Cytokine-induced killer cell activity-associated protein and application thereof

A cytokine and cell-killing technology, applied in peptide/protein components, allergic diseases, medical preparations containing active ingredients, etc.

Inactive Publication Date: 2015-02-04
曹劲松
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] According to previous reports, haptoglobin is mainly synthesized by the liver and is an acidic glycoprotein mainly present in serum, which can participate in the immune response process by regulating the activation of immune cells such as T cells (for example, refer to Delanghe JR, et al.Haptoglobin polymorphism : a key f

Method used

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  • Cytokine-induced killer cell activity-associated protein and application thereof
  • Cytokine-induced killer cell activity-associated protein and application thereof
  • Cytokine-induced killer cell activity-associated protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1, gentamicin stimulation induces cytokine-induced difference in killing cell activity

[0025] 1. Lymphocyte isolation and culture

[0026] Take 10ml of heparin sodium anticoagulated peripheral blood from healthy people, and centrifuge at 1,500rpm for 7min. Sterilize the supernatant at 56°C for 30 minutes, centrifuge at 2,000rpm for 10 minutes, remove the precipitate to obtain autologous plasma, and store it at 4°C for later use; resuspend the blood cell pellet in 0.9% normal saline to 20ml, and spread slowly along the wall of a 50ml centrifuge tube to 10ml Ficoll separation solution On the liquid surface, centrifuge at 1,800rpm for 20min, take the middle buffy coat, resuspend to 40ml in 0.9% normal saline, centrifuge at 1,600rpm for 6min, resuspend the pellet in 0.9% normal saline to 40ml, centrifuge at 1,500rpm for 6min, the pellet is lymphocytes.

[0027] The above cell pellet was resuspended in a T75 culture flask with an appropriate amount of initial lymp...

Embodiment 2

[0038] Example 2, Screening of proteins with differential CIK activity

[0039] Select the CIK cells on the eleventh day, centrifuge at 1,000 rpm for 10 min, wash the pellet twice with 0.9% normal saline, and resuspend in 100ul 0.9% normal saline. After the cell suspension was repeatedly frozen and thawed at -80°C to room temperature for 5 times, centrifuged at 12,000 rpm for 10 minutes, the supernatant was the CIK cell protein (for example, refer to Gao D, et al.Autologous tumor lysate-pulsed dendritic cell immunotherapy with cytokine-induced killer cells improves survival in gastric and colorectal cancer patients.PLoS One.2014, 9(4):e93886), and the protein concentration was determined by the Coomassie brilliant blue method. CIK protein was further separated by 1-DE (5% stacking gel, 8% separating gel).

[0040] The result is as Figure 4 As shown, there is a differentially expressed protein band (P43) between the gentamicin control group and the gentamicin experimental gr...

Embodiment 3

[0041] Example 3, Identification of CIK Activity Differential Proteins

[0042] The differential protein bands in Example 2 were identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The specific operation is as follows:

[0043] After reductive alkylation and trypsin digestion, 10ul samples were taken on the machine for LC-MS / MS detection and analysis. The specific parameters are as follows:

[0044] Liquid phase: prominence nano 2D (shimazhu) column material: C18, 5um, 150A (Eprogen); flow rate: 400 nl / min; liquid phase gradient: the linear gradient of buffer B is 5%-80%, of which A liquid is 100% h 2 O and 0.1% FA, liquid B is 100% ACN and 0.1% FA; mass spectrometry instrument: MicrOTOF-QII (BrukerDaltonics); data acquisition software: BrukerDaltonicsmicrOTOFcontrol; MS / MS scanning range: 50-2,200m / z; collision gas: argon gas; capillary voltage: 1,500V; drying gas temperature: 150°C. Data analysis software: Data Analysis Software; data retrieval: Mas...

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Abstract

The invention provides a cytokine-induced killer cell activity-associated protein and an application thereof, relates to an immune cell activity-associated protein and especially relates to influences of haptoglobin on variation of cell activity of cytokine-induced killer (CIK) cells. The expression of the haptoglobin at a protein level and a nucleic acid level is significantly increased with enhancement of the activity of the CIK cells, which means that a positive correlativity exists between the haptoglobin and the activity of the cytokine-induced killer cells. The invention provides an index of detection of an activity intensity of the CIK cells, or provides basis for researching a recombinant protein and a relative medicine which are used for improving an immune system with the haptoglobin as an active component.

Description

technical field [0001] The present invention relates to a significantly positively correlated protein in the enhancement of CIK cell activity - haptoglobin and its biological function, its expression at the protein level and nucleic acid level significantly increases with the enhancement of CIK cell activity, which can be used as a CIK activity detection Features: The application of human recombinant haptoglobin in improving the activity of human peripheral blood immune cells. Background technique [0002] CIK (cytokine-induced killer cell) refers to the killer cells induced and cultured by various cytokines in vitro. Because of its good curative effect on various tumors and no obvious side effects, it has become the fourth choice after surgery, radiotherapy and chemotherapy. Treatment of large tumors (see for example VivierE, et al. Targeting natural killer cells and natural killer T cells in cancer. Nat Rev Immunol. 2012, 12(4): 239-252; Jakel CE, et al. Clinical studies a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68A61K38/17A61P37/04
CPCA61K38/1709C12Q1/6876G01N33/68
Inventor 曹劲松陈聪
Owner 曹劲松
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