”。In the present invention, fructose-1,6-bisphosphate aldolase (abbreviated as FBP aldolase) is also abbreviated as aldolase in the present invention, and includes three isomers ALDOA, ALDOB and ALDOC. When referring to the amino acid sequence of Aldolase or Aldolase, it includes the full-length protein of Aldolase and also includes its fusion protein. However, those skilled in the art understand that in the amino acid sequence of Aldolase, mutations or mutations (including but not limited to substitutions, deletions and / or additions) can be naturally generated or artificially introduced without affecting its biological function. In one embodiment of the invention, Aldolase is human Aldolase. Preferably, Aldolase is any one, two or three selected from ALDOA, ALDOB and ALDOC. When Aldolase is ALDOA, ALDOB, and ALDOC, it is also expressed as "ALDOA-C".

人Aldolase A（ALDOA）的氨基酸序列如下：（364 AA） MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAASESLFVSNHAY （SEQ ID NO: 1）Human Aldolase A (ALDOA) an amino acid sequence as follows: (364 AA) MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAASESLFVSNHAY (SEQ ID NO: 1)

人Aldolase B（ALDOB）的氨基酸序列如下：（364 AA） MAHRFPALTQEQKKELSEIAQSIVANGKGILAADESVGTMGNRLQRIKVENTEENRRQFREILFSVDSSINQSIGGVILFHETLYQKDSQGKLFRNILKEKGIVVGIKLDQGGAPLAGTNKETTIQGLDGLSERCAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASICQQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVLAAVYKALNDHHVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAANKEATQEAFMKRAMANCQAAKGQYVHTGSSGAASTQSLFTACYTY （SEQ ID NO: 2）Human Aldolase B (ALDOB) an amino acid sequence as follows: (364 AA) MAHRFPALTQEQKKELSEIAQSIVANGKGILAADESVGTMGNRLQRIKVENTEENRRQFREILFSVDSSINQSIGGVILFHETLYQKDSQGKLFRNILKEKGIVVGIKLDQGGAPLAGTNKETTIQGLDGLSERCAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASICQQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVLAAVYKALNDHHVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAANKEATQEAFMKRAMANCQAAKGQYVHTGSSGAASTQSLFTACYTY (SEQ ID NO: 2)

人Aldolase C（ALDOC）的氨基酸序列如下：（364 AA） MPHSYPALSAEQKKELSDIALRIVAPGKGILAADESVGSMAKRLSQIGVENTEENRRLYRQVLFSADDRVKKCIGGVIFFHETLYQKDDNGVPFVRTIQDKGIVVGIKVDKGVVPLAGTDGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKISERTPSALAILENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHVYLEGTLLKPNMVTPGHACPIKYTPEEIAMATVTALRRTVPPAVPGVTFLSGGQSEEEASFNLNAINRCPLPRPWALTFSYGRALQASALNAWRGQRDNAGAATEEFIKRAEVNGLAAQGKYEGSGEDGGAAAQSLYIANHAY （SEQ ID NO: 3）Human Aldolase C (ALDOC) an amino acid sequence as follows: (364 AA) MPHSYPALSAEQKKELSDIALRIVAPGKGILAADESVGSMAKRLSQIGVENTEENRRLYRQVLFSADDRVKKCIGGVIFFHETLYQKDDNGVPFVRTIQDKGIVVGIKVDKGVVPLAGTDGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKISERTPSALAILENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHVYLEGTLLKPNMVTPGHACPIKYTPEEIAMATVTALRRTVPPAVPGVTFLSGGQSEEEASFNLNAINRCPLPRPWALTFSYGRALQASALNAWRGQRDNAGAATEEFIKRAEVNGLAAQGKYEGSGEDGGAAAQSLYIANHAY (SEQ ID NO: 3)

在本發明中，當提及aldolase基因時，其不僅包含編碼aldolase的核酸序列，還包含其簡並序列；進一步地，還可以包含讀碼框之外的調控序列。在本發明的一實施方案中，aldolase基因為人aldolase基因。In the present invention, when referring to the aldolase gene, it includes not only a nucleic acid sequence encoding the aldolase, but also a degenerate sequence thereof; further, it may also include a regulatory sequence outside the reading frame. In one embodiment of the present invention, the aldolase gene is a human aldolase gene.

編碼ALDOA的核酸序列（CDS）如下：（1095 BP） ATGCCCTACCAATATCCAGCACTGACCCCGGAGCAGAAGAAGGAGCTGTCTGACATCGCTCACCGCATCGTGGCACCTGGCAAGGGCATCCTGGCTGCAGATGAGTCCACTGGGAGCATTGCCAAGCGGCTGCAGTCCATTGGCACCGAGAACACCGAGGAGAACCGGCGCTTCTACCGCCAGCTGCTGCTGACAGCTGACGACCGCGTGAACCCCTGCATTGGGGGTGTCATCCTCTTCCATGAGACACTCTACCAGAAGGCGGATGATGGGCGTCCCTTCCCCCAAGTTATCAAATCCAAGGGCGGTGTTGTGGGCATCAAGGTAGACAAGGGCGTGGTCCCCCTGGCAGGGACAAATGGCGAGACTACCACCCAAGGGTTGGATGGGCTGTCTGAGCGCTGTGCCCAGTACAAGAAGGACGGAGCTGACTTCGCCAAGTGGCGTTGTGTGCTGAAGATTGGGGAACACACCCCCTCAGCCCTCGCCATCATGGAAAATGCCAATGTTCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCCATCGTGGAGCCTGAGATCCTCCCTGATGGGGACCATGACTTGAAGCGCTGCCAGTATGTGACCGAGAAGGTGCTGGCTGCTGTCTACAAGGCTCTGAGTGACCACCACATCTACCTGGAAGGCACCTTGCTGAAGCCCAACATGGTCACCCCAGGCCATGCTTGCACTCAGAAGTTTTCTCATGAGGAGATTGCCATGGCGACCGTCACAGCGCTGCGCCGCACAGTGCCCCCCGCTGTCACTGGGATCACCTTCCTGTCTGGAGGCCAGAGTGAGGAGGAGGCGTCCATCAACCTCAATGCCATTAACAAGTGCCCCCTGCTGAAGCCCTGGGCCCTGACCTTCTCCTACGGCCGAGCCCTGCAGGCCTCTGCCCTGAAGGCCTGGGGCGGGAAGAAGGAGAACCTGAAGGCTGCGCAGGAGGAGTATGTCAAGCGAGCCCTGGCCAACAGCCTTGCCTGTCAAGGAAAGTACACTCCGAGCGGTCAGGCTGGGGCTGCTGCCAGCGAGTCCCTCTTCGTCTCTAACCACGCCTATTAA （SEQ ID NO: 4）The nucleic acid sequence encoding ALDOA (CDS) as follows: (1095 BP) ATGCCCTACCAATATCCAGCACTGACCCCGGAGCAGAAGAAGGAGCTGTCTGACATCGCTCACCGCATCGTGGCACCTGGCAAGGGCATCCTGGCTGCAGATGAGTCCACTGGGAGCATTGCCAAGCGGCTGCAGTCCATTGGCACCGAGAACACCGAGGAGAACCGGCGCTTCTACCGCCAGCTGCTGCTGACAGCTGACGACCGCGTGAACCCCTGCATTGGGGGTGTCATCCTCTTCCATGAGACACTCTACCAGAAGGCGGATGATGGGCGTCCCTTCCCCCAAGTTATCAAATCCAAGGGCGGTGTTGTGGGCATCAAGGTAGACAAGGGCGTGGTCCCCCTGGCAGGGACAAATGGCGAGACTACCACCCAAGGGTTGGATGGGCTGTCTGAGCGCTGTGCCCAGTACAAGAAGGACGGAGCTGACTTCGCCAAGTGGCGTTGTGTGCTGAAGATTGGGGAACACACCCCCTCAGCCCTCGCCATCATGGAAAATGCCAATGTTCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCCATCGTGGAGCCTGAGATCCTCCCTGATGGGGACCATGACTTGAAGCGCTGCCAGTATGTGACCGAGAAGGTGCTGGCTGCTGTCTACAAGGCTCTGAGTGACCACCACATCTACCTGGAAGGCACCTTGCTGAAGCCCAACATGGTCACCCCAGGCCATGCTTGCACTCAGAAGTTTTCTCATGAGGAGATTGCCATGGCGACCGTCACAGCGCTGCGCCGCACAGTGCCCCCCGCTGTCACTGGGATCACCTTCCTGTCTGGAGGCCAGAGTGAGGAGGAGGCGTCCATCAACCTCAATGCCATTAACAAGTGCCCCCTGCTGAAGCCCTGGGCCCTGACCTTCTCCTACGGCCGAGCCCTGCAGGCCTCTGCCCTGAAGGCCTGG GGCGGGAAGAAGGAGAACCTGAAGGCTGCGCAGGAGGAGTATGTCAAGCGAGCCCTGGCCAACAGCCTTGCCTGTCAAGGAAAGTACACTCCGAGCGGTCAGGCTGGGGCTGCTGCCAGCGAGTCCCTCTTCGTCTCTAACCACGCCTATTAA (SEQ ID NO: 4)

編碼ALDOB的核酸序列（CDS）如下：（1095 BP） ATGGCCCACCGATTTCCAGCCCTCACCCAGGAGCAGAAGAAGGAGCTCTCAGAAATTGCCCAGAGCATTGTTGCCAATGGAAAGGGGATCCTGGCTGCAGATGAATCTGTAGGTACCATGGGGAACCGCCTGCAGAGGATCAAGGTGGAAAACACTGAAGAGAACCGCCGGCAGTTCCGAGAAATCCTCTTCTCTGTGGACAGTTCCATCAACCAGAGCATCGGGGGTGTGATCCTTTTCCACGAGACCCTCTACCAGAAGGACAGCCAGGGAAAGCTGTTCAGAAACATCCTCAAGGAAAAGGGGATCGTGGTGGGAATCAAGTTAGACCAAGGAGGTGCTCCTCTTGCAGGAACAAACAAAGAAACCACCATTCAAGGGCTTGATGGCCTCTCAGAGCGCTGTGCTCAGTACAAGAAAGATGGTGTTGACTTTGGGAAGTGGCGTGCTGTGCTGAGGATTGCCGACCAGTGTCCATCCAGCCTCGCTATCCAGGAAAACGCCAACGCCCTGGCTCGCTACGCCAGCATCTGTCAGCAGAATGGACTGGTACCTATTGTTGAACCAGAGGTAATTCCTGATGGAGACCATGACCTGGAACACTGCCAGTATGTTACTGAGAAGGTCCTGGCTGCTGTCTACAAGGCCCTGAATGACCATCATGTTTACCTGGAGGGCACCCTGCTAAAGCCCAACATGGTGACTGCTGGACATGCCTGCACCAAGAAGTATACTCCAGAACAAGTAGCTATGGCCACCGTAACAGCTCTCCACCGTACTGTTCCTGCAGCTGTTCCTGGCATCTGCTTTTTGTCTGGTGGCATGAGTGAAGAGGATGCCACTCTCAACCTCAATGCTATCAACCTTTGCCCTCTACCAAAGCCCTGGAAACTAAGTTTCTCTTATGGACGGGCCCTGCAGGCCAGTGCACTGGCTGCCTGGGGTGGCAAGGCTGCAAACAAGGAGGCAACCCAGGAGGCTTTTATGAAGCGGGCCATGGCTAACTGCCAGGCGGCCAAAGGACAGTATGTTCACACGGGTTCTTCTGGGGCTGCTTCCACCCAGTCGCTCTTCACAGCCTGCTATACCTACTAG （SEQ ID NO: 5）The nucleic acid sequence encoding ALDOB (CDS) as follows: (1095 BP) ATGGCCCACCGATTTCCAGCCCTCACCCAGGAGCAGAAGAAGGAGCTCTCAGAAATTGCCCAGAGCATTGTTGCCAATGGAAAGGGGATCCTGGCTGCAGATGAATCTGTAGGTACCATGGGGAACCGCCTGCAGAGGATCAAGGTGGAAAACACTGAAGAGAACCGCCGGCAGTTCCGAGAAATCCTCTTCTCTGTGGACAGTTCCATCAACCAGAGCATCGGGGGTGTGATCCTTTTCCACGAGACCCTCTACCAGAAGGACAGCCAGGGAAAGCTGTTCAGAAACATCCTCAAGGAAAAGGGGATCGTGGTGGGAATCAAGTTAGACCAAGGAGGTGCTCCTCTTGCAGGAACAAACAAAGAAACCACCATTCAAGGGCTTGATGGCCTCTCAGAGCGCTGTGCTCAGTACAAGAAAGATGGTGTTGACTTTGGGAAGTGGCGTGCTGTGCTGAGGATTGCCGACCAGTGTCCATCCAGCCTCGCTATCCAGGAAAACGCCAACGCCCTGGCTCGCTACGCCAGCATCTGTCAGCAGAATGGACTGGTACCTATTGTTGAACCAGAGGTAATTCCTGATGGAGACCATGACCTGGAACACTGCCAGTATGTTACTGAGAAGGTCCTGGCTGCTGTCTACAAGGCCCTGAATGACCATCATGTTTACCTGGAGGGCACCCTGCTAAAGCCCAACATGGTGACTGCTGGACATGCCTGCACCAAGAAGTATACTCCAGAACAAGTAGCTATGGCCACCGTAACAGCTCTCCACCGTACTGTTCCTGCAGCTGTTCCTGGCATCTGCTTTTTGTCTGGTGGCATGAGTGAAGAGGATGCCACTCTCAACCTCAATGCTATCAACCTTTGCCCTCTACCAAAGCCCTGGAAACTAAGTTTCTCTTATGGACGGGCCCTGCAGGCCAGTGCACTGGCTGCCTGG GGTGGCAAGGCTGCAAACAAGGAGGCAACCCAGGAGGCTTTTATGAGACGGGCCATGGCTAACTGCCAGGCGGCCAAAGGACAGTATGTTCACACGGGTTCTTCTGGGGCTGCTTCCACCCAGTCGCTCTTCACAGCCTGCTATACCTACTAG (SEQ ID NO: 5)

編碼ALDOC的核酸序列（CDS）如下：（1095 BP） ATGCCTCACTCGTACCCAGCCCTTTCTGCTGAGCAGAAGAAGGAGTTGTCTGACATTGCCCTGCGGATTGTAGCCCCGGGCAAAGGCATTCTGGCTGCGGATGAGTCTGTAGGCAGCATGGCCAAGCGGCTGAGCCAAATTGGGGTGGAAAACACAGAGGAGAACCGCCGGCTGTACCGCCAGGTCCTGTTCAGTGCTGATGACCGTGTGAAAAAGTGCATTGGAGGCGTCATTTTCTTCCATGAGACCCTCTACCAGAAAGATGATAATGGTGTTCCCTTCGTCCGAACCATCCAGGATAAGGGCATCGTCGTGGGCATCAAGGTTGACAAGGGTGTGGTGCCTCTAGCTGGGACTGATGGAGAAACCACCACTCAAGGGCTGGATGGGCTCTCAGAACGCTGTGCCCAATACAAGAAGGATGGTGCTGACTTTGCCAAGTGGCGCTGTGTGCTGAAAATCAGTGAGCGTACACCCTCTGCACTTGCCATTCTGGAGAACGCCAACGTGCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCTATTGTGGAACCTGAAATATTGCCTGATGGAGACCACGACCTCAAACGTTGTCAGTATGTTACAGAGAAGGTCTTGGCTGCTGTGTACAAGGCCCTGAGTGACCATCATGTATACCTGGAGGGGACCCTGCTCAAGCCCAACATGGTGACCCCGGGCCATGCCTGTCCCATCAAGTATACCCCAGAGGAGATTGCCATGGCAACTGTCACTGCCCTGCGTCGCACTGTGCCCCCAGCTGTCCCAGGAGTGACCTTCCTGTCTGGGGGTCAGAGCGAAGAAGAGGCATCATTCAACCTCAATGCCATCAACCGCTGCCCCCTTCCCCGACCCTGGGCGCTTACCTTCTCCTATGGGCGTGCCCTGCAAGCCTCTGCACTCAATGCCTGGCGAGGGCAACGGGACAATGCTGGGGCTGCCACTGAGGAGTTCATCAAGCGGGCTGAGGTGAATGGGCTTGCAGCCCAGGGCAAGTATGAAGGCAGTGGAGAAGATGGTGGAGCAGCAGCACAGTCACTCTACATTGCCAACCATGCCTACTGA （SEQ ID NO: 6）The nucleic acid sequence encoding ALDOC (CDS) as follows: (1095 BP) ATGCCTCACTCGTACCCAGCCCTTTCTGCTGAGCAGAAGAAGGAGTTGTCTGACATTGCCCTGCGGATTGTAGCCCCGGGCAAAGGCATTCTGGCTGCGGATGAGTCTGTAGGCAGCATGGCCAAGCGGCTGAGCCAAATTGGGGTGGAAAACACAGAGGAGAACCGCCGGCTGTACCGCCAGGTCCTGTTCAGTGCTGATGACCGTGTGAAAAAGTGCATTGGAGGCGTCATTTTCTTCCATGAGACCCTCTACCAGAAAGATGATAATGGTGTTCCCTTCGTCCGAACCATCCAGGATAAGGGCATCGTCGTGGGCATCAAGGTTGACAAGGGTGTGGTGCCTCTAGCTGGGACTGATGGAGAAACCACCACTCAAGGGCTGGATGGGCTCTCAGAACGCTGTGCCCAATACAAGAAGGATGGTGCTGACTTTGCCAAGTGGCGCTGTGTGCTGAAAATCAGTGAGCGTACACCCTCTGCACTTGCCATTCTGGAGAACGCCAACGTGCTGGCCCGTTATGCCAGTATCTGCCAGCAGAATGGCATTGTGCCTATTGTGGAACCTGAAATATTGCCTGATGGAGACCACGACCTCAAACGTTGTCAGTATGTTACAGAGAAGGTCTTGGCTGCTGTGTACAAGGCCCTGAGTGACCATCATGTATACCTGGAGGGGACCCTGCTCAAGCCCAACATGGTGACCCCGGGCCATGCCTGTCCCATCAAGTATACCCCAGAGGAGATTGCCATGGCAACTGTCACTGCCCTGCGTCGCACTGTGCCCCCAGCTGTCCCAGGAGTGACCTTCCTGTCTGGGGGTCAGAGCGAAGAAGAGGCATCATTCAACCTCAATGCCATCAACCGCTGCCCCCTTCCCCGACCCTGGGCGCTTACCTTCTCCTATGGGCGTGCCCTGCAAGCCTCTGCACTCAATGCCTGG CGAGGGCAACGGGACAATGCTGGGGCTGCCACTGAGGAGTTCATCAAGCGGGCTGAGGTGAATGGGCTTGCAGCCCAGGGCAAGTATGAAGGCAGTGGAGAATATTGTGGAGCAGCAGCACAGTCACTCTACATTGCCAACCATGCCTACTGA (SEQ ID NO: 6)

本發明中，術語“核酸構建體”，在文中定義為單鏈或雙鏈核酸分子，較佳是指人工構建的核酸分子。可選地，該核酸構建體還包含有可操作地連接的一或複數調控序列。In the present invention, the term "nucleic acid construct" is defined herein as a single-stranded or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises one or more regulatory sequences operably linked.

在本發明中，術語“可操作地連接”是指二或複數核苷酸區域或核酸序列的功能性的空間排列。該“可操作地連接”可以通過基因重組的手段實現。In the present invention, the term "operably linked" refers to the functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. This "operably linked" can be achieved by means of genetic recombination.

在本發明中，術語“載體”指的是，可將抑制某蛋白的多核苷酸插入其中的一種核酸運載工具。舉例來說，載體包括：質粒；噬菌粒；柯斯質粒；人工染色體如酵母人工染色體（YAC）、細菌人工染色體（BAC）或P1來源的人工染色體（PAC）；噬菌體如λ噬菌體或M13噬菌體及動物病毒等。用作載體的動物病毒種類有逆轉錄酶病毒（包括慢病毒）、腺病毒、腺相關病毒、皰疹病毒（如單純皰疹病毒）、痘病毒、桿狀病毒、乳頭瘤病毒、乳頭多瘤空泡病毒（如SV40）。一種載體可能含有多種控制表達的元件。In the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide that inhibits a protein is inserted. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; phages such as lambda phage or M13 phage And animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papilloma Vacuolar virus (such as SV40). A vector may contain multiple elements that control expression.

在本發明中，術語“宿主細胞”指的是導入載體的細胞，包括如下許多細胞類型，如大腸桿菌或枯草菌等原核細胞，如酵母細胞或曲黴菌等真菌細胞，如S2果蠅細胞或Sf9等昆蟲細胞，或者如纖維原細胞，CHO細胞，COS細胞，NSO細胞，HeLa細胞，BHK細胞，HEK 293細胞或人細胞的動物細胞。In the present invention, the term "host cell" refers to a cell introduced into a vector, and includes many cell types such as prokaryotic cells such as E. coli or subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Sf9 and other insect cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

術語“有效量”是指可在受試者中實現治療、預防、減輕及/或緩解本發明所述疾病或病症的劑量。The term "effective amount" refers to a dose that can achieve the treatment, prevention, reduction, and / or alleviation of a disease or condition described herein in a subject.

術語“疾病及/或病症”是指該受試者的一種身體狀態，該身體狀態與本發明所述疾病及/或病症有關。The term "disease and / or disorder" refers to a physical state of the subject, which is related to the disease and / or disorder described in the present invention.

術語“受試者”可以指患者或者其它接受本發明藥物組合物以治療、預防、減輕及/或緩解本發明所述疾病或病症的動物，特別是哺乳動物，例如人、狗、猴、牛、馬等。The term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, or cow, receiving a pharmaceutical composition of the invention to treat, prevent, reduce, and / or alleviate a disease or condition described in the invention. , Horses, etc.

本發明中，對於DNA或RNA的敲低包括但不限於，完全敲除和部分敲除。完全敲除是指將目標DNA或目標RNA的水準或其表達的蛋白的水準降低至幾乎檢測不到的水準（事實上，一般而言，很難將目標DNA或目標RNA 100％地敲除掉）。部分敲除是指敲除的程度大於零，小於完全敲除的情況。 發明的有益效果In the present invention, knockdown of DNA or RNA includes, but is not limited to, complete knockout and partial knockout. Complete knockout refers to reducing the level of target DNA or target RNA or the level of expressed proteins to a level that is almost undetectable (in fact, in general, it is difficult to knock out target DNA or target RNA 100% ). Partial knockout refers to the case where the degree of knockout is greater than zero and less than complete knockout. The beneficial effects of the invention

與先前技術相比，本發明應用aldolase的shRNA和蛋白突變體能夠顯著地直接啟動AMPK，從而實現了可以將aldolase作為靶點來開發藥物用來啟動AMPK，克服了先前技術中直接以AMPK為藥物靶點而存在的困難。Compared with the prior art, the shRNA and protein mutants using the aldolase of the present invention can significantly directly activate AMPK, thereby realizing that aldolase can be used as a target to develop a drug to start AMPK, which overcomes the direct use of AMPK as a drug in the prior art Target.

下面將結合實施例對本發明的實施方案進行詳細描述。本領域技術人員將會理解，下面的實施例僅用於說明本發明，而不應視為限定本發明的範圍。實施例中未注明具體技術或條件者，按照本領域內的文獻所描述的技術或條件（例如參考J.薩姆布魯克等著，黃培堂等譯的《分子克隆實驗指南》，第三版，科學出版社）或者按照產品說明書進行。所用試劑或儀器未注明生產廠商者，均為可以通過市購獲得的常規產品。 實施例1：shRNA抑制ALDOA-C基因的表達 1. 實驗材料和主要試劑The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Those who do not indicate specific techniques or conditions in the examples follow the techniques or conditions described in the literature in the art (for example, refer to the "Molecular Cloning Experiment Guide" translated by J. Sambrook et al., Huang Peitang et al., Third edition, Science Press) or in accordance with the product manual. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that are commercially available. Example 1: shRNA inhibits ALDOA-C gene expression 1. Experimental materials and main reagents

細胞株：小鼠成纖維細胞MEF，是永生化的MEF，即從小鼠胚胎分離出成纖維細胞之後，轉染SV40 T antigen使細胞永生。構建方法可參見文獻Lei Y, Methods Mol Biol. 2013;1031:59-64. Generation and culture of mouse embryonic fibroblasts。 人胚胎腎細胞HEK293T (cat. CRL-3216)，購自ATCC。 載體pLVX-IRES (cat. # 631849)，購自Clontech。 轉染試劑：Lipofectamine 2000 (cat. 11668-027)，購自Invitrogen公司。 強力黴素Doxycycline (cat. S4163)，購自Selleckchem。 Dulbecco’ s modified Eagle’ s medium (DMEM, Gibco, cat. 11965)，購自Thermofisher。 Western所用一抗： anti-ALDOA (cat. # 8060)，購自Cell Signaling Technology； anti-ALDOB (cat. 18065-1-AP)，購自Ptoteintech； anti-ALDOC (cat. AM2215b)，購自Abgent； anti-β-tubulin (cat. #2128)，購自Cell Signaling Technology。 Western所用二抗： HRP-conjugated goat anti-mouse IgG (cat. 115-035-003)和 HRP-conjugated goat anti-rabbit IgG (cat. 111-035-003)，均購自Jackson ImmunoResearch。 2. 實驗方法Cell line: Mouse fibroblast MEF is an immortalized MEF, that is, after fibroblasts are isolated from mouse embryos, SV40 T antigen is transfected to make the cells immortal. Construction methods can be found in the literature Lei Y, Methods Mol Biol. 2013; 1031: 59-64. Generation and culture of mouse embryonic fibroblasts. Human embryonic kidney cells HEK293T (cat. CRL-3216), purchased from ATCC. Vector pLVX-IRES (cat. # 631849) was purchased from Clontech. Transfection reagent: Lipofectamine 2000 (cat. 11668-027), purchased from Invitrogen. Doxycycline (cat. S4163), purchased from Selleckchem. Dulbecco ’s modified Eagle ’s medium (DMEM, Gibco, cat. 11965) was purchased from Thermofisher. Primary antibodies used in Western: anti-ALDOA (cat. # 8060), purchased from Cell Signaling Technology; anti-ALDOB (cat. 18065-1-AP), purchased from Ptoteintech; anti-ALDOC (cat. AM2215b), purchased from Abgent Anti-β-tubulin (cat. # 2128), purchased from Cell Signaling Technology. Secondary antibodies used in Western: HRP-conjugated goat anti-mouse IgG (cat. 115-035-003) and HRP-conjugated goat anti-rabbit IgG (cat. 111-035-003) were purchased from Jackson ImmunoResearch. Experimental method

已經有人報導敲低ALDOA能夠引起細胞死亡25 。本發明人的前期實驗結果也表明敲低ALDOB或者ALDOC能夠引起細胞死亡。為了避免上述細胞死亡對實驗結果造成影響，本發明人重新建立了一套新的實驗方法： （1） 構建誘導表達ALDOA-C的重組表達質粒It has been reported that knockdown ALDOA can cause cell death 25. The results of previous experiments by the inventors also show that knockdown of ALDOB or ALDOC can cause cell death. In order to prevent the above-mentioned cell death from affecting the experimental results, the inventors have re-established a new set of experimental methods: (1) Construction of a recombinant expression plasmid that induces the expression of ALDOA-C

構建強力黴素（Doxycycline，Dox）誘導表達的ALDOA-C的表達質粒（pLVX-IRES-ALDOA-C）。採用常規的克隆方法，可參見《分子克隆實驗指南》，其步驟如下：Construction of doxycycline (Dox) induced expression plasmid (ALDV-IRES-ALDOA-C) for ALDOA-C. For the conventional cloning method, please refer to the Molecular Cloning Experiment Guide. The steps are as follows:

通過PCR分別擴增ALDOA、ALDOB和ALDOC的CDS片段（SEQ ID NO: 4－6），同時利用限制性內切酶處理（EcoRI和BamHI雙酶切）載體pLVX-IRES，最後將CDS片段和酶切過的載體連接起來，得到pLVX-IRES-ALDOA-C。 （2）構建誘導表達ALDOA-C的MEF細胞株The CDS fragments (SEQ ID NO: 4-6) of ALDOA, ALDOB, and ALDOC were amplified by PCR, and the restriction enzyme treatment (EcoRI and BamHI double digestion) vector pLVX-IRES was used. Finally, the CDS fragment and enzyme The cut vectors were ligated to obtain pLVX-IRES-ALDOA-C. (2) Construction of MEF cell lines that induce the expression of ALDOA-C

將pLVX-IRES-ALDOA-C在HEK293T細胞中包裝成慢病毒，並用此病毒感染MEF細胞24小時以上。其中，慢病毒導入表達質粒的步驟如下：PLVX-IRES-ALDOA-C was packaged as a lentivirus in HEK293T cells, and MEF cells were infected with this virus for more than 24 hours. The steps for introducing the lentivirus into an expression plasmid are as follows:

接種1.5 x 106 個人胚胎腎細胞HEK293T至35 mm細胞培養皿，每皿加入2ml的DMEM細胞培養液，置於5％ CO2 培養箱中培養（培養條件為：5％ CO2 ，37℃），過夜，待細胞密度達到80％以上時，製備轉染試劑與表達質粒的轉染混合物，室溫放置20分鐘後加入到細胞培養液中，繼續培養48小時後收集含有慢病毒顆粒的上清。將含有慢病毒顆粒的上清加入到已接種小鼠成纖維細胞（MEFs）的35 mm細胞培養皿，繼續培養24小時以上，後收集細胞進行蛋白質免疫印跡實驗。Inoculate 1.5 x 10 6 human embryonic kidney cells HEK293T to a 35 mm cell culture dish, add 2 ml of DMEM cell culture solution to each dish, and culture in a 5% CO 2 incubator (culture conditions: 5% CO 2 , 37 ° C) Overnight, when the cell density reaches 80% or more, prepare a transfection mixture of the transfection reagent and the expression plasmid, leave it at room temperature for 20 minutes, add it to the cell culture solution, and continue to culture for 48 hours to collect the supernatant containing the lentiviral particles. . The supernatant containing the lentiviral particles was added to a 35 mm cell culture dish inoculated with mouse fibroblasts (MEFs), and the culture was continued for more than 24 hours, and then the cells were collected for Western blot experiments.

將細胞培養在含有100 ng/ml Dox的DMEM培養液中，如此構建成誘導表達ALDOA-C的MEF細胞株。 （3）shRNA序列設計The cells were cultured in a DMEM medium containing 100 ng / ml Dox, and thus a MEF cell line that induced the expression of ALDOA-C was constructed. (3) shRNA sequence design

靶向ALDOA： 5’-CCAAGTGGCGCTGTGTGCT-3’ （SEQ ID NO: 7），或 5’-GCCATGGGCCTTGACTTTC-3’ （SEQ ID NO: 8） 靶向ALDOB： 5’-GCTCTCTGAGCAGATCCAT-3’ （SEQ ID NO: 9），或 5’-GGCAGTTCCGAGAACTCCT-3’ （SEQ ID NO: 10） 靶向ALDOC： 5’-GAGTCTAGAGCTTATGTCT-3’ （SEQ ID NO: 11），或 5’-CAGTTACCCTTGATGGTAT-3’ （SEQ ID NO: 12） （4）細胞轉染和培養Targeting ALDOA: 5'-CCAAGTGGCGCTGTGTGCT-3 '(SEQ ID NO: 7), or 5'-GCCATGGGCCTTGACTTTC-3' (SEQ ID NO: 8) Targeting ALDOB: 5'-GCTCTCTGAGCAGATCCAT-3 '(SEQ ID NO: 9), or 5'-GGCAGTTCCGAGAACTCCT-3 '(SEQ ID NO: 10) targeting ALDOC: 5'-GAGTCTAGAGCTTATGTCT-3' (SEQ ID NO: 11), or 5'-CAGTTACCCTTGATGGTAT-3 '(SEQ ID NO: 12) (4) Cell transfection and culture

分別將靶向ALDOA、ALDOB或ALDOC的各兩條shRNA通過慢病毒導入上述MEF細胞株24小時以上，使得內源表達的ALDOA、ALDOB或ALDOC被阻斷。其中將慢病毒導入shRNA的具體步驟如下：The two shRNAs targeted to ALDOA, ALDOB or ALDOC were introduced into the above MEF cell line via lentivirus for more than 24 hours, so that the endogenously expressed ALDOA, ALDOB or ALDOC was blocked. The specific steps for introducing lentivirus into shRNA are as follows:

接種1.5 x 106 個人胚胎腎細胞HEK293T至35 mm細胞培養皿，每皿加入2ml的DMEM細胞培養液，置於5％ CO2 培養箱中培養（培養條件為：5％ CO2 ，37℃），過夜，待細胞密度達到80％以上時，製備轉染試劑與含有shRNA的質粒的轉染混合物，室溫放置20分鐘後加入到細胞培養液中，繼續培養48小時後收集含有慢病毒顆粒的上清。將含有慢病毒顆粒的上清加入到已接種小鼠成纖維細胞（MEFs）的35 mm細胞培養皿，繼續培養24小時以上。Inoculate 1.5 x 10 6 human embryonic kidney cells HEK293T to a 35 mm cell culture dish, add 2 ml of DMEM cell culture solution to each dish, and culture in a 5% CO 2 incubator (culture conditions: 5% CO 2 , 37 ° C) Overnight, when the cell density reaches 80% or more, prepare a transfection mixture of the transfection reagent and the shRNA-containing plasmid, leave it at room temperature for 20 minutes, add it to the cell culture solution, and continue to culture for 48 hours to collect the lentiviral particles. The supernatant. The supernatant containing the lentiviral particles was added to a 35 mm cell culture dish inoculated with mouse fibroblasts (MEFs), and the culture was continued for more than 24 hours.

最後，將細胞培養在不含Dox的DMEM培養基12小時，使得誘導表達的外源ALDOA-C消失，然後收集細胞，用於下面的蛋白質免疫印跡實驗。 （5）蛋白質免疫印跡實驗檢測ALDOA-C蛋白水準Finally, the cells were cultured in DMEM medium without Dox for 12 hours to cause exogenous ALDOA-C induced expression to disappear, and then the cells were collected for the following western blot experiments. (5) Western blot detection of ALDOA-C protein level

按照常規的Western Blot操作進行。 3. 實驗結果Follow the conventional Western Blot operation. 3. Experimental results

如第1圖所示。As shown in Figure 1.

結果顯示，shRNA處理組中ALDOA-C蛋白水準顯著下調。說明shRNA顯著抑制了ALDOA-C基因的表達。 實施例2：靶向ALDOA-C的shRNA能夠啟動AMPK 1. 實驗材料和主要試劑The results showed that the level of ALDOA-C protein was significantly down-regulated in the shRNA-treated group. This indicated that shRNA significantly inhibited the expression of ALDOA-C gene. Example 2: shRNA targeted to ALDOA-C can start AMPK 1. Experimental materials and main reagents

Western所用一抗： Rabbit 548 anti-phospho-AMPKα-T172 (cat #2535)，購自Cell Signaling Technology； anti-AMPKα (cat #2532, 1:1000 for IB)，購自Cell Signaling Technology； anti-phospho-ACC-Ser79 (cat. #3661, 1:1000 for IB)，購自Cell Signaling Technology； anti-ACC (cat.#3662, 1:1000 for IB)，購自Cell Signaling Technology。 其它用到的一抗和二抗如實施例1中所述。 不含葡萄糖的DMEM (Gibco, cat. 11966)購自Thermofisher。 2. 實驗方法Primary antibodies used in Western: Rabbit 548 anti-phospho-AMPKα-T172 (cat # 2535), purchased from Cell Signaling Technology; anti-AMPKα (cat # 2532, 1: 1000 for IB), purchased from Cell Signaling Technology; anti-phospho -ACC-Ser79 (cat. # 3661, 1: 1000 for IB), purchased from Cell Signaling Technology; anti-ACC (cat. # 3662, 1: 1000 for IB), purchased from Cell Signaling Technology. The other primary and secondary antibodies used were as described in Example 1. DMEM without glucose (Gibco, cat. 11966) was purchased from Thermofisher. Experimental method

（1） 首先參照如實施例1中所述的方法，製備得到如第1圖所示的ALDOA-C同時敲低的MEF細胞。其中，利用靶向GFP（Green Fluorescent Protein綠色螢光蛋白）的shRNA作為對照組（non-targeting對照組）,靶向GFP的shRNA序列5’-GGCACAAGCTGGAGTACAA-3’（ SEQ ID NO: 13），構建方法參考實施例1。(1) First refer to the method described in Example 1 to prepare MEF cells with simultaneous knockdown of ALDOA-C as shown in Figure 1. Among them, shRNA targeting GFP (Green Fluorescent Protein) was used as a control group (non-targeting control group), and shRNA sequence targeting GFP 5'-GGCACAAGCTGGAGTACAA-3 '(SEQ ID NO: 13) was used to construct For the method, refer to Example 1.

（2）用不含葡萄糖（Glc）的DMEM培養液處理該細胞2小時（啟動AMPK），同時用含葡萄糖的DMEM培養基作為對照。(2) Treat the cells with DMEM medium without glucose (Glc) for 2 hours (activate AMPK), while using DMEM medium with glucose as a control.

（3）裂解細胞，將裂解液用免疫印跡方法，檢測p-AMPK和p-ACC的水準從而衡量AMPK的啟動情況。 3. 實驗結果(3) Cells were lysed, and the lysate was subjected to immunoblotting to detect the levels of p-AMPK and p-ACC to measure the activation of AMPK. 3. Experimental results

如第2圖所示。As shown in Figure 2.

結果顯示，在排除細胞死亡的條件下，敲低ALDOA-C能夠顯著啟動AMPK。The results show that knocking down ALDOA-C can significantly activate AMPK under conditions that exclude cell death.

參考文獻： 1. Gil G, Sitges M, Bové J, Hegardt FG. Phosphorylation--dephosphorylation of rat liver 3-hydroxy 3-methylglutaryl coenzyme A reductase associated with changes in activity. FEBS Lett. 1980 Feb 11;110(2):195-9. 2. Yeh LA, Lee KH, Kim KH. Regulation of rat liver acetyl-CoA carboxylase. Regulation of phosphorylation and inactivation of acetyl-CoA carboxylase by the adenylate energy charge. J Biol Chem. 1980 Mar 25;255(6):2308-14. 3. Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol. 2012 Mar 22;13(4):251-62. 4. Chen S, Murphy J, Toth R, Campbell DG, Morrice NA, Mackintosh C. Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators. Biochem J. 2008 Jan 15;409(2):449-59. 5. Pehmøller C, Treebak JT, Birk JB, Chen S, Mackintosh C, Hardie DG, Richter EA, Wojtaszewski JF. Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle. Am J Physiol Endocrinol Metab. 2009 Sep;297(3):E665-75. 6. Ji C, Yang B, Yang YL, He SH, Miao DS, He L, Bi ZG. Exogenous cell-permeable C6 ceramide sensitizes multiple cancer cell lines to Doxorubicin-induced apoptosis by promoting AMPK activation and mTORC1 inhibition. Oncogene. 2010 Dec 16;29(50):6557-68. 7. Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004 Aug 5;430(7000):686-9. 8. Cantó C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, Elliott PJ, Puigserver P, Auwerx J. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature. 2009 Apr 23;458(7241):1056-60. 9. Rera M, Bahadorani S, Cho J, Koehler CL, Ulgherait M, Hur JH, Ansari WS, Lo T Jr, Jones DL, Walker DW. Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog. Cell Metab. 2011 Nov 2;14(5):623-34. 10. Greer EL, Dowlatshahi D, Banko MR, Villen J, Hoang K, Blanchard D, Gygi SP, Brunet A. An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans. Curr Biol. 2007 Oct 9;17(19):1646-56. 11. Stenesen D, Suh JM, Seo J, Yu K, Lee KS, Kim JS, Min KJ, Graff JM. Adenosine nucleotide biosynthesis and AMPK regulate adult life span and mediate the longevity benefit of caloric restriction in flies. Cell Metab. 2013 Jan 8;17(1):101-12. 12. Martin-Montalvo A, Mercken EM, Mitchell SJ, Palacios HH, Mote PL, Scheibye-Knudsen M, Gomes AP, Ward TM, Minor RK, Blouin MJ, Schwab M, Pollak M, Zhang Y, Yu Y, Becker KG, Bohr VA, Ingram DK, Sinclair DA, Wolf NS, Spindler SR, Bernier M, de Cabo R. Metformin improves healthspan and lifespan in mice. Nat Commun. 2013;4:2192. 13. Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol. 2011 Sep 2;13(9):1016-23. 14. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67-93. 15. Mottillo EP, Desjardins EM, Crane JD, Smith BK, Green AE, Ducommun S, Henriksen TI, Rebalka IA, Razi A, Sakamoto K, Scheele C, Kemp BE, Hawke TJ, Ortega J, Granneman JG, Steinberg GR. Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function. Cell Metab. 2016 Jul 12;24(1):118-29. 16. Martínez de Morentin PB, González-García I, Martins L, Lage R, Fernández-Mallo D, Martínez-Sánchez N, Ruíz-Pino F, Liu J, Morgan DA, Pinilla L, Gallego R, Saha AK, Kalsbeek A, Fliers E, Bisschop PH, Diéguez C, Nogueiras R, Rahmouni K, Tena-Sempere M, López M. Estradiol regulates brown adipose tissue thermogenesis via hypothalamic AMPK. Cell Metab. 2014 Jul 1;20(1):41-53. 17. Bartelt A, Heeren J. Adipose tissue browning and metabolic health. Nat Rev Endocrinol. 2014 Jan;10(1):24-36. 18. Toyama EQ, Herzig S, Courchet J, Lewis TL Jr, Losón OC, Hellberg K, Young NP, Chen H, Polleux F, Chan DC, Shaw RJ. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress. Science. 2016 Jan 15;351(6270):275-81. 19. Mishra P, Chan DC. Mitochondrial dynamics and inheritance during cell division, development and disease. Nat Rev Mol Cell Biol. 2014 Oct;15(10):634-46. 20. Fullerton MD, Galic S, Marcinko K, Sikkema S, Pulinilkunnil T, Chen ZP, O'Neill HM, Ford RJ, Palanivel R, O'Brien M, Hardie DG, Macaulay SL, Schertzer JD, Dyck JR, van Denderen BJ, Kemp BE, Steinberg GR. Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin. Nat Med. 2013 Dec;19(12):1649-54. 21. Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: from mechanisms of action to therapies. Cell Metab. 2014 Dec 2;20(6):953-66. 22. Grahame Hardie D. Regulation of AMP-activated protein kinase by natural and synthetic activators. Acta Pharm Sin B. 2016 Jan;6(1):1-19. 23. Cool B, Zinker B, Chiou W, Kifle L, Cao N, Perham M, Dickinson R, Adler A, Gagne G, Iyengar R, Zhao G, Marsh K, Kym P, Jung P, Camp HS, Frevert E. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 2006 Jun;3(6):403-16. 24. Benziane B, Björnholm M, Lantier L, Viollet B, Zierath JR, Chibalin AV. AMP-activated protein kinase activator A-769662 is an inhibitor of the Na(+)-K(+)-ATPase. Am J Physiol Cell Physiol. 2009 Dec;297(6):C1554-66. 25. Ritterson Lew C, Tolan DR. Targeting of several glycolytic enzymes using RNA interference reveals aldolase affects cancer cell proliferation through a non-glycolytic mechanism. J Biol Chem. 2012 Dec 14;287(51):42554-63. 26. Grandjean G, de Jong PR, James BP, Koh MY, Lemos R, Kingston J, Aleshin A, Bankston LA, Miller CP, Cho EJ, Edupuganti R, Devkota A, Stancu G, Liddington RC, Dalby KN, Powis G. Definition of a Novel Feed-Forward Mechanism for Glycolysis-HIF1α Signaling in Hypoxic Tumors Highlights Aldolase A as a Therapeutic Target. Cancer Res. 2016 Jul 15;76(14):4259-69.References: 1. Gil G, Sitges M, Bové J, Hegardt FG. Phosphorylation--dephosphorylation of rat liver 3-hydroxy 3-methylglutaryl coenzyme A reductase associated with changes in activity. FEBS Lett. 1980 Feb 11; 110 (2) : 195-9. 2. Yeh LA, Lee KH, Kim KH. Regulation of rat liver acetyl-CoA carboxylase. Regulation of phosphorylation and inactivation of acetyl-CoA carboxylase by the adenylate energy charge. J Biol Chem. 1980 Mar 25; 255 (6): 2308-14. 3. Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol. 2012 Mar 22; 13 (4): 251-62. 4. Chen S, Murphy J, Toth R, Campbell DG, Morrice NA, Mackintosh C. Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators. Biochem J. 2008 Jan 15; 409 (2): 449-59 5. Pehmøller C, Treebak JT, Birk JB, Chen S, Mackintosh C, Hardie DG, Richter EA, Wojtaszewski JF. Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle. Am J Physiol Endocrinol Metab. 2009 Sep; 297 (3): E665-75. 6. Ji C, Yang B, Yang YL, He SH, Miao DS, He L, Bi ZG. Exogenous cell-permeable C6 ceramide sensitizes multiple cancer cell lines to Doxorubicin-induced apoptosis by promoting AMPK activation and mTORC1 inhibition. Oncogene. 2010 Dec 16; 29 (50): 6557-68. 7. Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004 Aug 5; 430 (7000): 686-9. 8. Cantó C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC, Elliott PJ, Puigserver P, Auwerx J. AMPK regulates energy expenditure by modulating NAD + metabolism and SIRT1 activity. Nature. 2009 Apr 23; 458 (7241) : 1056-60. 9. Rera M, Bahadorani S, Cho J, Koehler CL, Ulgherait M, Hur JH, Ansari WS, Lo T Jr, Jones DL, Walker DW. Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog. Cell Metab. 2011 Nov 2; 14 (5): 623-34. 10. Greer EL, Dowlatshahi D, Banko MR, Villen J, Hoang K, Blanchard D, Gygi SP, Brunet A. An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans. Curr Biol. 2007 Oct 9; 17 (19): 1646-56. 11. Stenesen D, Suh JM, Seo J, Yu K, Lee KS, Kim JS, Min KJ, Graff JM. Adenosine nucleotide biosynthesis and AMPK regulate adult life span and mediate the longevity benefit of caloric restriction in flies. Cell Metab. 2013 Jan 8; 17 (1): 101-12. 12. Martin-Montalvo A, Mercken EM, Mitchell SJ, Palacios HH, Mote PL, Scheibye-Knudsen M, Gomes AP, Ward TM, Minor RK, Blouin MJ, Schwab M, Pollak M, Zhang Y, Yu Y, Becker KG, Bohr VA, Ingram DK, Sinclair DA, Wolf NS, Spindler SR, Bernier M, de Cabo R. Metformin improves healthspan and lifespan in mice. Nat Commun. 2013; 4: 2192. 13. Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Bi ol. 2011 Sep 2; 13 (9): 1016-23. 14. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009; 43: 67-93. 15. Mottillo EP, Desjardins EM , Crane JD, Smith BK, Green AE, Ducommun S, Henriksen TI, Rebalka IA, Razi A, Sakamoto K, Scheele C, Kemp BE, Hawke TJ, Ortega J, Granneman JG, Steinberg GR. Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function. Cell Metab. 2016 Jul 12; 24 (1): 118-29. 16. Martínez de Morentin PB, González-García I, Martins L, Lage R, Fernández-Mallo D, Martínez-Sánchez N, Ruíz-Pino F, Liu J, Morgan DA, Pinilla L, Gallego R, Saha AK, Kalsbeek A, Fliers E, Bisschop PH, Diéguez C, Nogueiras R, Rahmouni K, Tena-Sempere M, López M Estradiol regulates brown adipose tissue thermogenesis via hypothalamic AMPK. Cell Metab. 2014 Jul 1; 20 (1): 41-53. 17. Bartelt A, Heeren J. Adipose tissue browning and metabolic health. Nat Rev Endocrinol. 2014 Jan; 10 (1): 24-36 18. Toyama EQ, Herzig S, Courchet J, Lewis TL Jr, Losón OC, Hellberg K, Young NP, Chen H, Polleux F, Chan DC, Shaw RJ. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress. Science. 2016 Jan 15; 351 (6270): 275-81. 19. Mishra P, Chan DC. Mitochondrial dynamics and inheritance during cell division, development and disease. Nat Rev Mol Cell Biol. 2014 Oct; 15 (10 ): 634-46. 20. Fullerton MD, Galic S, Marcinko K, Sikkema S, Pulinilkunnil T, Chen ZP, O'Neill HM, Ford RJ, Palanivel R, O'Brien M, Hardie DG, Macaulay SL, Schertzer JD , Dyck JR, van Denderen BJ, Kemp BE, Steinberg GR. Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin. Nat Med. 2013 Dec; 19 (12): 1649-54. 21. Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: from mechanisms of action to therapies. Cell Metab. 2014 Dec 2; 20 (6): 953-66. 22. Grahame Hardie D. Regulation of AMP- activated protein kin ase by natural and synthetic activators. Acta Pharm Sin B. 2016 Jan; 6 (1): 1-19. 23. Cool B, Zinker B, Chiou W, Kifle L, Cao N, Perham M, Dickinson R, Adler A, Gagne G, Iyengar R, Zhao G, Marsh K, Kym P, Jung P, Camp HS, Frevert E. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 2006 Jun; 3 (6): 403-16. 24. Benziane B, Björnholm M, Lantier L, Viollet B, Zierath JR, Chibalin AV. AMP-activated protein kinase activator A-769662 is an inhibitor of the Na (+)- K (+)-ATPase. Am J Physiol Cell Physiol. 2009 Dec; 297 (6): C1554-66. 25. Ritterson Lew C, Tolan DR. Targeting of several glycolytic enzymes using RNA interference reveals aldolase affects cancer cell proliferation through a non-glycolytic mechanism. J Biol Chem. 2012 Dec 14; 287 (51): 42554-63. 26. Grandjean G, de Jong PR, James BP, Koh MY, Lemos R, Kingston J, Aleshin A, Bankston LA, Miller CP, Cho EJ, Edupuganti R, Devkota A, Sta ncu G, Liddington RC, Dalby KN, Powis G. Definition of a Novel Feed-Forward Mechanism for Glycolysis-HIF1α Signaling in Hypoxic Tumors Highlights Aldolase A as a Therapeutic Target. Cancer Res. 2016 Jul 15; 76 (14): 4259- 69.

儘管本發明的具體實施方式已經得到詳細的描述，本領域技術人員將會理解。根據已經揭露的所有教導，可以對那些細節進行各種修改和替換，這些改變均在本發明的保護範圍之內。本發明的全部範圍由所附申請專利範圍及其任何等同物給出。Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the invention is given by the scope of the appended claims and any equivalents thereof.

第1圖：shRNA抑制ALDOA-C基因表達的免疫印跡結果。其中，＃1和＃2分別代表靶向ALDOA、ALDOB或ALDOC的兩條shRNA中的一條。對於ALDOA：＃1代表SEQ ID NO: 7，＃2代表SEQ ID NO: 8。對於ALDOB：＃1代表SEQ ID NO: 9，＃2代表SEQ ID NO: 10。對於ALDOC：＃1代表SEQ ID NO: 11，＃2代表SEQ ID NO: 12。 第2圖：shRNA抑制ALDOA-C後AMPK的效果的檢測圖。其中，siGFP為對照組。Figure 1: Immunoblot results of shRNA inhibition of ALDOA-C gene expression. Among them, # 1 and # 2 respectively represent one of two shRNAs targeting ALDOA, ALDOB, or ALDOC. For ALDOA: # 1 represents SEQ ID NO: 7, and # 2 represents SEQ ID NO: 8. For ALDOB: # 1 represents SEQ ID NO: 9, and # 2 represents SEQ ID NO: 10. For ALDOC: # 1 represents SEQ ID NO: 11, and # 2 represents SEQ ID NO: 12. Figure 2: Detection of the effect of shRNA on AMPK after ALDOA-C inhibition. Among them, siGFP is the control group.

Claims (18)

選自如下的（1）至（6）項中的任一項在製備啟動AMPK的藥物或者在製備篩選啟動AMPK的藥物的模型中的用途： （1） Aldolase； （2） 編碼Aldolase的多核苷酸； （3） 核酸構建體，其含有用於完全敲除或者部分敲除Aldolase基因的多核苷酸；較佳地，該多核苷酸為siRNA例如shRNA，或者為用於CRISPR/Cas9系統的guide RNA； （4） 宿主細胞，其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除；較佳地，其含有第（3）項所述的核酸構建體； （5） 一抑制或阻斷Aldolase活性的藥物； （6） 一抑制或降低Aldolase基因表達水準的藥物。Use of any one selected from the following items (1) to (6) in the preparation of a drug that initiates AMPK or in a model for the screening of a drug that initiates AMPK: (1) Aldolase; (2) a polynucleoside encoding an Aldolase (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or a guide for the CRISPR / Cas9 system RNA; (4) a host cell in which the polynucleotide encoding Aldolase is completely or partially knocked out; preferably, it contains the nucleic acid construct according to item (3); (5) an inhibition or inhibition Drugs that interrupt Aldolase activity; (6) A drug that inhibits or reduces the level of Aldolase gene expression. 如申請專利範圍第1項所述的用途，其中，該抑制或阻斷Aldolase活性的藥物為抗Aldolase的一抗體或TDZD-8；較佳地，該抗體為一單克隆抗體。The use according to item 1 of the scope of patent application, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody. 如申請專利範圍第1項所述的用途，其中，該抑制或降低Aldolase基因表達水準的藥物選自siRNA例如shRNA，以及用於CRISPR-Cas9 系統的guide RNA。The use according to item 1 of the scope of patent application, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system. 選自如下的（1）至（6）項中的任一項在製備抑制膽固醇合成的藥物、降低脂肪酸合成的藥物、抗肥胖的藥物、預防及/或治療糖尿病的藥物、預防及/或治療腫瘤的藥物、預防及/或治療帕金森氏症的藥物、預防及/或治療阿茲海默症的藥物、抗衰老的藥物或者用於延長哺乳動物壽命的藥物中的用途： （1） Aldolase； （2） 編碼Aldolase的多核苷酸； （3） 核酸構建體，其含有用於完全敲除或者部分敲除Aldolase基因的多核苷酸；較佳地，該多核苷酸為siRNA例如shRNA，或者為用於CRISPR/Cas9系統的guide RNA； （4） 宿主細胞，其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除；較佳地，其含有第（3）項所述的核酸構建體； （5） 一抑制或阻斷Aldolase活性的藥物； （6） 一抑制或降低Aldolase基因表達水準的藥物； 較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。It is selected from any one of the following items (1) to (6) in the preparation of a drug for inhibiting cholesterol synthesis, a drug for reducing fatty acid synthesis, an anti-obesity drug, a drug for preventing and / or treating diabetes, a preventive and / or treating Use of a medicine for tumor, a medicine for preventing and / or treating Parkinson's disease, a medicine for preventing and / or treating Alzheimer's disease, an anti-aging medicine or a medicine for extending the life of a mammal: (1) Aldolase (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a polynucleotide for completely or partially knocking out the Aldolase gene; preferably, the polynucleotide is an siRNA such as shRNA, or Is a guide RNA for the CRISPR / Cas9 system; (4) a host cell in which a polynucleotide encoding Aldolase is completely or partially knocked out; preferably, it contains the nucleic acid construct according to item (3) (5) a drug that inhibits or blocks the activity of Aldolase; (6) a drug that inhibits or reduces the level of Aldolase gene expression; preferably, the tumor is selected from black Any one or more of melanoma, pancreatic cancer, ovarian cancer and breast cancer. 如申請專利範圍第4項所述的用途，其中，該抑制或阻斷Aldolase活性的藥物為抗Aldolase的一抗體或TDZD-8；較佳地，該抗體為單克隆抗體。The use according to item 4 of the scope of patent application, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody. 如申請專利範圍第4項所述的用途，其中，該抑制或降低Aldolase基因表達水準的藥物選自siRNA例如shRNA，以及用於CRISPR-Cas9 系統的guide RNA。The use according to item 4 of the scope of patent application, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system. 一種在體內或體外啟動AMPK的方法，包括抑制Aldolase活性或者下調Aldolase基因表達水準的步驟，例如，包括抑制有需求的受試者中或者細胞中的Aldolase的活性或者下調Aldolase基因的表達水準的步驟。A method for activating AMPK in vivo or in vitro, comprising the steps of inhibiting Aldolase activity or down-regulating Aldolase gene expression level, for example, including the step of inhibiting Aldolase activity or down-regulating Aldolase gene expression level in a subject or cell in need thereof . 一種篩選選自如下的藥物的方法，包括加入待測藥物，以及檢測Aldolase活性或者檢測Aldolase基因表達水準的步驟： 啟動AMPK的藥物、抑制膽固醇合成的藥物、降低脂肪酸合成的藥物、抗肥胖的藥物、預防及/或治療糖尿病的藥物、預防及/或治療腫瘤的藥物、預防及/或治療帕金森氏症的藥物、預防及/或治療阿茲海默症的藥物、抗衰老的藥物或者用於延長哺乳動物壽命的藥物； 較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。A method for screening a drug selected from the group consisting of adding a drug to be tested and detecting Aldolase activity or detecting an Aldolase gene expression level: a drug that activates AMPK, a drug that inhibits cholesterol synthesis, a drug that reduces fatty acid synthesis, and an anti-obesity drug Drugs for preventing and / or treating diabetes, drugs for preventing and / or treating tumors, drugs for preventing and / or treating Parkinson's disease, drugs for preventing and / or treating Alzheimer's disease, anti-aging drugs or using A drug for extending the life of a mammal; Preferably, the tumor is any one or more selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer. 一種重組載體，其含有下調Aldolase基因表達水準的siRNA例如shRNA，或者用於CRISPR-Cas9 系統的guide RNA；較佳地，該重組載體為重組慢病毒載體。A recombinant vector containing an siRNA such as shRNA that down-regulates the expression level of the Aldolase gene, or a guide RNA used in the CRISPR-Cas9 system; preferably, the recombinant vector is a recombinant lentiviral vector. 一種宿主細胞，其含有申請專利範圍第9項所述的重組載體，或者其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除。A host cell contains the recombinant vector described in item 9 of the patent application scope, or the polynucleotide encoding Aldolase therein is completely or partially knocked out. 一種藥物組合物，其包含申請專利範圍第9項所述的重組載體或者申請專利範圍第10項所述的宿主細胞，可選地，其還包含藥學上可接受的輔料。A pharmaceutical composition comprises the recombinant vector described in claim 9 or the host cell described in claim 10, and optionally, further comprises a pharmaceutically acceptable excipient. 選自如下的（1）至（4）項中的任一項，其用於製備啟動AMPK的藥物、抑制膽固醇合成的藥物、降低脂肪酸合成的藥物、抗肥胖的藥物、預防及/或治療糖尿病的藥物、預防及/或治療腫瘤的藥物、預防及/或治療帕金森氏症的藥物、預防及/或治療阿茲海默症的藥物、抗衰老的藥物或者用於延長哺乳動物壽命的藥物，或者用於製備篩選啟動AMPK的藥物的模型： （1） Aldolase； （2） 編碼Aldolase的一多核苷酸； （3） 核酸構建體，其含有用於完全敲除或者部分敲除Aldolase基因的多核苷酸；較佳地，該多核苷酸為siRNA例如shRNA，或者為用於CRISPR/Cas9系統的guide RNA； （4） 宿主細胞，其中的編碼Aldolase的多核苷酸被完全敲除或部分敲除；較佳地，其含有第（3）項所述的核酸構建體； 較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。Any one selected from the following items (1) to (4), which is used to prepare a drug that initiates AMPK, a drug that inhibits cholesterol synthesis, a drug that reduces fatty acid synthesis, an anti-obesity drug, and prevents and / or treats diabetes Drugs, drugs that prevent and / or treat tumors, drugs that prevent and / or treat Parkinson's disease, drugs that prevent and / or treat Alzheimer's disease, anti-aging drugs, or drugs that extend the life of mammals , Or a model for the preparation of a drug that activates AMPK: (1) Aldolase; (2) a polynucleotide encoding Aldolase; (3) a nucleic acid construct containing a complete or partial knockout of the Aldolase gene Preferably, the polynucleotide is an siRNA such as shRNA, or a guide RNA used in the CRISPR / Cas9 system; (4) a host cell in which the polynucleotide encoding Aldolase is completely knocked out or partially Knockout; preferably, it contains the nucleic acid construct according to item (3); preferably, the tumor is any one selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast cancer One or several. 一種抑制或阻斷Aldolase活性的藥物，或者一種抑制或降低Aldolase基因表達水準的藥物，其用於啟動AMPK、抑制膽固醇合成、降低脂肪酸合成、抗肥胖、預防及/或治療糖尿病、預防及/或治療一腫瘤、預防及/或治療帕金森氏症、預防及/或治療阿茲海默症、抗衰老或者用於延長哺乳動物壽命； 較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。A drug that inhibits or blocks Aldolase activity, or a drug that inhibits or reduces Aldolase gene expression level, and is used to activate AMPK, inhibit cholesterol synthesis, reduce fatty acid synthesis, anti-obesity, prevent and / or treat diabetes, prevent and / or Treating a tumor, preventing and / or treating Parkinson's disease, preventing and / or treating Alzheimer's disease, anti-aging or for prolonging the life of a mammal; preferably, the tumor is selected from the group consisting of melanoma, pancreatic cancer, Any one or several of ovarian cancer and breast cancer. 如申請專利範圍第13項所述的藥物，其中，該抑制或阻斷Aldolase活性的藥物為抗Aldolase的一抗體或TDZD-8；較佳地，該抗體為一單克隆抗體。The drug according to item 13 of the application, wherein the drug that inhibits or blocks Aldolase activity is an antibody against Aldolase or TDZD-8; preferably, the antibody is a monoclonal antibody. 如申請專利範圍第13項所述的藥物，其中，該抑制或降低Aldolase基因表達水準的藥物選自siRNA例如shRNA，以及用於CRISPR-Cas9 系統的guide RNA。The drug according to item 13 of the scope of patent application, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNA such as shRNA, and guide RNA for the CRISPR-Cas9 system. 一種治療及/或預防高膽固醇症、糖尿病、腫瘤、帕金森氏症或阿茲海默症的方法，或者一種抗肥胖（例如預防肥胖或者減肥）、抗衰老或延長哺乳動物壽命的方法，或者一種抑制膽固醇合成或降低脂肪酸合成的方法，包括抑制有需求的受試者中的Aldolase的活性或者下調有需求的受試者中的Aldolase基因的表達水準的步驟；較佳地，包括給予有需求的受試者以有效量的一抑制或阻斷Aldolase活性的藥物或者一抑制或降低Aldolase基因表達水準的藥物的步驟； 較佳地，該腫瘤為選自黑色素瘤、胰腺癌、卵巢癌和乳腺癌中的任意一種或幾種。A method of treating and / or preventing hypercholesterolemia, diabetes, tumors, Parkinson's disease, or Alzheimer's disease, or an anti-obesity (such as preventing obesity or losing weight), anti-aging, or extending the life of a mammal, or A method for inhibiting cholesterol synthesis or reducing fatty acid synthesis, comprising the steps of inhibiting the activity of Aldolase in a subject in need or down-regulating the expression level of Aldolase gene in a subject in need; preferably, the method comprises administering Step of the subject with an effective amount of a drug that inhibits or blocks Aldolase activity or a drug that inhibits or reduces the level of Aldolase gene expression; preferably, the tumor is selected from the group consisting of melanoma, pancreatic cancer, ovarian cancer, and breast Any one or more of the cancers. 如申請專利範圍第16項所述的方法，其中，該抑制或阻斷Aldolase活性的藥物為抗Aldolase的抗體或TDZD-8；較佳地，該抗體為單克隆抗體。The method according to item 16 of the application, wherein the drug that inhibits or blocks Aldolase activity is an anti-Aldolase antibody or TDZD-8; preferably, the antibody is a monoclonal antibody. 如申請專利範圍第16項所述的方法，其中，該抑制或降低Aldolase基因表達水準的藥物選自siRNA例如shRNA，以及用於CRISPR-Cas9 系統的guide RNA。The method of claim 16, wherein the drug that inhibits or reduces the expression level of the Aldolase gene is selected from siRNAs such as shRNA, and guide RNA for the CRISPR-Cas9 system.
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