CN115807071A - Application of detection reagent in preparation of type 2 diabetes with atherosclerosis diagnostic tool - Google Patents

Abstract

Translated fromChinese

本发明属于生物医学领域，具体涉及检测试剂在制备2型糖尿病伴动脉粥样硬化诊断工具中的应用。所述检测试剂为检测CD4基因和PLEK基因的表达量，或者检测CD4蛋白和PLEK蛋白的表达量的检测试剂。所述试检测剂选自特异性识别CD4基因和PLEK基因的寡核苷酸探针、特异性扩增CD4基因和PLEK基因的引物或特异性结合样本中CD4蛋白和PLEK蛋白的结合剂。本发明研究证实CD4和PLEK在疾病组中表达显著上调，同时，我们分析了CD4和PLEK诊断能力，ROC曲线结果显示CD4和PLEK的AUC值分别为0.733及0.790，结果表明，CD4和PLEK可作为动脉粥样硬化和2型糖尿病诊断生物标志物和干预靶点。

The invention belongs to the field of biomedicine, and in particular relates to the application of a detection reagent in the preparation of a diagnostic tool for type 2 diabetes accompanied by atherosclerosis. The detection reagent is a detection reagent for detecting the expression level of CD4 gene and PLEK gene, or detecting the expression level of CD4 protein and PLEK protein. The detection reagent is selected from oligonucleotide probes that specifically recognize CD4 gene and PLEK gene, primers that specifically amplify CD4 gene and PLEK gene, or binding agents that specifically bind CD4 protein and PLEK protein in samples. The study of the present invention confirmed that the expression of CD4 and PLEK was significantly up-regulated in the disease group. At the same time, we analyzed the diagnostic capabilities of CD4 and PLEK. The results of the ROC curve showed that the AUC values of CD4 and PLEK were 0.733 and 0.790, respectively. The results showed that CD4 and PLEK can be used as Atherosclerosis and type 2 diabetes diagnostic biomarkers and targets for intervention.

Description

Translated fromChinese

检测试剂在制备2型糖尿病伴动脉粥样硬化诊断工具中的应用The role of detection reagents in the preparation oftype 2 diabetes with atherosclerosis diagnostic toolsapplication

技术领域technical field

本发明属于生物医学领域，具体涉及检测试剂在制备2型糖尿病伴动脉粥样硬化诊断工具中的应用。The invention belongs to the field of biomedicine, and in particular relates to the application of a detection reagent in the preparation of a diagnostic tool fortype 2 diabetes accompanied by atherosclerosis.

背景技术Background technique

动脉粥样硬化(Atherosclerosis,AS)是一种慢性炎症性疾病，由脂质在动脉中积累导致狭窄和血栓形成，能够影响心脏、大脑和外周血管，是世界范围内致死致残的主要原因。研究表明，糖尿病与心血管疾病的关系密切，糖尿病患者，特别是在2型糖尿病患者，其动脉斑块坏死核心面积较大，动脉中膜钙化较多，相较于健康人群，糖尿病患者发生心血管疾病的概率会增加2到4倍。研究发现，低密度脂蛋白-胆固醇(LDL-C)和炎症因子被认为是糖尿病患者心血管疾病风险的因素之一。此外，胰岛素信号通路在动脉粥样硬化中起作用，平滑肌细胞中的胰岛素受体缺失会导致内膜增生减少。然而，对于动脉粥样硬化和2型糖尿病在基因水平上的联系，至今尚无系统的报道。因此，确定新的诊断标记物和治疗靶点对于动脉粥样硬化和2型糖尿病的早期诊断和特异性干预尤为重要。Atherosclerosis (Atherosclerosis, AS) is a chronic inflammatory disease caused by lipid accumulation in arteries leading to stenosis and thrombosis, which can affect the heart, brain and peripheral blood vessels, and is the main cause of death and disability worldwide. Studies have shown that diabetes is closely related to cardiovascular diseases. Diabetic patients, especially those withtype 2 diabetes, have a larger core area of arterial plaque necrosis and more calcification of the arterial media. The probability of vascular disease increases by 2 to 4 times. The study found that low-density lipoprotein-cholesterol (LDL-C) and inflammatory factors are considered as one of the risk factors for cardiovascular disease in diabetic patients. In addition, insulin signaling plays a role in atherosclerosis, and loss of insulin receptors in smooth muscle cells leads to reduced intimal hyperplasia. However, there is no systematic report on the connection between atherosclerosis andtype 2 diabetes at the genetic level. Therefore, identifying new diagnostic markers and therapeutic targets is particularly important for early diagnosis and specific intervention of atherosclerosis andtype 2 diabetes.

RNA-SEQ和微阵列技术是探索与疾病发病机制有关的基因、识别诊断生物标志物或探索疾病治疗靶点的有效工具。有研究者发现，在破裂斑块中，CCL4、CCL18、MMP9和SPP1水平升高，可作为介入靶点。ACLY、SERPING1和57ANPEP已被证实与2型糖尿病的发生相关。然而，对于动脉粥样硬化与2型糖尿病的联合研究目前仍在起步阶段。RNA-seq and microarray technologies are effective tools for exploring genes involved in disease pathogenesis, identifying diagnostic biomarkers, or exploring disease therapeutic targets. Some researchers have found that in ruptured plaques, the levels of CCL4, CCL18, MMP9 and SPP1 are increased, which can be used as intervention targets. ACLY, SERPING1 and 57ANPEP have been confirmed to be associated with the occurrence oftype 2 diabetes. However, joint research on atherosclerosis andtype 2 diabetes is still in its infancy.

发明内容Contents of the invention

为解决上述技术问题，本发明提供了一种检测试剂在制备2型糖尿病伴动脉粥样硬化诊断工具中的应用，所述检测试剂为检测CD4基因和PLEK基因的表达量，或者检测CD4蛋白和PLEK蛋白的表达量的检测试剂。In order to solve the above technical problems, the present invention provides an application of a detection reagent in the preparation of a diagnostic tool fortype 2 diabetes with atherosclerosis, the detection reagent is to detect the expression of CD4 gene and PLEK gene, or to detect CD4 protein and A reagent for detecting the expression level of PLEK protein.

更进一步的，所述试检测剂选自特异性识别CD4基因和PLEK基因的寡核苷酸探针、特异性扩增CD4基因和PLEK基因的引物或特异性结合样本中CD4蛋白和PLEK蛋白的结合剂。Further, the detection reagent is selected from oligonucleotide probes that specifically recognize CD4 gene and PLEK gene, primers that specifically amplify CD4 gene and PLEK gene, or specific binding to CD4 protein and PLEK protein in the sample. Binding agent.

更进一步的，特异性扩增CD4基因的引物序列如SEQ ID NO.1-2所示，特异性扩增PLEK基因的引物序列如SEQ ID NO.3-4所示。Furthermore, the primer sequences for specifically amplifying the CD4 gene are shown in SEQ ID NO.1-2, and the primer sequences for specifically amplifying the PLEK gene are shown in SEQ ID NO.3-4.

基于同一个发明构思，本发明还提供了一种诊断2型糖尿病伴动脉粥样硬化的试剂盒，所述试剂盒含有检测CD4基因和PLEK基因的表达量，或者检测CD4蛋白和PLEK蛋白的表达量的检测试剂；所述试检测剂选自特异性识别CD4基因和PLEK基因的寡核苷酸探针、特异性扩增CD4基因和PLEK基因的引物或特异性结合样本中CD4蛋白和PLEK蛋白的结合剂。Based on the same inventive concept, the present invention also provides a kit for diagnosingtype 2 diabetes mellitus with atherosclerosis. Amount of detection reagents; said detection reagents are selected from oligonucleotide probes for specific recognition of CD4 gene and PLEK gene, primers for specific amplification of CD4 gene and PLEK gene, or CD4 protein and PLEK protein in specific binding samples the binding agent.

基于同一个发明构思，本发明还提供了所述CD4蛋白和PLEK蛋白的表达抑制剂在制备治疗2型糖尿病伴动脉粥样硬化的药物中的应用。Based on the same inventive concept, the present invention also provides the application of the CD4 protein and PLEK protein expression inhibitors in the preparation of medicaments for treatingtype 2 diabetes with atherosclerosis.

基于同一个发明构思，本发明还提供了一种治疗2型糖尿病伴动脉粥样硬化的药物，有效成分为所述CD4蛋白和PLEK蛋白的表达抑制剂。Based on the same inventive concept, the present invention also provides a drug for treatingtype 2 diabetes with atherosclerosis, the active ingredient of which is the expression inhibitor of CD4 protein and PLEK protein.

本发明具有如下有益效果：The present invention has following beneficial effect:

本发明从GEO数据库中获得了4个数据集，其中包含动脉粥样硬化斑块和2型糖尿病相关的表达谱。在确定了这两种疾病之间共有的差异表达基因后，我们对基因进行了功能丰富分析。然后，基于蛋白质-蛋白质相互作用网络筛选了核心基因，并在其他两个验证数据集和临床样本中进行了验证。本研究表明，CD4和PLEK可作为动脉粥样硬化和2型糖尿病诊断生物标志物和干预靶点。The present invention obtains 4 data sets from the GEO database, which contain expression profiles related to atherosclerotic plaque andtype 2 diabetes. After identifying differentially expressed genes shared between the two disorders, we performed functional enrichment analysis of the genes. Then, the core genes were screened based on the protein-protein interaction network and validated in two other validation datasets and clinical samples. This study demonstrates that CD4 and PLEK can be used as diagnostic biomarkers and intervention targets for atherosclerosis andtype 2 diabetes.

附图说明Description of drawings

图1是动脉粥样硬化和2型糖尿病的共享基因。A为GSE28829数据集中所有差异表达基因的火山图，B为GSE20966数据集中所有差异表达基因的火山图，p值<0.05和|logFC|>0.5为差异表达显著基因，圆点表示上调基因，方框表示下调基因，三角形表示不显著基因；C为两个数据基中上调基因的维恩图，D为两个数据基中下调基因的维恩图。Figure 1. Shared genes for atherosclerosis andtype 2 diabetes. A is the volcano map of all differentially expressed genes in the GSE28829 dataset, B is the volcano map of all differentially expressed genes in the GSE20966 dataset, p-value <0.05 and |logFC|>0.5 are significantly differentially expressed genes, dots indicate up-regulated genes, boxes Represents down-regulated genes, triangles represent insignificant genes; C is the Venn diagram of up-regulated genes in two data bases, D is the Venn diagram of down-regulated genes in two data bases.

图2是GO分析生物过程(A)、细胞成分(B)和分子功能(C)，以及KEGG功能富集分析(D)结果。Figure 2 shows the results of GO analysis of biological processes (A), cellular components (B) and molecular functions (C), and KEGG functional enrichment analysis (D).

图3是蛋白相互作用网络(PPI)构建。利用STRING网站和Cytoscape软件构建PPI网络。五角星标记表示上调基因，四角星标记表示下调基因。Figure 3 is the construction of protein interaction network (PPI). The PPI network was constructed using the STRING website and Cytoscape software. Five-pointed star marks indicate up-regulated genes, and four-pointed star marks indicate down-regulated genes.

图4是验证关键基因CD4(A),CXCL8(B),DOCK8(C),ITGB2(D),NCF4(E),PLEK(F),THY1(G)和TYROBP(H)在动脉粥样硬化验证集GSE43292中的表达。*p<0.05,**p<0.01,***p<0.001,****p<0.0001。Figure 4 is the validation of key genes CD4(A), CXCL8(B), DOCK8(C), ITGB2(D), NCF4(E), PLEK(F), THY1(G) and TYROBP(H) in atherosclerosis Expression in the validation set GSE43292. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

图5是验证关键基因CD4(A),CXCL8(B),ITGB2(C),NCF4(D),PLEK(E),THY1(F)和TYROBP(G)在2型糖尿病验证集GSE25724中的表达。*p<0.05,**p<0.01,***p<0.001,****p<0.0001。Figure 5 is the verification of the expression of key genes CD4 (A), CXCL8 (B), ITGB2 (C), NCF4 (D), PLEK (E), THY1 (F) and TYROBP (G) in thetype 2 diabetes validation set GSE25724 . *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

图6是检测CD4和PLEK在血液中的表达及其诊断能力。A、B为qRT-PCR检测健康人群和动脉粥样硬化及2型糖尿病患者血液中CD4和PLEK的表达；C为ROC分析CD4和PLEK的诊断灵敏性。**p<0.01,***p<0.001。Figure 6 is the detection of the expression of CD4 and PLEK in blood and their diagnostic capabilities. A and B are qRT-PCR detection of CD4 and PLEK expression in the blood of healthy people and patients with atherosclerosis andtype 2 diabetes; C is the diagnostic sensitivity of ROC analysis of CD4 and PLEK. **p<0.01, ***p<0.001.

具体实施方式Detailed ways

下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，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。The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

实施例1Example 1

1方法1 method

1.1GEO数据集和数据处理1.1 GEO dataset and data processing

从GEO数据库中下载动脉粥样硬化和2型糖尿病的数据表达谱，将GSE28829(包含颈动脉斑块测序数据)和GSE20966数据集(包含2型糖尿病测序数据)纳入为训练集，GSE43292(包含颈动脉斑块测序数据)和GSE25724数据集(包含2型糖尿病测序数据)作为两个独立验证集。利用R软件将获得的测序原始数均一化处理，并将探针与基因名称进行转换，将表达矩阵进行log2对数转换，采用Limma包鉴定差异表达基因(筛选条件：p<0.05和|logFC|>0.5)。韦恩图展示2个数据集中的共享基因。利用在线软件对差异基因进行GO和KEGG富集分析。The data expression profiles of atherosclerosis andtype 2 diabetes were downloaded from the GEO database, GSE28829 (including carotid plaque sequencing data) and GSE20966 data sets (includingtype 2 diabetes sequencing data) were included as training sets, and GSE43292 (including carotid artery Arterial plaque sequencing data) and the GSE25724 dataset (containingtype 2 diabetes sequencing data) served as two independent validation sets. R software was used to normalize the obtained sequencing raw numbers, and the probes and gene names were converted, and the expression matrix was log2 logarithmically transformed, and the Limma package was used to identify differentially expressed genes (screening conditions: p<0.05 and |logFC| >0.5). Venn diagram showing shared genes in 2 datasets. GO and KEGG enrichment analysis of differential genes were performed using online software.

1.2蛋白互作分析及核心靶点的筛选1.2 Protein interaction analysis and screening of core targets

蛋白质相互作用网络(PPI)可以用来描述蛋白之间存在的物理联系，进一步理解疾病发生机制和药物发掘。根据STRING在线数据库，以交互分数>0.4获得PPI网络，并利用Cytoscape软件进行可视化。通过插件cytoHubba的8种算法(betweenness,bottleNeck,EPC,degree,MCC,MNC,radiality,stress)计算出了前10个核心基因，采用韦恩图分析核心基因。Protein Interaction Network (PPI) can be used to describe the physical connection between proteins, to further understand the mechanism of disease and drug discovery. According to the STRING online database, PPI networks were obtained with an interaction score >0.4 and visualized using Cytoscape software. The top 10 core genes were calculated through 8 algorithms (betweenness, bottleNeck, EPC, degree, MCC, MNC, radiality, stress) of the plug-in cytoHubba, and the core genes were analyzed by Venn diagram.

1.3共享核心基因的验证1.3 Verification of shared core genes

对上述取交集获得的核心基因，在GSE43292和GSE25724两个数据集中的表达量进行验证。将上述2个数据集中的数据进行归一化和标准化处理后，绘制箱线图检测核心基因的表达量，以P<0.05为差异具有显著性。For the core genes obtained by the above intersection, the expression levels in the two data sets GSE43292 and GSE25724 were verified. After normalizing and standardizing the data in the above two data sets, a box plot was drawn to detect the expression of core genes, and P<0.05 was regarded as a significant difference.

1.4RT-qPCR检测血液中核心基因的mRNA水平1.4 RT-qPCR detection of mRNA levels of core genes in blood

收集健康人和动脉粥样硬化及2型糖尿病患者的血液，提取RNA，逆转录后，通过荧光定量PCR仪进行扩增；引物由生工生物科技有限公司设计，序列如表1所示。PCR条件：95℃预变性3min，95℃变性10s、60℃退火及延伸34s，扩增45个循环。根据2-^△△Ct值进行结果分析。目的基因的相对量＝2-^△△Ct，△△Ct＝[Ct(待测样品)-Ct GAPDH(待测样品)]-[Ct(校正样品)-Ct GAPDH(校正样品)]。The blood of healthy people and patients with atherosclerosis andtype 2 diabetes was collected, RNA was extracted, and after reverse transcription, it was amplified by a fluorescent quantitative PCR instrument; the primers were designed by Sangon Biotechnology Co., Ltd., and their sequences are shown in Table 1. PCR conditions: pre-denaturation at 95°C for 3min, denaturation at 95°C for 10s, annealing and extension at 60°C for 34s, amplification for 45 cycles. The results were analyzed according to the 2-^ΔΔCt value. The relative amount of the target gene=2-^ΔΔCt , ΔΔCt=[Ct (test sample)-Ct GAPDH (test sample)]-[Ct (calibration sample)-Ct GAPDH (calibration sample)].

表1特异性扩增CD4和PLEK基因的引物Table 1 Primers for specific amplification of CD4 and PLEK genes

2.统计学处理2. Statistical processing

数据以均数±标准差

表示。两样本均数间比较采用两样本独立t检验，以P<0.05为差异有显著性。Data are presented as mean ± standard deviation

express. Two-sample means were compared using two-sample independent t-test, and P<0.05 was considered significant.

3.结果3. Results

3.1动脉粥样硬化和2型糖尿病共享基因3.1 Shared genes between atherosclerosis andtype 2 diabetes

对GEO数据集中的表达矩阵矫正、标准化处理后，从GSE28829数据集中鉴定出965个差异表达基因(602个上调和363个下调)，从GSE20966数据集中鉴定出644个差异表达基因(373个上调和271个下调)。使用维恩图提取2个数据集共有的差异表达基因作为潜在的交集基因，结果显示有34个基因在2个数据集都存在差异表达，其中28个上调，6个下调(图1)。After correcting and normalizing the expression matrix in the GEO data set, 965 differentially expressed genes (602 up-regulated and 363 down-regulated) were identified from the GSE28829 data set, and 644 differentially expressed genes (373 up-regulated and 363 down-regulated) were identified from the GSE20966 data set. 271 down). Using the Venn diagram to extract the differentially expressed genes common to the two data sets as potential intersection genes, the results showed that 34 genes were differentially expressed in both data sets, of which 28 were up-regulated and 6 were down-regulated (Figure 1).

3.2共享基因特征及功能3.2 Shared gene features and functions

动脉粥样硬化和2型糖尿病相关数据集中有34个基因重叠，为了探索共享基因的潜在功能，进行了GO富集分析。结果显示，在生物过程(BP)的变化中，大部分基因主要富集在细胞因子产生的调节、免疫应答和白细胞活化；在细胞成分(CC)中，大部分基因富集于分泌颗粒膜和细胞前缘；在分子功能(MF)中，大部分基因功能与磷脂酰肌醇结合和磷脂结合相关。KEGG主要富集的通路是：脂质与动脉粥样硬化、细胞因子-细胞因子受体相互作用(图2)。综上所述，这些结果表明，炎症通路和免疫激活参与了动脉粥样硬化和2型糖尿病的发展。There were 34 overlapping genes in the atherosclerosis andtype 2 diabetes related datasets, and GO enrichment analysis was performed to explore the potential functions of the shared genes. The results showed that in the changes of biological process (BP), most genes were mainly enriched in regulation of cytokine production, immune response and leukocyte activation; in cellular components (CC), most genes were enriched in secretory granule membrane and Cell leading edge; in molecular function (MF), most gene functions are related to phosphatidylinositol binding and phospholipid binding. The main KEGG-enriched pathways were: lipid and atherosclerosis, cytokine-cytokine receptor interaction (Fig. 2). Taken together, these results suggest that inflammatory pathways and immune activation are involved in the development of atherosclerosis andtype 2 diabetes.

3.3关键基因筛选及验证3.3 Screening and verification of key genes

利用STING数据库分析所有共享基因，构建PPI网络，共包括47个节点，根据Cytohubba插件，采用8种拓扑分析方法(betweenness,bottleNeck,EPC,degree,MCC,MNC,radiality,stress)分析前10位基因，最终交叉得到8个核心基因，即CD4,CXCL8,DOCK8,ITGB2,NCF4,PLEK,THY1和TYROBP(图3)。为了验证这些核心基因表达水平的可靠性，我们选择了另外两个动脉粥样硬化和2型糖尿病的数据集，并分析了这些核心基因的表达水平。结果表明，在2型糖尿病数据集GSE20966中，只有CD4，PLEK和THY1基因表达上调(图4)；在动脉粥样硬化相关数据集GSE43292中，所有核心基因表达均显著上调(图5)Use the STING database to analyze all shared genes, build a PPI network, including a total of 47 nodes, anduse 8 topological analysis methods (betweenness, bottleNeck, EPC, degree, MCC, MNC, radiality, stress) to analyze the top 10 genes according to the Cytohubba plug-in , and finally crossed to get 8 core genes, namely CD4, CXCL8, DOCK8, ITGB2, NCF4, PLEK, THY1 and TYROBP (Figure 3). To verify the reliability of these core gene expression levels, we selected two other datasets of atherosclerosis andtype 2 diabetes and analyzed the expression levels of these core genes. The results showed that in thetype 2 diabetes dataset GSE20966, only CD4, PLEK and THY1 genes were upregulated (Figure 4); in the atherosclerosis-related dataset GSE43292, all core gene expressions were significantly upregulated (Figure 5)

3.4RNA提取和RT-qPCR检测3.4 RNA extraction and RT-qPCR detection

收集健康人和2型糖尿病伴动脉粥样硬化患者的血液，提取RNA，采用RT-qPCR检测CD4，PLEK和THY1的表达，结果如图6所示，与健康人相比，CD4和PLEK在疾病组中表达显著上调，同时，我们分析了CD4和PLEK诊断能力，ROC曲线结果显示CD4和PLEK的AUC值分别为0.733及0.790，表明其具有较高诊断价值。The blood of healthy people and patients withtype 2 diabetes and atherosclerosis was collected, RNA was extracted, and the expression of CD4, PLEK and THY1 was detected by RT-qPCR. The results are shown in Figure 6. The expression in the group was significantly up-regulated. At the same time, we analyzed the diagnostic capabilities of CD4 and PLEK. The ROC curve results showed that the AUC values of CD4 and PLEK were 0.733 and 0.790, respectively, indicating that they have high diagnostic value.

尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (6)

1. The application of the detection reagent in preparing the diagnostic tool for type 2 diabetes mellitus with atherosclerosis is characterized in that the detection reagent is used for detecting the expression quantity of a CD4 gene and a PLEK gene or detecting the expression quantity of a CD4 protein and a PLEK protein.

2. The use of claim 1, wherein the test agent is selected from the group consisting of an oligonucleotide probe that specifically recognizes the CD4 gene and the PLEK gene, a primer that specifically amplifies the CD4 gene and the PLEK gene, and a binding agent that specifically binds to the CD4 protein and the PLEK protein in the sample.

3. The use of claim 2, wherein the primer sequence for specific amplification of the CD4 gene is shown as SEQ ID No.1-2, and the primer sequence for specific amplification of the PLEK gene is shown as SEQ ID No. 3-4.

4. A kit for diagnosing type 2 diabetes with atherosclerosis is characterized by comprising a detection reagent for detecting the expression quantity of a CD4 gene and a PLEK gene or the expression quantity of a CD4 protein and a PLEK protein; the test detection agent is selected from an oligonucleotide probe which specifically recognizes the CD4 gene and the PLEK gene, a primer which specifically amplifies the CD4 gene and the PLEK gene or a binding agent which specifically binds to the CD4 protein and the PLEK protein in a sample.

5. Use of an inhibitor of the expression of the CD4 protein and the PLEK protein of claim 1 in the manufacture of a medicament for the treatment of type 2 diabetes with atherosclerosis.

6. A medicament for treating type 2 diabetes with atherosclerosis, which comprises the effective components of the CD4 protein and the PLEK protein expression inhibitor in claim 1.

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