The Mechanism Of The Cik Cytotoxic Activity And Perforin N-terminal Peptide Of Radiation-induced Expression Study

Cytotoxic T lymphocyte and NK cells are two kinds of major effector cells engaged in anti-tumor immunity in vivo.Release of cytotoxic granules is a mechanism for CTL and NK cells to kill tumor cells.There are three kinds of effect molecules in cytotoxic granules:Perforin(PFN),granulysin(GNLY)and granzymes.CIK cells are the heterogenous cells generated by incubation of peripheral blood monocytes(PBMC)in the presence of various types of cytokines such as anti-CD3 MoAb,interleukin-2(IL-2), interleukin-1(IL-1)and interferon-gamma.It has also been efficiently employed as an adjuvant in anticancer immunotherapeutic strategy for the eradication of residual cancer cells and prevention or delay of tumor relapse.CIK obtained enhanced cytotoxic activity, but little is known about its molecular mechanism.We first studied the expression of PFN and GNLY in PBMC and CIK cells respectively as well as the anti-tomor activities of these two kinds of lymphocyte.After that we did more reseach in construct recombinant eukaryotic expression vector pcDNA3.1(+)/PFN-N and tried to control the expression of PFN-N in SPC-Al cells by X-ray.Part One:Study on molecular mechanism of enhanced cytotoxic activity of CIK cellsIn this study,we investigated the expression of perforin and granulysin in peripheral blood lymphocytes(PBMC)and CIK cells from cancer-bearing patients as well as healthy donors,and analysed the dependability between these two effectors expression level and the cell cytotoxic activity.We performed a simultaneous detection ofperforin and granulysin expression in PBMC(intracellular molecule)by flow cytometry and cytotoxic activity using an LDH release assay,again did the same detection of CIK cells after stimulation.The most interesting finding was a significant increase of granulysin expression but a impaired expression of perforin in cancer-bearing patient CIK cells(P＜0.05)which was also happened in healthy donors(P＜0.05)while cytolytic activities were both significant increased in this two group.The expression of perforin and granulysin was both higher in healthy donors compared with cancer-bearing patient in PBMC(P＜0.05),but after stimulation they were almost at the same level respectively in CIK cells between this two group of people.Our results clearly show the upregulation of granulysin expression and increased cytolytic activities of CIK cells but the down-regulation of perforin expression after PBMC be stimulated.These findings suggest that an un-known regulatory mechanism may exist to impair PFN expression in CIK cells while the expression level of other effectors and cytokines significantly increased.Part Two:Radiation-inducible expression of human Perforin N-terminal in lung cancer cellsEarly growth response-l promoter has 6 highly conservative motifs,inducing the downstream gene expression by radiation.Perforin(PFN)is the main effector molecule of NK cells and cytotoxic T lymphocyte and plays an important role in the tomor immunity.According to the findings above-mentioned the expression level of PFN in PBMC from cancer-bearing patients is much lower than healthy donors,and the down-regulation of PFN expression may associate with the development of tumor. Therefore,in our study of this part,the Egr-l promoter was ligated upstream of cDNA encording human PFN-N to construct a recombinant eukaryotic expression vector to construct radiation-inducible expression cell model.We first construct an eukaryotic radiation-inducible expressing vector of the human perforin N-terminal(hPFN-N).The gene hPFN-N was amplified by PCR from the plasmid pcDNA3.1(+)/hPFN and an enkaryotic radiation-inducible expression vector pcDNA3.1(+)/Egr-hPFN-N was constructed after DNA recombination.DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic radiation-inducible expressing vector pcDNA3.1(+)/Egr-hPFN-N had been constructed successfully.Then we investigated the distribution and the killing effect of human perforin N-terminal truncated 118 amino acid polypeptide(rhPFN-N,22-139aa)on tumor cells.After transfecting SPC-Al cells with this recombination vector via liposome mediation,the expression of the hPFN-N protein was detected by RT-PCR and Immunocytochemical method and the killing effect of hPFN-N protein was assessed by standard MTT chromatometry.The result of RT-PCR verified the expression of hPFN-N Mrna.The result of Immunocytochemical assay was positive and in MTT assay the killing activity of rhPFN-N on target cells was 29.2%.In conclusion,the expression of PFN-N gene can be induced by X ray and it was expressed on the cell membrane.The gene product also has the cytotoxic activity and the killing activity of rhPFN-N on target cells was 29.2%.