CN103656643A

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Info

Publication number: CN103656643A
Application number: CN201210359360.1A
Authority: CN
Inventors: 赵勇; 侯玉柱
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2012-09-24
Filing date: 2012-09-24
Publication date: 2014-03-26

Abstract

本发明提供一种p38-STAT1信号通路调节剂剂在制备调控HTRA1表达的产品中的应用，所述p38-STAT1信号通路调节剂包括激动剂和抑制剂，所述激动剂为干扰素γ和/或茴香霉素及其类似物，所述抑制剂磷酸激酶p38抑制剂或STAT1抑制剂；所述磷酸激酶p38抑制剂为SB203580及其类似物，或所述STAT1抑制剂为MTA及其类似物；所述丝氨酸蛋白酶HTRA1与炎症性疾病及其相关疾病或与肿瘤形成或迁移有关的疾病有关。因此，本发明开发了新的针对调控HTRA1基因表达的方法来进行HTRA1相关疾病的治疗，提供p38-STAT1信号通路调节剂的新用途，其p38-STAT1信号通路激动剂能够抑制丝氨酸蛋白酶HTRA1表达，从而使HTRA1相关疾病得以治疗。

The present invention provides an application of a p38-STAT1 signaling pathway regulator in the preparation of products regulating HTRA1 expression, the p38-STAT1 signaling pathway regulator includes agonists and inhibitors, and the agonists are interferon gamma and/or or anisomycin and its analogs, said inhibitor of phosphokinase p38 or STAT1 inhibitor; said phosphokinase p38 inhibitor is SB203580 and its analogs, or said STAT1 inhibitor is MTA and its analogs; The serine protease HTRA1 is related to inflammatory diseases and related diseases or diseases related to tumor formation or migration. Therefore, the present invention develops a new method for regulating HTRA1 gene expression to treat HTRA1-related diseases, and provides a new application of p38-STAT1 signaling pathway regulator, and its p38-STAT1 signaling pathway agonist can inhibit the expression of serine protease HTRA1, Thus, HTRA1-related diseases can be treated.

Description

Translated fromChinese

p38-STAT1信号通路调节剂在制备调控HTRA1表达的产品中的应用Application of p38-STAT1 signaling pathway regulator in the preparation of products regulating HTRA1 expression

技术领域technical field

本发明属于生物医药领域。本发明具体涉及一种信号通路调节剂的应用，尤其涉及一种p38-STAT1信号通路调节剂在制备调控HTRA1表达的产品中的应用，所述产品为药物或试剂盒。 The invention belongs to the field of biomedicine. The present invention specifically relates to the application of a signaling pathway regulator, in particular to the application of a p38-STAT1 signaling pathway regulator in the preparation of products for regulating HTRA1 expression, and the products are medicines or kits. the

背景技术Background technique

丝氨酸蛋白酶HTRA1（High temperature requirement A1）是一种分泌型蛋白酶。丝氨酸蛋白酶HTRA1能够降解纤连蛋白、聚集蛋白聚糖、核心蛋白聚糖、纤维调节素等多种胞外基质蛋白，并且参与调节TGF-β信号通路，因此其对于细胞粘附、运动以及胞外基质的稳态和代谢具有重要的调节作用。此外，有大量研究证实丝氨酸蛋白酶HTRA1与骨关节炎（OA）、风湿性关节炎（RA）、老年性黄斑变性（AMD）以及肿瘤形成与迁移等许多人类疾病密切相关。 Serine protease HTRA1 (High temperature requirement A1) is a secreted protease. The serine protease HTRA1 can degrade various extracellular matrix proteins such as fibronectin, aggrecan, decorin and fibromodulin, and participate in the regulation of TGF-β signaling pathway, so it plays an important role in cell adhesion, movement and extracellular Substrate homeostasis and metabolism have important regulatory roles. In addition, a large number of studies have confirmed that the serine protease HTRA1 is closely related to many human diseases such as osteoarthritis (OA), rheumatoid arthritis (RA), age-related macular degeneration (AMD), and tumor formation and migration. the

注射用重组人干扰素γ是一种临床用药，主要用于风湿性关节炎（RA）、骨髓增生异常综合症以及异位性皮炎和尖锐湿疣等疾病。 Recombinant human interferon gamma for injection is a clinical drug, mainly used for diseases such as rheumatoid arthritis (RA), myelodysplastic syndrome, atopic dermatitis and condyloma acuminatum. the

到目前为止，未有任何关于如何调节丝氨酸蛋白酶HTRA1基因表达的报道，也未有任何以HTRA1为靶点的临床治疗方案，更没有通过干扰素γ抑制丝氨酸蛋白酶HTRA1表达的报道。 So far, there is no report on how to regulate the expression of the serine protease HTRA1 gene, nor any clinical treatment plan targeting HTRA1, and there is no report on the inhibition of the expression of the serine protease HTRA1 by interferon gamma. the

发明内容Contents of the invention

因此，本发明的目的是针对目前未能从丝氨酸蛋白酶HTRA1基因表达调控来进行丝氨酸蛋白酶HTRA1相关疾病的治疗的不足，提供一种p38-STAT1信号通路调节剂在制备调节丝氨酸蛋白酶HTRA1表达的产品中的应用，所述产品为药物或试剂盒，为丝氨酸蛋白酶HTRA1相关疾病治疗药物提供重要分子靶点。 Therefore, the object of the present invention is to provide a p38-STAT1 signaling pathway regulator in the preparation of products that regulate the expression of serine protease HTRA1 in order to address the deficiency in the treatment of serine protease HTRA1-related diseases from the regulation of serine protease HTRA1 gene expression. The product is a drug or a kit, which provides an important molecular target for drugs for the treatment of diseases related to serine protease HTRA1. the

针对上述目的，本发明提供的技术方案如下： For above-mentioned purpose, the technical scheme that the present invention provides is as follows:

除非特别指明，本文中的“IFN-γ”均指“干扰素γ”。 Unless otherwise specified, "IFN-γ" herein refers to "interferon-γ". the

除非特别指明，本文中的“HTRA1”均指“丝氨酸蛋白酶HTRA1”。 Unless otherwise specified, "HTRA1" herein refers to "serine protease HTRA1". the

一方面，本发明提供一种p38-STAT1信号通路调节剂在制备调控丝氨酸蛋白酶HTRA1表达的药物或试剂盒中的应用。 In one aspect, the present invention provides an application of a p38-STAT1 signaling pathway regulator in the preparation of a drug or a kit for regulating the expression of serine protease HTRA1. the

优选地，所述p38-STAT1信号通路调节剂包括激动剂和抑制剂。 Preferably, the p38-STAT1 signaling pathway modulator includes agonists and inhibitors. the

优选地，所述激动剂为干扰素γ和/或茴香霉素及其类似物，用于抑制丝氨酸蛋白酶HTRA1表达。 Preferably, the agonist is interferon γ and/or anisomycin and its analogs, which are used to inhibit the expression of serine protease HTRA1. the

优选地，所述抑制剂为SB203580和/或MTA及其类似物，用于上调丝氨酸蛋白酶HTRA1表达。 Preferably, the inhibitor is SB203580 and/or MTA and its analogues, which are used to up-regulate the expression of serine protease HTRA1. the

优选地，所述丝氨酸蛋白酶HTRA1与炎症性疾病及其相关疾病或与肿瘤形成或迁移有关的疾病有关。 Preferably, said serine protease HTRA1 is related to inflammatory diseases and related diseases or diseases related to tumor formation or migration. the

优选地，所述炎症性疾病及其相关疾病选自骨关节炎、风湿性关节炎和老年性黄斑变性中的一种或多种。 Preferably, the inflammatory disease and related diseases are selected from one or more of osteoarthritis, rheumatoid arthritis and age-related macular degeneration. the

优选地，所述炎症性疾病为风湿性关节炎。 Preferably, the inflammatory disease is rheumatoid arthritis. the

优选地，所述与肿瘤形成或迁移有关的疾病为黑色素瘤。 Preferably, the disease associated with tumor formation or migration is melanoma. the

再一方面，本发明提供一种p38-STAT1信号通路调节剂在制备用于治疗丝氨酸蛋白酶HTRA1及其上游通路活化导致的HTRA1相关疾病的药物或试剂盒中的应用。 In yet another aspect, the present invention provides a p38-STAT1 signaling pathway regulator in the preparation of a drug or a kit for treating HTRA1-related diseases caused by the activation of the serine protease HTRA1 and its upstream pathway. the

优选地，所述激动剂选自干扰素γ和/或茴香霉素及其衍生物。 Preferably, the agonist is selected from interferon gamma and/or anisomycin and derivatives thereof. the

优选地，所述抑制剂为磷酸激酶p38抑制剂或STAT1抑制剂。 Preferably, the inhibitor is a phosphokinase p38 inhibitor or a STAT1 inhibitor. the

优选地，所述磷酸激酶p38抑制剂为SB203580及其类似物，或所述STAT1抑制剂为MTA及其类似物。 Preferably, the phosphokinase p38 inhibitor is SB203580 and its analogs, or the STAT1 inhibitor is MTA and its analogs. the

优选地，所述丝氨酸蛋白酶HTRA1相关疾病包括炎症性疾病及其相关疾病或与肿瘤形成或迁移有关的疾病。 Preferably, the serine protease HTRA1-related diseases include inflammatory diseases and related diseases or diseases related to tumor formation or migration. the

优选地，所述肿瘤形成或迁移有关的疾病为黑色素瘤。 Preferably, the disease related to tumor formation or migration is melanoma. the

还一方面，本发明提供一种用于调控丝氨酸蛋白酶HTRA1表达的药物组合物，所述药物组合物含有有效量的p38-STAT1信号通路调节剂和药学上可接受的载体。 In another aspect, the present invention provides a pharmaceutical composition for regulating the expression of serine protease HTRA1, said pharmaceutical composition comprising an effective amount of p38-STAT1 signaling pathway modulator and a pharmaceutically acceptable carrier. the

更优选地，所述激动剂为干扰素γ和/或茴香霉素及其类似物。 More preferably, the agonist is interferon gamma and/or anisomycin and its analogs. the

优选地，所述磷酸激酶p38抑制剂为SB203580及其类似物。 Preferably, the phosphokinase p38 inhibitor is SB203580 and its analogs. the

优选地，所述STAT1抑制剂为MTA及其类似物。 Preferably, the STAT1 inhibitor is MTA and its analogs. the

优选地，，所述有效量的干扰素γ及其类似物的浓度为0-100ng/ml。 Preferably, the effective amount of interferon gamma and its analogues has a concentration of 0-100 ng/ml. the

优选地，干扰素γ及其类似物的浓度为100ng/ml； Preferably, the concentration of interferon gamma and its analogs is 100ng/ml;

优选地，所述有效量的茴香霉素的浓度为1-10μM。 Preferably, the effective amount of anisomycin has a concentration of 1-10 μM. the

优选地，所述SB203580的有效浓度为10μM；或MTA的浓度为10μM。 Preferably, the effective concentration of SB203580 is 10 μM; or the concentration of MTA is 10 μM. the

本发明通过试验证实了p38-STAT1信号通路调节剂能够调控HTRA1的基因表达，其中激动剂能够通过抑制HTRA1的基因表达，从而有效治疗RA，此外还揭示p38-STAT1信号通路激动剂能够用于治疗骨关节炎（OA）、老年性黄斑变性（AMD）和黑色素瘤等HTRA1相关疾病，拓宽了重组干扰素γ的应用范围，而p38-STAT1信号通路抑制剂能够上调HTRA1的基因表达，从而为HTRA1相关疾病治疗药物提供了新药物靶分子。 The present invention confirms that the p38-STAT1 signaling pathway regulator can regulate the gene expression of HTRA1 through experiments, wherein the agonist can effectively treat RA by inhibiting the gene expression of HTRA1, and also reveals that the p38-STAT1 signaling pathway agonist can be used for the treatment of Osteoarthritis (OA), age-related macular degeneration (AMD) and melanoma and other HTRA1-related diseases have broadened the scope of application of recombinant interferon-γ, and p38-STAT1 signaling pathway inhibitors can up-regulate the gene expression of HTRA1, thereby providing HTRA1 Drugs for the treatment of related diseases provide new drug target molecules. the

附图说明Description of drawings

以下，结合附图来详细说明本发明的实施方案，其中： Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

图1表明干扰素γ能够显著降低小鼠成纤维细胞和巨噬细胞中HTRA1基因表达，其中，图1a为不同浓度分别为0、1、10、50、100ng/ml的干扰素γ处理后的成纤维细胞中HTRA1基因表达的试验结果，图1b为以浓度为100ng/ml的干扰素γ处理不同时间后的成纤维细胞中HTRA1基因表达的试验结果，图1c为不同浓度分别为0、1、10、50、100ng/ml的干扰素γ处理后的巨噬细胞中HTRA1基因表达的试验结果，图1d为以浓度为100ng/ml的干扰素γ处理不同时间后的巨噬细胞中HTRA1基因表达的试验结果，纵坐标表示以未用干扰素γ处理的细胞中HTRA1 mRNA表达水平为基准，在干扰素γ处理过程中细胞中HTRA1 mRNA表达水平； Figure 1 shows that interferon gamma can significantly reduce the expression of HTRA1 gene in mouse fibroblasts and macrophages, wherein, Figure 1a shows different concentrations of interferon gamma treated with 0, 1, 10, 50, 100ng/ml respectively The test results of HTRA1 gene expression in fibroblasts. Figure 1b shows the test results of HTRA1 gene expression in fibroblasts treated with interferon gamma at a concentration of 100ng/ml for different times. , 10, 50, 100ng/ml interferon gamma treatment results of HTRA1 gene expression in macrophages, Figure 1d is the HTRA1 gene in macrophages treated with 100ng/ml interferon gamma for different time The test results of expression, the ordinate represents the expression level of HTRA1 mRNA in cells during interferon gamma treatment based on the HTRA1 mRNA expression level in cells not treated with interferon gamma;

图2表明干扰素γ抑制小鼠关节和大肠部位HTRA1基因表达，纵坐标表示以未用任何物质处理的样品中HTRA1 mRNA表达水平为基准，在干扰素γ处理过程中样品中HTRA1 mRNA表达水平，图中，LPS表示脂多糖，其中，图2a显示干扰素γ抑制小鼠关节部位的HTRA1基因表达，1为加干扰素γ处理后小鼠关节部位的HTRA1 mRNA表达，2为加干扰素γ处理后小鼠关节部位的HTRA1蛋白表达，图中，LPS表示脂多糖，GAPDH表示磷酸甘油醛脱氢酶，作为内参； Figure 2 shows that interferon gamma inhibits mouse joint and large intestine site HTRA1 gene expression, and the ordinate represents the HTRA1 mRNA expression level in the sample that is not treated with any substance as a benchmark, and the HTRA1 mRNA expression level in the sample during interferon gamma treatment, In the figure, LPS represents lipopolysaccharide, wherein, Figure 2a shows that interferon gamma inhibits the expression of HTRA1 gene in the joints of mice, 1 is the expression of HTRA1 mRNA in the joints of mice after interferon gamma treatment, and 2 is the treatment with interferon gamma The expression of HTRA1 protein in the joints of the rear mice. In the figure, LPS represents lipopolysaccharide, and GAPDH represents glyceraldehyde phosphate dehydrogenase, which are used as internal reference;

图2b显示干扰素γ抑制小鼠大肠部位的HTRA1基因表达，1为加干扰素γ处理后小鼠大肠部位的HTRA1 mRNA表达，2为加干扰素γ处理后大肠部位的HTRA1蛋白表达，图中，LPS表示脂多糖，GAPDH表示磷酸甘油醛脱氢酶，作为内参； Figure 2b shows that interferon gamma inhibits the HTRA1 gene expression in the large intestine of mice, 1 is the HTRA1 mRNA expression in the large intestine of mice after interferon gamma treatment, and 2 is the HTRA1 protein expression in the large intestine after interferon gamma treatment, in the figure , LPS represents lipopolysaccharide, GAPDH represents glyceraldehyde phosphate dehydrogenase, as internal reference;

图2c显示干扰素γ敲除小鼠关节部位HTRA1基因表达升高，1为加干扰素γ处理后小鼠关节部位的HTRA1 mRNA表达，2为加干扰素γ处理后小鼠关节部位的HTRA1蛋白表达，图中LPS表示脂多糖，GAPDH表示磷酸甘油醛脱氢酶，IFN-γKO或KO表示IFN-γ基因敲除的小鼠，B6表示野生型B6小鼠，对照表示未注射LPS的小鼠（IFN-γKO和野生型B6小鼠）； Figure 2c shows that the expression of HTRA1 gene in the joints of interferon-γ knockout mice is increased, 1 is the HTRA1 mRNA expression in the joints of mice after interferon-γ treatment, and 2 is the HTRA1 protein in the joints of mice after interferon-γ treatment Expression, LPS in the figure means lipopolysaccharide, GAPDH means glyceraldehyde phosphate dehydrogenase, IFN-γKO or KO means IFN-γ knockout mice, B6 means wild-type B6 mice, control means mice without LPS injection (IFN-γKO and wild-type B6 mice);

图2d显示干扰素γ敲除小鼠大肠部位HTRA1基因表达升高，1为加干扰素γ处理后小鼠大肠部位的HTRA1 mRNA表达，2为加干扰素γ处理后大肠部位的HTRA1蛋白表达，GAPDH表示磷酸甘油醛脱氢酶，IFN-γKO表示IFN-γ基因敲除的小鼠，B6表示野生型B6小鼠，对照表示未注射LPS的小鼠（IFN-γKO和野生型B6小鼠）； Figure 2d shows that the expression of HTRA1 gene in the large intestine of interferon-γ-knockout mice is increased, 1 is the expression of HTRA1 mRNA in the large intestine of mice treated with interferon-γ, and 2 is the expression of HTRA1 protein in the large intestine of mice treated with interferon-γ, GAPDH indicates glyceraldehyde phosphate dehydrogenase, IFN-γKO indicates IFN-γ knockout mice, B6 indicates wild-type B6 mice, and control indicates mice not injected with LPS (IFN-γKO and wild-type B6 mice) ;

图3表明干扰素γ抑制小鼠关节HTRA1表达及关节炎病征，其中，图3a显示野生型和干扰素γ基因敲除小鼠诱导关节炎的患病率，图中，B6表示诱导关节炎的野生型B6小鼠，IFN-γKO表示诱导关节炎的IFN-γ基因敲除小鼠； Figure 3 shows that interferon gamma inhibits mouse joint HTRA1 expression and arthritis symptoms, wherein, Figure 3a shows the prevalence of arthritis induced by wild type and interferon gamma gene knockout mice, in the figure, B6 represents the induction of arthritis Wild-type B6 mice, IFN-γKO means IFN-γ knockout mice that induce arthritis;

图3b显示野生型和干扰素γ基因敲除小鼠诱导关节炎后小鼠后肢的表型，图中，WT是指野生型B6小鼠，WT CIA是指诱导关节炎的野生型B6小鼠，IFN-γKO表示IFN-γ基因敲除小鼠，IFN-γKO CIA是指诱导关节炎的IFN-γ基因敲除小鼠； Figure 3b shows the phenotype of the hindlimbs of wild-type and interferon-γ knockout mice after induction of arthritis. In the figure, WT refers to wild-type B6 mice, and WT CIA refers to wild-type B6 mice that induce arthritis , IFN-γKO means IFN-γ knockout mice, IFN-γKO CIA means IFN-γ knockout mice that induce arthritis;

图3c显示野生型和干扰素γ基因敲除小鼠诱导关节炎后小鼠前后后肢关节HE染色的照片，图中，WT是指野生型B6小鼠，WT CIA是指诱导关节炎的野生型B6小鼠，IFN-γKO表示IFN-γ基因敲除小鼠，IFN-γCIA是指诱导关节炎的IFN-γ基因敲除小鼠； Figure 3c shows the photos of HE staining of the front and rear limb joints of wild-type and interferon-γ knockout mice after arthritis was induced. In the figure, WT refers to wild-type B6 mice, and WT CIA refers to wild-type mice that induced arthritis B6 mice, IFN-γKO means IFN-γ knockout mice, IFN-γCIA means IFN-γ knockout mice that induce arthritis;

图3d显示野生型和干扰素γ基因敲除小鼠诱导关节炎的HTRA1表达的抑制，其中1为HTRA1表达的结果，图中，WT是指野生型B6小鼠，WT CIA是指诱导关节炎的野生型B6小鼠，IFN-γKO表示IFN-γ基因敲除小鼠，IFN-γKO CIA是指诱导关节炎的IFN-γ基因敲除小鼠；2为蛋白印迹结果，图中，对照为未诱导关节炎的小鼠，CIA表示诱导关节炎后的小鼠，其中，B6表示B6小鼠，KO表示IFN-γ基因敲除小鼠，纵坐标表示以野生型B6小鼠中HTRA1 mRNA表达水平为基准，在诱导关节炎的过程中小鼠中HTRA1 mRNA表达水平； Figure 3d shows the inhibition of HTRA1 expression induced by wild-type and interferon-γ gene knockout mice, where 1 is the result of HTRA1 expression, in the figure, WT refers to wild-type B6 mice, and WT CIA refers to the induction of arthritis wild-type B6 mice, IFN-γKO means IFN-γ gene knockout mice, IFN-γKO CIA means IFN-γ gene knockout mice that induce arthritis; 2 is the result of western blot, in the figure, the control is Mice without induced arthritis, CIA means mice after induction of arthritis, among them, B6 means B6 mice, KO means IFN-γ gene knockout mice, and the ordinate means HTRA1 mRNA expression in wild type B6 mice Level as a benchmark, HTRA1 mRNA expression level in mice during the induction of arthritis;

图3e显示对诱导关节炎的野生型小鼠注射干扰素γ后的患病率，图中，CIA对照是指诱导关节炎（CIA）的野生型B6小鼠；CIA+IFN-γ是指诱导关节炎（CIA），同时注射干扰素的野生型B6小鼠，CIA+LPS是指诱导关节炎（CIA），同时注射LPS的野生型B6小鼠，CIA+IFN-γ+LPS是指诱导关节炎（CIA），同时注射干扰素和LPS的野生型B6小鼠； Figure 3e shows the prevalence of interferon-γ injections to wild-type mice that induce arthritis. In the figure, CIA control refers to wild-type B6 mice that induce arthritis (CIA); CIA+IFN-γ refers to induced arthritis (CIA) wild-type B6 mice; Arthritis (CIA), wild-type B6 mice injected with interferon at the same time, CIA+LPS means induced arthritis (CIA), wild-type B6 mice injected with LPS at the same time, CIA+IFN-γ+LPS means induced joint Inflammation (CIA), wild-type B6 mice injected with interferon and LPS at the same time;

图3f显示对诱导关节炎的野生型小鼠注射干扰素γ后的小鼠后肢的表型，图中，Con是指诱导关节炎（CIA）的野生型B6小鼠，IFN-γ是指诱导关节炎（CIA），同时注射干扰素治疗的野生型B6小鼠，LPS是指诱导关节炎（CIA），同时注射LPS的野生型B6小鼠，IFN-γ+LPS是指诱导关节炎（CIA），同时注射干扰素和LPS的野生型B6小鼠； Figure 3f shows the phenotype of the hind limbs of mice injected with interferon-γ to wild-type mice that induced arthritis. In the figure, Con refers to wild-type B6 mice that induced arthritis (CIA), and IFN-γ refers to the Arthritis (CIA), wild-type B6 mice treated with interferon at the same time, LPS refers to induced arthritis (CIA), wild-type B6 mice injected with LPS at the same time, IFN-γ+LPS refers to induced arthritis (CIA ), wild-type B6 mice injected with interferon and LPS at the same time;

图3g显示对诱导关节炎的野生型小鼠注射干扰素γ后的小鼠前后后肢关节HE染色的照片，图中，Con是指诱导关节炎（CIA）的野生型B6小鼠，IFN-γ是指，同时注射干扰素的野生型B6小鼠，LPS是指诱导关节炎（CIA），同时注射LPS的野生型B6小鼠，IFN-γ+LPS是指诱导关节炎（CIA），同时注射干扰素和LPS的野生型B6小鼠； Figure 3g shows the photos of HE staining of the front and rear limb joints of mice after injecting interferon γ to wild-type mice that induce arthritis. In the figure, Con refers to wild-type B6 mice that induce arthritis (CIA), and IFN-γ Refers to wild-type B6 mice injected with interferon at the same time, LPS refers to induced arthritis (CIA), and wild-type B6 mice injected with LPS at the same time, IFN-γ+LPS refers to induced arthritis (CIA), injected at the same time Wild-type B6 mice with interferon and LPS;

图3h显示对诱导关节炎的野生型小鼠注射干扰素γ后小鼠中的HTRA1表达，其中1为HTRA1 mRNA表达的结果，2为蛋白印迹结果，图中，对照是指野生型B6小鼠（未诱导关节炎），CIA是指诱导关节炎（CIA）的野生型B6小鼠，CIA+IFN-γ+LPS是指诱导关节炎（CIA），同时注射干扰素和LPS的野生型B6小鼠，CIA+LPS是指诱导关节炎（CIA），同时注射LPS的野生型B6小鼠，CIA+IFN-γ+LPS是指诱导关节炎（CIA），同时注射干扰素和LPS的野生型B6小鼠纵坐标表示以野生型B6小鼠（未诱导关节炎）中的HTRA1 mRNA的表达为标准，在诱导关节炎和注射干扰素γ的过程中小鼠中的HTRA1 mRNA的表达，GAPDH 表示磷酸甘油醛脱氢酶，作为内参； Figure 3h shows the expression of HTRA1 in mice after injecting interferon gamma to wild-type mice that induce arthritis, wherein 1 is the result of HTRA1 mRNA expression, and 2 is the result of Western blot. In the figure, the control refers to wild-type B6 mice (no arthritis induced), CIA refers to the wild-type B6 mice with induced arthritis (CIA), CIA+IFN-γ+LPS refers to the induced arthritis (CIA), and the wild-type B6 mice injected with interferon and LPS at the same time Mice, CIA+LPS refers to the induction of arthritis (CIA), while injecting LPS wild-type B6 mice, CIA+IFN-γ+LPS refers to the induction of arthritis (CIA), while injecting interferon and LPS wild-type B6 mice The mouse ordinate indicates the expression of HTRA1 mRNA in wild-type B6 mice (without induction of arthritis) as a standard, the expression of HTRA1 mRNA in mice during the induction of arthritis and injection of interferon-γ, and GAPDH indicates phosphoglycerol Aldehyde dehydrogenase, as an internal reference;

图4表明干扰素γ通过活化蛋白激酶p38 STAT1通路抑制HTRA1表达，其中，图4a显示磷酸激酶p38抑制剂（SB203580）处理的巨噬细胞系RAW264.7中HTRA1 mRNA的表达，图中，DMSO表示体积比浓度为1‰的DMSO处理的巨噬细胞系RAW264.7，作为对照，SB203580表示SB203580处理的巨噬细胞系RAW264.7，IFN-γ表示IFN-γ处理的巨噬细胞系RAW264.7，IFN-γ+SB203580表示IFN-γ和SB203580处理的巨噬细胞系RAW264.7，纵坐标表示以DMSO处理的巨噬细胞系RAW264.7中的HTRA1 mRNA的表达为标准，在加干扰素γ或SB203580处理的过程中RAW264.7中的HTRA1 mRNA的表达； Figure 4 shows that interferon gamma inhibits the expression of HTRA1 by activating the protein kinase p38 STAT1 pathway, wherein, Figure 4a shows the expression of HTRA1 mRNA in the macrophage cell line RAW264.7 treated with the phosphokinase p38 inhibitor (SB203580), in the figure, DMSO represents The macrophage cell line RAW264.7 treated with DMSO at a volume ratio concentration of 1‰, as a control, SB203580 represents the macrophage cell line RAW264.7 treated with SB203580, and IFN-γ represents the macrophage cell line RAW264.7 treated with IFN-γ , IFN-γ+SB203580 represents the macrophage cell line RAW264.7 treated with IFN-γ and SB203580, and the ordinate represents the expression of HTRA1 mRNA in the macrophage cell line RAW264.7 treated with DMSO as a standard. or the expression of HTRA1 mRNA in RAW264.7 during SB203580 treatment;

图4b表示磷酸激酶p38抑制剂在干扰素γ抑制HTRA1基因表达中的作用，图中，对照为巨噬细胞系RAW264.7，p38 shRNA为p38基因RNA干扰的RAW264.7细胞，1为未加干扰素γ处理的巨噬细胞系RAW264.7，2为加干扰素γ处理的巨噬细胞系RAW264.7，3为未加干扰素γ处理的p38 shRNA为p38基因RNA干扰的RAW264.7细胞，4为加干扰素γ处理的p38 shRNA为p38基因RNA干扰的RAW264.7细胞，纵坐标表示以巨噬细胞系RAW264.7中的HTRA1 mRNA的表达为标准，在加干扰素γ处理的过程中RAW264.7或p38 shRNA中的HTRA1 mRNA的表达； Figure 4b shows the effect of phosphokinase p38 inhibitors on the inhibition of HTRA1 gene expression by interferon gamma. The macrophage cell line RAW264.7 treated with interferon-γ, 2 is the macrophage cell line RAW264.7 treated with interferon-γ, 3 is the p38 shRNA without interferon-γ-treated RAW264.7 cells with p38 gene RNA interference , 4 is the p38 shRNA treated with interferon gamma is the RAW264.7 cell of p38 gene RNA interference, and the ordinate indicates the expression of HTRA1 mRNA in the macrophage cell line RAW264. Expression of HTRA1 mRNA in RAW264.7 or p38 shRNA;

图4c表示茴香霉素处理的巨噬细胞系RAW264.7中HTRA1 mRNA的表达，图中，DMSO表示体积比浓度为1‰的DMSO处理的巨噬细胞系RAW264.7，作为对照，1μM表示1μM茴香霉素处理的巨噬细胞系RAW264.7，10μM表示10μM茴香霉素处理的巨噬细胞系RAW264.7，+SP600125是指在10μM茴香霉素+SP600125处理的巨噬细胞系RAW264.7，纵坐标表示以DMSO处理的巨噬细胞系RAW264.7中的HTRA1 mRNA的表达为标准，在加茴香霉素或SP600125处理的过程中RAW264.7中的HTRA1 mRNA的表达； Figure 4c shows the expression of HTRA1 mRNA in the macrophage cell line RAW264.7 treated with anisomycin. In the figure, DMSO represents the macrophage cell line RAW264.7 treated with DMSO at a volume ratio concentration of 1‰. As a control, 1 μM represents 1 μM The macrophage cell line RAW264.7 treated with anisomycin, 10 μM means the macrophage cell line RAW264.7 treated with 10 μM anisomycin, +SP600125 refers to the macrophage cell line RAW264.7 treated with 10 μM anisomycin+SP600125, The ordinate represents the expression of HTRA1 mRNA in the macrophage cell line RAW264.7 treated with DMSO as a standard, and the expression of HTRA1 mRNA in RAW264.7 during the process of adding anisomycin or SP600125;

图4d表示显示STAT1抑制剂（MTA）处理的巨噬细胞系RAW264.7中HTRA1 mRNA的表达，图中，对照表示体积比浓度为1‰的DMSO处理的巨噬细胞系RAW264.7；IFN-γ表示IFN-γ处理的巨噬细胞系RAW264.7；IFN-γ+MTA表示IFN-γ和MTA处理的巨噬细胞系RAW264.7；MTA表示MTA处理的巨噬细胞系RAW264.7，纵坐标表示以对照中HTRA1 mRNA的表达为基准，在加干扰素γ或MTA处理的过程中巨噬细胞系RAW264.7中HTRA1 mRNA的表达； Figure 4d shows the expression of HTRA1 mRNA in the macrophage cell line RAW264.7 treated with STAT1 inhibitor (MTA). γ denotes the macrophage cell line RAW264.7 treated with IFN-γ; IFN-γ+MTA denotes the macrophage cell line RAW264.7 treated with IFN-γ and MTA; MTA denotes the macrophage cell line RAW264.7 treated with MTA, longitudinal Coordinates represent the expression of HTRA1 mRNA in the macrophage cell line RAW264.7 in the process of adding interferon gamma or MTA treatment based on the expression of HTRA1 mRNA in the control;

图4e表示STAT1在干扰素γ抑制HTRA1基因表达中的作用，图中，对照为巨噬细胞系RAW264.7，STAT1 shRNA为STAT1基因RNA干扰的RAW264.7细胞，1为未加干扰素γ处理的巨噬细胞系RAW264.7，2为加干扰素γ处理的巨噬细胞系RAW264.7，3为未加干扰素γ处理的STAT1基因RNA干扰的RAW264.7细胞，4为加干扰素γ处理的STAT1基因RNA干扰的RAW264.7细胞，纵坐标表示以未加干扰素γ处理的巨噬细胞系RAW264.7中HTRA1 mRNA的表达为基准，在加干扰素γ处理的过程中巨噬细胞系RAW264.7或STAT1 shRNA中HTRA1 mRNA的表达； Figure 4e shows the role of STAT1 in the inhibition of HTRA1 gene expression by interferon gamma. In the figure, the control is the macrophage cell line RAW264.7, STAT1 shRNA is the RAW264.7 cell line interfered with by STAT1 gene RNA, and 1 is no interferon gamma treatment The macrophage cell line RAW264.7, 2 is the macrophage cell line RAW264.7 treated with interferon γ, 3 is the RAW264.7 cell line of STAT1 gene RNA interference without interferon γ treatment, 4 is the RAW264.7 cell line with interferon γ The treated RAW264.7 cells with RNA interference of STAT1 gene, the ordinate indicates the expression of HTRA1 mRNA in the macrophage cell line RAW264.7 without interferon-γ treatment, and the macrophages in the process of interferon-γ treatment Expression of HTRA1 mRNA in RAW264.7 or STAT1 shRNA;

图5为通过流式细胞仪分选p38 RNA干扰细胞系和STAT1 RNA干扰细胞系的结果，其中，图5a为流式细胞仪分选p38干扰细胞系的流式细胞仪分选结果，图5b为STAT1 RNA干扰细胞系的流式细胞仪分选结果，图中，M1为流式细胞仪分选的GFP阳性（GFP+）细胞系； Figure 5 is the result of sorting p38 RNA interference cell lines and STAT1 RNA interference cell lines by flow cytometry, wherein Figure 5a is the flow cytometry sorting results of p38 interference cell lines sorted by flow cytometry, and Figure 5b is the flow cytometry sorting result of STAT1 RNA interference cell line, in the figure, M1 is the GFP positive (GFP+) cell line sorted by flow cytometry;

图6为干扰素γ降低风湿性关节炎患者滑膜液细胞的HTRA1表达水平。其中，图6a表示干扰素γ处理来自8位关节炎患者的滑膜液细胞24小时后，HTRA1 mRNA的表达水平，图中左边的点表示对照，右边的点表示干扰素γ处理后的患者的HTRA1 mRNA的表达水平，纵坐标表示以对照中HTRA1 mRNA的表达水平为基准，干扰素γ处理后的患者的HTRA1 mRNA的表达水平； Fig. 6 shows that interferon gamma reduces the expression level of HTRA1 in synovial fluid cells of patients with rheumatoid arthritis. Wherein, Fig. 6 a shows that interferon gamma is processed from the synovial fluid cell of 8 arthritis patients after 24 hours, the expression level of HTRA1 mRNA, the dot on the left side of the figure represents control, the dot on the right represents the patient's after interferon gamma processing The expression level of HTRA1 mRNA, the ordinate represents the expression level of HTRA1 mRNA in patients treated with interferon gamma based on the expression level of HTRA1 mRNA in the control;

图6b显示磷酸激酶p38抑制剂（SB203580）处理的滑膜液细胞中HTRA1 mRNA的表达，图中，DMSO表示体积比浓度为1‰的DMSO处理的滑膜液细胞，作为对照，SB203580表示SB203580处理的滑膜液细胞，IFN-γ表示IFN-γ处理的滑膜液细胞，IFN-γ+SB203580表示IFN-γ和SB203580处理的滑膜液细胞，纵坐标表示以DMSO处理的滑膜液细胞中的HTRA1 mRNA的表达为标准，在加干扰素γ或SB203580处理的过程中滑膜液细胞中的HTRA1 mRNA的表达； Figure 6b shows the expression of HTRA1 mRNA in synovial fluid cells treated with a phosphokinase p38 inhibitor (SB203580). In the figure, DMSO indicates the synovial fluid cells treated with DMSO at a volume ratio concentration of 1‰. As a control, SB203580 indicates the treatment with SB203580 synovial fluid cells, IFN-γ means synovial fluid cells treated with IFN-γ, IFN-γ+SB203580 means synovial fluid cells treated with IFN-γ and SB203580, and ordinate means synovial fluid cells treated with DMSO The expression of HTRA1 mRNA was taken as the standard, and the expression of HTRA1 mRNA in synovial fluid cells was treated with interferon γ or SB203580;

图6c显示STAT1抑制剂（MTA）处理的滑膜液细胞中HTRA1 mRNA的表达，图中，DMSO表示体积比浓度为1‰的DMSO处理的滑膜液细胞，作为对照，MTA表示MTA处理的滑膜液细胞，IFN-γ表示IFN-γ处理的滑膜液细胞，IFN-γ+MTA表示IFN-γ和MTA处理的滑膜液细胞，纵坐标表示以DMSO处理的滑膜液细胞中的HTRA1 mRNA的表达为标准，在加干扰素γ或MTA处理的过程中滑膜液细胞中的HTRA1 mRNA 的表达； Figure 6c shows the expression of HTRA1 mRNA in synovial fluid cells treated with STAT1 inhibitor (MTA). In the figure, DMSO indicates the synovial fluid cells treated with DMSO at a volume ratio concentration of 1‰. As a control, MTA indicates the synovial fluid cells treated with MTA. Membrane fluid cells, IFN-γ indicates synovial fluid cells treated with IFN-γ, IFN-γ+MTA indicates synovial fluid cells treated with IFN-γ and MTA, and the ordinate indicates HTRA1 in synovial fluid cells treated with DMSO The expression of mRNA is the standard, and the expression of HTRA1 mRNA in the synovial fluid cells during the process of adding interferon γ or MTA treatment;

图7为通过试验证明的干扰素γ通过活化p38激酶，进而活化转录因子STAT1的信号通路示意图； Figure 7 is a schematic diagram of the signal pathway of the activation of the transcription factor STAT1 through the activation of interferon gamma through the activation of p38 kinase;

图8为p38-STAT1信号通路图。 Figure 8 is a diagram of the p38-STAT1 signaling pathway. the

具体实施方式Detailed ways

除非特别指明，以下实施例中所用的小鼠品种为B6野生型小鼠（购自北京大学实验动物中心），IFN-γ基因敲除小鼠为B6背景，购自美国JACKSON实验室（The Jackson Laboratory，公司网址：http://www.jax.org/），IFN-γ基因敲除小鼠货号为002287；小鼠详细信息请参考http://jaxmice.jax.org/strain/002287.html。 Unless otherwise specified, the mouse species used in the following examples are B6 wild-type mice (purchased from the Experimental Animal Center of Peking University), and the IFN-γ gene knockout mice are B6 background, purchased from the US Jackson laboratory (The Jackson Laboratory, company website: http://www.jax.org/), IFN-γ gene knockout mouse product number is 002287; for details of the mouse, please refer to http://jaxmice.jax.org/strain/002287.html . the

除非特别指明，以下实施例中所用的试剂均为分析纯级试剂，且可从正规渠道商购获得。 Unless otherwise specified, the reagents used in the following examples are of analytical grade and commercially available from formal channels. the

实施例1干扰素γ能够在体外降低丝氨酸蛋白酶HTRA1的表达Example 1 Interferon gamma can reduce the expression of serine protease HTRA1 in vitro

通过常规方法获取小鼠（购自北京大学实验动物中心，品种为B6小鼠）成纤维细胞和巨噬细胞，具体为常规方法处死实验小鼠，剖开小鼠腹壁，用预冷的磷酸盐缓冲液（PBS）冲洗小鼠腹腔，并收集冲洗液，1700rpm离心5分钟，沉淀即为小鼠腹腔巨噬细胞。将12.5天-14.5天孕鼠无菌条件下取出子宫，并取出小鼠胚胎，减去头部和四肢，PBS洗涤3次；将小鼠躯干剪成约1立方毫米小块，加入胰酶，培养箱中孵育15分钟，反复吹打，加入细胞培养基终止消化反应，1700rpm离心5分钟，弃上清，加入新鲜培养基，将沉底物重悬，并移至培养皿中，培养箱中培养3-7天，PBS冲洗两次，胰酶-EDTA消化、传代，该细胞即为小鼠胚胎成纤维细胞（MEF）。 The fibroblasts and macrophages of mice (purchased from the Experimental Animal Center of Peking University, type B6 mice) were obtained by conventional methods. Specifically, the experimental mice were killed by conventional methods, the abdominal wall of the mice was cut open, and pre-cooled phosphate The mouse peritoneal cavity was washed with buffer solution (PBS), and the washing fluid was collected, centrifuged at 1700rpm for 5 minutes, and the pellet was mouse peritoneal macrophages. Take out the uterus of 12.5-14.5-day-old pregnant mice under aseptic conditions, and take out the mouse embryos, subtract the head and limbs, wash 3 times with PBS; cut the mouse trunk into small pieces of about 1 cubic millimeter, add trypsin, Incubate in the incubator for 15 minutes, pipette repeatedly, add cell culture medium to terminate the digestion reaction, centrifuge at 1700rpm for 5 minutes, discard the supernatant, add fresh medium, resuspend the sinking substrate, and transfer it to a petri dish, culture in the incubator After 3-7 days, wash twice with PBS, digest with trypsin-EDTA, and passage, the cells are mouse embryonic fibroblasts (MEFs). the

将上述两种细胞移至培养板中培养，经贴壁纯化后，使用浓度梯度分别为0、1、10、50、100ng/ml干扰素γ（购自美国Peprotech公司）处理细胞24小时及浓度为100ng/ml干扰素γ处理不同时间后，分别使用TRIzol试剂盒（购自美国Invitrogen公司），按照试剂盒说明书操作，提取细胞总RNA（参考文献：Olson J H;Xiang X;et al.Allurin,a 21-kDa sperm chemoattractant from Xenopus egg jelly,is related to mammalian sperm-binding proteins.Proc Natl Acad Sci USA.2001 Sep25;98(20):11205-10.）；然后，再按照试剂盒说明书操作将其逆转录为cDNA，具体为取上述提取的1微克总RNA，加入0.5微升Oligo dT（购自日本TAKARA公司），1微升RNA酶抑制剂，AMV逆转录酶以及AMV缓冲液（均购自日本TAKARA公司），用水将体积补至25微升。将反应物置于42℃水浴锅孵育1小时，所得反应产物即cDNA。 The above two kinds of cells were transferred to culture plates for culture, and after adherence and purification, the cells were treated with concentration gradients of 0, 1, 10, 50, and 100 ng/ml interferon gamma (purchased from Peprotech, USA) for 24 hours and concentration After treating with 100ng/ml interferon gamma for different time, use the TRIzol kit (purchased from Invitrogen, USA) respectively, and follow the instructions of the kit to extract the total cellular RNA (references: Olson J H; Xiang X; et al. Allurin , a 21-kDa sperm chemoattractant from Xenopus egg jelly, is related to mammalian sperm-binding proteins.Proc Natl Acad Sci USA.2001 Sep25;98(20):11205-10.); Its reverse transcription into cDNA, specifically, take 1 μg of total RNA extracted above, add 0.5 μl Oligo dT (purchased from Japan TAKARA company), 1 μl RNase inhibitor, AMV reverse transcriptase and AMV buffer (both purchased (from Japan TAKARA Company), make up the volume to 25 microliters with water. The reaction was incubated in a water bath at 42°C for 1 hour, and the resulting reaction product was cDNA. the

然后按照试剂盒说明书操作，使用Realtime-PCR试剂盒（购自日本TAKARA公司），检测成纤维细胞和巨噬细胞中HTRA1基因表达。具体为以提取的cDNA为模板，PCR反应程序为95℃3分钟；95℃30秒；58℃30秒；72℃30秒，循环数为35。PCR反应在实时定量PCR仪中进行（CFX96，购自美国Biorad公司），扩增HTRA1 mRNA的基因编码序列（SEQ ID NO:1）中的一部分序列（SEQ ID NO:2），实验结果以每个样品的转磷酸核糖基酶（HPRT，phosphoribosyltransferase）为内参进行比对得出。 Then follow the instructions of the kit, and use the Realtime-PCR kit (purchased from Japan TAKARA Company) to detect the expression of HTRA1 gene in fibroblasts and macrophages. Specifically, the extracted cDNA was used as a template, the PCR reaction program was 95°C for 3 minutes; 95°C for 30 seconds; 58°C for 30 seconds; 72°C for 30 seconds, and the number of cycles was 35. The PCR reaction was carried out in a real-time quantitative PCR instrument (CFX96, purchased from Biorad, USA), and a part of the sequence (SEQ ID NO: 2) in the gene coding sequence (SEQ ID NO: 1) of HTRA1 mRNA was amplified. Phosphoribosyltransferase (HPRT, phosphoribosyltransferase) of each sample was used as an internal reference for comparison. the

HTRA1检测引物及扩增产物序列： HTRA1 detection primers and amplified product sequences:

上游引物(Htra1 Sense)：5′-CAAGGATGTGGATGAAAAGGC-3′（SEQ IDNO:3） Upstream primer (Htra1 Sense): 5′-CAAGGATGTGGATGAAAAGGC-3′ (SEQ IDNO:3)

下游引物(Htra1 Reverse)：5′-ATGATAGCGTCTGTCTGAATGTAGTC-3′（SEQ ID NO:4） Downstream primer (Htra1 Reverse): 5′-ATGATAGCGTCTGTCTGAATGTAGTC-3′ (SEQ ID NO: 4)

扩增产物为：caagga tgtggatgaa aaggcggaca ttgcgcttat caagattgac caccagggaaagctgccagt cctgctgctc ggccgctcct cagagctgag acctggagaa tttgtagttg ccattggaagccccttttct cttcaaaaca cagtcaccac tgggatcgtc agcaccaccc agcgaggcgg caaagagctgggacttcgga actccgatat ggactacatt cagacagacg ctatcat（SEQ ID NO:2）扩增产物为：caagga tgtggatgaa aaggc ggaca ttgcgcttat caagattgac caccagggaaagctgccagt cctgctgctc ggccgctcct cagagctgag acctggagaa tttgtagttg ccattggaagccccttttct cttcaaaaca cagtcaccac tgggatcgtc agcaccaccc agcgaggcgg caaagagctgggacttcgga actccgatat ggactacatt cagacagacg ctatcat （SEQ ID NO:2）

HPRT检测引物及扩增产物序列： HPRT detection primers and amplified product sequences:

上游引物(HPRT Sense)：5′-AGTACAGCCCCAAAATGGTTAAG-3′（SEQID NO:7） Upstream primer (HPRT Sense): 5′-AGTACAGCCCCAAAATGGTTAAG-3′ (SEQ ID NO: 7)

下游引物(HPRT Reverse)：5′-CTTAGGCTTTGTATTTGGCTTTTC-3′（SEQ ID NO:8） Downstream primer (HPRT Reverse): 5′-CTTAGGCTTTGTATTTGGCTTTTC-3′ (SEQ ID NO: 8)

扩增产物为HPRT mRNA的基因编码序列（SEQ ID NO:10）中的部分序列： agtacagccc caaaatggtt aaggttgcaa gcttgctggt gaaaaggacc tctcgaagtg ttggatacaggccagacttt gttggatttg aaattccaga caagtttgtt gttggatatg cccttgacta taatgagtac ttcagggatt tgaatcacgt ttgtgtcatt agtgaaactg gaaaagccaa atacaaagcc taag（SEQID NO:9）扩增产物为HPRT mRNA的基因编码序列（SEQ ID NO:10）中的部分序列：agtacagccc caaaatggtt aag gttgcaa gcttgctggt gaaaaggacc tctcgaagtg ttggatacaggccagacttt gttggatttg aaattccaga caagtttgtt gttggatatg cccttgacta taatgagtac ttcagggatt tgaatcacgt ttgtgtcatt agtgaaactggaaaagccaa atacaaagcc taag （SEQID NO:9）

结果如图1所示，显示通过Realtime-PCR技术证实干扰素γ能够显著降低小鼠和人成纤维细胞和巨噬细胞中HTRA1基因表达，且具有时间与剂量依赖性。 The results are shown in Figure 1, which shows that interferon-γ can significantly reduce the expression of HTRA1 gene in mouse and human fibroblasts and macrophages by Realtime-PCR technology in a time- and dose-dependent manner. the

实施例2干扰素γ能够在体内降低丝氨酸蛋白酶HTRA1的表达Example 2 Interferon gamma can reduce the expression of serine protease HTRA1 in vivo

通过常规方法对小鼠（野生型B6小鼠，购自北京大学实验动物中心）和干扰素γ基因敲除小鼠（该小鼠为野生型B6小鼠背景，购自美国JACKSON实验室（The Jackson Laboratory，公司网址：http://www.jax.org/），IFN-γ基因敲除小鼠货号为002287；小鼠详细信息请参考http://jaxmice.jax.org/strain/002287.html）分别进行干扰素γ腹腔注射（5μg/小鼠/天，共注射5天），其中LPS腹腔注射剂量为100ng/小鼠/5天，5天后手术获取小鼠关节部位及大肠部位，提取组织RNA并逆转录成cDNA（操作方法同实施例1），再以提取的cDNA为模板，使用Realtime-PCR试剂盒（购自日本TAKARA公司），按照说明书操作，检测小鼠关节部位和大肠部位的样品中HTRA1基因表达。 Mice (wild-type B6 mice, purchased from the Experimental Animal Center of Peking University) and interferon-γ gene knockout mice (the mice were wild-type B6 mice, purchased from the American Jackson Laboratory (The Jackson Laboratory, company website: http://www.jax.org/), the IFN-γ knockout mouse product number is 002287; for details of the mouse, please refer to http://jaxmice.jax.org/strain/002287. html) were injected intraperitoneally with interferon gamma (5 μg/mouse/day, for a total of 5 days), and the dose of LPS intraperitoneally injected was 100ng/mouse/5 days. Organize the RNA and reverse transcribe it into cDNA (the operation method is the same as in Example 1), then use the extracted cDNA as a template, use the Realtime-PCR kit (purchased from Japan TAKARA Company), and operate according to the instructions to detect the mouse joints and large intestines HTRA1 gene expression in the samples. the

同时提取获取的鼠关节部位及大肠部位的组织蛋白质，采用免疫杂交印记（Western-Blot）(一抗为HTRA1特异性抗体购自美国Santa Cruz公司)检测HTRA1蛋白水平，具体方法如下： At the same time, the tissue proteins of the acquired mouse joints and large intestine were extracted, and the protein level of HTRA1 was detected by Western-Blot (the primary antibody was HTRA1-specific antibody purchased from Santa Cruz, USA). The specific method was as follows:

1．将200微升预冷细胞裂解液RIPA（购自北京赛弛生物科技公司）到装有关节或大肠组织的1.5毫升离心管中，反复吹打，充分裂解细胞，置于冰上15分钟；13000rpm离心15分钟；再将将收取的蛋白样品用北京赛弛生物科技公司BCA蛋白浓度检测试剂盒定量细胞裂解液的蛋白浓度，调节各样品的蛋白浓度一致，然后加入6×上样缓冲液，沸水浴中煮5-10分钟；2．13000rpm，4℃离心10分钟，取上清；3．每孔加入等量的样品（约30微克），聚丙烯酰胺凝胶电泳（SDS-PAGE）分离蛋白质，95V稳压电泳2-2.5小时，再经电转移将蛋白质转移至PVDF膜上；4．转好PVDF膜经5%脱脂牛奶/TBST（TBS＋0.05%Tween20）封闭（室温，1小时），加HTRA1或内参GAPDH（磷酸甘油醛脱氢酶）的特异性抗体，4℃温和振荡过夜，倾倒去除游离一抗后，室温TBST洗涤三次，每次15 分钟，再与辣根过氧化物酶（HRP）标记的二抗室温振荡孵育1小时，最后再用PBST洗涤三次，于Millipore公司的显色底物孵育3分钟，于暗室曝光显影。 1. Put 200 microliters of pre-cooled cell lysate RIPA (purchased from Beijing Saichi Biotechnology Co., Ltd.) into a 1.5ml centrifuge tube containing joint or large intestine tissue, pipette repeatedly to fully lyse the cells, and place it on ice for 15 minutes; centrifuge at13000rpm 15 minutes; then use the collected protein samples to quantify the protein concentration of the cell lysate with the BCA protein concentration detection kit of Beijing Saichi Biotechnology Co. Boil for 5-10 minutes; 2. Centrifuge at 13000rpm, 4°C for 10 minutes, and take the supernatant; 3. Add an equal amount of sample (about 30 micrograms) to each well, separate the protein by polyacrylamide gel electrophoresis (SDS-PAGE), electrophoresis at 95V for 2-2.5 hours, and then transfer the protein to the PVDF membrane by electrotransfer; 4. Turn the PVDF membrane and block it with 5% skimmed milk/TBST (TBS+0.05% Tween20) (room temperature, 1 hour), add the specific antibody of HTRA1 or the internal reference GAPDH (glyceraldehyde phosphate dehydrogenase), shake gently overnight at 4°C, After pouring to remove the free primary antibody, wash three times with TBST at room temperature, each time for 15 minutes, and then incubate with horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour at room temperature shaking, and finally wash three times with PBST, in the Millipore company's display Incubate with chromatic substrate for 3 minutes and expose to develop in dark room. the

结果如图2所示，表明动物体内实验也证实，腹腔注射重组干扰素γ（5μg/小鼠/天）能显著降低小鼠大肠及关节部位HTRA1的表达；与之相反，干扰素γ敲除HTRA1的小鼠HTRA1的表达相应升高。 The results are shown in Figure 2, indicating that animal experiments also confirmed that intraperitoneal injection of recombinant interferon gamma (5 μg/mouse/day) can significantly reduce the expression of HTRA1 in the large intestine and joints of mice; in contrast, interferon gamma knockout Expression of HTRA1 is correspondingly elevated in HTRA1 mice. the

实施例3干扰素γ降低关节炎病征Example 3 Interferon gamma reduces arthritis symptoms

为了证实干扰素γ对HTRA1基因表达抑制作用的临床意义，我们建立小鼠关节炎模型（Collagen-Induced Arthritis,CIA），以下试验中所用的小鼠为野生型B6小鼠和干扰素γ基因敲除的B6小鼠。 In order to confirm the clinical significance of the inhibitory effect of interferon γ on HTRA1 gene expression, we established a mouse arthritis model (Collagen-Induced Arthritis, CIA). The mice used in the following experiments were wild-type B6 mice and interferon γ gene knockout Except for B6 mice. the

小鼠关节炎模型（CIA）的建立：将鸡II型胶原（购自美国Sigma公司）与完全弗氏佐剂（购自美国Sigma公司）充分乳化，使其浓度为1mg/ml，再使用该乳化剂分别于第1天和第21天，以100μl/小鼠的量对实验小鼠进行皮下注射，并在第18天开始监测小鼠四肢关节肿胀程度；同时在用野生型B6小鼠和干扰素γ基因敲除的B6小鼠诱导关节炎的过程中注射干扰素（5μg/小鼠/天，共注射5天）或LPS（脂多糖，其为革兰氏阴性菌壁成分，能激活免疫细胞，注射量为100μg/小鼠，仅在第一天注射一次）；在第50天将实验小鼠处死，并进行关节部位的HE染色检测（参考文献：Damo Xu,Hui-Rong Jiang,et al.IL-33 Exacerbates Autoantibody-Induced Arthritis.J.Immunol.2010;184;2620-2626）和进行HTRA1基因表达检测。HTRA1基因表达检测分别采用实时定量PCR（Realtime-PCR）（具体操作方法同实施例1）和蛋白质杂交印迹（Western-Blot）技术（具体操作方法同实施例2）。 Establishment of mouse arthritis model (CIA): fully emulsify chicken type II collagen (purchased from Sigma, USA) with complete Freund's adjuvant (purchased from Sigma, USA) to a concentration of 1 mg/ml, and then use the On the 1st day and the 21st day, the emulsifier was injected subcutaneously to the experimental mice at the amount of 100 μl/mouse, and the degree of swelling of the joints of the limbs of the mice was monitored on the 18th day; at the same time, wild-type B6 mice and Interferon γ knockout B6 mice were injected with interferon (5 μg/mouse/day for 5 days) or LPS (lipopolysaccharide, which is a Gram-negative bacterial wall component, can activate Immune cells, the injection volume was 100 μg/mouse, only injected once on the first day); on the 50th day, the experimental mice were sacrificed, and the HE staining of the joints was performed (references: Damo Xu, Hui-Rong Jiang, et al.IL-33 Exacerbates Autoantibody-Induced Arthritis.J.Immunol.2010;184;2620-2626) and HTRA1 gene expression detection. HTRA1 gene expression was detected by real-time quantitative PCR (Realtime-PCR) (the specific operation method is the same as in Example 1) and protein blot (Western-Blot) technology (the specific operation method is the same as in Example 2). the

结果如图3所示，表明腹腔注射重组干扰素γ能显著降低小鼠关节部位HTRA1的表达，进而降低关节炎病征；与之相反，干扰素γ基因敲除的小鼠HTRA1的表达相应升高，关节炎病征更为严重。 The results are shown in Figure 3, indicating that intraperitoneal injection of recombinant interferon-γ can significantly reduce the expression of HTRA1 in the joints of mice, thereby reducing the symptoms of arthritis; in contrast, the expression of HTRA1 in interferon-γ gene knockout mice increased correspondingly , the symptoms of arthritis are more severe. the

实施例4干扰素γ通过p38-STAT1通路抑制HTRA1的基因表达Example 4 Interferon gamma inhibits the gene expression of HTRA1 through the p38-STAT1 pathway

为了研究干扰素γ抑制HTRA1基因表达的分子机制，我们采用了通路激动剂/抑制剂和RNA干扰等技术。 In order to study the molecular mechanism of IFN-γ inhibiting HTRA1 gene expression, we used techniques such as pathway agonists/inhibitors and RNA interference. the

为研究干扰素γ抑制HTRA1基因表达的分子机制，采用巨噬细胞系 RAW264.7（购自美国模式培养物集存库，ATCC）为研究对象。使用干扰素γ（注射浓度为100ng/ml）和p38抑制剂SB203580（购自德国Merk公司，货号为559389）（注射浓度为10μM）或STAT1抑制剂MTA（购自美国Sigma公司，货号为D5011）（注射浓度为10μM），及茴香霉素（注射浓度为1μM、10μM）处理RAW264.7细胞，24小时后收取细胞并提取RNA，采用实时定量PCR（Realtime-PCR）试剂盒（购自日本TAKARA公司），按照说明书操作，检测HTRA1基因表达，具体操作方法同实施例1。 In order to study the molecular mechanism of interferon-γ inhibiting HTRA1 gene expression, the macrophage cell line RAW264.7 (purchased from the American Type Culture Collection, ATCC) was used as the research object. Use interferon-γ (injection concentration of 100ng/ml) and p38 inhibitor SB203580 (purchased from Merk, Germany, product number 559389) (injection concentration of 10 μM) or STAT1 inhibitor MTA (purchased from American Sigma Company, product number is D5011) (injection concentration of 10 μM), and anisomycin (injection concentration of 1 μM, 10 μM) to treat RAW264.7 cells, harvest the cells after 24 hours and extract RNA, using real-time quantitative PCR (Realtime-PCR) kit (purchased from Japan TAKARA company), operate according to the instructions, and detect the expression of HTRA1 gene, and the specific operation method is the same as that in Example 1. the

为了进一步证实p38和STAT1在干扰素γ抑制HTRA1基因表达中的作用，我们制备了p38和STAT1的RNA干扰细胞系（p38 shRNA和STAT1shRNA），具体方法是：将p38和STAT1的目标干扰序列（STAT1干扰序列为5′-GCC GAG AAC ATA CCA GAGAAT-3′（SEQ ID NO:5）；p38干扰序列为5′-GAA CTT CGC AAA TGT ATT T-3′（SEQ ID NO:6））通过酶切方法嵌入逆转录病毒载体质粒中（所使用的载体质粒为LentiLox 3.7，购自美国Addgene公司）。然后将上述逆转录病毒载体分别与psPAX2和pMD2.G（均购自Addgene公司）共转染至293细胞（购自美国模式培养物集存库，ATCC）中，48小时后收集培养上清，并用此上清加入到RAW264.7细胞培养液中，培养48小时后，流式细胞仪分选GFP阳性细胞，即为p38 RNA干扰和STAT1 RNA干扰细胞系（结果如图5所示），再加干扰素γ分别处理p38和STAT1 RNA干扰细胞系，并用未加干扰素γ处理的p38和STAT1 RNA干扰细胞系作为对照。 In order to further confirm the role of p38 and STAT1 in the inhibition of HTRA1 gene expression by interferon gamma, we prepared p38 and STAT1 RNA interference cell lines (p38 shRNA and STAT1 shRNA), the specific method is: the target interference sequence of p38 and STAT1 (STAT1 The interference sequence is 5′-GCC GAG AAC ATA CCA GAGAAT-3′ (SEQ ID NO:5); the p38 interference sequence is 5′-GAA CTT CGC AAA TGT ATT T-3′ (SEQ ID NO:6)) through the enzyme Embedded in the retroviral vector plasmid by cutting method (the vector plasmid used is LentiLox 3.7, purchased from Addgene, USA). Then, the above retroviral vectors were co-transfected with psPAX2 and pMD2.G (both purchased from Addgene) into 293 cells (purchased from American Type Culture Collection, ATCC), and the culture supernatant was collected 48 hours later. And use this supernatant to add to the RAW264.7 cell culture medium, after culturing for 48 hours, sort the GFP-positive cells by flow cytometry, which are the p38 RNA interference and STAT1 RNA interference cell lines (the results are shown in Figure 5), and then The p38 and STAT1 RNA interference cell lines were treated with interferon-γ, respectively, and the p38 and STAT1 RNA interference cell lines without interferon-γ treatment were used as controls. the

结果如图4所示，图4a表明干扰素γ能够抑制HTRA1基因的表达，且在SB203580（磷酸激酶p38的抑制剂）的共同作用下，抑制作用减弱；图4b表明干扰素γ在p38 RNA干扰细胞系中抑制HTRA1基因的表达减弱；图4c表明茴香霉素也能够抑制HTRA1基因的表达，并且在SP600125（SP600125为磷酸激酶JNK抑制剂，购自美国Invivogen公司，货号tlrl-sp60）的作用下，抑制作用加强；图4d表明干扰素γ能够抑制HTRA1基因的表达，且在MTA（转录因子STAT1抑制剂，能够抑制其转录活性，购自美国SIGMA公司，货号为D5011）的共同作用下，抑制作用减弱；图4e表明干扰素γ在STAT1 RNA干扰细胞系中抑制HTRA1基因的表达减弱。 The results are shown in Figure 4. Figure 4a shows that interferon-γ can inhibit the expression of HTRA1 gene, and under the combined action of SB203580 (an inhibitor of phosphokinase p38), the inhibitory effect is weakened; Figure 4b shows that interferon-γ can inhibit the expression of p38 RNA interference Inhibition of the expression of the HTRA1 gene in the cell line was weakened; Figure 4c shows that anisomycin can also inhibit the expression of the HTRA1 gene, and under the action of SP600125 (SP600125 is a phosphokinase JNK inhibitor, purchased from Invivogen, USA, Cat. No. tlrl-sp60) , the inhibitory effect is strengthened; Figure 4d shows that interferon γ can inhibit the expression of HTRA1 gene, and under the combined action of MTA (transcription factor STAT1 inhibitor, which can inhibit its transcriptional activity, purchased from SIGMA Company of the United States, product number D5011), inhibit The effect is weakened; Figure 4e shows that interferon-γ inhibits the expression of HTRA1 gene in the STAT1 RNA interference cell line. the

因而，如图7所示，结果证实干扰素γ与细胞表面干扰素γ受体结合，将活化信号传递给细胞质内的磷酸激酶p38，活化p38激酶，进而活化转录因子STAT1。活化的STAT1能够从细胞质进入细胞核，并结合在HTRA1基因转录启动区，从而抑制HTRA1基因转录。 Therefore, as shown in Figure 7, the results confirm that interferon gamma binds to the cell surface interferon gamma receptor, transmits the activation signal to the phosphokinase p38 in the cytoplasm, activates the p38 kinase, and then activates the transcription factor STAT1. Activated STAT1 can enter the nucleus from the cytoplasm and bind to the transcription promoter region of the HTRA1 gene, thereby inhibiting the transcription of the HTRA1 gene. the

实施例5干扰素γ抑制人滑膜液细胞中HTRA1的基因表达Example 5 Interferon gamma inhibits gene expression of HTRA1 in human synovial fluid cells

为了确定干扰素γ是否对风湿性关节炎患者的HTRA1基因表达具有同样的抑制的作用，我们使用浓度为100ng/ml的干扰素γ处理了8位风湿性关节炎患者的关节滑膜液细胞（取自北京大学人民医院风湿免疫科，并由患者提供签署了知情同意书），然后通过Realtime-PCR方法检测细胞内HTRA1 mRNA表达水平。干扰素γ处理时间为24小时，RNA提取及检测方法同实施例1，试验结果以每个样品的转磷酸核糖基酶（HPRT，phosphoribosyltransferase）为内参进行比对得出。人HTRA1以及HPRT检测引物及扩增产物序列如下： In order to determine whether interferon γ has the same inhibitory effect on HTRA1 gene expression in patients with rheumatoid arthritis, we used interferon γ at a concentration of 100ng/ml to treat the synovial fluid cells of 8 patients with rheumatoid arthritis ( Obtained from the Department of Rheumatology and Immunology, Peking University People's Hospital, and the patient provided and signed the informed consent), and then the expression level of HTRA1 mRNA in the cells was detected by Realtime-PCR method. The interferon gamma treatment time was 24 hours, and the RNA extraction and detection methods were the same as in Example 1. The test results were obtained by comparing the phosphoribosyltransferase (HPRT, phosphoribosyltransferase) of each sample as an internal reference. The sequences of human HTRA1 and HPRT detection primers and amplification products are as follows:

人HTRA1检测引物及扩增产物序列： Human HTRA1 detection primers and amplified product sequences:

上游引物(Htra1 Sense)：5′-CAAGGATGTGGATGAGAAAGCAGACA-3′（SEQ ID NO:11） Upstream primer (Htra1 Sense): 5′-CAAGGATGTGGATGAGAAAGCAGACA-3′ (SEQ ID NO: 11)

下游引物(Htra1 Reverse)：5′-ATGATGGCGTCGGTCTGGATGTAGTC-3′（SEQ ID NO:12） Downstream primer (Htra1 Reverse): 5′-ATGATGGCGTCGGTCTGGATGTAGTC-3′ (SEQ ID NO: 12)

扩增产物为HTRA1 mRNA的基因编码序列（SEQ ID NO:13）中的部分序列：caaggat gtggatgaga aagcagacat cgcactcatc aaaattgacc accagggcaagctgcctgtc ctgctgcttg gccgctcctc agagctgcgg ccgggagagt tcgtggtcgc catcggaagcccgttttccc ttcaaaacac agtcaccacc gggatcgtga gcaccaccca gcgaggcggcaaagagctgg ggctccgcaa ctcagacatg gactacatcc agaccgacgc catcat（SEQ IDNO:14）扩增产物为HTRA1 mRNA的基因编码序列（SEQ ID NO:13）中的部分序列：caaggat gtggatgaga aagcagaca t cgcactcatc aaaattgacc accagggcaagctgcctgtc ctgctgcttg gccgctcctc agagctgcgg ccgggagagt tcgtggtcgc catcggaagcccgttttccc ttcaaaacac agtcaccacc gggatcgtga gcaccaccca gcgaggcggcaaagagctgg ggctccgcaa ctcagacatggactacatcc agaccgacgc catcat （SEQ IDNO:14 )

人HPRT检测引物及扩增产物序列： Human HPRT detection primers and amplified product sequences:

上游引物(HPRT Sense)：5′-CAGTATAATCCAAAGATGGTCAA-3′（SEQ ID NO:15） Upstream primer (HPRT Sense): 5′-CAGTATAATCCAAAGATGGTCAA-3′ (SEQ ID NO: 15)

下游引物(HPRT Reverse)：5′-TTAGGCTTTGTATTTTGCTTTTCC-3′（SEQ ID NO:16） Downstream primer (HPRT Reverse): 5′-TTAGGCTTTGTATTTTGCTTTTCC-3′ (SEQ ID NO: 16)

扩增产物为HPRT mRNA的基因基因编码序列（SEQ ID NO:17）中的部分序列：cagtataatc caaagatggt caaggtcgca agcttgctgg tgaaaaggac cccacgaagt gttggatata agccagactt tgttggattt gaaattccag acaagtttgt tgtaggatat gcccttgactataatgaata cttcagggat ttgaatcatg tttgtgtcat tagtgaaact ggaaaagcaa aatacaaagcctaa（SEQ ID NO:18）扩增产物为HPRT mRNA的基因基因编码序列（SEQ ID NO:17）中的部分序列：cagtataatc caaagatggt caa ggtcgca agcttgctgg tgaaaaggac cccacgaagt gttggatata agccagactt tgttggattt gaaattccag acaagtttgt tgtaggatat gcccttgactataatgaata cttcagggat ttgaatcatg tttgtgtcat tagtgaaactggaaaagcaa aatacaaagcctaa （SEQ ID NO:18）

结果如图6a所示，在8位风湿性关节炎患者的关节滑膜液细胞中，干扰素γ均能显著降低其HTRA1基因表达水平。 The results are shown in Figure 6a, in the joint synovial fluid cells of 8 patients with rheumatoid arthritis, interferon-γ can significantly reduce the expression level of HTRA1 gene. the

此外，为研究干扰素γ在人类细胞中抑制HTRA1基因表达的分子机制，还使用浓度为100ng/ml的干扰素γ和浓度为10μM的p38抑制剂SB203580（购自德国Merk公司，货号为559389）或浓度为10μM的STAT1抑制剂MTA（购自美国Sigma公司，货号为D5011）处理滑膜液细胞，24小时后收取细胞并提取RNA，采用实时定量PCR（Realtime-PCR）试剂盒（购自日本TAKARA公司），按照说明书操作，检测HTRA1基因表达，具体操作方法同实施例1，具体引物设计同上。 In addition, in order to study the molecular mechanism of interferon gamma inhibiting HTRA1 gene expression in human cells, interferon gamma at a concentration of 100 ng/ml and p38 inhibitor SB203580 at a concentration of 10 μM (purchased from Merk, Germany, catalog number 559389) were also used Or the synovial fluid cells were treated with STAT1 inhibitor MTA (purchased from Sigma, USA, product number: D5011) at a concentration of 10 μM, and the cells were harvested after 24 hours to extract RNA. Real-time quantitative PCR (Realtime-PCR) kit (purchased from Japan TAKARA Company), according to the instructions, to detect the expression of HTRA1 gene, the specific operation method is the same as in Example 1, and the specific primer design is the same as above. the

结果表明干扰素γ对风湿性关节炎患者的HTRA1基因表达具有同样的抑制的作用，并且这种抑制作用是通过活化p38-STAT1通路实现的。 The results showed that interferon-gamma had the same inhibitory effect on HTRA1 gene expression in patients with rheumatoid arthritis, and this inhibitory effect was achieved by activating the p38-STAT1 pathway. the