WO2019100503A1 - 试剂在制备药物中的用途、筛选药物的方法以及药物组合物 - Google Patents

试剂在制备药物中的用途、筛选药物的方法以及药物组合物 Download PDF

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WO2019100503A1
WO2019100503A1 PCT/CN2017/117832 CN2017117832W WO2019100503A1 WO 2019100503 A1 WO2019100503 A1 WO 2019100503A1 CN 2017117832 W CN2017117832 W CN 2017117832W WO 2019100503 A1 WO2019100503 A1 WO 2019100503A1
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microrna
obesity
pharmaceutical composition
inhibition
diabetes
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French (fr)
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李蓬
李祺琪
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清华大学
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Definitions

  • the present invention relates to the field of biology, and in particular, to the use of an agent in the preparation of a medicament, a method of screening a medicament, and a pharmaceutical composition.
  • MicroRNAs are a class of endogenous small RNAs of about 20-24 nucleotides in length that have a variety of important regulatory roles in cells. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene. This complex regulatory network can regulate the expression of multiple genes through a single miRNA, or it can fine-regulate the expression of a gene through a combination of several miRNAs. It is speculated that miRNA regulates one-third of human genes. Recent studies indicate that approximately 70% of mammalian miRNAs are located in the TUs region (TUs) (Rodriguez et al, 2004), and most of them are located in intron regions (Kim & Nam, 2006).
  • TUs TUs region
  • miRNAs are not only conserved at gene positions, but also exhibit high homology in sequence (Pasquinelli et al, 2000; Ruvkun et al, 2001; Lee & Ambros, 2001).
  • the high degree of conservation of miRNAs is closely related to the importance of their function.
  • the miRNA is closely related to the evolution of its target gene, and its evolutionary history helps to further understand its mechanism and function.
  • mice used two mouse models available in the laboratory: ob (leptin knockout) mice and ob/fsp27-/- (leptin and fsp27 genes simultaneously knockout) mice, both mice They showed two phenotypes of obesity and leanness.
  • the inventors isolated the intra-abdominal fat of these two mice, then extracted RNA, performed miRNA microarray and statistical analysis, realtime PCR and functional verification, and found that microRNA-708 can Significantly increase the intracellular triglyceride content.
  • the invention provides the use of an agent for the preparation of a medicament for inhibiting microRNA-708 for use in at least one of the following: reducing intracellular triglycerides Content, inhibit the differentiation of fat cells, resist obesity, promote insulin sensitivity, increase respiratory metabolic rate, increase energy consumption, High mitochondrial number, up-regulates oxidative phosphorylation or heat-producing genes, relieves insulin resistance, and treats or prevents type 2 diabetes.
  • drugs prepared by using microRNA-708 inhibiting agents can be effectively used to reduce intracellular triglyceride levels, inhibit adipocyte differentiation, resist obesity, promote insulin sensitivity, increase respiratory metabolic rate, and increase energy. Consumption, increase the number of mitochondria, up-regulate oxidative phosphorylation or heat-producing genes, relieve insulin resistance, treat or prevent type 2 diabetes, and resist fatty liver.
  • the above use may further include at least one of the following additional technical features:
  • the inhibition is achieved by at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpfl and zinc finger nuclease.
  • shRNA antisense nucleic acid
  • ribozyme dominant negative mutation
  • CRISPR-Cas9 CRISPR-Cpfl
  • zinc finger nuclease zinc finger nuclease
  • the inhibition is achieved by a shRNA comprising a first nucleic acid, the first nucleic acid having SEQ ID NO: 1.
  • the first nucleic acid having the above nucleotide sequence can effectively knock out or knock down the microRNA-708, thereby effectively inhibiting the microRNA-708.
  • White adipose tissue has obvious browning, mitochondria increased significantly, and genes related to oxidative phosphorylation and heat production were significantly up-regulated.
  • the inhibition is achieved by an antisense nucleic acid comprising a second nucleic acid having the nucleotide sequence set forth in SEQ ID NO:2.
  • the second nucleic acid having the above nucleotide sequence is effective for inhibiting the function of microRNA-708.
  • the inventors found that after antisense nucleic acid treatment in obese mice, the degree of obesity in the mice was significantly decreased, and the symptoms of insulin resistance were also significantly alleviated.
  • the obesity is high fat induced obesity.
  • the microRNA-708-inhibiting agent has a more pronounced inhibitory effect on high-fat-induced obesity.
  • the oxidative phosphorylation or heat production-related gene comprises at least one of ucp1, cidea, pgc1a, ppara, and Dio2.
  • the inventors found that the above-mentioned oxidative phosphorylation or heat-producing genes in microRNA-708 knockout mice can be significantly up-regulated, suggesting that microRNA-708 can inhibit the expression of oxidative phosphorylation or heat-producing genes, thereby inhibiting respiratory metabolism of cells. Rate and energy consumption.
  • the invention provides a method for screening a medicament for use in at least one of the following: reducing intracellular triglyceride content, inhibiting differentiation of fat cells, resisting obesity, and promoting insulin Sensitivity, increase respiratory metabolic rate, increase energy consumption, increase mitochondria number, up-regulate oxidative phosphorylation or heat-producing genes, Relieve insulin resistance, treat or prevent type 2 diabetes, and resist fatty liver.
  • the method comprises: contacting the candidate drug with a disease model, wherein the microRNA-708 is inhibited in the disease model as an indication of the candidate drug as the target drug.
  • the target drug obtained by the above method according to the embodiment of the present invention can be effectively used for reducing intracellular triglyceride content, inhibiting adipocyte differentiation, resisting obesity, promoting insulin sensitivity, increasing respiratory metabolic rate, and increasing energy consumption. Increase the number of mitochondria, up-regulate oxidative phosphorylation or heat-producing genes, relieve insulin resistance, treat or prevent type 2 diabetes, and resist fatty liver.
  • the above method may further include at least one of the following additional technical features:
  • the microRNA-708 is inhibited to include downregulation of microRNA-708 expression or inhibition of microRNA-708 function. Furthermore, microRNA-708 is effectively repressed by regulatory functions such as oxidative phosphorylation or heat-producing genes.
  • the disease model is an adipocyte or obese mouse model.
  • mirRNA-708 has a specific high expression in adipose tissue, and microRNA-708 is highly expressed in adipose tissue of obese humans (relative to normal human adipose tissue), and the disease model selects fat cells or is small in obesity.
  • the drug can inhibit mirRNA-708, the inhibitory effect will be effectively amplified, and the target drug selected for screening will have high credibility.
  • the invention proposes a pharmaceutical composition.
  • the pharmaceutical composition comprises a reagent as defined above.
  • the pharmaceutical composition according to the embodiment of the present invention can be effectively used for reducing intracellular triglyceride content, inhibiting differentiation of fat cells, resisting obesity, promoting insulin sensitivity, increasing respiratory metabolic rate, increasing energy consumption, and increasing mitochondria quantity. , up-regulate oxidative phosphorylation or heat-producing genes, relieve insulin resistance, treat or prevent type 2 diabetes.
  • the above pharmaceutical composition may further comprise at least one of the following additional technical features:
  • the other drug comprises at least one of orlistat, oxazolidinedione (TZD) and the like.
  • fillers, anticoagulants, lubricants, humectants, fragrances and preservatives may also be included in the pharmaceutical compositions of the invention.
  • the invention provides a method of treating or preventing type 2 diabetes, obesity.
  • the method comprises administering to the patient a pharmaceutical composition as described above.
  • the pharmaceutical composition of the present invention can reduce intracellular triglyceride content, inhibit adipocyte differentiation, promote insulin sensitivity, increase respiratory metabolic rate, increase energy consumption, increase mitochondria quantity, and up-regulate
  • the oxidative phosphorylation or heat-producing genes alleviate insulin resistance, and thus, the pharmaceutical composition of the present invention can be administered when treating or preventing type II diabetes and obesity.
  • administering refers to introducing a predetermined amount of a substance into a patient in some suitable manner.
  • the pharmaceutical composition of the present invention can be administered by any conventional route as long as it can reach the intended tissue.
  • Each administered Modes are contemplated, including peritoneal, venous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplary modes of administration.
  • the compositions of the invention may be administered as an injectable preparation.
  • the pharmaceutical compositions of the invention may be administered using a particular device that delivers the active ingredient to the target cells.
  • the frequency and dosage of the pharmaceutical composition of the present invention can be determined by a number of relevant factors including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease as well as the active ingredient. Type of drug. According to some embodiments of the invention, the daily dose may be divided into 1 dose, 2 doses or multiple doses in a suitable form for administration once, twice or more times throughout the time period, as long as a therapeutically effective amount is achieved. .
  • terapéuticaally effective amount refers to an amount of a compound that is sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • a drug or compound that reduces, prevents, delays, inhibits, or arrests any symptoms of a disease or condition should be therapeutically effective.
  • a therapeutically effective amount of a drug or compound does not require a cure for the disease or condition, but will provide a treatment for the disease or condition such that the onset of the disease or condition of the individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the disease or The duration of the condition is altered, or for example the disease or condition becomes less severe, or the recovery is accelerated.
  • treatment is used to mean obtaining the desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing the disease and/or the adverse effects caused by the disease.
  • treatment encompasses the treatment of diseases in mammals, particularly humans (mainly type II diabetes, obesity), including: (a) prevention of disease in individuals who are susceptible to disease but have not yet been diagnosed (eg, prevention of type II) (diabetes) or a condition occurs; (b) inhibiting the disease, such as retarding the progression of the disease; or (c) relieving the disease, for example, alleviating the symptoms associated with the disease.
  • treatment encompasses any administration of a medicament or compound to an individual for treating, curing, ameliorating, ameliorating, ameliorating or inhibiting a disease in an individual, including, but not limited to, administering a pharmaceutical composition comprising a pharmaceutical composition described herein to an individual in need thereof.
  • the pharmaceutical composition of the invention may be used in combination with conventional methods of treatment and/or therapy, or may be used separately from conventional methods of treatment and/or therapy.
  • the pharmaceutical compositions of the present invention are administered in combination therapy with other drugs, they can be administered to the individual sequentially or simultaneously.
  • the pharmaceutical compositions of the invention may comprise a combination of the agents or pharmaceutically acceptable excipients of the invention and other therapeutic or prophylactic agents known in the art.
  • FIG. 1 is a graph showing the results of mirRNA-708 which can significantly increase the content of intracellular triglyceride according to an embodiment of the present invention
  • FIG. 2 is a graph showing the results of specific high expression of mirRNA-708 in adipose tissue according to an embodiment of the present invention
  • FIG. 3 is a graph showing the results of gradual increase of mirRNA-708 expression as adipose cells differentiate according to an embodiment of the present invention
  • FIG. 4 is a knockout mouse model of mirRNA-708, which is heavy when fed on a high fat diet, in accordance with an embodiment of the present invention.
  • the increase in the amount is significantly lower than that of normal mice;
  • Figure 5 is a graph showing the results of the body fat content of the knockout mouse model of mirRNA-708 when fed on a high-fat diet, which is significantly lower than that of normal mice, according to an embodiment of the present invention
  • Figure 6 is a graph showing the statistical results of glucose tolerance of mirRNA-708 knockout mice significantly higher than that of normal mice according to an embodiment of the present invention
  • Figure 7 is a graph showing the statistical results of insulin resistance of mirRNA-708 knockout mice significantly higher than that of normal mice according to an embodiment of the present invention.
  • Figure 8 is a graph showing the respiratory metabolic rate of mirRNA-708 knockout mice is significantly higher than that of normal mice, and the energy consumption is also significantly higher than that of normal mice according to an embodiment of the present invention
  • Figure 9 is a graph showing the results of significant browning of subcutaneous white adipose tissue of mirRNA-708 knockout mice according to an embodiment of the present invention.
  • Figure 10 is a graph showing the results of oxidative phosphorylation and heat-producing genes significantly up-regulated in mirRNA-708 knockout mice according to an embodiment of the present invention.
  • Figure 11 is a graph showing the results of significant relief of fatty liver produced by mirRNA-708 knockout mice relative to normal mice in the case of high fat feeding according to an embodiment of the present invention.
  • Figure 12 is a graph showing the results of a significant decrease in lipid content in the liver of mirRNA-708 knockout mice relative to normal mice in the case of high fat feeding according to an embodiment of the present invention.
  • Figure 13 is a structural diagram showing a significant decrease in the degree of obesity in mice after mirRNA-708 reverse nucleic acid treatment of obese mice according to an embodiment of the present invention
  • Figure 14 is a graph showing the results of significant relief of symptoms of glucose tolerance after mirRNA-708 reverse nucleic acid treatment of obese mice in accordance with an embodiment of the present invention
  • Figure 15 is a graph showing the results of significant relief of insulin sensitivity symptoms after mirRNA-708 reverse nucleic acid treatment of obese mice in accordance with an embodiment of the present invention
  • Figure 16 is a graph showing the results of mirRNA-708 significantly high expression (relative to normal human adipose tissue) in adipose tissue of obese humans in accordance with an embodiment of the present invention.
  • Example 1 microRNA-708 can significantly increase intracellular triglyceride content
  • the inventors extracted RNA from various tissues of mice, and then detected the distribution of microRNA-708 in various tissues of mice by Realtime-PCR, and found that microRNA-708 has a specific high expression in adipose tissue.
  • the inventors isolated the mouse primary pro-adipocyte cell line by centrifugation and induced it to differentiate into mature adipocytes in vitro.
  • the inventors found that the expression of microRNA-708 gradually increased with the differentiation of adipocytes.
  • the side demonstrates that microRNA-708 is closely related to the function of adipose tissue or fat cells. The results are shown in Figures 2 and 3.
  • the inventors purchased a knockout mouse model of microRNA-708 from the Nanjing Model Biology Institute, which was completed using the cas9 gene editing technique. The inventors then began normal diet and knockout mice from normal diet (ND) and high fat diet (HFD) for ten weeks from week 10, and the weight of the mice was measured weekly. The inventors found that the knock-over mice gained significantly less weight when fed on a high-fat diet than normal mice, indicating that the mice were resistant to high-fat-induced obesity. The results are shown in Figures 4 and 5.
  • the inventors Since obesity is closely related to type 2 diabetes, the inventors also examined the role of microRNA-708 in insulin sensitivity and the like.
  • the inventors used insulin resistance test (ITT) and glucose tolerance test (GTT) to detect insulin sensitivity in the mouse.
  • ITT insulin resistance test
  • GTT glucose tolerance test
  • the weight was weighed, the serial number was marked, the blood glucose was measured before the injection of insulin, and the injection volume was calculated according to the body weight.
  • the blood glucose was measured at 15 min, 30 min, 45 min, and 60 min. After the experiment, each cage was supplemented with feed. Then, the amount of glucose in the mouse glucose tolerance test is generally 1.5-2 g/kg, such as 2 g/kg of glucose, and a 20% glucose solution is prepared with physiological saline. 1g/kg, 0, 15, 45, 75, 105min blood glucose, 10% glucose injection, injection volume 0.1mg/10g body weight. The food was fasted at 5 pm the previous day, and the drinking water was normal at 16 am the next day at 9 am. Blood glucose was measured before injection, and glucose was injected intraperitoneally. 0.01 ml per g was injected, and each interval was 1 min.
  • mice tested the oxygen consumption and carbon dioxide emissions per unit minute and unit weight of mice by experiments in mouse metabolic cages.
  • the inventors found that knockout mice have the function of resisting obesity, mainly due to the call of the mouse.
  • the rate of aspiration metabolism was significantly higher than that of normal mice. The result is shown in Figure 8.
  • the inventors found that the subcutaneous fat of the mouse was fixed with 10% paraformaldehyde, and the subcutaneous white adipose tissue of the mouse was obviously browned by hematoxylin-eosin staining and transmission electron microscopy.
  • the lipid droplets change from one large to many small lipid droplets, and the number of mitochondria increases significantly.
  • the inventors extracted RNA from knockout mice and normal mouse adipose tissue, obtained cDNA by reverse transcription, and then detected by Realtime-PCR, and found that oxidative phosphorylation and heat-producing genes were significantly up-regulated. The results are shown in Figures 9 and 10.
  • the inventors designed the reverse complement RNA of the microRNA-708, namely CCCAGCUAGACUUGUAAGUCCUU (SEQ ID NO: 2), and the inventors gave high fat-induced obese mice for 2 months by subcutaneous injection of a reverse amount of 1 OD/g.
  • Complementary RNA once a week for 4 weeks, after the inventors treated the obese mice, the degree of obesity in the mice decreased significantly, and the symptoms of insulin resistance were also significantly alleviated. The results are shown in Figures 13, 14 and 15.
  • the inventors obtained fat from different parts of normal and obese patients (including abdominal fat, abdominal subcutaneous fat, and groin subcutaneous fat) from the Department of Endocrinology, Shanghai Sixth Hospital. The inventors homogenized these tissues and extracted the total RNA of the tissue by the TRIZOL method as follows:
  • the inventors then reversed the expression of mir-708 in these tissues by Realtime-PCR after inversion using the full-scale miRNA reverse transcription kit.
  • the inventors found that the microRNA is highly expressed in adipose tissue of obese humans (relative to normal human adipose tissue). The result is shown in Fig. 16.

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Abstract

本发明提供了试剂在制备药物中的用途，该试剂用于抑制microRNA-708，该药物用于下列的至少之一：降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，抵御肥胖，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提高线粒体数量，上调氧化磷酸化或产热相关基因，缓解胰岛素抵抗，抵御脂肪肝，治疗或预防II型糖尿病。

Description

试剂在制备药物中的用途、筛选药物的方法以及药物组合物
优先权信息
本申请请求2017年11月23日向中国国家知识产权局提交的、专利申请号为2017111854466的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及生物领域，具体地，本发明涉及试剂在制备药物中的用途、筛选药物的方法以及药物组合物。
背景技术
MicroRNA(miRNA)是一类内生的、长度约为20-24个核苷酸的小RNA，其在细胞内具有多种重要的调节作用。每个miRNA可以有多个靶基因，而几个miRNA也可以调节同一个基因。这种复杂的调节网络既可以通过一个miRNA来调控多个基因的表达，也可以通过几个miRNA的组合来精细调控某个基因的表达。据推测，miRNA调节着人类三分之一的基因。最近的研究表明大约70％的哺乳动物miRNA是位于TUs区(transcriptionunits,TUs)(Rodriguez et al,2004)，且其中大部分是位于内含子区(Kim&Nam,2006)。一些内含子miRNA的位置在不同的物种中是高度保守的。miRNA不仅在基因位置上保守,序列上也呈现出高度的同源性(Pasquinelli etal，2000；Ruvkun et al，2001；Lee&Ambros，2001)。miRNA高度的保守性与其功能的重要性有着密切的关系。miRNA与其靶基因的进化有着密切的联系,研究其进化历史有助于进一步了解其作用机制和功能。
microRNA在调控生命活动的多个方面起着至关重要的调控作用。但microRNA与肥胖以及糖尿病的关系还有待科学家进一步地探索和发现。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
发明人利用了实验室已有的两种小鼠模型：即ob(瘦素基因敲除)小鼠和ob/fsp27-/-(leptin和fsp27基因同时敲除)小鼠，这两种小鼠分别表现为肥胖和瘦的两种表型，发明人将这两种小鼠的腹腔内脂肪分离出来，然后提取RNA，进行miRNA microarray和统计学分析、realtime PCR以及功能验证，发现microRNA-708能显著地提高细胞内甘油三酯的含量。
为此，在本发明的第一方面，本发明提出了试剂在制备药物中的用途，所述试剂用于抑制microRNA-708，所述药物用于下列的至少之一：降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，抵御肥胖，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提 高线粒体数量，上调氧化磷酸化或产热相关基因，缓解胰岛素抵抗，治疗或预防II型糖尿病。发明人发现，利用抑制microRNA-708的试剂所制备的药物可有效用于降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，抵御肥胖，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提高线粒体数量，上调氧化磷酸化或产热相关基因，缓解胰岛素抵抗，治疗或预防II型糖尿病，抵御脂肪肝。
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述抑制是通过shRNA，反义核酸，核酶，显性负突变，CRISPR-Cas9，CRISPR-Cpf1和锌指核酸酶至少之一实现的。进而有效实现敲除或敲低microRNA-708或抑制microRNA-708的功能。
根据本发明的实施例，所述抑制是通过shRNA实现的，所述试剂包含第一核酸，所述第一核酸具有SEQ ID NO：1。
AAGGAGCUUACAAUCUAGCUGGG(SEQ ID NO：1)。
具有上述核苷酸序列的第一核酸可有效敲除或敲低microRNA-708，从而有效抑制microRNA-708。发明人发现，microRNA-708敲除小鼠可抵御高脂诱导的肥胖的产生，葡萄糖耐受性和胰岛素的耐受性显著提高，呼吸代谢速率和能量消耗量也显著提高，该小鼠的皮下白色脂肪组织有明显的棕色化现象，线粒体明显增加，氧化磷酸化和产热相关基因明显被上调。
根据本发明的实施例，所述抑制是通过反义核酸实现的，所述试剂包含第二核酸，所述第二核酸具有SEQ ID NO：2所示的核苷酸序列。
CCCAGCUAGACUUGUAAGUCCUU(SEQ ID NO：2)。
具有上述核苷酸序列的第二核酸可有效抑制microRNA-708的功能。发明人发现，对肥胖小鼠进行反义核酸处理后，小鼠的肥胖程度明显下降，并且胰岛素抵抗性的症状也明显缓解。
根据本发明的实施例，所述肥胖为高脂诱导的肥胖。所述抑制microRNA-708的试剂对高脂诱导的肥胖的抑制效果更加显著。
根据本发明的实施例，所述氧化磷酸化或产热相关基因包括ucp1，cidea，pgc1a，ppara，Dio2的至少之一。发明人发现，microRNA-708敲除小鼠中上述的氧化磷酸化或产热相关基因可明显上调，暗示microRNA-708可抑制上述氧化磷酸化或产热相关基因的表达，进而抑制细胞的呼吸代谢速率和能量消耗量。
在本发明的第二方面，本发明提出了一种筛选药物的方法，所述药物用于下列的至少之一：降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，抵御肥胖，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提高线粒体数量，上调氧化磷酸化或产热相关基因， 缓解胰岛素抵抗，治疗或预防II型糖尿病，抵御脂肪肝。根据本发明的实施例，所述方法包括：使候选药物与疾病模型接触，接触后，所述疾病模型中microRNA-708被抑制是候选药物作为目标药物的指示。根据本发明实施例的上述方法筛选获得的目标药物能够有效用于降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，抵御肥胖，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提高线粒体数量，上调氧化磷酸化或产热相关基因，缓解胰岛素抵抗，治疗或预防II型糖尿病，抵御脂肪肝。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述microRNA-708被抑制包括microRNA-708表达量下调或microRNA-708功能被抑制。进而microRNA-708对如氧化磷酸化或产热相关基因的调节功能受到有效阻遏。
根据本发明的实施例，所述疾病模型为脂肪细胞或肥胖小鼠模型。发明人发现，mirRNA-708在脂肪组织内有特异性的高表达，且microRNA-708在肥胖人的脂肪组织内明显高表达(相对于正常人的脂肪组织)，疾病模型选择脂肪细胞或肥胖小鼠，如果药物能够抑制mirRNA-708，则抑制效果会有效放大，筛选所得目标药物的可信度高。
在本发明的第三方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括试剂，所述试剂如前面所限定的。根据本发明实施例的药物组合物能够有效用于降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，抵御肥胖，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提高线粒体数量，上调氧化磷酸化或产热相关基因，缓解胰岛素抵抗，治疗或预防II型糖尿病。
根据本发明的实施例，上述药物组合物还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，进一步包括其它药物，所述其它药物用于治疗或预防II型糖尿病、肥胖。根据本发明的具体实施例，所述其它药物包括奥利司他(orlistat)，唑烷二酮(TZD)等至少之一。根据本发明的另一些实施例，本发明的药物组合物中还可以包括填充剂，抗凝血剂，润滑剂，保湿剂，芳香剂和防腐剂。
在本发明的第四方面，本发明提出了一种治疗或预防II型糖尿病、肥胖的方法。根据本发明的实施例，所述方法包括给予患者前面所述的药物组合物。
根据本发明的实施例，本发明的药物组合物能够降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提高线粒体数量，上调氧化磷酸化或产热相关基因，缓解胰岛素抵抗，因而，本发明的药物组合物可以在治疗或预防II型糖尿病、肥胖时被给药。
在本文中所使用的术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的药物组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各 种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。优选地，本发明的组合物可以注射制剂被给药。此外，本发明的药物组合物可以使用将活性成分传送到靶细胞的特定器械来给药。
本发明的药物组合物的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。
术语“治疗有效量”是指化合物足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。例如，在II型糖尿病治疗中，减少、预防、延缓、抑制或阻滞疾病或病症的任何症状的药物或化合物应当是治疗有效的。治疗有效量的药物或化合物不需要治愈疾病或病症，但将为疾病或病症提供治疗，使得个体的疾病或病症的发作被延缓、阻止或预防，或者疾病或病症的症状得以缓解，或者疾病或病症的期限被改变，或者例如疾病或病症变得不严重，或者加速康复。
术语“治疗”用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病(主要指II型糖尿病、肥胖)的治疗，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病(例如预防II型糖尿病)或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或化合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述药物组合物给予有需要的个体。
根据本发明的实施例，本发明的药物组合物可与常规治疗方法和/或疗法相结合使用，或者可与常规治疗方法和/或疗法分开使用。当本发明的药物组合物在采用与其它药物的联合疗法中给药时，它们可序贯地或同时地给予个体。或者，本发明的药物组合物可包含本发明的所述试剂或药学上可接受的赋形剂以及本领域已知的其它治疗药或预防药的组合。
附图说明
图1是根据本发明实施例的mirRNA-708能显著地提高细胞内甘油三酯的含量的结果图；
图2是根据本发明实施例的mirRNA-708在脂肪组织内有特异性的高表达的结果图；
图3是根据本发明实施例的随着脂肪细胞的分化，mirRNA-708的表达逐渐增加的结果图；
图4是根据本发明实施例的mirRNA-708的敲除小鼠模型在高脂饮食喂养的时候重 量的增加明显低于正常小鼠的统计结果图；
图5是根据本发明实施例的mirRNA-708的敲除小鼠模型在高脂饮食喂养的时候体脂的含量明显低于正常小鼠的结果图；
图6是根据本发明实施例的mirRNA-708敲除小鼠的葡萄糖耐受性明显高于正常小鼠的统计结果图；
图7是根据本发明实施例的mirRNA-708敲除小鼠的胰岛素的耐受性明显高于正常小鼠的统计结果图；
图8是根据本发明实施例的mirRNA-708敲除小鼠的呼吸代谢速率明显高于正常小鼠，能量消耗量也明显高于正常小鼠的结果图；
图9是根据本发明实施例的mirRNA-708敲除小鼠的皮下白色脂肪组织有明显的棕色化现象的结果图；
图10是根据本发明实施例的mirRNA-708敲除小鼠的氧化磷酸化和产热相关基因明显被上调的结果图；
图11是根据本发明实施例的mirRNA-708敲除小鼠相对于正常小鼠在高脂喂养情况下产生的脂肪肝得到了明显缓解的结果图。
图12是根据本发明实施例的mirRNA-708敲除小鼠相对于正常小鼠在高脂喂养情况下的肝脏的脂含量明显下降的结果图。
图13根据本发明实施例的对肥胖小鼠进行mirRNA-708反向核酸处理后，小鼠的肥胖程度明显下降的结构图；
图14是根据本发明实施例的对肥胖小鼠进行mirRNA-708反向核酸处理后，葡萄糖耐受性的症状明显缓解的结果图；
图15是根据本发明实施例的对肥胖小鼠进行mirRNA-708反向核酸处理后，胰岛素敏感性的症状明显缓解的结果图；以及
图16是根据本发明实施例的mirRNA-708在肥胖人的脂肪组织内明显高表达(相对于正常人的脂肪组织)的结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
实施例1 microRNA-708能显著地提高细胞内甘油三酯的含量
发明人在上海吉玛公司合成mirRNA-708的成熟的序列，用lipofectamine 2000将其转染进小鼠原代脂肪细胞内。48小时后，细胞用PBS洗2-3次，再用2mlPBS吹起，移入15ml falcon tube,加入8ml(Hexane:Iso＝3:2)。长时间剧烈振荡(可至2于摇床上过夜)，然后静置10分钟，当中层无白色沉淀即可抽出上层有机相至玻璃管。将上层有机相底部用70℃加热，上面用氮气吹干，然后溶入100ul甲苯中。将下层含蛋白的水溶液离心，4000rpm,20min,去 掉水溶液，将沉淀60℃烘干至粉末状(置于超净台里的heat block上)，然后加入1ml 0.2M KOH溶过夜。第二天测蛋白浓度，用于做对照。将收集到的样品和标准脂溶液跑TLC(Hexane:乙醚：醋酸＝70：30：1)，然后用硫酸铜浸泡20秒左右，拿出后稍微吹干，直至未见明显液滴，再置于100—120℃烘5-10分钟。最后用凝胶成像系统进行定量分析。发明人发现mirRNA-708能显著地提高细胞内甘油三酯的含量，结果如图1所示。
实施例2 microRNA-708在脂肪代谢中的作用
首先，发明人提取了小鼠各组织的RNA，然后用Realtime-PCR的方法检测microRNA-708在小鼠各个组织中的分布，发现microRNA-708在脂肪组织内有特异性的高表达。同时，发明人通过离心的方法分离小鼠原代前脂肪细胞系，在体外将其诱导分化成成熟的脂肪细胞的过程中，发明人发现随着脂肪细胞的分化，microRNA-708的表达逐渐增加，侧面证明microRNA-708与脂肪组织或脂肪细胞的功能密切相关。结果如图2和图3所示。
然后，发明人从南京模式生物研究所购买到microRNA-708的敲除小鼠模型，该小鼠是利用cas9基因编辑技术来完成的。然后发明人对正常和敲除小鼠从第十周开始正常饮食喂养(ND)和高脂饮食喂养(HFD)十周，并且每周检测小鼠的重量。发明人发现敲除小鼠在高脂饮食喂养的时候重量的增加明显低于正常小鼠，说明该小鼠能够抵御高脂诱导的肥胖的产生。结果如图4和图5所示。
由于肥胖与II型糖尿病密切关联，所以发明人也检测了microRNA-708在胰岛素敏感性等方面的作用。发明人用胰岛素耐受实验(ITT)和葡萄糖耐受实验(GTT)两个方法来检测该小鼠的胰岛素敏感性。首先，小鼠胰岛素耐受实验的胰岛素用量一般为0.5-1.2U/kg，胰岛素用生理盐水稀释，如用量0.5U/kg，配制浓度0.5U/ml，0、15、30、60min测血糖；0.027U/10g＝2.7U/kg上午禁食4小时，正常饮水。下午试验，称体重，标记序号，注射胰岛素前测血糖，胰岛素按体重计算注射量，在15min、30min、45min、60min分别测血糖，实验完毕，每笼补充上饲料。然后，小鼠葡萄糖耐受实验的葡萄糖用量一般为1.5-2g/kg，如葡萄糖2g/kg，用生理盐水配制20％葡萄糖溶液。1g/kg，0、15、45、75、105min测血糖，10％葡萄糖注射液，注射量0.1mg/10g体重。前一天下午5点禁食，16h即次日上午9点，饮水正常。注射前测血糖，腹腔注射葡萄糖，每g注射0.01ml，每只间隔1min，在15min、30min、60min、90min、120min分别测血糖，实验完毕，每笼补充上饲料。发明人发现在正常饮食和高脂饮食的情况下，敲除小鼠的葡萄糖耐受性和胰岛素的耐受性都明显高于正常小鼠。说明敲除或敲低microRNA-708，能有效地促进胰岛素的敏感性，是II型糖尿病的良好治疗靶点。结果如图6和图7所示。
同时，发明人通过小鼠代谢笼的实验，检测单位分钟和单位重量小鼠的氧气消耗量和二氧化碳的排放量。发明人发现敲除小鼠之所以有抵抗肥胖的功能，主要是由于该小鼠的呼 吸代谢速率明显高于正常小鼠。结果如图8所示。
并且发明人通过对小鼠的皮下脂肪用10％多聚甲醛进行固定后，通过苏木精-伊红染色法和透射电镜观察发现该小鼠的皮下白色脂肪组织有明显的棕色化现象，即脂滴由一个大的变成很多小脂滴，线粒体数目明显增加。发明人提取了敲除小鼠和正常小鼠脂肪组织的RNA，通过反转录得到cDNA，然后用Realtime-PCR的方法检测，发现氧化磷酸化和产热相关基因明显被上调了。结果如图9和图10所示。
发明人通过对高脂喂养的正常和敲除小鼠的肝脏用10％多聚甲醛进行固定后，通过苏木精-伊红染色法观察发现敲除小鼠的脂肪肝程度明显低于正常小鼠。同时发明人提取了高脂喂养的正常和敲除小鼠肝脏的总脂。通过TLC的检测发现，高脂喂养的敲除小鼠的肝脏的甘油三酯含量明显低于高脂喂养的正常小鼠。结果如图11和12所示。
同时发明人设计了该microRNA-708的反向互补RNA，即CCCAGCUAGACUUGUAAGUCCUU(SEQ ID NO：2)，发明者给高脂诱导2个月的肥胖小鼠，通过皮下注射了1OD/g量的反向互补RNA，每周打一次，共4周，发明人对肥胖小鼠进行此处理后，小鼠的肥胖程度明显下降，并且胰岛素抵抗性的症状也明显缓解。结果如图13，14和15所示。
实施例3 microRNA-708在人类肥胖模型中的变化
发明人从上海第六医院内分泌科获得正常和肥胖病人的不同部位脂肪(包括腹部脂肪，腹部皮下脂肪和腹股沟皮下脂肪)。发明人将这些组织进行匀浆，用TRIZOL法提取组织的总RNA，步骤如下：
1，细胞或组织加Trizol后，室温放置5min，使其充分裂解；
2、12,000rpm离心5min，弃沉淀；
3、按200ul氯仿/ml Trizol加入氯仿，振荡混匀后室温放置15min；
4、4℃12,000g离心15min；
5、吸取上层水相，至另一离心管中；
6、按0.5ml异丙醇/ml Trizol加入异丙醇混匀，室温放置5-10min；
7、4℃12,000g离心10min，弃上清，RNA沉于管底；
8、按1ml 75％乙醇/ml Trizol加入75％乙醇，温和振荡离心管，悬浮沉淀；
9、4℃8,000g离心5min，尽量弃上清；
10、室温晾干或真空干燥5-10min。
然后发明人用全式金公司的miRNA反转录试剂盒进行反转后，通过Realtime-PCR检测mir-708在这些组织中的表达水平。发明人发现该microRNA在肥胖人的脂肪组织内明显高表达(相对于正常人的脂肪组织)。结果如图16所示。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (13)

  1. 试剂在制备药物中的用途，所述试剂用于抑制microRNA-708，所述药物用于下列的至少之一：
    降低细胞内甘油三脂的含量，
    抑制脂肪细胞的分化，
    抵御肥胖，
    促进胰岛素的敏感性，
    提高呼吸代谢速率，
    提高能量消耗量，
    提高线粒体数量，
    上调氧化磷酸化或产热相关基因，
    缓解胰岛素抵抗，
    抵御脂肪肝，
    治疗或预防II型糖尿病。
  2. 根据权利要求1所述的用途，其特征在于，所述抑制是通过shRNA，反义核酸，核酶，显性负突变，CRISPR-Cas9，CRISPR-Cpf1和锌指核酸酶的至少之一实现的。
  3. 根据权利要求1所述的用途，其特征在于，所述抑制是通过shRNA实现的，所述试剂包含第一核酸，所述第一核酸具有SEQ ID NO：1所示的核苷酸序列。
  4. 根据权利要求1所述的用途，其特征在于，所述抑制是通过反义核酸实现的，所述试剂包含第二核酸，所述第二核酸具有SEQ ID NO：2所示的核苷酸序列。
  5. 根据权利要求1所述的用途，其特征在于，所述肥胖为高脂诱导的肥胖。
  6. 根据权利要求1所述的用途，其特征在于，所述氧化磷酸化或产热相关基因包括ucp1，cidea，pgc1a，ppara，Dio2的至少之一。
  7. 一种筛选药物的方法，所述药物用于下列的至少之一：
    降低细胞内甘油三脂的含量，抑制脂肪细胞的分化，抵御肥胖，促进胰岛素的敏感性，提高呼吸代谢速率，提高能量消耗量，提高线粒体数量，上调氧化磷酸化或产热相关基因，缓解胰岛素抵抗，抵御脂肪肝，治疗或预防II型糖尿病，其特征在于，包括：
    使候选药物与疾病模型接触，
    接触后，所述疾病模型中microRNA-708被抑制是候选药物作为目标药物的指示。
  8. 根据权利要求7所述的方法，其特征在于，所述microRNA-708被抑制包括microRNA-708表达量下调或microRNA-708功能被抑制。
  9. 根据权利要求7所述的方法，其特征在于，所述疾病模型为脂肪细胞或肥胖小鼠模型。
  10. 一种药物组合物，其特征在于，包括：
    试剂，所述试剂如权利要求1～4任一项所限定的。
  11. 根据权利要求10所述的药物组合物，其特征在于，进一步包括其它药物，所述其它药物用于治疗或预防II型糖尿病、肥胖。
  12. 根据权利要求11所述的药物组合物，其特征在于，所述其它药物包括选自奥利司他，唑烷二酮至少之一。
  13. 一种治疗或预防II型糖尿病、肥胖的方法，其特征在于，给予患者权利要求10～12任一项所述的药物组合物。
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