The Study Of Biological Features And Clinical Application On CIK Cells From Different Resource

Background: At present, surgery, radiotherapy, chemotherapy are three main traditional means for the treatment of tumor, but there are their own shortcomings. Biological treatment, which is regarded as the fourth means for the treatment and at the same time paid more and more attention, has been applied to clinic. Adoptive immunotherapy, which is one of the important branch of biotherapy,transfer the product of the immune response for the disease(such as antibodies, small molecule peptides and immune effoctor cells) to other individuals, or transfuse autologous cells after treatment in vitro, and has therapeutic effect. Compared with other anti-tumor drugs, CIK cells that are new adoptive immunotherapy force can kill tumor cells directly,adjust and reinforce function of immune system, in the absence of damage on the structure and function of the immune system. At present, the technique of autologous peripheral blood CIK cells from tumor patients has been widely used in clinical, and achieved certain effects. However, the general view of the therapeutic effect of autologous CIK cells was not satisfactory and there was only about 30% efficiency. The reason might be "immune editing", the theory argue that the immune system kill the tumor,and at the same time promote the development of malignant tumors, resulting in immune escape of malignant tumors, or immunotolerance to tumor of immune cells. As a result, the tumor occur,develop and relapse. This means that the immune system lose its role in the destruction of its own tumor cells, and as such is the main reason for refractory tumors. Inspired by the theory of "immune editing", we treated advanced tumor patients who had been accepted other treat method but failure by using of allogeneic(healthy people) CIK cells. Some tumor patients achieved unexpected good results. In order to provide the corresponding theoretical basis and clinical basis for the clinical application of the novel technology, we carried out the corresponding research including the biological characteristics of CIK cells from healthy people and tumor patients, and the clinical application of CIK cells from healthy donors.objective: To compare the proliferation in vitro, phenotype and cytotoxicity of CIK cells from peripheral blood of healthy donors and tumors patients. To observe the precaution and treatment of CIK cells from healthy donors to nude mouse transplanted tumor in vivo. To observe the role of clinical application on CIK cells from healthy donors to advanced tumors patients.Methods:PartⅠ: The proliferation in vitro, phenotype and cytotoxicity of CIK cells from peripheral blood of healthy donors and tumors patients1.To induce CIK cells: 10 shares of peripheral blood of healthy donors and tumors patients were respectively harvested.The lymphocytes were isolated by density gradient centrifugation and suspended in medium with CD3mAb, rIL-2 and IFN-γas inducing agents to prepare CIK cells.2.The proliferation of the cells were compared by cell counting .3.The phenotype of CIK cells were analysed by flow cytometry.4.The cytotoxic activities to K562 cells and LOVO cells were determined by MTT method.PartⅡ: The precaution and treatment of CIK cells from healthy donors to nude mouse transplanted tumor in vivo1. Control group:six nude mice, inoculate A549 cells 1×106/0.2ml in back subcutaneously in the first day, from the second day,inject isotonic Na chloride 0.2ml into caudal vein every day, continuous 5 days, observe the growth of transplantation tumor.2. Precaution function:6 nude mice inject CIK cells 2×10~7 /0.2ml into caudal vein every day, continuous 5 days,the sixth day inoculate A549 cells 1×10~7/0.2ml in back subcutaneously, observe the growth of transplantation tumor.3.Treat function: 18 nude mice, inoculate A549 cells 1×10~6/0.2ml in back subcutaneously of nude mice, the second day , separate the nude mice into 3 group, six nude mice each group,all treat 5 days,observe the growth of transplantation tumor.A: Inject CIK cells 2×10~7 /0.2ml into inoculation part every day.B: Inject CIK cells 2×10~7 /0.2ml into caudal vein every day.C: Inject CIK cells 2×10~7 /0.2ml into caudal vein and inject CIK cells 2×10~7 /0.2ml into inoculation part every day.4. Nude mice were killed after four weeks, measuring tumor size, weighing, calculating the tumor volume and inhibitory rates, and having pathological examinations.PartⅢ:The role of clinical application on CIK cells from healthy donors to advanced tumors patientsWe selected 12 cases of advanced tumor patients who had been accepted adoptive immunotherapy by CIK cells, and compared the symptoms before and after treatment, KPS score, efficacy and anti-K562 activity of PBMC to change with own control method.Results:PartⅠ: The proliferation speed of CIK cells originated from healthy donors were significantly higher than those from tumors patients (P < 0.05); The phenotype of CIK cells which analysed by flow cytometry shows that the percentage of CD3+CD8+,CD3+CD56+ of CIK cells originated from healthy donors were significantly higher than those from tumors patients (P <0.05);the anti-tumor effect to K562 and LOVO cell line of CIK cells originated from healthy donors were significantly higher than those from tumors patients (P < 0.05).PartⅡ: The transplantation tumor of mice appear early ,grow repid and the volume is big in control group, the transplantation tumor of mice appear late and grow slow in precaution group , the transplantation tumor of treat group appear later, grow slower, volume smaller, the treatment effect of combination treatment group is the best(P < 0.05).PartⅢ:The symptoms of the 12 advanced tumor patients who accepted CIK cells adoptive immunotherapy 11 example release, 8 patients PS score is enhanced, curative effect CR 2 example,PR 3 example,MR 2 example,SD 1 example,PD 4 example, the effective power(CR+PR+MR=58.3%), the anti-tumor effect to K562 cell line is enhanced 16.47%.Conclusion:1. In vitro experiment, the proliferation, the percentage of CD3+CD8+, CD3+CD56+ and the cytotoxicity of CIK cells originated from peripheral blood of healthy donors are higher than the CIK cells originated from tumor patients.2. In animal experiment, CIK cells originated from healthy donors have the precaution and therapeutical effect to transplantation tumor that inoculate in nude mice.3. The date of clinic research indicate that the CIK cells from healthy donors can cure tumor patients that accepted other treat method(including autologous CIK cells therapy) but failure, it mainly show that tumor deflate or disappear the symptom improve,the KPS score raise,the cytotoxicity of PBMC reinforce.