Establishment Of The Culture System Of Cytokine-induced Killer(CIK) Cells And The Study Of Cytotoxicity Of CIK Cells From Bama Suckling Pig

Collecting jugular vein blood from Bama suckling pig, and isolating peripheral blood lymphocytes using percoll density gradient centrifugation method. And porcine CIK cell culture system was established through the contrast test. The phenotype of porcine CIK cells was identified by flow cytometry and qRT-PCR method, and cytotoxic activity test was carried out. Eventually, a method was established for efficiently obtaining a large number of pig lymphocyte populations which have a cytolytic activity for tumor cells. By using the method, the sufficient amount of effector cells can be provided in anti tumor immunit y and the mechanism, and can also provide research ideas for in vitro cellular immunity induced by pathogenic microorganisms and immune evasion. Through the implementation of this study, the results obtained are as follows:(1)Establishing a culture system for porcine CIK cells through the optimization test on cytokines and culture medium. After 5 days’ culturing porcine peripheral blood lymphocytes with this system, can make the total number of lymphocytes growing nearly 8 times. And it can provide a large number of immune cells which have a phenotype of NK cells for study of in vitro cell immunity with this culture system.(2)Analyzing the changes of porcine CIK cell ratio during culture process with flow cytometry. The result shows that after 5 days’ culturing, the proportion of lymphocytes with a phenotype of NK cells(CD2+/CD8+/CD3-)increased 5.59 times than originally isolated lymphocytes. And the result of q RT-PCR also shows that, the expression of NK cell surface markers related(CD2, CD3, CD8α, SLA and PRF1)reached the peak at 5d, which is the same with the flow cytometry results.(3)The result of the cytotoxic activity of porcine CIK cell shows that, the cultured 5d CIK cells reached the best cytolytic activity to YAC-1 at the effect target ratio of 12:1, with a killing rate of nearly 80%, however the killing rate of initial separated lymphocytes to YAC-1 was only 56.86%.In summary, a porcine CIK cell in vitro culture system was established through this study, and we can obtain a large number of porcine CIK cells which have a NK cell cytolytic activity in vitro in a short time. And then a large number of high-quality effector cells can be provided for the study on antitumor immunity of tumor cells in vitro and the study on cellular imm unity induced by pathogenic microorganisms, which shows that the application prospect is broad.