DC and CIC cultured a study of the lethal activity of liver cancer cells

" summary The purpose of the study was to observe the proliferation activity, ideopaedic change >s and effects of DC-CIK cells after >" and "> cytokine induced killer cells< (CIK) and iso-degenerative dexterous cells (DC). Collecting a healthy patient's exoskeleton blood single nuclear cell (MNC), placed at 37 degrees C, 5% CO2 culture box culture for 2 hours, collecting non-wall cells used to induce the culture of CIK cells, wall cells induced differentiation of mature DC, mature DC and CIK cells in a ratio of 1:5 mixed culture for 3 days, using MTT method to detect DC-CIK co-culture cell killer SMMC-7721 liver cancer cell activity. The results showed that after DC and CIK cells were cultured together, the proliferation activity and killing activity of DC-CIK cell group was higher than that of pure CIK cells. Conclusion: DC and CIC coculture cells are immunoactive cells with higher proliferation activity and cytotoxic activity than CPK cells.

"Keywords" liver cancer cells

In recent years, with the development of cytoi immunology and> molecular biology", cell immunotherapy of malignant tumors has begun to be used in clinical applications, and achieved certain efficacy. Cytokineed induced killer cell (CIK) is an efficient, new type of immunoactive cell, which is one of the important means of superimmune therapy of malignant tumors. Dendritic cells (DC) are the most powerfulantigen presenting cells (APCs) found so far, which mediate the body's immune response by processing and presenting antigens. Because many tumor hosts lack functional DC and cannot induce antigen-specific T-cell response, in-body induced functional DC is of important clinical application value for active immunotherapy. In this experiment, mature DC and CIK cells were cultured together to observe the proliferation activity and ideo-shaped changes of DC-CIK cell population, and its in-body killing effect on liver cancer cells.

materials and methods

main reagents and instruments

IMDM full media for Gibco products, 10% AB inactivationserum < for Nanjing Red Cross Blood Center products, AIM-V serum-free media for Gibco products, lymphocyte separation fluid purchased from Shanghai Second Pharmaceutical Factory, CD3 monoclonalantibodyfrom Ortho Biotech, IFN-alpha;1b from Shanghai Institute of Biological Products, rhIL-2 from Chiron, rhGM-CSF, rhIL-1 The samp;beta;, rhIL-4, rhIL-6, TNF-alpha; and PGE-2 are all purchased from PEPRO Tech, MTT from R and D Systems). Normal extraterroral blood was taken from 4 healthy providers. The liver cancer cell strain SMMC-7721 is a laboratory liquid nitrogen preservation strain. Streaming cytometers are products of Becton Dickinson of the United States and transmission mirrors are products of HITACHI.

In-body induction and amplification of CIK cells

A single nuclear cell (MNC) of outer blood is collected from a healthy provider through a blood cell separator, MNC is isolated by lymphocyte isolation fluid (Ficoll), and the concentration of CELLs in AIM-V serum-free cultured tone cells is (4-6) ×106/ml, placed in a 37c, 5% CO2 culture tank for 2 hours. Collect non-walled cells, adjust cell concentrations of (1-2) ×106/ml with IMDM with 10% human AB serum, and add 1,000 U/ml of IFN-and-alpha; suspended culture, 24 hours later to add 1 samp ;mu;g/ml CD3 monoclonal antibody and 1,000 U/ml rhIL-2, 1 change of fluid every 3 days and a half, supplement rhIL-2, adjust the cell concentration is always (1-2) ×106/ml.

DC in-body culture

in the wall cells 2 hours after PBMNC culture, with 1,000 U/ml of GM-CSF and 1,000 U/ml of IL-4 AIM-V culture, 37 degrees C, 5% CO2 culture tank culture. 1 change of fluid every 3 days and supplement cytokines, the 6th day to add the above cytokines also added IL-1 and beta;, TNF-and-alpha;, PGE-2 and other cytokines induced DC maturation, a total of 8-9 days.

DC and CPK cells cultured

-killing rate s1-(A experimental hole-A effect hole/A target cell hole)) ×100%

DC transmission mirror observation

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Results

CIK and DC-CIK Proliferation Activity

DC proliferation

DC's ultra-microstructural observation

cells

DC-CIK co-culture and CIK cell cytotoxic activity

discussed

a tree protrusion cell (DC) is the most important and powerful antigen delivery cell (APC) in the body, originally discovered in 1973 by Steinman and Cohn in isolation from mouse spleen tissue. DC is characterized by the ability to activate initial T-cell proliferation and establish a primary immune response, and DC stimulates T-cell proliferation and antigen delivery 100-1,000 times more than macrophages and B-cells. The extraterrestorial blood monocyte is DC pregenuular cell-induced DC, which is an important way to generate DC in large quantities outside of the body. GM-CSF promotes myelin cell development and is the most fundamental cytokine for DC development and differentiation; IL-4 inhibits granulocyte and macrophage production, promotes the transformation of monocytes into DC, and maintains DC maturation; TNF-alpha; and PGE2 prevents granular differentiation to stimulate DC's maturation and enhances its antigen delivery.

marten and others co-cultureDS CPK and co-source DC, it was found that DC secretion of IL-12 increased significantly, and CPK cells significantly increased the activity of tumor cells in cytotoxicity. Blocking the effect of IL-12 on CIC cells can significantly reduce CIC lethality. Our experimental studies show that the DC-CIK cell population has a stronger proliferation activity than the same source CIK cells, and the amplification multiplle of DC-CIK after 1 week of culture is about 1 times higher than that of the same source CIK. Zoll and other studies have shown that IL-2 and IL-12 have proliferative effects on CPK cells, and IL-12 makes CPK cells highly expressed CD56 plus cells that play an important role in tumor killing activity. Mature DC itself secretes high levels of cytokines such as IL-2, IL-12 and IFN-gamma;

the co-culture process between CIK and CIK, the proportion of CD3-CD8-plus and CD3-CD56-plus in CIK cells increased to varying degrees compared to pure CIK cells, indicating that a large number of mature DC further promoted the maturation of CIK cells. At present, the principle of CIC killer target cells has not been fully clarified, it may be through the adhesion factor LFA/ICAM-1 pathway and tumor cells, secreted a large number of BLT esterase particles, these particles can penetrate the target cell membrane, resulting in the cleavage of tumor cells, and CPK cells can also secrete IL-2, IL-6, T