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Monocyte chemoattractant protein (MCP) 2 used as marker for tubercular hydrothorax detection, and applications thereof

A tuberculosis and protein technology, applied in the field of medicine and diagnosis, can solve the problems of lack of analysis of immune cells and immune molecular systems, and the discovery of new diagnostic markers.

Inactive Publication Date: 2014-06-25
SHANGHAI PULMONARY HOSPITAL AFFILIATED TO TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are sporadic reports on the expression levels and diagnostic value of certain cytokines such as IL-2, TNF-α, IL-1β and chemokines such as IP-10 and MCP-1 in tuberculous pleural effusions, there is still no evidence yet. There is a lack of systematic analysis of immune cells and immune molecules in pleural effusions, therefore, no new diagnostic markers superior to IFN-γ have been found

Method used

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  • Monocyte chemoattractant protein (MCP) 2 used as marker for tubercular hydrothorax detection, and applications thereof
  • Monocyte chemoattractant protein (MCP) 2 used as marker for tubercular hydrothorax detection, and applications thereof
  • Monocyte chemoattractant protein (MCP) 2 used as marker for tubercular hydrothorax detection, and applications thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0105] RNA preparation, reverse transcription PCR and real-time quantitative PCR

[0106] After total RNA was isolated with TRIzol reagent, reverse transcription PCR (RT-PCR) was performed using MMLV reverse transcription system (Invitrogen). The primers for real-time quantitative PCR (qRT-PCR) of MCP-2 / CCL8 are forward primer 5'-TCTACGCAGTGCTTCTTTGCC-3' (SEQ ID NO:3) and reverse primer 5'-AAGGGGGATCTTCAGCTTTAGTA-3' (SEQ ID NO: 4). Applied Biosystems7300 real-time PCR system for real-time quantitative PCR and Green real-time PCR master mix (TOYOBO) was performed. All PCR experiments were performed in 3 parallel groups, and each experiment was repeated at least 3 times.

[0107] Flow Cytometry

[0108] The PE obtained from the patient was centrifuged at 1000g for 5min, and the cell pellet was resuspended in red blood cell lysate to remove red blood cells, and then washed twice with PBS containing 3.7% PFA. Flow cytometry analysis of cell surface markers CD3, CD4, CD8 and ...

Embodiment 1

[0114] Example 1. Detection of expression of MCP-2 / CCL8 in pleural fluid of tuberculosis patients

[0115] In order to understand the pathogenesis of pulmonary tuberculosis, the inventors carried out protein chip analysis to detect the expression of 40 different cytokines between the pleural effusion of tuberculosis patients and non-tuberculosis patients, including cancer, pneumonia, and pulmonary hypertension patients ( figure 1 A, Table 1). The concentrations of eight cytokines, I-309 / CCL1, RANTES / CCL5, MCP-2 / CCL8, IL-8 / CXCL8, MIG / CXCL9, MCP-1 / CCL2, IFN-γ and PDGF-BB, were significantly increased in the pleural effusion of tuberculosis patients. improve( figure 1 B). The expression of these cytokines was detected by ELISA in 13 tuberculosis patients and 12 non-tuberculosis patients. It was found that IFN-γ was highly expressed in the pleural fluid of tuberculosis patients ( figure 1 C). At the same time, MCP-2 / CCL8 was also highly expressed in the pleural effusion of tu...

Embodiment 2

[0120] Example 2. Detection of CD4 expressing CCR5 in the pleural effusion of tuberculosis patients + percentage of T cells

[0121] MCP-2 / CCL8 is a pro-inflammatory chemokine that activates different cell types, including CCR5, through different chemokine receptors. CCR5 can recognize a variety of ligands, especially the important receptors for activated T cells to recognize MCP-2 / CCL8. Consistent with previous findings, the pleural fluid of tuberculosis patients was rich in lymphocytes, especially T cells (68.8%±26.7%, n=6) ( figure 2 A). CD4 + and CD8 + T cells exist in the pleural fluid of tuberculosis patients, mainly CD4 + T cells (49.6%±19.2% vs 19.6%±12.8%, n=6)( figure 2 A). CCR5 in pleural fluid CD3 of tuberculosis patients + Highly expressed on T lymphocytes, while on CD3 - Rarely expressed on T cells ( figure 2 B), but CD4 + and CD8 + T cells express CCR5. CD3 expressing CCR5 in pleural fluid of tuberculosis patients + and CD4 + The percentage of ...

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Abstract

The invention relates to a MCP-2 / CCL8 protein used as a marker for identifying and diagnosing tubercular hydrothorax, and applications of the protein. The invention provides applications of the MCP-2 / CCL8 protein in preparation of diagnostic reagents or kits for identifying and diagnosing tubercular hydrothorax. The invention also provides a corresponding detection kit. The invention also provides a method for detecting or diagnosing tubercular hydrothorax.

Description

technical field [0001] The present invention relates to the fields of medicine and diagnostics. More specifically, the present invention relates to a pleural fluid marker useful for detecting tuberculosis and its use. Background technique [0002] Currently, tuberculosis is still the infectious disease with the highest morbidity and mortality worldwide. Tuberculous pleurisy (Tuberculosis Pleuritis, TP) is the body in a state of hypersensitivity, and the inflammatory reaction to Mycobacterium tuberculosis and its metabolites in the pleura is the result of primary or secondary tuberculosis involving the pleura. TP is one of the most common forms of tuberculosis infection in extrapulmonary tuberculosis, second only to lymph node tuberculosis. About 5% of tuberculosis patients will develop TP, which is often acute. About 1 / 3 of the patients develop symptoms within 1 week, and 2 / 3 of the patients develop symptoms within 1 month. The most common symptoms were pleuritic pain (75...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/532
CPCG01N33/6893G01N2800/12
Inventor 戈宝学刘海鹏刘忠华
Owner SHANGHAI PULMONARY HOSPITAL AFFILIATED TO TONGJI UNIV
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