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Application of fibroblast-like synoviocytes in preparation of rheumatoid arthritis (RA) diagnosis reagent

A technology of synoviocytes, diagnostic reagents, applied in the field of medicine

Inactive Publication Date: 2015-03-18
李立
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the pathogenesis of RA, it is agreed that the excessive proliferation of fibroblast-like synoviocytes and the destruction of cartilage are the root causes of joint pathological changes. The application of cells in the preparation of RA diagnostic reagents has not been reported. For those skilled in the art, it is not obvious that fibroblast-like synoviocytes can be used to diagnose RA

Method used

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  • Application of fibroblast-like synoviocytes in preparation of rheumatoid arthritis (RA) diagnosis reagent
  • Application of fibroblast-like synoviocytes in preparation of rheumatoid arthritis (RA) diagnosis reagent
  • Application of fibroblast-like synoviocytes in preparation of rheumatoid arthritis (RA) diagnosis reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Isolation, identification and primary culture of human fibroblast-like synoviocytes

[0047] 1. Source of synovium

[0048] Synovium was obtained from patients with RA who underwent knee arthroplasty or synovectomy and whose diagnosis met the 1987 American College of Rheumatology classification criteria.

[0049] 2. Isolation and culture of synoviocytes

[0050] The synovial tissue obtained during the operation was cut into pieces with sterile scissors, digested with 1.0 mg / mL type Ⅰ collagenase (GIBCO) at 37°C for 4 hours, centrifuged to collect cells, and added to DMEM culture medium (GIBCO) containing 20% ​​fetal bovine serum (GIBCO). ), placed at 37℃, 5%CO 2 Adherent growth in the incubator. When the cells grew to 80% of the bottom of the culture flask, they were digested with 0.25% trypsin (Sigma), terminated with calf serum, washed and centrifuged with 0.01M PBS, the cells were collected, and subcultured. Synoviocytes proliferate rapidly after passaging, an...

Embodiment 2

[0128] Example 2 The use of chondrocytes as antigen substrates is not suitable for clinical auxiliary diagnosis of RA

[0129] 1. Isolation and culture of RA chondrocytes

[0130] (1) Material collection: the articular cartilage of RA patients undergoing knee arthroplasty was taken, the fascia and perichondrium covering the cartilage tissue were peeled off, and put into a petri dish filled with PBS solution;

[0131] (2) Cut the cartilage tissue into fine pieces of 0.5-0.8mm, wash with PBS solution containing penicillin and streptomycin;

[0132] (3) Digest with 0.25% trypsin at 37°C for 2 hours and stop;

[0133] (4) Digest overnight at 37°C with 0.02% type II collagenase;

[0134] (5) Observe under an inverted microscope, when a single cell is dissociated, add DMEM medium to dilute type II collagenase to terminate digestion;

[0135] (6) After blowing the cell mass into a single cell, filter it with a 200-mesh filter, collect the filtrate, and centrifuge at 1500r / min fo...

Embodiment 3

[0171] Example 3 The use of Th17 positive cells as antigen substrates is not suitable for clinical auxiliary diagnosis of RA

[0172] 1. Magnetic bead sorting of Th17 cells in peripheral blood of RA patients

[0173] (1) Mononuclear cells (PBMC) were isolated from RA peripheral blood by gradient centrifugation.

[0174] (2) Select the Th17 cell positive sorting column of German Metienne to sort the PBMC in the peripheral blood of RA according to the instructions, and obtain Th17 positive cells.

[0175] 2. Th17 cell protein total protein extraction

[0176] Add protease inhibitors to prevent protein degradation, and disrupt cells by ultrasonic lysis to obtain total cell protein.

[0177] 3. Western-blot method to detect anti-Th17 cell antibody in serum of RA patients and normal people

[0178] This part of the method is the same as that used in Example 2 to detect anti-chondrocyte antibodies in serum by Western-blot method. After the above experiments, no reaction bands ...

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Abstract

The invention provides the application of fibroblast-like synoviocytes in preparation of a rheumatoid arthritis (RA) diagnosis reagent such as a kit or a cell chip. The application proves that a specific antibody which can be combined with the fibroblast-like synoviocytes exists in the serum of a RA patient. An indirect immunofluorescence method taking the fibroblast-like synoviocytes as a substrate antigen and a cell chip detection method are established, the application of the fibroblast-like synoviocytes in preparation of the RA diagnosis reagent has higher sensitivity and specificity for RA diagnosis, and a new diagnosis reagent and a new diagnosis method are provided for the RA diagnosis.

Description

technical field [0001] The invention relates to the technology of diagnostic reagents for rheumatoid arthritis, in particular to the application of fibroblast-like synoviocytes in the preparation of diagnostic reagents for rheumatoid arthritis, and belongs to the medical field. Background technique [0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease, mainly involving the peripheral joints, manifested as chronic, symmetrical, progressive arthritis. The pathological changes of RA initially manifested as joint synovial hyperplasia, which gradually invaded cartilage, bone and surrounding tendons, eventually leading to joint destruction, joint deformity and loss of function. RA mostly occurs in middle-aged women, with a prevalence rate of 0.32% to 0.36% in my country and as high as 1% in European and American countries. The etiology of RA is still unclear, but it is mainly related to infection factors, genetic factors and changes in hormone levels. Its clinical m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N21/64G01N33/533
CPCG01N33/543G01N2800/102
Inventor 李立
Owner 李立
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