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Application of vegf-c in the preparation of diagnostic reagents for sepsis and severe bacterial infection

A VEGF-C, 1. VEGF-C technology, applied in the field of biomedicine and disease diagnosis, can solve the problems of time-consuming and false positive in bacteriological methods

Active Publication Date: 2016-08-17
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, bacteriological methods have the disadvantages of time-consuming and false positives, which have great limitations on timely diagnosis and medication

Method used

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  • Application of vegf-c in the preparation of diagnostic reagents for sepsis and severe bacterial infection
  • Application of vegf-c in the preparation of diagnostic reagents for sepsis and severe bacterial infection
  • Application of vegf-c in the preparation of diagnostic reagents for sepsis and severe bacterial infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Experimental method

[0044] 1.1 Cell experiments

[0045] The primary macrophages (PEM) induced and enriched by 3% Thioglycollate (THG, purchased from Sigma, B2551) were isolated from the peritoneal cavity of wild-type mice, and treated with 10% FBS and double antibody (penicillin 100 U / ml, chain Mycin 100 μg / ml, purchased from Invitrogen, 15140), cultured cells in DMEM medium, planted in a 12-well plate, the number of cells per well was 0.5mln, and the volume of culture medium was 1ml. The cells in each well were stimulated with DMEM culture medium containing 1ug / ml LPS (purchased from Sigma Company), and the supernatants were collected after stimulating for different times, and the mouse VEGF-C and VEGF-D detection ELISA kits (purchased from Shanghai Xitang Biotechnology Company, Cat. Nos. F11672 and F11673 respectively) were used to detect the contents of VEGF-C and VEGF-D in the supernatant of the culture medium. Standard VEGF-C dilutions of 4000pg / ml and 500p...

Embodiment 2

[0052] 1. Experimental method

[0053] 1.1 Real-time PCR (RT-PCR) method

[0054] Bone marrow (bone marrow) cells were isolated from wild-type adult mice, induced in vitro by DMEM medium containing 10 ng / ml M-CSF (Peprotech, Cat. No. 315-02-10) to differentiate into mature bone marrow-derived Macrophages (BMMs). The cells were stimulated with DMEM full culture solution containing 1ug / ml LPS, and after 24 hours, the cells were lysed with Trizol, RNA was extracted, and reverse transcribed into cDNA. The relative content of VEGFR-3 in the cells was detected by specific VEGFR-3 primers (sequence shown in SEQ ID NO: 1-2 in Table 1) and RT-PCR technology, and the internal reference gene was selected as beta-Actin (internal reference primer sequence as shown in Shown in SEQ ID NO: 3-4 in Table 1), the experimental results are shown in Figure 5 .

[0055] Table 1 PCR primer sequence

[0056]

[0057] 1.2 Immunofluorescence and flow cytometry

[0058] Peritoneal macrophages (...

Embodiment 3

[0063] 1. Experimental method

[0064] The peripheral blood samples of 20 patients with confirmed sepsis were collected as the experimental group, and the peripheral blood samples of 24 healthy people were collected as the control group. Each blood sample was collected in an EDTA anticoagulant tube with a volume of about 5ml, and placed in a refrigerator at 4°C. In a 15ml centrifuge tube, add an equal volume of Ficoll separation solution with a density of 1.077g / ml, then add the blood sample to the upper layer of the separation solution, and centrifuge at room temperature at 800g for 20 minutes with a horizontal rotor. After stratification, the light yellow part of the uppermost layer is the serum, and the concentrations of VEGF-C, VEGF-D and IL-6 in the upper serum are detected by ELISA kits (VEGF-C enzyme-linked immunosorbent assay kits were purchased from Shanghai Xitang Biotechnology Co., Ltd. Technology company, product number F03080, VEGF-D enzyme-linked immunosorbent a...

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Abstract

The invention relates to the fields of biomedicine and disease diagnosis, and concretely relates to identification of a novel diagnosis marker of sepsis and severe bacterial infection, and a clinical application of the marker in disease progression monitoring as a diagnosis index. The invention concretely discloses a use of the VEGF-C in the preparation of a sepsis or severe bacterial infection diagnosis and disease course monitoring reagent, and a use of VEGF-C and VEGFR-3 and / or IL-6 in the preparation of a sepsis or severe bacterial infection diagnosis and disease course monitoring reagent. A VEGF-C protein has the advantages of sensitivity, high efficiency and stability as a sepsis and severe bacterial infection serum marker, provides a theoretical basis and a new detection method for the rapid diagnosis and monitoring of sepsis or severe bacterial infection, and is of important significance to basic research and clinical application.

Description

technical field [0001] The invention relates to the fields of biomedicine and disease diagnosis, in particular to the application of vascular endothelial growth factor-C in the preparation of sepsis and severe bacterial infection detection reagents and related diagnostic reagents. Background technique [0002] Innate immunity, as an important part of the body's immune system, quickly exerts an immune response when the body is invaded by pathogenic microorganisms, and is the key defense system for the body to eliminate pathogenic microorganisms. However, excessive activation or persistent inflammatory response can also cause severe body damage and related inflammatory diseases, such as sepsis (Sepsis) caused by pathogenic microorganisms such as bacteria. Sepsis or septic shock (septic shock) is a common clinical emergency and severe disease with high mortality rate. So far, there is still no clinical method with high efficacy and few side effects. Bacterial infection is the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/96
CPCG01N33/6893G01N2333/71G01N2800/26
Inventor 王红艳张彦波路瑶马丽魏滨
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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