WO2019137138A1 - 人gpnmb蛋白或其拮抗剂或激动剂的用途 - Google Patents

人gpnmb蛋白或其拮抗剂或激动剂的用途 Download PDF

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WO2019137138A1
WO2019137138A1 PCT/CN2018/120616 CN2018120616W WO2019137138A1 WO 2019137138 A1 WO2019137138 A1 WO 2019137138A1 CN 2018120616 W CN2018120616 W CN 2018120616W WO 2019137138 A1 WO2019137138 A1 WO 2019137138A1
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gpnmb
protein
antagonist
agonist
mice
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宋保亮
李云峰
魏健
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武汉大学
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    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
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Definitions

  • the invention relates to the field of biomedical technology, in particular to the application of the protein Glycoprotein nonmetastaticmelanoma protein B (GPNMB) in the diagnosis and treatment of metabolic diseases (such as obesity, diabetes, insulin resistance, hyperlipidemia, etc.).
  • GPNMB protein Glycoprotein nonmetastaticmelanoma protein B
  • GPNMB is a type I transmembrane protein that is located on chromosome 7 of the human body and consists of 572 amino acids.
  • the intact transmembrane protein can be cleaved by extracellular proteases such as ADAM8 and ADAM10, releasing the extracellular domain of GPNMB into the bloodstream to function as a cytokine.
  • GPNMB can be detected in normal bone, hematopoietic system and skin tissues, and can also be detected at high levels in malignant hyperplasia tissues such as breast cancer, melanoma, and hepatocellular carcinoma. .
  • liver fatty acid synthesis of liver tissue Scap or gp78-deficient mice decreased, but at the same time, fatty acid synthesis of white adipose tissue increased.
  • a serum study found that substances secreted by the liver of mice can up-regulate the expression of genes involved in fat synthesis. Subsequently, a total of 301 genes with up-regulated expression in the liver of the deficient mice were identified, and 12 protein-coding genes with N-terminal signal peptides were identified, and further quantitative PCR was performed to determine four expression levels relative to wild-type liters.
  • Another object of the present invention is to provide a use of GPNMB protein for the diagnosis, prevention and treatment of obesity and related metabolic diseases.
  • the invention identifies that GPNMB secreted by the rat liver can significantly enhance the fat synthesis ability of white fat, reduce energy metabolism of mice, and cause obesity, hyperlipidemia and insulin resistance in mice.
  • the level of GPNMB in the blood was significantly higher than that in normal mice.
  • Analysis of the population showed that the level of GPNMB in human blood was positively correlated with indicators such as body weight, obesity, blood pressure, insulin resistance, diabetes and hyperlipidemia.
  • GPNMB may be a molecular marker for insulin resistance, diabetes, and hyperlipidemia. It may also be a drug target for the treatment of obesity, etc., which can reduce Gpnmb expression by gene therapy, or inhibit GPNMB protein by using an antagonist or the like. active.
  • a human GPNMB protein or an antagonist or agonist thereof wherein the GPNMB protein or an antagonist or agonist thereof is used for diagnosing a metabolic disease, or the GPNMB protein or its antagonist Agent or agonist for the preparation of a medicament for the treatment and/or prevention of a metabolic disease, the GPNMB protein comprising:
  • Antagonists or agonists for the GPNMB protein of the invention are selected from:
  • altering Gpnmb gene expression includes, but is not limited to, inhibiting expression of a gene encoding a GPNMB protein, for example, an agent that inhibits expression of a Gpnmb gene or decreases its expression level, including: inhibition of Gpnmb gene transcriptional activity Reagents, agents that inhibit the transcriptional level of Gpnmb mRNA, agents that promote Gpnmb mRNA degradation, siRNAs against Gpnmb genes, agents that inhibit the translation of Gpnmb mRNA, and targeting nucleic acids that specifically recognize the Gpnmb gene and cut to reduce their levels
  • the reagent may also be an agent that causes mutation of the Gpnmb gene sequence, such that the expressed GPNMB protein is inactivated, such as a gene editing vector, a homologous recombinant vector, and the like.
  • an antagonist which reduces the activity of a GPNMB protein which may be, for example, a specific antibody or a small molecule having inhibitory activity.
  • the use of antibodies to neutralize GPNMB has been shown to prevent and treat obesity, reduced fat accumulation, decreased blood lipids, increased energy metabolism, increased insulin sensitivity, and enhanced blood glucose clearance.
  • Preferred antibodies for therapeutic purposes may be, for example, but not limited to, those described in US Pat. No. 8,039,593, B2, US Pat. No. 1,130,156, 784, A1, US Pat. No. 8,848, 873 B2, WO ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI> ⁇ / RTI>
  • a gene encoding a GPNMB protein wherein the amino acid sequence of the GPNMB protein is as shown in SEQ ID NO. 1, the metabolic disease is selected from the group consisting of obesity, diabetes , insulin resistance and hyperlipidemia.
  • the Gpnmb gene/GPNMB protein is used as a molecular marker for clinical diagnosis of the progression of metabolic diseases such as insulin resistance, diabetes, hyperlipidemia and obesity.
  • the reagent for detecting the Gpnmb gene, RNA or protein includes, but is not limited to, various primers and probes for detecting the Gpnmb gene, and/or specific antibodies for detecting the GPNMB protein, and the like, and the reagent includes the Gpnmb gene, RNA or Samples of the protein and other reagents used in performing the assay, such as solvents, etc., include, but are not limited to, various reagents required for performing PCR and the like. These reagents are well known to those skilled in the art and can be prepared according to the relevant references.
  • GPNMB gene, RNA and protein can be used as indicators and therapeutic targets for the diagnosis of metabolic diseases.
  • the present invention proves for the first time that GPNMB protein has a very important pathological function in obesity and its related metabolic diseases, and can use GPNMB gene, RNA and protein as biomarkers for diagnosing metabolic diseases such as obesity, diabetes, insulin resistance and hyperlipidemia. And as a drug target for the treatment of the above diseases.
  • Figure 1 Functional study of adeno-associated virus (AAV) overexpression system on hepatic secretion of GPNMB to upregulate the lipogenic capacity of white adipose tissue;
  • AAV adeno-associated virus
  • Figure 2 Antibody neutralization of blood GPNMB against high fat diet induced obesity
  • a Schematic diagram of antibody treatment of food-induced obesity; b. day 18 mouse body weight; c. food intake; d. white adipose tissue weight; e. white adipose tissue HE staining and cell size statistics; f. white adipose tissue fat production Related gene changes; g. mouse oxygen consumption; h. mouse respiratory entropy; i. blood insulin concentration; j. blood glucose concentration; k. glucose tolerance test; l. insulin resistance test; m. brown fat Tissue differentiation and changes in genes related to heat production.
  • FIG. 1 AAV-specific knockdown of liver Gpnmb expression improves diet-induced obesity
  • FIG. 4 Study of the correlation between the concentration of GPNMB in the blood and obesity and its associated metabolic diseases
  • Serum concentration of GPNMB in WT and diet-induced-obesity (DIO) mice after 4 weeks of a.chow diet or high-fat diet; b. serum GPNMB concentration of WT and OB mice at 8 weeks old; The relationship between serum GPNMB concentration in obese population (BMI> 28) and non-obese population (BMI ⁇ 28); d. Correlation between blood GPNMB and BMI in the population.
  • Example 1 GPNMB secreted by the liver can up-regulate the lipogenic ability of white adipose tissue
  • Quantitative-PCR was used to determine the effect of GPNMB secreted by liver in mice on the lipogenic ability of white adipose tissue. After the mice were subjected to experimental treatment, the mice were killed and the tissues were transferred to the tissue disruption tube, which was then used for mRNA extraction. Reverse transcription synthesis of cDNA and real-time quantitative PCR, the specific steps are as follows:
  • RNA Take 2 ⁇ l of RNA into 98 ⁇ l of water, mix and measure the absorbance at 260 nm with a spectrophotometer (Eppendorf), calculate the sample concentration, determine the ratio of absorbance at 260 nm to 280 nm, calculate the purity of the sample, and finally adjust the sample concentration to 1 ⁇ g. / ⁇ l.
  • a spectrophotometer Eppendorf
  • cDNA synthesis was performed using Promega's M-MLV reverse transcriptase kit. Each 50 ⁇ l system contained 4 ⁇ g of RNA, 1 ⁇ g of OligodT, and a final concentration of 0.4 mM dNTPs.
  • RNA For each 0.2 ml PCR tube, add 4 ⁇ l of RNA at a concentration of 1 ⁇ g/ ⁇ l, 1 ⁇ l of 1 ⁇ g/ ⁇ l of OligodT, 15 ⁇ l of DEPC water, and mix.
  • Realtime PCR uses Shengyuan Bio's Sharpvue 2x Universal qPCR Master Mix reagent.
  • Sharpvue 2x Mix 10 ⁇ l, a primer with a final concentration of 0.5 ⁇ M, 2 ⁇ l of template cDNA (diluted 4 times for reverse transcription), and supplemented with water to 20 ⁇ l. After the reaction system is configured, mix and mix, and each sample has 3 duplicate wells.
  • mice required fasting pretreatment before the experiment.
  • the Glucose tolerance test (GTT) was fasted overnight, and the Insulin tolerance test (ITT) was fasted for 4 hours.
  • GTT Glucose tolerance test
  • ITT Insulin tolerance test
  • 2 g/kg glucose was intraperitoneally injected; ITT was injected intraperitoneally with 0.75 U/kg insulin (Sigma).
  • Blood was collected at the tip of the nose at 15, 30, 60, and 120 minutes.
  • Onetouch Ultra blood glucose monitoring systerm-LifeScan was measured using the American Johnson & Johnson Blood Glucose Test Strip.
  • Control mice and experimental mice were transferred to a comprehensive experimental animal monitoring system (Columbus Instruments, Columbus, OH). Adapted in the metabolic cage and continuously monitored according to the instrument operating guidelines until the experimental data is stable. This process takes about 24 hours, which is called the animal adaptation period. The instrument then monitors changes in mouse O 2 depletion volume, CO 2 production volume, and exercise within 24–48 hours. Statistics and calculation of respiratory entropy (O 2 consumption volume: CO 2 generation volume).
  • Mouse GPNMB is a type I secreted protein, including 574 amino acid residues, of which 1-502 amino acids belong to the GPNMB ectodomain (ECD).
  • the adenoviral vector plasmid was constructed by ObioTechnology Co., Ltd. (Shanghai), and the AAV-CMV-Gpnmb-ECD-3 ⁇ Flag plasmid was constructed using pAOV-CMV-3 ⁇ Flag vector and packaged for expression mainly in the liver.
  • Type 8 adeno-associated virus AAV8-Gpnmb-ECD. Mice were injected with AAV8-Gpnmb-ECD and each mouse was injected at a dose of 5 x 10 11 pfu.
  • mice All of which were C57BL/6 in the mice without special instructions
  • the body weight and food intake of the mice were monitored at different time points during the feeding process. It was found that with the prolonged feeding time, the weight of mice overexpressing GPNMB-ECD increased faster than that of control mice (injected with AAV8-control virus), and there was a significant difference in the detection points after 4 weeks of injection, and between the two groups. There was no significant change in food intake (Fig. 1a, b). Decreased metabolic rate is an important indicator of obese mice, and then functional analysis of metabolic cages showed that the oxygen consumption (VO 2 ) of overexpressed GPNMB-ECD mice decreased, indicating that the metabolic rate of mice did decrease.
  • VO 2 oxygen consumption
  • Fig. 1c Analysis of the results of various metabolic indicators in mice showed (Fig. 1c) that there was no significant inflammation (alginate aminotransferase (ALT), aspartate aminotransferase (AST)) in both groups of mice.
  • the weight of the mice in the experimental group was significantly increased, and the weight of the liver was There was no change in liver specific gravity, the weight of adipose tissue, and the proportion of adipose tissue and body weight were significantly up-regulated and statistically different.
  • the levels of serum cholesterol and triglyceride did not change significantly, but blood sugar and insulin levels were significantly up-regulated. Elevated blood glucose and insulin levels suggest that insulin resistance may be present in overexpressing GPNMB-ECD mice (Fig. 1g, h).
  • the next GTT and ITT experiments further demonstrate our speculation (Fig. 1i, j).
  • Example 2 Antibody Neutralization Blood GPNMB is resistant to high fat diet induced obesity
  • the measurement method is the same as in Example 1.
  • mice white adipose tissue was removed, rinsed with cold PBS, and fixed in 4% PFA for 4 hours;
  • each rabbit can collect about 50-100 mL of plasma;
  • mice fed a high-fat diet for 4 weeks were intraperitoneally injected with GPNMB neutralizing antibodies.
  • the mice were injected every 3 days and the body weight and other physiological indicators of the mice were monitored at different time points (Fig. 2a).
  • the results showed that the mice in the GPNMB-negative mice had a slower weight gain and a difference on the 6th day after the injection, and the difference was significant with the injection time.
  • Increase Figure 2b, c
  • lipid synthesis related genes Srebp1c, Acs, Acc, Acl and Fasn
  • Fasn was down-regulated most
  • the weight of the adipose tissue was also significantly reduced (Fig. 2d), while the HE staining of the WAT paraffin sections showed that the fat cells were significantly smaller (Fig. 2e).
  • the brown adipose tissue significantly increased the thermogenic gene (Ucp-1) on the premise that the adipocyte differentiation efficiency did not change significantly, which may be the reason for the increase in metabolic rate in the metabolic cage experiment (Fig. 2m).
  • antibody neutralization of GPNMB can enhance the metabolic rate of diet-induced obese mice, increase the heat production of mice, reduce the rate of lipid synthesis in white adipose tissue, improve insulin resistance, and ultimately resist obesity induced by high-fat diet.
  • Example 3 AAV-specific knockdown of liver Gpnmb expression improves diet-induced obesity
  • the measurement method is the same as in the first embodiment.
  • Gpnmb-shRNA also written as AAV8-Gpnmb-shRNA, AAV8-Gpnmb-shRNA, AAV-shGpnmb.
  • mice injected with AAV-Gpnmb-shRNA mediated expression of Gpnmb-shRNA knocked down Gpnmb expression in the liver (Fig. 3a).
  • lipid synthesis-related genes (Srebp1c, Acs, Acc, Acl, and Fasn) were significantly decreased in white adipose tissue of mice after knockdown of Gpnmb expression in the liver (Fig. 3d).
  • the expression of the heat-related gene (Ucp-1) was significantly increased, which was consistent with the results of the previous metabolic cage experiment, indicating an increase in the energy consumption of the mouse (Fig. 3e).
  • liver Gpnmb expression also enhances the metabolic rate of diet-induced obese mice, increases mouse heat production, reduces white adipose tissue lipid synthesis rate, improves insulin resistance, and ultimately resists high-fat diet induction. obesity.
  • GPNMB is present in mice and humans, and the concentration of GPNMB in the blood is positively correlated with metabolic diseases such as obesity, diabetes, insulin resistance, and hyperlipidemia.
  • the data displayed is the median (range).
  • BMI body mass index WHR waist-to-hip ratio, WH waist-to-height ratio, SBP systolic blood pressure, DBP diastolic blood pressure, TG triglyceride, TC cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol Ogtt Oral glucose tolerance test, HbA1C hemoglobin a1c, HOMA-IR steady state model to evaluate insulin resistance.
  • the diagnosis of BMI>30 is obesity.
  • GPNMB glycoprotein nmb

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Abstract

本发明涉及人GPNMB蛋白或其拮抗剂或激动剂的用途，本发明发现GPNMB基因、RNA和蛋白可用作诊断肥胖、糖尿病、胰岛素抵抗和高脂血症等代谢性疾病的生物标志物，以及作为治疗上述疾病的药物靶标。

Description

人GPNMB蛋白或其拮抗剂或激动剂的用途 技术领域
本发明涉及生物医药技术领域，具体涉及蛋白Glycoprotein nonmetastaticmelanoma protein B(GPNMB)在代谢性疾病(如：肥胖、糖尿病、胰岛素抵抗、高脂血症等)的诊断与治疗中的应用。
背景技术
近年来，肥胖及相关疾病(包括：肥胖、糖尿病、胰岛素抵抗、高脂血症等)大量增加，并逐渐变成影响公众健康及其生活质量的主要问题。根据世界卫生组织统计，超过1亿的成年人体重超重(身体质量指数BMI>25)，其中，300多万成年人是严重肥胖(BMI>30)。在亚洲国家，2型糖尿病患者的数量急剧上升，而肥胖就是发展为2型糖尿病的主要危险因素。事实上，仅在过去的十年中，中国糖尿病患者的数量几乎增加了一倍，据统计有1.26亿患者。
因为肥胖往往是由于能量摄入超过能量消耗所导致的，所以目前治疗肥胖的方法主要为减少能量的摄入，如食欲抑制和节食，但是该方法的长期执行比较难，治疗效果差；药物治疗对胃肠、肾脏以及心脏具有较强的副作用。目前治疗肥胖的最有效的方法是手术，由于价格昂贵而未能得到广泛的应用。随着我国人们饮食结构变化及生活习惯的变化，肥胖及肥胖引起的各类代谢性疾病发生越来越多，迫切需要加强自主知识产权的新型药靶系统与创新药的研究开发。
GPNMB是一个I型跨膜蛋白，基因位于人体第七号染色体，由572个氨基酸构成。完整的跨膜蛋白可以被胞外蛋白酶如ADAM8和ADAM10剪切，释放出GPNMB的胞外区段进入血液发挥细胞因子的作用。GPNMB在正常骨、造血系统和皮肤等组织内都能被检测到，另外也能在乳腺癌，黑色素瘤，肝细胞癌等恶性增生组织高水平检测到。。
发明内容
发明人在进行脂肪代谢研究中发现，肝脏组织Scap或gp78缺陷型小鼠(L-Scap-/-,L-gp78-/-)的肝脂肪酸合成下降，但同时白色脂肪组织的脂肪酸合成上升。进行血清研究发现，确认小鼠肝脏分泌的物质能上调脂肪合成相关基因表达。随后，鉴定出了 缺陷小鼠肝脏中表达上调了的一共301个基因，再找出12个带有N端信号肽的蛋白编码基因，进一步定量PCR，确定了4个表达水平相对于野生型升高30倍以上的基因，即Gpnmb、Timp1、Ly6d、Lcn2，最后构建腺病毒表达载体验证，确认是基因Gpnmb表达上调引起了白色脂肪组织的脂肪酸合成上升。
本发明的一个目的是提供Gpnmb基因在诊断、预防和治疗肥胖及其相关代谢性疾病药物中的应用。
本发明的另一个目的是提供GPNMB蛋白在诊断、预防和治疗肥胖及其相关代谢性疾病药物中的应用。
本发明鉴定出，鼠肝脏分泌的GPNMB能显著的增强白色脂肪的脂肪合成能力，降低小鼠能量代谢，引起小鼠的肥胖、高脂血症和胰岛素抵抗。而在肥胖的小鼠中，血液中GPNMB的水平相对于正常小鼠都有明显升高。对人群分析表明，人血液中的GPNMB水平与体重、肥胖、血压、胰岛素抵抗、糖尿病和高血脂等指标正相关。
这表明GPNMB有可能是胰岛素抵抗、糖尿病和高血脂等的分子标记物，也可能作为治疗肥胖症等的药物靶点，既可以通过基因治疗降低Gpnmb表达，也可以通过采用拮抗剂等抑制GPNMB蛋白活性。
根据本发明的一个方面，提供人GPNMB蛋白或其拮抗剂或激动剂的用途，其中将所述GPNMB蛋白或其拮抗剂或激动剂用于诊断代谢性疾病，或将所述GPNMB蛋白或其拮抗剂或激动剂用于制备治疗和/或预防代谢性疾病的药物，所述GPNMB蛋白包括：
1)具有SEQ ID NO:1所示的氨基酸序列的蛋白；或
2)将SEQ ID NO:1所示氨基酸序列经过一个或数个氨基酸残基的取代、缺失或添加而形成的，且具有诊断、预防和治疗代谢性疾病功能的由1)衍生的蛋白。
用于本发明的GPNMB蛋白的拮抗剂或激动剂选自：
i)能与GPNMB蛋白结合的化合物或蛋白；
ii)能改变GPNMB蛋白降解的化合物或蛋白；
iii)能改变GPNMB蛋白合成的化合物或蛋白；
iv)能改变GPNMB蛋白活性的化合物或蛋白；和
v)能改变GPNMB的RNA的翻译或稳定性的化合物或蛋白；和
vi)能改变编码GPNMB蛋白的基因表达的化合物或蛋白。
根据本发明的一个实施方案，改变Gpnmb基因表达包括但不限于抑制GPNMB 蛋白的编码基因的表达，例如，可给予抑制Gpnmb基因表达或降低其表达水平的试剂，这些试剂包括：抑制Gpnmb基因转录活性的试剂，抑制Gpnmb mRNA的转录水平的试剂，促进Gpnmb mRNA降解的试剂，针对Gpnmb基因的siRNA，抑制Gpnmb mRNA的翻译的试剂，和特异性识别Gpnmb基因的导向核酸并进行剪切以降低其水平的试剂，还可以是引起Gpnmb基因序列突变的试剂，使得表达出来的GPNMB蛋白失活，如基因编辑载体、同源重组载体等。
根据本发明的另一个实施方案，提供降低GPNMB蛋白活性的拮抗剂，可以是例如特异性抗体或具有抑制活性的小分子。利用抗体中和GPNMB后发现，可预防和治疗肥胖、脂肪堆积减少、血脂下降、能量代谢增强、胰岛素敏感性增加、血液葡萄糖清除能力增强。用于治疗目的的优选的抗体可以是例如：US8039593B2、US20130156784A1、US8846873B2、WO2017046061A1、US20180064809A1、US20180208655A1中所描述的抗体，但不限于这些抗GPNMB抗体。
根据本发明的再一个方面，提供编码GPNMB蛋白的基因在诊断代谢性疾病中的用途，其中所述GPNMB蛋白的氨基酸序列如SEQ ID NO.1所示，所述代谢性疾病选自肥胖、糖尿病、胰岛素抵抗和高脂血症。
以Gpnmb基因/GPNMB蛋白作分子指标，在临床上用于诊断胰岛素抵抗、糖尿病、高血脂症、肥胖等代谢性疾病的病程发展。检测Gpnmb基因、RNA或蛋白的试剂包括但不限于用于检测Gpnmb基因的各种引物和探针，和/或用于检测GPNMB蛋白的特异性抗体等，这类试剂包括含Gpnmb基因、RNA或蛋白的样品以及实施检测过程中使用的其它试剂，如溶剂等，包括但不限于实施PCR等所需的各种试剂。这些试剂均为本领域技术人员熟知，可以根据相关的参考文献制备。
实验结果表明GPNMB基因、RNA和蛋白可以作为代谢性疾病诊断的指标与治疗的靶点。本发明首次证明GPNMB蛋白在肥胖及其相关代谢疾病中具有十分重要的病理功能，可将GPNMB基因、RNA和蛋白用作诊断肥胖、糖尿病、胰岛素抵抗和高脂血症等代谢性疾病的生物标志物，以及作为治疗上述疾病的药物靶标。
附图说明
图1：腺相关病毒(AAV)过表达系统对肝分泌GPNMB上调白色脂肪组织的产脂能力的功能研究；
a.体重检测；b.进食量；c.小鼠代谢参数；d.qPCR分析小鼠白色脂肪的脂 合成相关基因；e.小鼠氧气消耗量；f.棕色脂肪组织分化及产热相关基因水平变化；g.血糖水平；h.血胰岛素水平；i.葡萄糖耐受实验；j.胰岛素耐受实验。
图2：抗体中和血液GPNMB抵抗高脂饮食诱导肥胖的研究；
a.抗体治疗食物诱导肥胖的示意图；b.day 18的小鼠体重；c.进食量；d.白色脂肪组织重量；e.白色脂肪组织HE染色及细胞大小统计；f.白色脂肪组织产脂相关基因的变化；g.小鼠氧气消耗量；h.小鼠呼吸熵；i.血胰岛素浓度；j.血葡萄糖浓度；k.葡萄糖耐受实验；l.胰岛素耐受实验；m.棕色脂肪组织分化及产热相关基因水平变化。
图3：AAV特异敲低肝脏Gpnmb表达改善饮食诱导的肥胖的研究；
a.实验流程图；b.肝脏的Gpnmb表达水平；c.Western blotting检测血清中GPNMB的量；d.白色脂肪组织中脂肪合成相关基因的表达水平；e.白色脂肪组织与棕色脂肪组织的产热相关基因的表达水平；f.代谢笼的小鼠氧气消耗量；g.葡萄糖耐受实验。
图4：血液中GPNMB的浓度与肥胖及其相关代谢性疾病相关性的研究；
a.chow diet或high-fat diet饲养小鼠4周后，WT和diet-induced-obesity(DIO)小鼠的血清GPNMB浓度；b.8周大的WT和OB小鼠的血清GPNMB浓度；c.肥胖人群(BMI>＝28)与非肥胖人群(BMI<28)中血清GPNMB浓度；d.人群中血液GPNMB与BMI的相关性。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。
实施例1肝分泌的GPNMB能上调白色脂肪组织的产脂能力
1.1定量PCR测定方法：
Quantitative-PCR测定小鼠肝分泌的GPNMB对白色脂肪组织的产脂能力的影响，取小鼠进行实验处理后，杀小鼠，将组织转移到组织破碎管管中，随后用于mRNA的提取、逆转录合成cDNA以及荧光实时定量PCR，具体步骤如下：
1.1.1、mRNA的提取和定量
1)每1ml TRIzol中加入200μl的氯仿，涡旋仪上剧烈振荡混匀，室温静置15分 钟。
2)4℃，13200rpm离心10分钟，转移500μl的上层水相至新的1.5ml Eppendorf管中。
3)每份样品中加入600μl异戊醇，来回颠倒混匀。
4)4℃，13200rpm离心10分钟，弃去上清，管底可见乳白色RNA沉淀。每份样品中加入1ml的70％乙醇(用DEPC处理过的去离子水稀释)，颠倒混匀。
5)4℃，13200rpm离心10分钟，弃去上清，沉淀室温晾干，加入30μl DEPC处理过的去离子水，充分溶解。
6)取2μl RNA到98μl水中，混匀后用分光光度计(Eppendorf)测定260nm的光吸收值，计算样品浓度，测定260nm与280nm光吸收值的比值，计算样品纯度，最终调整样品浓度为1μg/μl。
1.1.2、逆转录合成cDNA
cDNA合成采用Promega公司的M-MLV逆转录酶试剂盒。每50μl体系含有4μg的RNA，1μg的OligodT，终浓度各为0.4mM的dNTP。
1)每个0.2ml PCR管中，加入4μl浓度为1μg/μl的RNA，1μl 1μg/μl的OligodT，15μl DEPC水，混匀。
2)70℃变性5分钟，冰上骤冷。
3)每个反应体系中加入17μl DEPC水，10μl 5×MLV缓冲液，1μl dNTP(各10mM)，2μl M-MLV逆转录酶，混匀。
4)37℃反应1小时。
5)70℃变性10分钟。
6)10℃降温5分钟，加入200μl水稀释cDNA。
1.1.3、荧光实时定量PCR
Realtime PCR采用盛元生物公司的Sharpvue 2x Universal qPCR Master Mix试剂。每20μl体系中，Sharpvue 2x Mix:10μl，终浓度为0.5μM的引物，2μl模板cDNA(逆转录产物稀释4倍用)，用水补至20μl。反应体系配置好后，混匀，每个样品3个复孔。在Stratagene Mx30005P实时荧光定量PCR仪上按照以下程序运行：a.95℃热激活5分钟，b.95℃变性30秒，c.60℃退火30秒，d.72℃延伸30秒，延伸结束后采集荧光信号，e.b-d循环40次，f.溶解曲线分析：95℃15秒，60℃1分钟，以1％的速度上升到95℃，95℃持续15秒，60℃上升到95℃的过程中采集荧光信号。运用比较 Ct法计算mRNA的相对含量(相对于Cyclophilin)，并以WT小鼠组标准化为1。本实施例中荧光定量PCR采用的引物序列见表1。
表1本实施例中荧光定量PCR所用引物的信息
Figure PCTCN2018120616-appb-000001
1.2葡萄糖耐受和胰岛素耐受试验测定方法：
实验进行之前，小鼠需要禁食预处理。葡萄糖耐受试验(Glucose tolerance test,GTT)禁食过夜，胰岛素耐受试验(Insulin tolerance test，ITT)禁食4小时。在GTT实验中，腹腔注射2g/kg葡萄糖；ITT实验腹腔注射0.75U/kg胰岛素(Sigma)。分别在15、30、60及120分钟时间点尾尖采血。用美国强生稳豪血糖试纸测量血液葡萄糖浓度(Onetouch Ultra blood glucose monitoring systerm-LifeScan)。
1.3代谢笼试验测定方法
对照组小鼠和实验组小鼠转移至综合性实验动物监视系统(Columbus Instruments,Columbus,OH)。在代谢笼内适应并按照仪器操作指南进行连续监视，一直到实验数据呈现稳定状态。此过程大约耗时约24小时，这段时间被称为动物适应期。此后仪器监测24–48小时内小鼠O 2消耗体积、CO 2生成体积的变化以及运动情况。统计数据 并计算呼吸熵(O 2消耗体积：CO 2生成体积)。
2.通过腺病毒载体在肝脏表达GPNMB胞外分泌段对小鼠的影响研究
选用腺相关病毒过表达系统。AAV具有感染效率高、表达时效性长(最长达6个月)以及免疫反应小等优点。小鼠GPNMB属于Ⅰ型分泌蛋白，包括574个氨基酸残基，其中1-502氨基酸属于GPNMB胞外分泌段(ectodomain，ECD)。委托ObioTechnology Co.,Ltd.(上海)构建腺病毒载体质粒，采用pAOV-CMV-3×Flag载体，构建了AAV-CMV-Gpnmb-ECD-3×Flag质粒，并选择包装成主要在肝脏表达的8型腺相关性病毒(AAV8-Gpnmb-ECD)。对小鼠进行AAV8-Gpnmb-ECD注射，每只小鼠注射5×10 11pfu的剂量。
在注射1周后，喂食小鼠(本文中小鼠无特殊说明，则全为C57BL/6)60％的高脂饲料，在喂食过程中的不同时间点对小鼠的体重和进食量进行监测，发现随着喂食时间延长，过表达GPNMB-ECD的小鼠体重同比增长比对照小鼠(注射AAV8-对照病毒)快，并在注射4周后的检测点中出现明显差异，同时两组之间的进食量没有显著变化(图1a,b)。代谢速率下降是肥胖小鼠的一个重要指标，进而进行代谢笼功能性分析，结果显示过表达GPNMB-ECD小鼠的氧耗量(VO 2)下降，这表明小鼠的代谢率确实有所下降(图1e)。接下来，Quantitative-PCR分析了棕色脂肪组织的成脂分化基因(422/ap2)及促进能耗产热的基因(Ucp-1)的表达，发现在脂肪细胞分化效率没有明显变化的前提下产热基因明显下调(图1f)，这可能是代谢率下降的原因。同样，对小鼠白色脂肪组织中的脂质合成基因(Srebp1c、Acs、Acc、Acl以及Fasn)进行了分析，分析结果显示GPNMB-ECD促进了WAT中脂质合成相关基因显著上调(图1d)。
分析小鼠的各项代谢指标结果显示(图1c)，两组小鼠均无明显炎症(谷丙转氨酶(ALT)，谷草转氨酶(AST))，实验组小鼠的体重明显增加，肝脏的重量以及肝比重没有变化，脂肪组织的重量以及脂肪组织与体重的比重明显上调且有统计差异，血清胆固醇以及甘油三酯的含量没有明显改变，然而血糖以及胰岛素的含量显著上调。血糖和胰岛素水平的升高提示过表达GPNMB-ECD小鼠可能存在胰岛素抵抗(图1g,h)。接下来的GTT和ITT实验也进一步证明了我们的推测(图1i,j)。
因此，在高脂诱导肥胖的过程中，过表达GPNMB-ECD小鼠的体重增加，代谢率降低，产热减少，胰岛素抵抗加重。
实施例2抗体中和血液GPNMB能抵抗高脂饮食诱导肥胖
测定方法与实施例1中一致
2.1小鼠白色脂肪组织的HE染色
(1)小鼠白色脂肪组织取出后经过冷的PBS漂洗后置于4％PFA中固定4小时；
(2)PBS洗3次；
(3)使用梯度乙醇脱水，二甲苯透明后包埋；
(4)使用莱卡石蜡切片机手动切片，切片厚度4-10m；
(5)贴片于多聚赖氨酸处理过的载玻片上，37℃烤片过夜；
(6)进行HE染色。
2.2GPNMB中和抗体制备
1)真核表达纯化Gb1蛋白，0.22μm滤膜过滤，得到不少于50mg真核蛋白。
2)第一天，第一次免疫，取1mg蛋白(2mg/mL)每只兔子与0.5mL完全弗氏佐剂(Freundadjuvant)充分混合，乳化，在兔子背部皮下注射；
3)第三天，第二次免疫。取1mg蛋白(2mg/mL)每只兔子与0.5mL完全弗氏佐剂充分混合，乳化，在兔子背部皮下注射；
4)第28天，第三次免疫。取1mg蛋白(2mg/mL)每只兔子与0.5mL不完全弗氏佐剂充分混合，乳化，在兔子背部皮下注射；
5)7天后，耳缘静脉采血，western blot检测抗体特异性；
6)7天后，第四次免疫。取1mg蛋白(2mg/mL)每只兔子与0.5mL不完全弗氏佐剂充分混合，乳化，在兔子背部皮下注射；
7)以后每14天免疫一次，根据需要决定处死兔子采血时间，通常为5–7次免疫后；
8)当最后一次免疫后7–10天，颈动脉采血，每只兔子大约能够采集50-100mL血浆左右；
9)全血4℃放置过夜，4℃，1500g离心15分钟，分离血清；
10)Western Blot检测，选择较好的抗血清备用。
2.3给予GPNMB中和抗体对高脂饮食诱导肥胖的抵抗作用
对高脂饮食喂食4周的野生型小鼠腹腔注射GPNMB中和抗体。每3天注射一次， 在不同的时间点监测小鼠的体重及其它生理指标(图2a)。结果显示，在进食量没有显著变化的前提下，中和GPNMB的小鼠同比对照组小鼠体重增长减慢，并在注射后第6天呈现出差异，随着注射时间的延长，差异显著性增加(图2b,c)。在从开始注射中和抗体后的第15天，对小鼠进行了代谢笼检测，结果显示治疗组小鼠的代谢率(氧气消耗量VO2)显著上升(图2g,h)。分析血糖水平和胰岛素的浓度，两者均有下调，这提示胰岛素抵抗可能得到了改善(图2i,j)。一致的是，GTT和ITT检测结果显示中和GPNMB后的小鼠胰岛素抵抗症状明显改善(图2k,l)。
对白色脂肪组织的脂质合成基因表达进行了检测，结果显示脂质合成相关基因(Srebp1c、Acs、Acc、Acl以及Fasn)均有不同程度的下调，其中Fasn下调最明显(图2f)，相应的脂肪组织的重量也明显减轻(图2d)，同时WAT石蜡切片的HE染色结果显示脂肪细胞显著变小(图2e)。而棕色脂肪组织在脂肪细胞分化效率没有明显变化的前提下产热基因(Ucp-1)明显上升，这可能是代谢笼实验中代谢率上升的原因(图2m)。
综上所述，抗体中和GPNMB能够增强饮食诱导肥胖小鼠的代谢率，增加小鼠产热，降低白色脂肪组织脂质合成速率，改善胰岛素抵抗，最终抵抗高脂饮食诱导的肥胖。
实施例3AAV特异敲低肝脏Gpnmb表达能改善饮食诱导的肥胖
测定方法同实施例1
委托Obio Technology Co.,Ltd.(上海)构建腺病毒干扰载体，使用pAKD-CMV-bGlobin-EGFP-H1-shRNA，构建了Gpnmb-shRNA(也写作AAV8-Gpnmb-shRNA、AAV8-Gpnmb-shRNA、AAV-shGpnmb)。
如图3a所示，使用AAV-Gpnmb-shRNA注射小鼠，介导表达的Gpnmb-shRNA敲低了肝内的Gpnmb表达(图3a)。研究表明，AAV8-Gpnmb-shRNA能显著敲低肝内Gpnmb的表达(图3b)，进一步降低血液中GPNMB的浓度(图3c)。
在从开始注射AAV的第二周后，对小鼠进行了代谢笼检测，结果显示治疗组小鼠的代谢率显著上升(图3f)。GTT检测结果显示敲低肝内Gpnmb表达后的小鼠胰岛素抵抗症状明显得到了改善(图3g)。
在敲低肝内Gpnmb表达后的小鼠的白色脂肪组织中，脂质合成相关基因(Srebp1c、Acs、Acc、Acl以及Fasn)表达水平都有显著下降(图3d)。而在棕色脂肪组织和腹股 沟间脂肪中，产热相关的基因(Ucp-1)表达显著上升，这与前面的代谢笼实验结果相符，表明小鼠的能量消耗增加(图3e)。
此实施例证明，AAV8特异敲低肝脏Gpnmb表达也能增强饮食诱导肥胖小鼠的代谢率，增加小鼠产热，降低白色脂肪组织脂质合成速率，改善胰岛素抵抗，最终抵抗高脂饮食诱导的肥胖。
实施例4人群血液GPNMB浓度与肥胖及其相关代谢性疾病相关性分析
为研究GPNMB与肥胖的关系,我们分析了DIO(diet-induced obesity)和OB(obesity)小鼠血清中的GPNMB的浓度(图4a，b)，结果显示饮食诱导肥胖小鼠和OB小鼠血清中GPNMB浓度显著高于各自的对照WT小鼠浓度。
同样，我们收集了318例人血清样本，按照BMI值将人群分为肥胖组和非肥胖组，通过ELISA分析发现肥胖患者体内的GPNMB含量显著高于非肥胖组(图4c，d)。我们全面分析了血清中GPNMB的浓度与人类肥胖相关指标的关系。在收集的318血清样本中(收集的不同BMI的人血清样本，均在收集血清前未进行任何药物及手术治疗)，按照GPNMB的浓度分成三等分位，我们分析了各个代谢指标随GPNMB浓度变化的关系，并做了统计分析(表2)。通过分析，我们发现许多肥胖相关的指标(尤其是BMI和HOMA-IR)与GPNMB的浓度呈正相关，并且关联紧密。
因此，在正常生理条件下GPNMB存在于小鼠和人体中，并且血液中GPNMB的浓度与肥胖、糖尿病、胰岛素抵抗和高脂血症等代谢性疾病具有正相关性。
表2.不同BMI指数对象的Gpnmb(ng/ml)三分组的代谢物成分比较
Figure PCTCN2018120616-appb-000002
Figure PCTCN2018120616-appb-000003
显示的数据是中位数(范围).
*p<0.05vs.Low group(Gpnmb<10.16ng/mL)
BMI身体质量指数,WHR腰臀围比,WH腰围身高比,SBP收缩压,DBP舒张压,TG甘油三酯,TC胆固醇,HDL-C高密度脂蛋白胆固醇,LDL-C低密度脂蛋白胆固醇Ogtt口服葡萄糖耐受实验,HbA1C血红蛋白a1c,HOMA-IR稳态模型评价胰岛素抵抗.BMI>30的诊断为肥胖。
附注：人GPNMB氨基酸序列如SEQ ID NO:1所示，即如下：
Homo sapiens glycoprotein nmb(GPNMB)
NM_001005340.1
Figure PCTCN2018120616-appb-000004
Figure PCTCN2018120616-appb-000005

Claims (7)

  1. 人GPNMB蛋白或其拮抗剂或激动剂的用途，其中将所述GPNMB蛋白或其拮抗剂或激动剂用于诊断代谢性疾病，或将所述GPNMB蛋白或其拮抗剂或激动剂用于制备治疗和/或预防代谢性疾病的药物，所述GPNMB蛋白包括：
    1)具有SEQ ID NO:1所示的氨基酸序列的蛋白；或
    2)将SEQ ID NO:1所示氨基酸序列经过一个或数个氨基酸残基的取代、缺失或添加而形成的，且具有诊断、预防和治疗代谢性疾病功能的由1)衍生的蛋白。
  2. 根据权利要求1所述的应用，其中所述代谢性疾病选自肥胖、糖尿病、胰岛素抵抗和高脂血症。
  3. 根据权利要求1的应用，其中所述GPNMB蛋白的拮抗剂或激动剂选自：
    i)能与GPNMB蛋白结合的化合物或蛋白；
    ii)能改变GPNMB蛋白降解的化合物或蛋白；
    iii)能改变GPNMB蛋白合成的化合物或蛋白；
    iv)能改变GPNMB蛋白活性的化合物或蛋白；
    v)能改变GPNMB的RNA的翻译或稳定性的化合物或蛋白；和
    vi)能改变编码GPNMB蛋白的基因表达的化合物或蛋白。
  4. 根据权利要求3所述的应用，其中所述拮抗剂为中和GPNMB蛋白的抗体。
  5. 根据权利要求3所述的应用，其中所述GPNMB蛋白的拮抗剂或激动剂为可敲低Gpnmb基因表达的腺病毒相关病毒表达载体。
  6. 根据权利要求3所述的应用，其中所述GPNMB蛋白的拮抗剂或激动剂为可敲低Gpnmb基因表达的RNA干扰剂或反义核苷酸。
  7. 编码GPNMB蛋白的基因在诊断代谢性疾病中的用途，其中所述GPNMB蛋白的氨基酸序列如SEQ ID NO.1所示，所述代谢性疾病选自肥胖、糖尿病、胰岛素抵抗和高脂血症。
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Publication number Priority date Publication date Assignee Title
WO2024026447A1 (en) 2022-07-29 2024-02-01 Alector Llc Anti-gpnmb antibodies and methods of use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017046061A1 (en) * 2015-09-14 2017-03-23 F. Hoffmann-La Roche Ag Anti-gpnmb antibodies and diagnostic uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
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CN101780268B (zh) * 2008-12-03 2013-08-21 中国科学院上海生命科学研究院 用于诊断,预防和治疗胰岛素抵抗的方法和试剂

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017046061A1 (en) * 2015-09-14 2017-03-23 F. Hoffmann-La Roche Ag Anti-gpnmb antibodies and diagnostic uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHOI, M.S. ET AL.: "High-fat Diet Decreases Energy Expenditure and Expression of Genes Controlling Lipid Metabolism, Mitochondrial Function and Skeletal System Development in the Adipose Tissue, along with Increased Expression of Extracellular Matrix Remodelling-and Inflammation-related Genes", BRITISH JOURNAL OF NUTRITION, vol. 113, no. 6, 6 March 2015 (2015-03-06), pages 867 - 877, XP055626146 *
KATAYAMA, A. ET AL.: "Benefcial Impact of Gpnmb and Its Signifcance as A Biomarker in Nonalcoholic Steatohepatitis", SCIENTIFIC REPORTS, vol. 19, no. 5, 19 November 2015 (2015-11-19), pages 1 - 15, XP055626140 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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