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OBJECTIVE@#To assess the activation function of specific tumor polypeptide for dendritic cell vaccine on lymphocytes proliferation, production of cytokines and killing activity in vitro by using dendritic cells as antigen presenting vector.@*METHODS@#Peripheral blood dendritic cells (DC) and cytokine-induced killer (CIK) were isolated and cultured by adherent culture method; CCK-8 method was used to assess the proliferation function of lymphocytes and the killing function of lymphocytes to tumor cells; enzyme-linked immunospot assay method was used to evaluate the secretion function of cytokines. The experiment was divided into tumor polypeptide group (peptide with DC-CIK), DC-CIK group and CIK group.@*RESULTS@#With presence of interleukin-2 (IL-2) in the culture system, the lymphocyte proliferation of the three groups was obvious. The absorbance at 450 nm of tumor polypeptide group was significantly higher than that of CIK group at the time points day 4 and day 6 (day 4: Z=-3.79, P < 0.001; day 6: Z =-2.95, P < 0.01). The absorbance at 450 nm of group tumor polypeptide was significantly higher than that of DC-CIK group on day 4 (Z=-2.02, P < 0.05). Without IL-2 in the culture system, lymphocytes proliferated slowly in all the three groups, and there was no significant difference in 450 nm absorbance at each time point. The levels of IL-4 (Z=-2.61, P < 0.01), granulocyte-macrophage colony-stimulation factor (GM-CSF, Z=-3.85, P < 0.001), interferon- γ (IFN- γ, Z=-3.56, P < 0.001) and tumor necrosis factor-α (TNF-ɑ, Z=-3.40, P < 0.001) of tumor polypeptide group were higher than those of CIK group. There was no significant difference in the production of cytokines except IL-4 (Z=-2.15, P < 0.05) when tumor polypeptide group was compared with DC-CIK group. The production of IFN-γ (Z=-2.44, P < 0.05), TNF-ɑ (Z=-2.26, P < 0.05) and GM-CSF (Z=-3.73, P < 0.001) in DC-CIK group were higher than those of CIK group. Although there was no significant difference in killing activity between tumor polypeptide group, DC-CIK group and CIK group at hour 18 and hour 24, and the killing activity of tumor polypeptide group was higher than that of the other two groups.@*CONCLUSION@#Tumor peptide combined with dendritic cells can improve the proliferation activity of CIK cells in vitro, and increase the secretion of several cytokines.
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Objective: To explore the synergetic effect and observe the safety of Astragalus Polysaccharide Injection combined with cytokine-induced killer cells (CIK cells) in the treatment of advanced NSCLC patients with qi deficiency syndrome. Methods: A total of 75 advanced NSCLC patients with qi deficiency syndrome in oncology department of First Teaching Hospital of Tianjin University of TCM enrolled in the study were randomized into two groups: the control group (CIK cells group) and the combined group (Astragalus Polysaccharide Injection + CIK cells group) by the random number table method. The control group: CIK cells were transfused in vein (100 mL each time, once every Monday, Wednesday, and Friday, five times in total, the total number of cells > 1× 1010/mL). The combined group: CIK cells treatment combined with astragalus polysaccharide injection intravenous drip (250 mg Qd, 10 d). Ten days is one cycle, both groups were received the second cycle treatment in a month later. Results: The combined group's disease control rate (DCR=CR + PR + SD) was 69.4%, higher than that of control group's (36.1%), and the difference was statistically significant (P < 0.05).The KPS of combined group's effective rate was 77.8%, higher than 55.6% in the control group, the difference was statistically significant (P < 0.05). After treatment, shortness of breath, spiritlessness, hypodynamia, spontaneous perspiration and speaking reluctantly had taken a turn for the better in combined group, especially in spiritlessness, hypodynamia, spontaneous perspiration, gospeaking reluctantlyt to improve obviously, and the difference was statistical significance (P < 0.05). The haematologic toxicity in II, III, IV degree and hepatic and renal toxicity in III, IV degree did not appear in two groups. Conclusion: The Astragalus Polysaccharide Injection combined with CIK cells could control tumor lesion's progress, and improve the patient's immune function, the symptom of qi deficiency syndrome, and body functional status due to itsbetter security.
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Objective To observe the influence of IL-12,IL-15 on CIK cell in the normal culture;to observe the anti-tumor effect in the circumstance of different combination of cytokines,and to provide a new insight for preparing high effective and qualified CIK cell in vitro.Methods The optimal concentrations of IL-2,IL-12 and IL-15 were determined,respectively.After the peripheral blood from healthy blood donors was collected,monocytes were selected and co-cultured with different cytokines into different groups,as group A(IL-2 normal culture group),group B(IL-2 and IL-12 group),group C(IL-2 and IL-15 group),group D(IL-2,IL-12 and IL-15 group),and group E(cytokine control group).The monocytes in different groups were calculated by globulimeter,the activity of cells was detected by Trypan blue staining,positive ratio of CD3,CD8,CD56 on the celluar membrane was detected by flow cytometry,and the anti-tumor effect of CIK to SMMC-7721 was detected by MTSmethod,inthedayof0,5,10,15,20 after the culture.Results Statistical analysis indicated that,the proliferation multiplication of CIK cells was significantly higher in group B,group C and group D after 10,15 and 20 days of culture than those in group A(P＜0.05);and group D had higher proliferation multi-plication than that of group C(P＜0.05).The percentage of CD3 + CD8 +,CD3 + CD56+ in CIK cell membrane in group B,C,D was significantly higher than that in group A after 15 and 20 days of culture (P＜0.05).The percentage of CD3+ CD8+,CD3+ CD56+ in CIK cell membrane in group D was significantly higher than that in group B after 15 and 20 days of culture (P＜0.05).The killing rate of CIK cells for liver cancer in each group at 10,15,20 days of culture was significantly higher than that of group A when the target target ratio was 5 ∶ 1 (P＜0.05).The killing rate of CIK cells for liver cancer in group D,C at 10,15,20 days of culture was significantly higher than that of group B(P＜0.05).Conclusion IL-12and IL-15 could improve the proliferation of CIK cells,and IL-15 also has the effect of enhancing CIK cells the tumor-killing to SMMC-7721 activity.
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Objective:To investigate the effect on the phenotype and function of CIK cells induced from splenic lymphocyte in mice treated with carboxylated single-walled carbon nanotubes.Methods:CIK cells were treated with carboxylated single-walled carbon nanotubes at different concentrations.Cell growth status was observed using inverted fluorescence microscopy.The killing effect was de-termined by MTS method.Immunophenotype was analyzed by flow cytometry.Results: With the increase of the effect target ratio,the killer rate of CIK was gradually enhanced and the optimal effect target ratio was 20:1.With the increase of the dose of carboxylated single-walled carbon nanotubes,the proportion of Treg cells decreased.When CIK cells were treated with 0.5 μg/ml carboxylated single-walled carbon nanotubes(CNTs),the proportion of CD3+CD4+and CD3+CD8+was significantly higher than that of the control group(P<0.05),and the killing effects of CIK anchieved best results to B16 cells,H22 cells and RM-1cells.Conclusion: The carboxylated single-walled carbon nanotubes enhanced the ability of CIK cells to kill tumor cells,significantly which provide has potential value in tumor drug development.
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Objective To observe the clinical effect of DC-CIK cells combined with S-1 in the treatment of non-small-cell lung cancer.Methods 76 patients with non-small-cell lung cancer were randomly divided into the observation group and the control group on average.The control group was treated with S-1,and the observation group was treated with DC-CIK cells combined with S-1.For the observation group,the peripheral venous blood was collected before the treatment for DC cells and CIK cells cultivation.The expression of CD4+/CD8+,CD4+ and NK cells in the peripheral blood of the two groups was detected by flow cytometry before the treatment and after one week of the treatments.Besides,the number of white blood cells and platelet count were also measured and the symptoms of non-small-cell lung cancer were observed.Results The percentage of CD4+/CD8+,CD4+ and NK cells in the peripheral blood of the observation group after the treatment was (1.65±1.03),(34.56±8.90) and (18.68±7.98),respectively,which was significantly higher than (1.32±0.70),(29.07±7.15) and (15.28±8.23) in the control group (all P＜0.05).There was no significant difference in the percentage of CD8+ cells between the observation group (25.56± 8.90) and the control group (26.64±6.77) (P＞0.05).The vomiting,leukopenia and thrombocytopenia in the observation group were less than those in the control group (all P＜0.05).The one-year survival rate was 52.63％ in the observation group which was significant higher than 42.11％ in the control group (P＜0.05).Conclusions DC-CIK cells combined with S-1 is a safe and effective treatment for non-small-cell lung cancer,which can effectively improve the clinical efficacy and prolong the survival time of patients.
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In recent years,considerable progress has been made in the treatment of hematologic malig-nancies.However,most patients will eventually relapse and suffer from severe side effects caused by chemotherapy or radiation.Thus,as a well tolerated,safe,effective and innovative therapy,adoptive cellular immunotherapy e-merges at this very moment.This therapy can kill tumors or control recurrence by reinfusing the in vitro amplifi-cated or treated immune effective cells into the patients.It has been actively used in the treatment of various he-matological malignancies and achieved significant effects,which brings hope for more and more patients with re-lapsed or refractory hematological diseases.
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Cytokine-induced killer (CIK) cells play important roles in the treatment of solid tumors.In particular,they have broad application prospects for the patients with recurrence or metastasis.Although recently CIK treatment strategy has achieved encouraging results in preclinical studies,there are still some difficulties in the clinical applications.Some key issues remain to be resolved,such as how to ensure that the antitumor immune effector can be a lot of amplified and can stay longer in the body,how to selectively kill tumor cells,and how to ensure the security of application.
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Objective To evaluate the clinical effect of cytokine induced killer cellular immunotherapies ( CIK) on the patients with advanced lung cancer.Methods Peripheral blood mononuclear cell from 32 cases of patients with advanced lung cancer were isolated.Cells were cultured in DMEM supplemented with IFN-γ,IL-2,anti-CD3 monoclonal antibody and other cytokines,and transfused back into the patients in several times after 15 days of culture.Subsequently,we observed the size of tumor,the change of immunologic function by CIK treat-ment,the status of clinical symptoms,and the toxic and side effect.The clinical curative effect was also evaluated. Results The symptoms in 32 patients who received CIK therapy were reduced,and the side effects were light. Immunophenotype analysis showed that CD3 /CD4 coexpression were found in 34.4%patients,CD3 /CD8 co-expression in 68.2%,and CD3 /CD56 co-expression in 13.7% patients.CT images about 15 days after CIK cell transfusion showed that pleural effusion were decreased.Clinical evaluation effective rate was 56.0%,and disease control rate( CR +PR +SD) was 84.3%.Conclusion CIK cell immunotherapies of cancer patients can improve immunologic function of cancer patients and inhibit tumor growth and relieve the clinical symptom of patients,and improve the quality of life.
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Objective To investigate the efficacy and safety of decitabine in ultra-low dose combining with autologous cytokine-induced killer (CIK) cells for treatment of elderly patients with acute myelocytic leukemia (AML) transformed from myelodysplastic syndrome (MDS). Methods An 83-year-old patient with initial diagnosis of MDS-RA was treated with amifostine combined with erythropoietin (EPO), and hemogram was improved nearly to normal lasting about three and a half years. Then the patient's morbidity transformed to chronic myelomonocytic leukemia (AML-M4) two months later. Then the patient was treated with an ultra-low dose of decitabine combining with autologous CIK cells. The detailed treatment included decitabine 10mg, d1-5, and CIK cells transfusion (2×109-8×109 each time) d14; rhIL-2 2mU d15-19. 28-day treatment was accounted as one treatment course. Side effects, changes in hemogram, signs of recuperation and duration of survival were systematically observed. Further, the literature concerning decitabine for the treatment of elderly patients with MDS/AML was reviewed. Results In total, 8 cycles of decitabine combining with autologous CIK cells were given in addition to the best supportive treatments available. During treatment, side reactions including I/II grade bone marrow inhibition and grade I gastrointestinal reaction were observed. No obvious signs of dysfunction were found in liver and kidney. Leukocytosis appeared during 2nd, 3rd and 8th cycles of treatment, with the highest white blood cells count of 141.95×109/L, and it could be controlled by giving etoposide (50mg, d1-3). Hemoglobin content varied between 77 and 138g/L in the context of intermittent blood transfusion. During 3rd treatment, platelets started to increase in number, but reaching normal level during 6th treatment cycle. It was evaluated as partial remission. Finally, the patient died due to deterioration in leukemia and pneumonia. The overall survival duration was 22 months from diagnosis of AML to death, and it was significantly longer than that reported before. Conclusion The treatment consisting of ultra-low dose of decitabine combining with autologous CIK cells, is safe and effective in treatment of elderly patients with AML transformed from MDS. Further investigation with more patients is warranted in the future.
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Purpose To evaluate the tumor tropism of cytokine-induced killer ( CIK) cells, the movement track in nude mice bearing breast carcinoma and the influence on major organs of nude mice. Methods Separated and prepared CIK cells using human peripheral blood. The transwell migration assay was used to study the migratory response of CIK cells to human MDA-MB-435 breast carcinoma cells. A nude mouse xenograft model ( BALB/c) was established by injection of human MDA-MB-435 breast carcinoma cells. CIK cells labelled with DiI were injected into caudal vein of the nude mice bearing transplantation tumor. Movement track of CIK cells in vi-vo and influence on major organs were observed by living imaging technology, histopathology and immunohistopathology. Results When cultured in vitro during 14 ~20 days, CIK cells reached the peak level in proliferating stage with the maximum proportion of CD3 +CD56 + T cells. Transwell migration assay showed that the migrating number of CIK cells was increasing along with the increasing concentration of tumor cell cultural supernatants. Living imaging technology showed that the fluorescence signal began to appear 24 hours after injection of CIK cells and was strongest at 48 hours. Immunohistochemical technique and hematoxylin-eosin stain showed CIK cells tended to gather around tumor tissue 6 hours after injection, the most at 48 hours, and with some of the remaining cells on 14 day. In the meantime, no pathological damage caused by CIK cells was observed. Conclusion CIK cells have good tropism to the tumor tissue and safety to the normal tissue, and could be used as a promising cell vector for targeted therapy of cancer.
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ObjectiveTo observe the effect of betulinic acid(BetA) on the growth of human cytokine induced killer(CIK) cells and the killing activity of CIK cells on the gastric cancer cells in vitro before and after induced by betulinic acid,explore its mechanism.MethodsPeripheral blood mononuclear cell (PBMC) were separated form the healthy and were induced with various of cytokine to become CIK cells in vitro.CIK cells were collected on the tenth day and were induced with betulinic acid in different concentrations,followed by 48 h,the colorimetric methyl thiazolyl tetrazolium(MTT) method assay the proliferation rate of human CIK cells.Flow cytometry (FCM) was used to detect the expression changes of perforin,granzyme B and CD107a of human CIK cells before and after betulinic acid-induced.Lactate dehydrogenase (LDH) release assay was used to measure the influence on cytotoxic activity of CIK cells induced by betulinic acid against gastric cancer cell line SGC-7901 in vitro.Western blot assay was used to measure the extracellular signal-regulated kinase1/2 (ERK1/2),and adapter proteins SH2-domain containing leukocyte protein of 76KD(SLP-76) and linker for activative of T cells(LAT) expression changes of human CIK cells before and after drug-induced.ResultsBetulinic acid can promote CIK cells growth when the concentration were in 0.08-10 μg/ml,the expression of perforin,granzyme B and CD107a of CIK cells were significantly higher than control group(P＜0.05) when the concentration of betulinic acid were in 0.3 μg/ml.In the meanwhile,the cytotoxic activity of CIK cells in vitro against gastric cancer cell line SGC-7901 were also remarkably higher than the control group (P＜0.05).The expression of SLP-76,LAT and ERK1/2 were significantly increased to a certain extent than the control group( P＜0.05 ),when CIK cells were treated with betulinic acid.ConclusionThese results suggest that betulinic acid can promote CIK cells growth in some concentrations and increase the cytotoxic activity of CIK cells against gastric cancer cell line SGC-7901,its mechanism may related with two factors,on the one hand,enhancing the activity of SLP-76,LAT and ERK1/2,on the other hand,increasing the expression of perforin,granzyme B and CD107a on the surface of CIK cells.
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Objective To confirm the difference in the biological characteristics between V?24 natural killer T cells(NKT) and the cytokine-induced killer cell(CIK).Methods V?24 NKT cells and CIK cells were expanded from human peripheral blood mononuclear cells.Purified TCRV?24~+ NKT cells and CD3~+CD56~+CIK cells were obtained by using Dynal beads.The phenotype and the levels of cytokine and necrosis-related factors expression on the purified NKT cells and CIK cells were determined by flow cytometry.The cytotoxicity of the purified NKT cells and CIK cells were measured by means of DIOC18 staining and flow cytometry.Results The proportions of TCRV?24~+V?11~+NKT cells and CD3~+CD56~+CIK cells were elevated up to 90% after purification.The majority of V?24 NKT cells were CD4~+ and DN NKT cells.They demonstrated high levels of expression of TCRV?24,V?11,CD3 and CD161 and a low level of expression of CD56,but most purified CD3~+CD56~+CIK cells were CD8~+ T cells,with high levels of CD3 and CD56 expression,low level CD161 expression and little TCRV?24 and V?11expression.After antigen stimulation,the purified CD3~+CD56~+CIK cells showed higher levels of expression of IFN-?,TNF-?,Perforin,FasL and TRAIL,compared to the NKT cells.CD3~+CD56~+CIK cells secreted little IL-4,whereas,V?24~+NKT cells secreted high level of IL-4.In addition,the cytotoxic effect of the CD3~+CD56~+CIK cells on tumor cell lines K562,U937 and Jurkat were more intense than that of the NKT cells.Conclusion It is evident that TCRV?24~+ NKT cells and CD3~+CD56~+CIK cells differ absolutely in many ways and might play different roles in anti-tumor immunity and immune regulation.
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Objective To investigate the proliferation and the change of antigen on the cord blood CIK cells. Methods Cord blood CIK cells were generated by culture cord blood mononuclear cells in the presence of ?-IFN on day 0 and rhIL-2, antiCD3 MCAb on day 1. CIK cultures were analysed on different time point by FACS(Fluorescence activated cell sorter). The killing efficacy in tumor cell lines and primary leukemia cells were detected by MTT assay. Results After 2~3 weeks of culturing cord blood CIK cells expanded about 64.14?16 folds and CD+3 CD+56 cells which were the major cytolytic effector in CIK cells proliferated about 900 folds. Cord blood CIK cells have great cytotoxity against tumor cell lines as well as primary leukemia cells. Conclusions Cord blood MNCs can be cultured to CIK cells and the times of expansion are very high. Cord Blood CIK cells are highly efficient cytolytic effector cells.
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Objective:To study the anti proliferation and inducing apoptosis effects of cytokine induced killer cells CIK cells on MGC 803 gastric cancer cell lines and to probe its underlying mechanism.Methods:To detect the anti proliferation and the cytotoxicity of CIK cells on MGC 803 gastric cancer line by MTT assay.The morphological changes of the apoptosis cell were observed by HE stain, scanning and transmission electron microscope. The positive expression of p53, p16,C myc were determined by immunocytochemistry (ICC).Results:MTT assay showed that the inhibitive rate inhanced obviously with the addition of Effect/Target rate and extension of time ( P