Analysis On CIK Cell's Amplification Ability And Cytotoxic Activity In Vitro Between Adults And Tumor Patients

PrefaceCancer is the greatest threat to human's health and life.Operation chemotherapy,and radiotherapy are the traditional treatments,but respectvely have some shortages. Adoptive immunotherapy could provide the ready-made immunnity for patients through inputting immunologically competent cells which are from autogeneic or allogenic body to gain therapeutic aim.It can kill tumor cells directly,regulate and enhance the immune function,without hurting the immune system construction and function. The cell used in adoptive immunotherapy must have strong cytotoxic and proliferative ability,so CIK cell is thought of the new generation anti-tumor adoptive immunity treatment. Our laboratory has got the abundant experience to CIK research,and has cured more than 400 tumor patients successfully with CIK So far. This experiment observes the difference of amplification ability and the ability of killing tumor cells of PBMC induced by various factors in vitro between the normal persons and tumor patients.Materials and Methods1,Materials(1) Clinical Datas10 healthy adults'peripheral blood samples,5 samples (25-30 years old) and 5 samples(50-60 years old) from my unit' male healthy volunteers;33 patients adults'peripheral blood samples(41-75 years old)who are male lung squamous carcinoma patients from the first hospital affiliated China Medical College and total patho-specimen were finally diagnosed by two doctors.(2) Tumor Cell LineHuman Breast Cancer Line(MDA-MB-231),Lung Cancer line(BE-1),Carcinoma of Prostate Cell Line(PC-3)(3) ReagentFetal Calf Serum,0.25%Trypsin,RPMI-1640 Medium,MTT,Ficoll-Paque Plus,MabCD₃(Anti-CD₃ Monoclonal Antibody),IL-2(Interleukin-2),IL-1α(interleukin-1α),IFN-γ(interferon-γ)(4) Test RigInverted Fluorescent Microscope,Centrifuge,CO2 gas Incubator,Super Clean Bench,Computer Steam Sterilizer2,Method(1) CIK PreparationMononuclear cells were isolated from peripheral blood with ficoll solution,CIK cells were induced with different cytokines(IFN-γ,IL-2, IL-1, CD3McAb)(2) CIK ProliferationCIK cells amplification ability is tested by blood cell recording board(3) Tumor Cell CultureThree tumor cell lines were grown in RPMI 1640 medium containing 10% fetal calf serum.(4) Cytotoxic Activity of CIK Cells by MTT AssayCIK cells cultured for 14 days as effector cells and three tumor cell lines as target cells were seeded in 96-well tissue culture plates at 20:1 ratio.We setted up alone target cell group and effector cell group,and every group had three rep.wells.The mixed cells were incubated for 48 hours and then analyzed by MTT assay.From these assays, the killed rate(k) can be calculated according to the formula: k =[1-( experimental group OD-alone effector cell group OD) / alone target cell OD ]×100%(5) Statistical AnalysisResults1,CIK Cells AmplicationCompared with health adults',tumor patients'CIK proliferated slower and its max proliferative multiple was lower(P＜0.01).Meanwhile,the amplification ability was related to age(P＜0.01).2,Cytotoxic Activity of CIK Cells by MTT assay(1) Comparision of health adults' and tumor patients' CIK cytotoxic activity against three tumor cell linesCIK cells cultured for 14 days as effector cells and three tumor cell lines as target cells were seeded in 96-well tissue culture plates at 20:1 ratio. The killed rates of health adults to three tumor cell llines (MDA-MB-231,BE-l,PC-3)were respectively 75.80±10.1, 74.2±8.5, 81.3±9.6,which was obviously higher than tumor patients' (P＜0.01).(2) Comparison of different age group tumor patients' CIK cytotoxic acvtivity against tumor cell linesGrouping tumor patients according to age, we found that the cytotoxic activity of A group(＜50 years old) is stronger than B(50-60 years old),C(60-70 years old),and D(＞70 years old) group (P＜0.05); There was no obvious difference between B and C group in the cytotoxic activity(.P＞0.05),but B and C group was stronger than D group(P＜0.05);So we think that CIK cytotoxic activity was related to age.DiscussionThis experiment used the similar stimulating factor to PBMC of normal human and tumor patients under the same condition, induced CIK cells,then found the proliferative ability and the killing ability of CIK cells from different individuals were different, and the normal humans' were stronger than the tumor patients'. Du QingYou has observed the generation of CIK cells of normal human and the tumor patients in vitro,then found hepatoma patients' CIK cells multiplicate slower than health adults,and its max amplification multiple is lower too. The reason may be the nature of patient cell itself. Tumor immunology express that the organism immune function may decrease with the tumor growing, especially the advanced stage tumor can obviously repress special and nonspecial cellular and humoral immune function.Many foreign researches pointed out that many tumor patients' T cell subgroup is usually abnormal and disproportional,and its ratio of CD4 and CD8 is related to the process degree. With the tumor growing,the tumor antigen which enter in blood could form a compound with specific antibody.The compound can inhibit the discrimination and aggression of T cell to tumor cell to reduce CIK killing ability. Otherwise, organism could induce suppressor cell with the tumor antigen which can repress T cell generating and the antibody forming. These factors could suppress the ability of killing tumor cells of CIK cell. The experiment also found that the ability of killing tumor cells of 50-year younger tumor patients is higher than that of 50-year older tumor patients,which was concerned with hormonal control of internal secretion system.In a word, The CIK treatment is a new main kind of adoptive immunotherapy, and has the extensive treatment foreground.ConclusionCIK amplification ability and cytotoxic activity in vitro are related to age;The proliferative ability and killing activity of normal human CIK in vitro is stronger than that of tumor patients.