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Method for detecting immune regulation function of neural stem cells

A technology of neural stem cells and immune regulation, applied in the field of stem cell technology research and immunology, to achieve the effect of reducing operation steps, reducing costs, and being easy to obtain

Pending Publication Date: 2020-12-29
北京银丰鼎诚生物工程技术有限公司
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Problems solved by technology

Therefore, a better understanding of the immunomodulatory effects of neural stem cells from different sources, different passages, and different culture methods is crucial for the further development of effective neural stem cell therapeutic drugs. Detection method of stem cell immune regulation
[0005] Animal models have been widely used in the field of stem cell technology, but there are species differences between animal models and humans, and there are certain limitations in the detection of immune regulation function of neural stem cells in humans. It is necessary to develop an in vitro model that is closer to humans or method

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  • Method for detecting immune regulation function of neural stem cells
  • Method for detecting immune regulation function of neural stem cells
  • Method for detecting immune regulation function of neural stem cells

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Embodiment 1

[0050] Example 1 Detection of the immune regulation function of neural stem cells

[0051] Neural stem cells are human-derived neurospheres cultured in suspension, and the detection indicators are the proliferation of CD3+, CD3+CD4+, CD3+CD4- cell subsets, the ratio of Th and Treg subsets, and the level of TNF-α. Specific steps are as follows:

[0052] (1) Suspension neurosphere adherent culture: Coat the 24-well plate with human laminin at a final concentration of 10 μg / ml for 3 hours, collect the neural stem cell spheres in suspension culture in a 50ml centrifuge tube, centrifuge at 500g for 5 minutes, discard Add 1ml of accutase digestive enzyme to digest at 37°C for 15 minutes, add 20ml of normal saline to dilute the digestion solution, centrifuge at 500g for 5 minutes, and discard the supernatant; resuspend the neutralized neural stem cells in NSC medium and inoculate them on the well-coated well plate , 1×10 5 / well; 37°C, 5.0% CO 2 Incubator for 16 hours.

[0053] (...

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Abstract

The invention discloses a method for detecting an immune regulation function of neural stem cells. The method comprises the following steps of (1) conducting adherent culture of suspended neural spheres; (2) inactivating the neural stem cells; (3) marking hPBMC by CFSE; (4) conducting co-culture; (5) conducting lymphocyte subpopulation and proliferation detection; (6) conducting cytokine detection; (7) conducting data processing; and (8) conducting result analysis. According to the method, the hPBMC is utilized to simulate human immune response in vitro, and immunomodulatory effects of the neural stem cells with different sources, different generations, different culture modes and the like are studied, so that a basis is provided for process development of the neural stem cells. Accordingto the method, the nerve spheres subjected to suspension culture are digested into single cells, and then wall attachment is carried out, so that quantity control and inactivation of the single cellsare facilitated, and the proliferation condition of specific cell subsets can be visually detected by using a flow cytometry in combination with different lymphocyte subset antibodies by utilizing thecharacteristic that the CFSE is averagely distributed in a filial generation along with cell division. Various detection indexes are combined, so that operation steps are reduced, experimental materials are saved, and the cost is reduced.

Description

technical field [0001] The invention relates to a method for detecting the immune regulation function of neural stem cells, which belongs to the fields of stem cell technology research and immunology. Background technique [0002] Neural stem cell (NSC) refers to the existence in the nervous system, which has the potential to differentiate into neurons, astrocytes and oligodendrocytes, so that it can produce a large number of brain cell tissues and can carry out self-organization. Cell populations that are new and sufficient to provide a large number of brain tissue cells. Neural stem cell technology has developed rapidly in recent years, and it has great potential for clinical application in central nervous system injury diseases and other aspects. Not only that, neural stem cell therapy has obvious advantages, such as safety, sufficient sources of treatment materials, and a wide range of diseases to be treated. Many countries regard it as one of the important research and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N15/14C12N5/0783
CPCG01N33/6863G01N33/56966G01N15/1434C12N5/0637C12N5/0636C12N2501/2302C12N2501/59C12N2501/999C12N2502/088C12N2506/11C12N2533/52G01N2333/525
Inventor 和会娟杨印祥杨敏董白翔
Owner 北京银丰鼎诚生物工程技术有限公司
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