Soluble CD40L stimulation human peripheral blood B cell long-period culture method

Abstract

The invention discloses a long-term culturing method with soluble CD40 ligand (sCD40L) to stimulate human peripheral blood B cell, which comprises the following steps: asepsis-collecting human peripheral blood; separating human peripheral blood from separating liquid of lymphocyte; placing individual nuclear cell of human peripheral blood in culturing liquid of 10% human AB serum, transferring and insulin; hanging load cell; putting into incubator to culture; adding into 2ug / ml 1L-4 of sCD40L, CsA 625ug / ml and 2ng / ml; stimulating the culture continually. The active B cell can high express MHCI, MHC II, CD80, CD86 and so on necessary molecular with submitting antigen, which can effectively submit tumour cell cracking matter, antigen peptide, antigen cDNA and RNA to induce immune response of specificity cell.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a long-term culture method using soluble CD40 ligand (sCD40L) to stimulate human peripheral blood B cells. Background technique [0002] In the field of anti-HBV-specific active immunotherapy, dendritic cells (DC), as a powerful antigen-presenting cell (APC), play a central role in anti-viral immunity. Scientific research has proved that DC functional defects or reduced numbers are directly related to chronic hepatitis B or chronic carrier status. Therefore, the use of activated DC can induce HBsAg-specific killing effect, which is a very promising method for active immunotherapy against hepatitis virus. However, this method has the following defects: (1) the number of dendritic cells is very small in a natural state, only accounting for 0.1%-0.5% of peripheral blood mononuclear cells, and it is difficult to culture in vitro, and the survival time is short. In 10-15 days, the number of ...

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Problems solved by technology

However, this method has the following defects: (1) the number of dendritic cells is very small in a natural state, only accounting for 0.1%-0.5% of peripheral blood mononuclear cells, and it is difficult to culture in vitro, and the survival time is short. In 10-15 days, the number of cells needed to reach the therapeutic dose is large, so in repeated immunotherapy, the patient is damaged and the clinical application is inconvenient

(2) In patients with chronic hepatitis B or virus infection, the function of dendritic cells is significantly defective or the number is reduced, and the efficiency of in vitro culture is not high or the function is insufficient after culture

(3) The cost of in vitro culture of dendritic cells is relatively high

This method not only introduces exogenous cells, which increases the interference factors of the experiment, but also cannot directly act on the human body and cannot be used in clinical practice.

Therefore, although it can cultivate human peripheral blood B cells in vitro, it cannot solve the problems of clinical diseases.

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