WO2021243951A1 - 定量检测bst1、stab1和tlr4基因表达水平的方法及应用 - Google Patents

定量检测bst1、stab1和tlr4基因表达水平的方法及应用 Download PDF

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WO2021243951A1
WO2021243951A1 PCT/CN2020/126875 CN2020126875W WO2021243951A1 WO 2021243951 A1 WO2021243951 A1 WO 2021243951A1 CN 2020126875 W CN2020126875 W CN 2020126875W WO 2021243951 A1 WO2021243951 A1 WO 2021243951A1
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bst1
stab1
lung cancer
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tlr4
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吴式琇
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  • the present invention relates to the technical field of gene diagnosis, in particular to the application of a set of gene profiles expressed in tumor-interacting T cells in early lung cancer diagnosis reagents, and more specifically to the establishment of a transcriptome micro-database to detect a very small amount of CD8+PD-1+T Differentially expressed genes in cells, the gene profiles BST1, STAB1 and TLR4 are used to develop detection reagents for the diagnosis of early lung cancer.
  • Peripheral blood T cells also have abnormalities in the PD-1/PD-L1 pathway, which are often expressed as PD1 positive expression of CD8-positive T lymphocytes (CD8+T cells), which may interact with the secretion of T cells and tumors.
  • CD8+T cells CD8-positive T lymphocytes
  • the positive expression of PD1 on CD8+ T cells can also be found.
  • CD8+ T cells will also express PD1. Therefore, the tumor-interacting T cell gene expression profile that distinguishes normal population and early lung cancer can be used to detect early lung cancer, and the peripheral blood facilitates the development of practical diagnostic reagents.
  • the second-generation sequencing technology is one of the important methods for high-throughput screening of molecular markers in recent years, and has been widely used in the study of the mechanism of tumorigenesis and development.
  • lung cancer markers such as carcinoembryonic antigen (CEA), SCC, cytokeratin cytokeratins (CYFRA, TPA, and TPS), etc.
  • CEA carcinoembryonic antigen
  • SCC SCC
  • cytokeratin cytokeratins CYFRA, TPA, and TPS
  • NSE neuron-specific enolization Enzymes
  • ProGRP neuron-specific enolization Enzymes
  • the purpose of the present invention is to provide a method and application for quantitatively detecting the expression levels of BST1, STAB1 and TLR4 genes and the application of a preparation for detecting BST1, STAB1 and TLR4 genes in preparing a preparation for diagnosis of lung cancer.
  • the lung cancer diagnostic preparation includes the use of trace RNA library building, RNA-seq sequencing, and fluorescence quantitative PCR methods to detect the expression of BST1, STAB1 and TLR4 genes in peripheral blood CD8+PD-1+T cells.
  • the invention aims to find a bio-marker with strong specificity and high sensitivity, which is expected to improve the early diagnosis of lung cancer.
  • PD-1 positive CD8 positive T lymphocytes referred to as CD8+PD-1+T cells
  • CD8+PD-1+T cells PD-1 positive CD8 positive T lymphocytes
  • RNA-seq sequencing RNA-seq sequencing.
  • the sequencing results were verified by RT-qPCR technology;
  • the sample size was expanded to determine the differentially expressed gene profiles (BST1, STAB1 and TLR4) in CD8+PD-1+T cells, and the BST1, STAB1 and TLR4 gene profiles As a new target for the early diagnosis and treatment of lung cancer.
  • a method for quantitatively detecting the expression levels of BST1, STAB1 and TLR4 genes in CD8+PD-1+T cells including: extracting and separating (through flow sorting) from isolated peripheral blood samples to obtain CD8+PD-1+ T cells, using fluorescent quantitative PCR method to detect the expression levels of BST1, STAB1 and TLR4 genes in CD8+PD-1+T cells;
  • the BST1 gene is a human BST1 gene.
  • the BST1 gene sequence information is provided by the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • the gene is located on chromosome 4, the genome number is NC_000004.12, and the sequence information website is (https:// www.ncbi.nlm.nih.gov/gene/683);
  • the STAB1 gene is a human STAB1 gene.
  • the sequence information of the STAB1 gene is provided by the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • the gene is located on chromosome 3.
  • the genome number is NC_000003.12.
  • the sequence information website is (https:// www.ncbi.nlm.nih.gov/gene/23166);
  • the TLR4 gene is a human TLR4 gene.
  • the TLR4 gene sequence information is provided by the National Center for Biotechnology Information (NCBI). The gene is located on chromosome 9 and the genome number is NC_000009.12. The sequence information website is (https:// www.ncbi.nlm.nih.gov/gene/7099).
  • the described fluorescence quantitative PCR method detects the CDS regions of BST1, STAB1 and TLR4 genes, and contains three pairs of specific amplification primers:
  • the upstream primer sequence of the primer of the BST1 gene is TGGGAAAATAGCCACCTCCT, and the downstream primer sequence is CCCTGCCATACAGAACATCG;
  • the upstream primer sequence of the STAB1 gene primer is CAACATTAGTGGGAGGGTCTGG, and the downstream primer sequence is GGGCACAAAGATGGTGTAGGC;
  • the upstream primer sequence of the primer of the TLR4 gene is AGACCTGTCCCTGAACCCTATG, and the downstream primer sequence is TTAGACCTGTCCCTGAACCCTA.
  • the diagnostic agent for lung cancer adopts a fluorescent quantitative PCR method to detect the expression of BST1, STAB1 and TLR4 genes.
  • the described application, fluorescent quantitative PCR method to detect the CDS region of BST1, STAB1 and TLR4 genes contains three pairs of specific amplification primers: the sequence of the upstream primer of the BST1 gene is TGGGA AAATAGCCACCTCCT, and the sequence of the downstream primer is CCCTGCCATACAGAACATCG; the sequence of the STAB1 gene
  • the upstream primer sequence of the primer is CAACATTAGTGGGAGGGTCTGG, and the downstream primer sequence is GGGCACAAAGATGGTGTAGGC; the upstream primer sequence of the TLR4 gene primer is AGACCTGTCCCTGAACCCTATG, and the downstream primer sequence is TTAGACCTGTCCCTGAACCCTA.
  • the present invention has the following advantages:
  • peripheral blood samples as genetic testing objects is more convenient and practical.
  • the present invention uses T cells that interact with lung cancer as a research object based on the occurrence and development mechanism of lung cancer, and provides a new perspective for the diagnosis of lung cancer. Firstly, it is planned to detect the differentially expressed genes in CD8+PD-1+T cells from the peripheral blood of patients with early lung cancer and normal people through transcriptome sequencing technology; secondly, the sequencing results will be performed by transcriptome micro-banking and RT-qPCR technology. Validation; once again expand the sample size, determine the gene profile of differential expression in CD8+PD-1+T cells, and provide new markers for the early diagnosis of lung cancer.
  • Figure 1 is a schematic diagram of the proportion of CD8+PD1+ cells in peripheral blood of 248 tumor patients, 272 pulmonary tuberculosis patients, and 620 healthy subjects to CD8+ cells;
  • Figure 2 is a classification analysis diagram of the support vector machine algorithm for 17 patients with early lung cancer and 9 healthy patients on physical examination; among them, Batch represents batch, a total of three batches; health represents healthy people on physical examination, lung represents early lung cancer patients;
  • Figure 3 shows the high expression of BST1, STAB1 and TLR4 genes in CD3+CD8+PD1+ cells in peripheral blood of 31 cases of lung cancer.
  • Example 1 The present invention found that compared with healthy people and tuberculosis patients on physical examination, the peripheral blood CD8+PD-1+T cells of tumor patients are highly expressed.
  • Ficoll-Paque Lymphocyte Separation Solution GE
  • Red Blood Cell Lysis Solution BD PMG, Item No. 555899
  • Flow Cytometry Antibody CD3 APC-H7 BD PMG, Item No. 560176,
  • CD4 FITC BD PMG, Item No. 556615
  • CD8 PerCP BD IS, article number 652829
  • PD1 PE-Cy TM 7 (BD PMG, article number 561272), of which IgG1 ⁇ PE-Cy TM 7 (BD PMG, article number 557872) is used as Isotype Control.
  • Material collection and preparation Collect peripheral blood samples of 248 tumor patients, 620 healthy subjects and 272 pulmonary tuberculosis patients. Take 5ml of venous blood and place it in a blood routine tube after heparin anticoagulation, and then separate it with lymphocyte separation solution. The narrow band of white cloud layer at the middle interface is monocytes (including lymphocytes and monocytes). After the red blood cell lysis solution is processed, the pellet is resuspended in PBS, and 20ul is taken for cell count. Perform the same operation as above.
  • Flow cytometric antibody staining and machine detection staining according to 1 ⁇ 10 6 /test, set single positive group, Isotype Control group and sample group, (single positive tube: CD3/5ul or CD4/20ul or CD8/20ul or PD1/ 5ul; Isotype Control tube IgG1 ⁇ PE-Cy TM 7/1ul; sample tube: CD3/5ul+CD4/20ul+CD8/20ul+PD1/5ul). Resuspend the monocytes with an appropriate amount of 3% BSA solution and transfer them to a flow tube. Use BD FCS AriaIII for analysis to calculate and collect the number of CD3+CD8+PD1+ cells.
  • Example 2 The present invention found that the BST1, STAB1 and TLR4 genes are highly expressed in CD8+PD-1+ cells in the peripheral blood of patients with early lung cancer.
  • Example 2 Materials collection and preparation: We selected the peripheral blood samples of 17 patients with early lung cancer and 9 healthy healthy persons in Example 1, and the follow-up processing was the same as that in Example 1. 5ml of venous blood was drawn and placed in the blood routine tube after heparin anticoagulation. , Use the lymphocyte separation solution to separate the narrow band of the white cloud layer located at the upper and middle layer interface, that is, monocytes (including lymphocytes and monocytes). After the red blood cell lysis solution is processed, the pellet is resuspended in PBS, and 20ul is taken for cell count.
  • Flow cytometric antibody staining and machine detection The staining steps are the same as in Example 1, using BD FCS AriaIII to analyze, count and sort CD3+CD8+PD1+ cells, collect CD3+CD8+PD1+ cells, mix them with RNAlatter reagent and freeze them Keep at -80°C for later use.
  • Transcriptome sequencing complete transcriptome RNA extraction, Dr GenTLE precipitation, double-stranded cDNA synthesis, end repair, addition of A and adaptors, fragment selection and PCR amplification, XP magnetic bead purification of amplified products, library quality testing, and after the products are qualified , According to Illumina company's HiSeq 2500 high-throughput sequencing platform standard process on-machine sequencing.
  • FPKM expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced
  • RPKM is the number of reads aligned to the gene divided by the number of reads aligned to the genome (in millions) and the length of RNA (in kb).
  • the correlation coefficient is approximately 1, indicating that the higher the similarity of the expression patterns between samples, it also indicates the reliability of the experiment and the rationality of sample selection. According to the FPKM value of all genes in each sample, the correlation coefficient of samples within and between groups was calculated using the Pearson Correlation method.
  • the analysis data of gene differential expression is the read data in gene expression level analysis, not FPKM.
  • DESeq Anders et al, 2010
  • FoldChange based on the standardized read count average, and also calculates the significance p value of each gene in all comparison combinations.
  • pval pval
  • p-value qvalue/padj
  • Count the number of differential genes (up-regulated and down-regulated genes) for each comparison combination, and then set the log2FoldChange threshold and padj value to screen for differentially expressed genes.
  • the threshold setting is generally:
  • SVM Support vector machine
  • SVM support vector machine algorithm
  • the peripheral blood of 31 cases of lung cancer, 4 cases of pulmonary granulomatous inflammation and 17 cases of pulmonary tuberculosis were collected.
  • the collection standard is shown in Example 1.
  • Example 1 The processing of peripheral blood, flow cytometry antibody staining and machine detection are shown in Example 1. After flow cytometry, CD3+CD8+PD1+ cells are obtained.
  • the total RNA was extracted according to the standard operation of QIAGEN RNeasy Micro Kit. After sorting, the cells were added with 350 ⁇ l ⁇ -ME-containing Buffer RLT and 350 ⁇ L 70% ethanol, vigorously shaken and mixed, passed through the MinElute filter column, centrifuged at ⁇ 10000rpm for 15s, discarded Waste liquid. Add 350 ⁇ L Buffer RW1, centrifuge for 15s at ⁇ 10000rpm, and discard the waste solution. Add 80 ⁇ L DNase I (10 ⁇ L) and RDD (70 ⁇ L) to the filter column membrane, let it stand for 15min, add 350 ⁇ L Buffer RW1 ⁇ 10000rpm and centrifuge for 15s, discard the waste liquid.
  • RNA is obtained, the micro-library is built, the steps are as follows: first add 1/10 volume of 3M sodium Acetate (PH 5.2), 2.5 times volume of absolute ethanol, 4 ⁇ L Dr GenTLE, mix well, 12000rpm, 4°C, centrifuge for 15min, Discard the supernatant and leave a white precipitate. Add 70% ethanol, 12000rpm, 4°C, centrifuge for 5min, discard the supernatant, and dry the pellet. Reconstitute with 2 ⁇ L of water and wait for SMART-seq2. Mix the Dr GenTLE precipitation product with 1 ⁇ L 10mM dNTP, 1 ⁇ L 10uM Oligo-dT Primer and add 2 ⁇ L of lysis buffer. Place the 4 ⁇ L system in a PCR machine and incubate at 72°C for 3min. The heating cover temperature is 75°C. Immediately after the lysis is complete Place on ice for 1 min.
  • Reaction system 500ng total RNA (volume calculated by concentration), 2 ⁇ L 5 ⁇ SuperScript II First-Strand Buffer, 2 ⁇ L 5M Betaine, 0.9 ⁇ L 100mM MgCl 2 , 0.25 ⁇ L 100mM DTT, 0.1 ⁇ L 100uM TSO, 0.25 ⁇ L 40U/ ⁇ L RNAse inhibitor , 0.5 ⁇ L 200U/ ⁇ L SSII, the reaction system is 6 ⁇ L, the hot lid temperature is 75°C, the reaction conditions are as follows, as shown in Table 1:
  • Amplification system 10 ⁇ L reverse transcription product, 12.5 ⁇ L 5X KAPA HiFi HotStart ReadyMix, 0.25 ⁇ L 10uM IS PCR Primer, 2.25 ⁇ L Nuclease-free water. 25 ⁇ L of the total system, pre-amplified according to the following conditions, as shown in Table 2:
  • Resuspend AmPure XP Beads at room temperature take 25 ⁇ L in a 1:1 ratio into a 1.5mL centrifuge tube, add the pre-amplified product in the 25 ⁇ L centrifuge tube to the magnetic beads and gently pipette to mix 10 times, and incubate for 8min at room temperature; Place the centrifuge tube on a magnetic stand for 5 minutes and absorb after the liquid is clarified; add 200 ⁇ L of 80% ethanol without disturbing the magnetic beads, let it stand for 30 seconds, aspirate, and repeat the washing; dry at room temperature for 3 ⁇ 5min, after the magnetic beads are slightly cracked, remove the centrifuge tube, add 19 ⁇ L of water, pipetting and mixing, and let stand for 2min at room temperature; magnetic stand for 2min, take out the liquid (19ul) into a clean centrifuge tube; take 1ul for Qubit and 1ul are used for 2100 detection.
  • the qualified result is: the band ranges from 500 bp to 7 kb, among which a peak appears near 1.5 to 2 k
  • RT-qPCR Real Time quantitative polymerase chain reaction
  • RT-qPCR can be performed on the obtained reverse transcription samples, the system and steps are as follows:
  • the PCR amplification procedure is as follows, as shown in Table 4:
  • RT-qPCR products should be analyzed by 2% agarose gel electrophoresis to identify the correct size fragments to ensure the accuracy and specificity of the PCR products.
  • the relative quantitative analysis of gene expression was performed with Microsoft Excel. The expression of target gene (T) was used as a reference with the expression of reference gene (R), and the data was calculated as 2 Ct R-Ct T.
  • BST1 also called ADP-ribosyl cyclase 2
  • BST1 is a bifunctional extracellular enzyme for lipid misspecification, which can catalyze the ribonucleotide ring ⁇ hydrolysis.
  • BST1 is expressed in acute myeloid leukemia, more than 90% of primary epithelial ovarian cancer and malignant pleural mesothelioma, and its expression level is related to the prognosis of the disease, and CD157 is currently being studied as an acute myeloid cell The target of immunotherapy for sexual leukemia.
  • STAB1 is a type of scavenger receptor, its expression affects the ability of macrophages to phagocytosis, and has been confirmed to be a receptor for phagocytosis, involved in apoptosis and phagocytosis of senescent cells.
  • TLR4 is the most studied member of the Toll-like receptor family. It is mainly expressed on cell membranes, especially dendritic cells and macrophages. It recognizes lipopolysaccharides in the form of lipopolysaccharide-binding protein-CD14-TLR4 trimers and combines Inflammation signals are introduced into cells, which are involved in the evolution of many diseases, especially in inflammation and host defense responses.
  • BST1 (CD157) is also called ADP-ribosyl cyclase 2, which is a bifunctional extracellular enzyme of lipid misspecification. It can catalyze the cyclization of ribonucleotides. And hydrolysis. BST1 is expressed in acute myeloid leukemia, more than 90% of primary epithelial ovarian cancer and malignant pleural mesothelioma, and its expression level is related to the prognosis of the disease, and CD157 is currently being studied as an acute myeloid cell The target of immunotherapy for sexual leukemia.
  • STAB1 is a type of scavenger receptor, its expression affects the ability of macrophages to phagocytosis, and has been confirmed to be a receptor for phagocytosis, involved in apoptosis and phagocytosis of senescent cells.
  • TLR4 is the most studied member of the Toll-like receptor family. It is mainly expressed on cell membranes, especially dendritic cells and macrophages. It recognizes lipopolysaccharides in the form of lipopolysaccharide-binding protein-CD14-TLR4 trimers and combines Inflammation signals are introduced into cells, which are involved in the evolution of many diseases, especially in inflammation and host defense responses.

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Abstract

提供一种定量检测BST1、STAB1和TLR4基因表达水平的方法及应用，具体涉及BST1、STAB1和TLR4基因在诊断早期肺癌试剂中的应用。肺癌是绝大多数国家主要的癌症死亡原因，早期诊断和早期治疗尤为重要。为了根据肺癌的发生和发展机制，将与肺癌相互作用的T细胞作为研究对象，为肺癌的诊断提供全新视角。首先拟通过转录组测序技术检测早期肺癌病人、正常人外周血经分选后获得CD8+PD-1+T细胞中差异表达基因；其次通过转录组微量建库和RT-qPCR技术对测序结果进行验证；再次扩大样本量，确定CD8+PD-1+T细胞中差异性表达的基因谱，为肺癌的早期诊断提供新的标志物。

Description

定量检测BST1、STAB1和TLR4基因表达水平的方法及应用 技术领域
本发明涉及基因诊断技术领域，具体涉及一组表达于肿瘤相互作用T细胞的基因谱在早期肺癌诊断试剂中的应用，更具体涉及转录组微量建库实现检测极少量CD8+PD-1+T细胞中差异性表达基因，将该基因谱BST1、STAB1和TLR4应用于开发诊断早期肺癌的检测试剂。
背景技术
2018年国际癌症研究机构公布的最新全球癌症评估报告显示，肺癌是世界上发病率和死亡率都位居前列的恶性肿瘤之一。我国国家癌症中心2017年的统计数据同样显示肺癌是高居癌症发病率和死亡率之首。目前，早期患者通过手术切除，并辅助以化疗，效果良好；但对晚期已转移的患者尚缺乏有效的治疗手段，且预后差。因此，亟待寻找早期诊断肺癌病变的手段以减少肺癌发病率和死亡率，如何将分子生物学的技术用于肺癌早期诊断，提高早期肺癌的检出率是当前研究的热点之一。
近年来研究，对细胞程序性死亡受体1(PD-1；也称为CD279)及其配体PD-L1(B7-H1或CD274)通路在肿瘤免疫逃逸的作用有了深入理解，在肺癌外周血的T细胞也有PD-1/PD-L1通路的异常，常表现为CD8阳性T淋巴细胞(CD8+T细胞)的PD1阳性表达，可能与T细胞与肿瘤的分泌相互作用而形成，在早期肺癌的形成过程中，也可发现CD8+T细胞的PD1阳性表达。此外对于正常人群有慢病毒感染的，CD8+T细胞也会表达PD1。因此区分正常人群和早期肺癌的与肿瘤相互作用T细胞基因表达谱可以用于发现早期肺癌，外周血液方便研制实用的诊断试剂。
随着人类基因组学的解密，第二代测序技术是近年来高通量筛选分子标志物的重要方法之一，已被广泛应用到肿瘤发生、发展的机制研究中。
以胸部X光片、螺旋CT、支气管内镜、痰液细胞学等检测手段用于肺癌的筛检和早诊已有较多报道，但以上检查手段在敏感性、特异性、适用度等方面存在局限性，近年来国内外研究人员对有关肺癌早期诊断的分子标志 物做了大量有益的探索。肺癌的标志物已有很多报道，如癌胚抗原(CEA)、SCC、细胞角蛋白cytokeratins(CYFRA,TPA,and TPS)等可作为非小细胞肺癌的早诊标志物，神经元特异性烯醇化酶(NSE)、ProGRP则可用于小细胞肺癌的早期诊断，但这些标志物并没有很理想，原因如下：不是肺癌特异性的，在其他肿瘤中也存在异常表达；灵敏度较差；标志物之间是否存在相互联系不得而知。
发明内容
本发明的目的在于提供了一种定量检测BST1、STAB1和TLR4基因表达水平的方法及应用及检测BST1、STAB1和TLR4基因的制剂在制备诊断肺癌制剂中的应用。进一步，所述的肺癌诊断制剂包括用微量RNA建库、RNA-seq测序、荧光定量PCR方法检测外周血CD8+PD-1+T细胞中BST1、STAB1和TLR4基因的表达。
本发明旨在找到特异性强，灵敏度高的bio-marker，有望提高肺癌的早期诊断。首先拟通过转录组微量建库结合RNA-seq测序检测早期肺癌病人、正常人外周血经分选后获得PD-1阳性CD8阳性T淋巴细(简称CD8+PD-1+T细胞)中差异表达基因；其次通过RT-qPCR技术对测序结果进行验证；扩大样本量，确定CD8+PD-1+T细胞中差异性表达的基因谱(BST1、STAB1和TLR4)，将BST1、STAB1和TLR4基因谱作为肺癌的早期诊断和治疗提供新的靶点。
一种定量检测CD8+PD-1+T细胞中BST1、STAB1和TLR4基因表达水平的方法，包括：从离体的外周血样本中提取分离(通过流式分选)得到CD8+PD-1+T细胞，采用荧光定量PCR方法检测检测CD8+PD-1+T细胞中BST1、STAB1和TLR4基因的表达水平；
所述的BST1基因为人源BST1基因，BST1基因序列信息由美国国立生物技术信息中心(NCBI)提供，该基因位于四号染色体，基因组编号为NC_000004.12，序列信息网站为(https://www.ncbi.nlm.nih.gov/gene/683)；
所述的STAB1基因为人源STAB1基因，STAB1基因序列信息由美国国立生物技术信息中心(NCBI)提供，该基因位于三号染色体，基因组编号为NC_000003.12，序列信息网站为(https://www.ncbi.nlm.nih.gov/gene/23166)；
所述的TLR4基因为人源TLR4基因，TLR4基因序列信息由美国国立生物技术信息中心(NCBI)提供，该基因位于九号染色体，基因组编号为 NC_000009.12，序列信息网站为(https://www.ncbi.nlm.nih.gov/gene/7099)。
所述的荧光定量PCR方法检测BST1、STAB1和TLR4基因的CDS区域，含有三对特异性扩增的引物：
所述的BST1基因的引物上游引物序列为TGGGAAAATAGCCACCTCCT，下游引物序列为CCCTGCCATACAGAACATCG；
所述的STAB1基因的引物上游引物序列为CAACATTAGTGGGAGGGTCTGG，下游引物序列为GGGCACAAAGATGGTGTAGGC；
所述的TLR4基因的引物上游引物序列为AGACCTGTCCCTGAACCCTATG，下游引物序列为TTAGACCTGTCCCTGAACCCTA。
定量检测CD8+PD-1+T细胞中BST1、STAB1和TLR4基因表达水平的制剂在制备肺癌的诊断制剂(即肺癌早诊制剂)中的应用。
所述的应用，肺癌的诊断制剂采用荧光定量PCR方法检测BST1、STAB1和TLR4基因的表达。所述的应用，荧光定量PCR方法检测BST1、STAB1和TLR4基因的CDS区域，含有三对特异性扩增的引物：BST1基因的引物上游引物序列为TGGGA AAATAGCCACCTCCT，下游引物序列为CCCTGCCATACAGAACATCG；STAB1基因的引物上游引物序列为CAACATTAGTGGGAGGGTCTGG，下游引物序列为GGGCACAAAGATGGTGTAGGC；TLR4基因的引物上游引物序列为AGACCTGTCCCTGAACCCTATG，下游引物序列为TTAGACCTGTCCCTGAACCCTA。
与现有技术相比，本发明具有如下优点：
一、以免疫细胞为目标细胞，是肿瘤诊断新机制。
二、微量建库方法筛选差异基因有利于早期发现差异基因谱。
三、由于免疫细胞的异质性不大避免了肿瘤细胞的高异质性带来的敏感性和特异性问题。
四、利用外周血样本作为基因检测对象，较方便实用。
五、本发明为了根据肺癌的发生和发展机制，将与肺癌相互作用的T细胞作为研究对象，为肺癌的诊断提供全新视角。首先拟通过转录组测序技术检测早期肺癌病人、正常人外周血经分选后获得CD8+PD-1+T细胞中差异表 达基因；其次通过转录组微量建库和RT-qPCR技术对测序结果进行验证；再次扩大样本量，确定CD8+PD-1+T细胞中差异性表达的基因谱，为肺癌的早期诊断提供新的标志物。
附图说明
图1为248例肿瘤患者、272例肺结核患者与620例健康体检者外周血CD8+PD1+细胞占CD8+细胞的比例的示意图；
图2为支持向量机算法对17例早期肺癌患者及9例体检健康者的分类分析图；其中，Batch代表批次，共三个批次；health代表体检健康者，lung代表早期肺癌患者；
图3为31例肺癌外周血CD3+CD8+PD1+细胞中高表达BST1、STAB1和TLR4基因图。
具体实施方式
实施例1 本发明发现与体检健康的正常人、肺结核患者相比，肿瘤患者外周血CD8+PD-1+T细胞高表达。
试剂和材料
Ficoll-Paque淋巴细胞分离液(GE)，红细胞裂解液(BD PMG，货号555899)，流式抗体CD3 APC-H7(BD PMG，货号560176，)，CD4 FITC(BD PMG，货号556615)，CD8 PerCP(BD IS，货号652829)，PD1 PE-Cy TM7(BD PMG，货号561272)，其中IgG1κPE-Cy TM7(BD PMG，货号557872)作为Isotype Control。
实验方法：
材料的采集与准备：抽取248例肿瘤患者，620例健康体检者与272例肺结核患者的外周血样本，抽取5ml静脉血置于血常规管子肝素抗凝后，用淋巴细胞分离液分离出位于上中层界面处的那一白色云雾层的窄带，即单核细胞(包括淋巴细胞和单核细胞)。经红细胞裂解液处理后用PBS重悬沉淀，取20ul进行细胞计数。如上进行相同操作。
流式抗体染色与上机检测：按1×10 6/test进行染色，设置单阳组，Isotype Control组和样本组，(单阳管：CD3/5ul或者CD4/20ul或者CD8/20ul或者PD1/5ul；Isotype Control管IgG1κPE-Cy TM7/1ul；样本管：CD3/5ul+CD4/20ul+CD8/20ul+PD1/5ul)。用适量3％BSA溶液重悬单核细胞， 并转移至流式管内，采用BD FCS AriaIII进行分析，计算并收集CD3+CD8+PD1+细胞数量。
实验结果
我们发现健康者与肺结核患者的CD3+CD8+PD1+细胞占CD8+总量的平均值分别为0.19，0.23，肿瘤患者CD3+CD8+PD1+细胞比例显著高于体检健康者(2.02，p<0.001)，详见图1，如图1所示，流式结果显示肿瘤患者外周血PBMC中CD3+CD8+PD1+细胞数量明显高于正常人、。
实施例2 本发明发现早期肺癌患者外周血CD8+PD-1+细胞中高表达BST1、STAB1和TLR4基因。
试剂和材料
见实施例1。
实验方法：
材料的采集与准备：我们选取实施例1中17例早期肺癌患者及9例体检健康的正常人的外周血样本，后续处理同实施例1，抽取5ml静脉血置于血常规管子肝素抗凝后，用淋巴细胞分离液分离出位于上中层界面处的那一白色云雾层的窄带，即单核细胞(包括淋巴细胞和单核细胞)。经红细胞裂解液处理后用PBS重悬沉淀，取20ul进行细胞计数。
流式抗体染色与上机检测：染色步骤同实施例1，采用BD FCS AriaIII进行CD3+CD8+PD1+细胞的分析计数与分选，收集CD3+CD8+PD1+细胞，并用RNA latter试剂混匀后冻于-80℃备用。
转录组测序：进行全转录组RNA提取、Dr GenTLE沉淀、双链cDNA合成、末端修复、加A和接头、片段选择和PCR扩增、XP磁珠纯化扩增产物、文库质量检测，产物合格后，按照Illumina公司的HiSeq 2500高通量测序平台标准流程上机测序。
数据过滤和参考基因组比对
首先，为了保证数据分析的质量及可靠性，需要对raw reads进行过滤，去除含有接头adapter reads和去除低质量reads等，得到每个样本约12Gb clean data，Q30不低于99.9％。
然后，利用HISAT2软件(http://ccb.jhu.edu/software/hisat2/faq.shtml)将clean reads与UCSCH.sapiens参考基因组(hg19)进行快速精确的比对，进而获得reads在人参考基因组上的定位信息。同时，采用subread软件中的 featureCounts□具分别过滤掉比对质量值低于10的reads，非成对比对上的reads，以及比对到基因组多个区域的reads。该部分本项目所产生的测序reads成功比对率不低于70％(total_map>70％)。
基因表达水平分析
为了消除测序深度和基因长度因素对基因的表达量的影响，因此需要用一个参数FPKM(expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced)进行校正。RPKM是将比对到基因的reads数目除以比对到基因组上的所有reads数目(以million为单位)与RNA的长度(以kb为单位)。首先，利用软件Cufflinks v1.0.32将mapped reads进行FPKM标准化转化，一般FPKM>1认为基因表达的阈值。
为了衡量生物学重复性，我们需要进行样本间相关性分析，相关性系数约接近1，表明样品之间的表达模式的相似度越高，也说明实验的可靠性和样本选择的合理性。根据各样本所有基因的FPKM值用Pearson correlation方法计算组内及组间样本的相关性系数。
基因差异表达分析
基因差异表达的分析数据为基因表达水平分析中的reads数据，而不是FPKM。对于有生物学重复的样品，我们通常采用DESeq(Anders et al,2010)方法进行分析，其根据标准化的reads count均值计算FoldChange，同时也会对每个基因在所有比较组合的差异显著性p value(pval)以及多重假设检验校正后p值(qvalue/padj)进行分析。对每个比较组合的差异基因(上调和下调基因)进行数目统计，再设置log2FoldChange阈值和padj值来筛选差异表达的基因。对有生物学重复试验的差异基因进行筛选，阈值设定一般为:|log2(FoldChange)|>1且qvalue<0.05。
支持向量机(SVM)算法进行分类
我们使用支持向量机算法(SVM)对样本(健康与疾病)进行分类，该分类器是使用mlr包在R中实现的。
实验结果
与健康者相比，我们发现早期肺癌患者外周血CD8+PD-1+T细胞中14个差异表达基因BST1、STAB1、TLR4、ASIC1、ARHGEF10、CHST13、CLEC12A、CTSS、GCA、KLF4、PLEKHS1、PURA、TCN2、TLR8基因，且依赖于这14个基因，肿瘤与健康者有明显的分类趋势，且平均错误分类误差(mmce)小于0.01(图2)。这三个差异表达明显基因是我们研究的重 点。
实施例3 RT-qPCR验证肺癌CD3+CD8+PD1+细胞中14个差异基因的表达水平
试剂和材料
采集31例肺癌外周血、4例肺部肉芽肿性炎症及17例肺结核患者外周血，采集标准见实施例1。
实验方法
外周血的处理，流式抗体染色与上机检测见实施例1，流式分选后得到CD3+CD8+PD1+细胞。
RNA的提取
按凯杰RNeasy Micro Kit的标准操作抽提总RNA，分选后细胞加入配好含350μlβ-ME的Buffer RLT和350μL 70％乙醇，剧烈震荡混匀后过MinElute过滤柱，≥10000rpm离心15s，弃废液。加入350μL Buffer RW1，≥10000rpm离心15s，弃废液。滤柱膜上加入80μL DNase I(10μL)与RDD(70μL)，静置15min，加入350μL Buffer RW1≥10000rpm离心15s，弃废液。加入配好的RPE 500μL(RPE与4倍体积的乙醇混合)，≥10000rpm，离心15s，弃废液。加入80％的乙醇500μL，≥10000rpm，离心2min，弃废液。把滤柱放入新的1.5mL收集管，向膜的中心加入14μL无RNA水，静置2min，离心2min。
微量建库
Dr GenTLE沉淀
得到RNA后进行微量建库，步骤如下：首先加入1/10体积的3M sodium Acetate(PH 5.2)，2.5倍体积的无水乙醇，4μL Dr GenTLE，充分混匀，12000rpm，4℃，离心15min，弃上清，留白色沉淀。加入70％乙醇，12000rpm，4℃，离心5min，弃上清，干燥沉淀。2μL水复溶，待SMART-seq2。将Dr GenTLE沉淀产物与1μL 10mM dNTP，1μL 10uM Oligo-dT Primer混合后加入2μL裂解液，该4μL的体系置于PCR仪内，72℃，3min孵育，热盖温度为75℃，裂解完成后立即置于冰上1min。
逆转录
反应体系：500ng总RNA(按浓度计算体积)，2μL 5×SuperScript II First-Strand Buffer，2μL 5M Betaine，0.9μL 100mM MgCl 2，0.25μL 100mM  DTT，0.1μL 100uM TSO，0.25μL 40U/μL RNAse inhibitor，0.5μL 200U/μL SSII，反应体系为6μL，热盖温度75℃，反应条件如下，如表1所示：
表1
Figure PCTCN2020126875-appb-000001
此步后，所有mRNAs的第一链cDNA合成完毕。
PCR预扩增
扩增体系：10μL逆转录产物，12.5μL5X KAPA HiFi HotStart ReadyMix，0.25μL 10uM IS PCR Primer，2.25μL Nuclease-free water。总体系25μL，按下述条件预扩增，如表2所示：
表2
Figure PCTCN2020126875-appb-000002
XP磁珠纯化
室温下重悬AmPure XP Beads，按1:1比例取25μL到1.5mL离心管中，将25μL离心管中的预扩增产物加入到磁珠中轻轻吹打混匀10次，室温下孵育8min；将离心管置于磁力架上吸附5min，待液体澄清后，吸去；在不扰动磁珠的情况下加入200μL 80％乙醇，静置30s后，吸去，重复洗涤一遍；室温下，干燥3～5min，磁珠有轻微裂痕后即可取下离心管，加入19μL水，吹打混匀，室温静置2min；磁力架吸附2min，取出液体(19ul)至一洁净的离心管中；取1ul用于Qubit、1ul用于2100检测。合格结果为：条带范围在500bp～7kb，其中，在1.5～2kb附近会出现一个峰。
Real Time荧光定量PCR(Real time quantitative polymerase chain reaction，RT-qPCR)
得到的逆转录样品进行可进行RT-qPCR，体系及步骤如下：
在冰上配制如下反应体系，如表3所示：
表3
Figure PCTCN2020126875-appb-000003
PCR扩增程序如下，如表4所示：
表4
Figure PCTCN2020126875-appb-000004
使用新引物时需要验证引物的溶解曲线是否良好，要将RT-qPCR产物进行2％琼脂糖凝胶电泳分析，鉴定出正确大小的片段才能保证PCR扩增产物的准确性和特异性。用Microsoft Excel进行基因表达的相对定量分析，目的基因(target gene，T)的表达量以内参基因(reference gene，R)的表达量作为参照，数据计算为2 Ct R-Ct T。
实验结果：
BST1、STAB1、TLR4三个基因具有许多重要的功能，其中BST1(CD157)又叫ADP-核糖基环化酶2，是脂质错定的双功能胞外酶，它能够催化核糖核巧酸环化和水解。BST1在急性髓细胞性白血病、90％以上的原发性上皮性卵巢癌和恶性胸膜间皮瘤等中均有表达，且其表达水平与疾病的预后相关，且目前正在研究CD157作为急性髓细胞性白血病免疫疗法的靶标。STAB1是一类清道夫受体，其表达影响巨噬细胞噬菌作用的能力，并被证实是噬菌 作用的受体，参与凋亡和吞噬衰老细胞。TLR4是Toll样受体家族中研究最多的成员，它主要表达于细胞膜上，尤其是树突状细胞和巨噬细胞，以脂多糖结合蛋白-CD14-TLR4三聚体形式识别脂多糖，并将炎症信号传导入细胞，其参与多种疾病的演变过程，尤其在炎症和宿主防御反应等方面起着至关重要的作用。
我们研究发现31例肺癌外周血CD3+CD8+PD1+细胞中高表达BST1、STAB1和TLR4基因(ct值17-25)，4例肺部肉芽肿性炎症与17例肺结核患者外周血CD3+CD8+PD1+细胞中BST1、STAB1和TLR4基因低表达(ct值29-检测不到)，详见图3。该结果提示通过检测周血CD3+CD8+PD1+细胞中BST1、STAB1和TLR4基因组合的表达水平，可以区分肺癌和非肺癌患者。而这三个基因还具有其他许多重要的功能，其中BST1(CD157)又叫ADP-核糖基环化酶2，是脂质错定的双功能胞外酶，它能够催化核糖核巧酸环化和水解。BST1在急性髓细胞性白血病、90％以上的原发性上皮性卵巢癌和恶性胸膜间皮瘤等中均有表达，且其表达水平与疾病的预后相关，且目前正在研究CD157作为急性髓细胞性白血病免疫疗法的靶标。STAB1是一类清道夫受体，其表达影响巨噬细胞噬菌作用的能力，并被证实是噬菌作用的受体，参与凋亡和吞噬衰老细胞。TLR4是Toll样受体家族中研究最多的成员，它主要表达于细胞膜上，尤其是树突状细胞和巨噬细胞，以脂多糖结合蛋白-CD14-TLR4三聚体形式识别脂多糖，并将炎症信号传导入细胞，其参与多种疾病的演变过程，尤其在炎症和宿主防御反应等方面起着至关重要的作用。

Claims (5)

  1. 一种定量检测CD8+PD-1+T细胞中BST1、STAB1和TLR4基因表达水平的方法，其特征在于，包括：提取分离离体的外周血样本，通过流式分选得到CD8+PD-1+T细胞，采用荧光定量PCR方法检测检测CD8+PD-1+T细胞中BST1、STAB1和TLR4基因的表达水平。
  2. 根据权利要求1所述的定量检测CD8+PD-1+T细胞中BST1、STAB1和TLR4基因表达水平的方法，其特征在于，所述的荧光定量PCR方法检测BST1、STAB1和TLR4基因的CDS区域，含有三对特异性扩增的引物：
    所述的BST1基因的引物上游引物序列为TGGGAAAATAGCCACCTCCT，下游引物序列为CCCTGCCATACAGAACATCG；
    所述的STAB1基因的引物上游引物序列为CAACATTAGTGGGAGGGTCTGG，下游引物序列为GGGCACAAAGATGGTGTAGGC；
    所述的TLR4基因的引物上游引物序列为AGACCTGTCCCTGAACCCTATG，下游引物序列为TTAGACCTGTCCCTGAACCCTA。
  3. 定量检测CD8+PD-1+T细胞中BST1、STAB1和TLR4基因表达水平的制剂在制备肺癌早诊制剂中的应用。
  4. 根据权利要求3所述的应用，其特征在于，肺癌的诊断制剂采用荧光定量PCR方法检测BST1、STAB1和TLR4基因的表达。
  5. 根据权利要求4所述的应用，荧光定量PCR方法检测BST1、STAB1和TLR4基因的CDS区域，含有三对特异性扩增的引物：
    BST1基因的引物上游引物序列为TGGGA AAATAGCCACCTCCT，下游引物序列为CCCTGCCATACAGAACATCG；STAB1基因的引物上游引物序列为CAACATTAGTGGGAGGGTCTGG，下游引物序列为GGGCACAAAGATGGTGTAGGC；TLR4基因的引物上游引物序列为AGACCTGTCCCTGAACCCTATG，下游引物序列为TTAGACCTGTCCCTGAACCCTA。
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