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Method for in-vitro preparation of semimature dendritic cells

A dendritic cell, semi-mature technology, applied in the fields of biology and medicine, can solve the problems of complicated preparation technology, different mechanisms and functions of DCs differentiation, difficult to control the semi-mature degree of dendritic cells, etc. the effect of

Inactive Publication Date: 2011-08-17
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, some need to treat iDCs with two or more factors that induce maturation and inhibit maturation, so that the preparation technology is more complicated, and the semi-maturity of dendritic cells depends on two factors with completely opposite effects. Difficult to control, the mechanism and function of DCs differentiation are also different

Method used

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  • Method for in-vitro preparation of semimature dendritic cells
  • Method for in-vitro preparation of semimature dendritic cells
  • Method for in-vitro preparation of semimature dendritic cells

Examples

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Embodiment 1

[0032] Example 1 semi-mature DCs Jagged-1 preparation of

[0033] Balb / c (provided by Guangdong Provincial Medical Experimental Animal Center) was killed by cervical spinal dislocation, the complete femur and tibia were aseptically separated, muscle and connective tissue were removed, soaked in 75% alcohol for 2 minutes, and rinsed with PBS. Cut off both ends of the bone, rinse the bone marrow cavity with a syringe filled with cold PBS, gently blow and beat the bone marrow cell suspension placed in a plate on an ice bath, filter through a 200-mesh stainless steel mesh, collect the bone marrow cell suspension and centrifuge (4°C, 300 × g, 3min). After resuspended in 300 μl of cold PBS, 5 ml of erythrocyte lysate (Sigma-Aldrich) was added, protected from light at 37.0° C. for 8 minutes, and washed 3 times with cold PBS (4° C., 300×g, 3 minutes). Finally, the cells were resuspended in RPMI1640 complete medium (GibcoBRL) containing 10% fetal bovine serum, and the cell density wa...

Embodiment 2

[0034] Example 2 semi-mature DCs Jagged-1 preparation of

[0035] C57BL / 6 mice (provided by Guangdong Medical Experimental Animal Center) were sacrificed by cervical spinal dislocation, the complete femur and tibia were aseptically isolated, muscle and connective tissue were removed, soaked in 75% alcohol for 2 minutes, and rinsed with PBS. Cut off both ends of the bone, rinse the bone marrow cavity with a syringe filled with cold PBS, gently blow and beat the bone marrow cell suspension placed in a plate on an ice bath, filter through a 200-mesh stainless steel mesh, collect the bone marrow cell suspension and centrifuge (4°C, 300 × g, 3min). After resuspended in 300 μl of cold PBS, 6 ml of erythrocyte lysate (Sigma-Aldrich) was added, protected from light at 37.0° C. for 9 minutes, and washed three times with cold PBS (4° C., 300×g, 3 minutes). Finally, the cells were resuspended in RPMI1640 complete medium (GibcoBRL) containing 10% fetal bovine serum, and the cell density...

Embodiment 3

[0036] Example 3 semi-mature DCs Jagged-1 preparation of

[0037] C57BL / 6 mice (provided by Guangdong Medical Experimental Animal Center) were sacrificed by cervical spinal dislocation, the complete femur and tibia were aseptically isolated, muscle and connective tissue were removed, soaked in 75% alcohol for 2 minutes, and rinsed with PBS. Cut off both ends of the bone, rinse the bone marrow cavity with a syringe filled with cold PBS, gently blow and beat the bone marrow cell suspension placed in a plate on an ice bath, filter through a 200-mesh stainless steel mesh, collect the bone marrow cell suspension and centrifuge (4°C, 300 × g, 3min). After resuspended in 300 μl of cold PBS, 4.5 ml of erythrocyte lysate (Sigma-Aldrich) was added, protected from light at 37.0° C. for 9 minutes, and washed 3 times with cold PBS (4° C., 300×g, 3 minutes). Finally, the cells were resuspended in RPMI1640 complete medium (GibcoBRL) containing 10% fetal bovine serum, and the cell density w...

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Abstract

The invention discloses a method for in-vitro preparation of semi-mature dendritic cells, which comprises: collecting suspension of mouse bone marrow cells, centrifuging, lysing, suspending cells in a Roswell Park Memorial Institute (RPMI) 1610 complete culture medium, adding recombinant murine granulocyte macrophage colony stimulating factor (rmGM-CSF) at a concentration of 10 to 20 mu g / L, recombinant murine interleukin 4 (rmIL-4) at a concentration of 2.5 to 10 mu g / L and Jagged-1 / Fc at a concentration of 1 to 5mg / L, incubating, collecting loose anchorage-dependent cells, resuspending cells in a complete culture medium, adding the rmGM-CSF, rmIL-4 and Jagged-1 / Fc in the same amount, collecting loose anchorage-dependent cells, adding the rmGM-CSF and rmIL-4 in half amount and Jagged-1 / Fc in the same amount in the 6th to 8th day, and collecting loose anchorage-dependent cells, namely semi-mature DCsJagged-1. The cells are only treated by Jagged-1 / Fc, and can reduce the immune response capability of allogeneic lymphocytes and help to induce immune tolerance.

Description

technical field [0001] The invention belongs to the technical fields of biology and medicine, and particularly relates to a method for preparing semi-mature dendritic cells. Background technique [0002] Dendritic cells (DCs) are one of the two cores of the body's cellular immunity. Different subtypes of dendritic cells guide the differentiation of different effector T cells, thus determining whether the body's immune system performs an immune response in recognizing foreign antigens. Immune tolerance serves two distinct functions. A large number of studies have proved that immature dendritic cells (iDCs), fully mature dendritic cells (mDCs) and semi-mature dendritic cells (semi-mature DCs) play an important role in mediating the immune system to exercise immune tolerance or immune response play a completely different decisive role. For the purpose of solving tissue and organ transplant rejection and treating autoimmune diseases, people are making unremitting efforts to fi...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/0784
Inventor 邢飞跃于哲刘静
Owner JINAN UNIVERSITY
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