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Method for quantitatively detecting expression levels of BST1, STAB1 and TLR4 genes and application

A gene expression level, STAB1 technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of poor sensitivity

Active Publication Date: 2020-10-30
吴式琇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports on the markers of lung cancer, such as carcinoembryonic antigen (CEA), SCC, cytokeratins (CYFRA, TPA, and TPS), etc. can be used as markers for early diagnosis of non-small cell lung cancer, neuron-specific enolization Enzyme (NSE) and ProGRP can be used for the early diagnosis of small cell lung cancer, but these markers are not ideal for the following reasons: they are not specific to lung cancer, and have abnormal expression in other tumors; the sensitivity is poor; Whether there is a relationship between the

Method used

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  • Method for quantitatively detecting expression levels of BST1, STAB1 and TLR4 genes and application
  • Method for quantitatively detecting expression levels of BST1, STAB1 and TLR4 genes and application
  • Method for quantitatively detecting expression levels of BST1, STAB1 and TLR4 genes and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 The present invention found that compared with healthy normal people and tuberculosis patients, the peripheral blood CD8+PD-1+T cells of tumor patients are highly expressed.

[0028] Reagents and Materials

[0029] Ficoll-Paque lymphocyte separation medium (GE), red blood cell lysate (BD PMG, catalog number 555899), flow cytometry antibody CD3 APC-H7 (BD PMG, catalog number 560176), CD4 FITC (BD PMG, catalog number 556615), CD8 PerCP (BDIS, Cat. No. 652829), PD1 PE-Cy TM 7 (BD PMG, Cat. No. 561272), where IgG1κPE-Cy TM 7 (BD PMG, Cat. No. 557872) as Isotype Control.

[0030] experimental method:

[0031] Collection and preparation of materials: 248 cases of tumor patients, 620 cases of healthy persons and 272 cases of pulmonary tuberculosis were collected from peripheral blood samples, and 5ml of venous blood was collected and placed in a blood routine tube after anticoagulation with heparin, and then separated with lymphocyte separation fluid. The narrow ...

Embodiment 2

[0035] Example 2 The present invention found that BST1, STAB1 and TLR4 genes were highly expressed in peripheral blood CD8+PD-1+ cells of patients with early lung cancer.

[0036] Reagents and Materials

[0037] See Example 1.

[0038] experimental method:

[0039] Collection and preparation of materials: We selected peripheral blood samples from 17 patients with early lung cancer and 9 normal persons who underwent physical examination in Example 1. The follow-up treatment was the same as in Example 1. 5ml of venous blood was drawn and placed in a blood routine tube after anticoagulation with heparin. , use lymphocyte separation fluid to separate the narrow band of the white cloud layer located at the interface between the upper and middle layers, that is, mononuclear cells (including lymphocytes and monocytes). After being treated with erythrocyte lysate, the pellet was resuspended with PBS, and 20ul was taken for cell counting.

[0040] Flow cytometry antibody staining an...

Embodiment 3

[0054] Example 3 RT-qPCR verification of expression levels of 14 differential genes in lung cancer CD3+CD8+PD1+ cells

[0055] Reagents and Materials

[0056] 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.

[0057] experimental method

[0058] See Example 1 for the processing of peripheral blood, flow cytometry antibody staining and on-machine detection, and obtain CD3+CD8+PD1+ cells after flow cytometry sorting.

[0059] RNA extraction

[0060] Extract total RNA according to the standard operation of Qiagen RNeasy Micro Kit. After sorting, add Buffer RLT containing 350 μl β-ME and 350 μL 70% ethanol to the cells, shake vigorously and mix well, pass through MinElute filter column, centrifuge at ≥10,000 rpm for 15 seconds, discard waste liquid. Add 350μL Buffer RW1, centrifuge at ≥10000rpm for 15s, and discard the waste. Ad...

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Abstract

The invention discloses a method for quantitatively detecting expression levels of BST1, STAB1 and TLR4 genes and application, and particularly relates to application of the BST1, STAB1 and TLR4 genesin a reagent for diagnosing early lung cancer. Lung cancer is the main cause of cancer death in most countries, and early diagnosis and early treatment are particularly important. According to the occurrence and development mechanism of lung cancer, T cells interacting with lung cancer are used as research objects, and a brand-new visual angle is provided for lung cancer diagnosis. The method comprises the following steps of: firstly, detecting an early lung cancer patient through a transcriptome sequencing technology, and sorting peripheral blood of a normal person to obtain differential expression genes in CD8 + PD-1 + T cells; secondly, verifying a sequencing result through transcriptome trace library building and an RT-qPCR technology; and increasing the sample size again, and determining the gene spectrum of differential expression in CD8 + PD-1 + T cells. And a new marker is provided for early diagnosis of lung cancer.

Description

technical field [0001] The invention relates to the technical field of gene diagnosis, in particular to the application of a group of gene profiles expressed in tumor-interacting T cells in early lung cancer diagnostic reagents, and more specifically to the construction of transcriptome trace databases to detect a very small amount of CD8+PD-1+T Differentially expressed genes in the cells, and the gene spectrum BST1, STAB1 and TLR4 were applied to the development of detection reagents for the diagnosis of early lung cancer. Background technique [0002] According to the latest global cancer assessment report released by the International Agency for Research on Cancer in 2018, lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world. Statistics from the National Cancer Center of my country in 2017 also show that lung cancer ranks first in cancer incidence and mortality. At present, early stage patients are treated with surgical resecti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2531/113C12Q2563/107
Inventor 吴式琇
Owner 吴式琇
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