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Objective:To investigate the cytotoxic effects of CTL cell induced by DCs loaded with exosomes derived from hepatoma Huh-7 cells(T-exo).Methods: Exosomes derived from hepatoma Huh-7 cells were isolated and purified by combination of ultrafiltration centrifugation and sucrose density gradient centrifugation.Morphology of exosomes was observed under transmission electron microscopy and the expression of CD 9,CD63,HSP70 and AFP was detected by Western blot.DCs were induced with peripheral blood monocytes isolated from healthy donors.Flow cytometry was used to analysis surface markers of the DCs loaded with T-exo.WST-1 light absorption measurement was adopted to evaluate the T cell proliferation ability.Annexin-V/PI Flow cytometry were respectively used to examined cytotoxicity against the tumor cells.Results:Exosomes isolated and extracted from culture supernatant of Huh-7 cells presented as circular or elliptical vesicle with bilayer membrane , unequal in size , and with diameter of 50 to 100 nm.Western blot showed that the T-exo expressed CD9,CD63,HSP70 and AFP molecules.DCs loaded with T-exo caused significantly higher T cell pro-liferation and cytotoxic effect against AFP positive Huh-7 cells as compare to gainst AFP negative SMMC 7721 cells and un-loaded control group ( P<0.05 ).Conclusion: T-exosome loaded-DC can promote proliferation and induce significant cytotoxic effect of CTL against Huh-7 cells.
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Objective: To explore the significance of Th17 in hepatocellular carcinoma, expecially with HBV infection.Methods:Cytometric bead array(CBA) was employed to detect 5 cytokines(IL-2,IL-4,IL-6,IFN-γ,IL-17A)from 39 tumor and non-tumor tissues of HCC and combined clinical data for comparative statistic analysis.Results:The expression of IL-2,IL-4,IFN-γin liver cancer tissue[(4.61±0.28),(3.37±0.58),(3.08±1.08)pg/ml,respectively] was significant lower than non-cancer tissue [(5.57±0.59),(3.77±0.70),(3.69±1.20)pg/ml,respectively].Otherwise,the expression of IL-6,IL-17A in cancer tissue [(280.09±254.68), (2.66±1.66) pg/ml, respectively] was higher than non-cancer [(6.58 ±1.92), (1.49 ±0.98) pg/ml, respectively].And,whatever cancer or non-cancer tissue,the expression of IL-17A in tissue[(3.45±1.86)pg/ml] with high HBV load (>1 000 U/ml) was significant higher than tissue with low HBV load[(1.97±1.16)pg/ml].Conclusion: IL-17A was highly expressed in HCC,and IL-2,IL-4,IFN-γmay inhibit its expression,and IL-6 may promote it.Hepatitis B virus infection may promote Th17 expression,thereby reducing patient′s prognosis.
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Objective To induce the immune tolerance of heart grafts with infusion of isogeneic bone marrow mesenchymal stem cells (BMSCs) in heart transplant rats.Method Donor Wistar rats and recipient F344 rats were randomly divided into 4 groups:acute rejection group (group A),Wistar rats as the donors and F344 rats as the recipients for heart transplantation; low dose cyclosporin A(CsA) group (group B),recipient F344 rats given low dose CsA; BMSCs group (group C),recipient F344 rats given isogeneic BMSCs; BMSC and low dose CsA group (group D),the recipient F344 rats given isogeneic BMSCs and low dose CsA.The serum cytokine levels were determined,and the donor heart pathological changes and survival were observed postoperatively.The relative level of Foxp3 mRNA expression in the spleen of the recipient F344 rats was also observed.Result The blood levels of interleukin-2 (IL-2) and interferon-γ(INF-γ) were significantly reduced,but IL-4 and IL-10 levels were increased (P＜0.05),and the survival time of donor heart was significantly prolonged in group D as compared with groups A,B and C (P＜0.05 for all).Heart pathological examination revealed a mild acute rejection in group D,moderate acute rejection in groups B and C group,and severe acute rejection in group A respectively.The expression of Foxp3 mRNA was significantly lower in group A than in groups B,C and D (P＜0.05 for all),and that in group D was significantly higher than in groups B and C (P＜0.05 for both),but there was no significant difference between between groups B and C (P＞0.05).Conclusion Intravenous administration of BMSCs can alleviate immunorejection in heterotopic rat heart transplantation.Low-dose CsA acts synergistically with BMSCs to significantly inhibit acute rejection after heart transplantation.The partial mechanisms involve the suppressive effect of BMSCs on the expression of Foxp3 mRNA and modulation on cytokine.
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Objective:To explore the role of NKG2D ligand MHC-I related molecule A (MICA) in chemotherapy combined with NK cell immunotherapy in patients with advanced esophageal cancer after surgery. Methods:A total of 90 patients with esophageal cancer from Fujian Provincial Tumor Hospital were divided into three groups after surgery:40 patients of chemotherapy alone, 25 patients of chemotherapy combined with NK cell therapy with negative expression of MICA (MICA-group), and 25 patients of chemotherapy combined with NK cells therapy with positive expression of MICA (MICA+group). The efficacy was then compared. Results:Compared with the chemotherapy alone and MICA-groups, the positive rates of CD3+, CD4+T cells, NK cells, and the CD4+/CD8+ratio in peripheral blood from MICA+group were higher than those before treatment (64.2%± 6.4%vs. 51.3%± 5.6%, 39.8%± 8.2%vs. 29.5%± 3.2%, 25.3%± 2.1%vs. 16.4%±4.3%, 1.4%± 0.5%vs. 1.1%± 0.7%;P0.05). Conclusion:Treatment with chemotherapy and autologous NK cells on patients with advanced esophageal carcinoma and MICA positive expression can be safely transfused with only minor side effects and can effectively improve a patient's immune system, quality of life, and survival.
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Objective To explore the relevance of expression of MHC class Ⅰ-related molecules A (MICA) molecule and NK cells immunotherapy in esophageal cancer patients after operation.To analyze the significance of MICA expression in NK cell immunotherapy.Methods 100 patients of esophageal cancer were divided into 3 group,surgical alone group,MICA negative with NK therapy group (MICA-group) and MICA positive with NK therapy group (MICA+ group).The immunity indicators and tumor markers including the levels of CD3+,CD4+ T cells,ratio of CD4+/CD8+, NK cells,Treg cells,the levels of Th1/Th2/Th17 cytokine,the antibody IgA,IgM,IgG and the tumor markers of CEA,SCC,CA199,CYFRA21-1 were detected before treatment and after treatment 60 days.Results The positives rates of CD3+,CD4+ T cells,NK cells and the ratio of CD4+/CD8+ in peripheral blood from MICA+ patients group were higher than those of before treatment [(68.3±7.6) ％ vs (56.2±4.1) ％,(39.8±8.2) ％ vs (30.8±4.7) ％,(22.2±4.7) ％ vs (18.7±5.5) ％,(1.49±0.30) vs (1.15±0.61),P ＜ 0.05],meanwhile the levels of Treg cells was lower than those of before treatment [(8.1± 4.0) ％ vs (13.4±4.5) ％,P ＜ 0.05].There was no statistical significant difference of positive rate of CD8+ T cells [(26.9±6.2) ％ vs (27.8±7.1) ％,P ＞ 0.05].The levels of Th1 cytokin (IL-2,IFN-γand TNF-α) increased and Th2 cytokin (IL-4,IL-6 and IL-10) decreased after treatment.The level of Th17 cytokine was not different significantly (P ＞ 0.05).The content of IgA,IgM,IgG in MICA+ group were effectively improved after treatment.The tumor markers CEA,SCC,CA199,CYFRA21-1 had no statistically change before and after treatment.Conclusion The results indicate that NK cells immunotherapy can enhance the cellular immunity and humoral immunity of MICA positive esophageal cancer patients after operation.
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Background and purpose: Renal-cell carcinoma (RCC) is susceptible to immune therapy including the use of the nonmyeloablative allogeneic transplantation(NAT). However, NST can produce severe toxicity, so it might not be appropriate for many patients with metastatic RCC. Other novel allogeneic immunotherapies have been designed to induce an autologous immune response directed against the malignancy. This study evaluated the efficacy and safety of infusions of partially HLA-matched irradiated allogeneic blood mononuclear cells for advanced renal-cell carcinoma. Methods: Patients with histologically proven diagnosis of advanced RCC received infusions of partially HLA-matched allogeneic blood mononuclear cells. Repeat infusions were given every 8 weeks. Treatment was continued until disease progressed, unacceptable toxicity, or patient (or donor) choice. Results: Eight patients were enrolled. After every infusion, 6 patients received an oral administration of thalidomide daily with 100-300 mg/d for 2 months. One patient had durable complete response. Five stable diseases and two progress diseases were observed. In eight patients, time to progression and survival were 320 and 879+days, respectively. Severe toxicity was not observed. Conclusion: Infusions of partially HLA-matcbed irradiated allogeneic blood mononuclear cells for advanced RCC may induce some antitumor effects and deserves further study.
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Objective To observe the effect of jiaweibugan decoction on the expression of p-JNK mitogen-activated protein kinase in sciatic nerve of experimental diabetic rats and explore the preventive and therapeutic effects of Jiaweibugan decoction on peripheral neuropa-thy in experimental diabetic rats. Methods The diabetic rat model was established by streptozotocin (STZ). The rats were killed on the 4th or 8th week from the beginning of treatment, and the expression of p-JNK mitogen-activated protein kinase in sciatic nerve was detected by immunohistochemistry. Serum SOD was determined by xanthine oxidasemethod. Results The immunohistochemistry results showed that the expression of p-iNK in the model group on the 4th or 8th week was higher than that in normal control group (P<0.05) , while p-JNK in jiaweibugan decoction group was markedly lower than that in model rats(P<0.05). The results showed that SOD in the model group on the 4th or 8th week was lower than that in the normal control group (P<0.05) , while SOD in jiaweibugan decoction group was markedly high-er than that in model rats(P<0.05). Conclusion Jiaweibugan decoction has a preventive and therapeutic effect on peripheral neuropathy in experimental diabetic rats.
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Objective To observe the expression of cytotoxicity and receptors on NK cells in breast cancer,and investigate the impact of soluble MICA(sohble MHC class Ⅰ-related molecules A,sMICA) on NK cells receptors expression and cytotoxicity.Methods ELISA was used to examine the sMICA in peripheral blood.The expressions of activated receptor(NKG2D),killer inhibitory receptor (KIR)(CD158b) and NK cells were identified by flow cytometry(FCM).Cytotoxicity of NK cells to breast cancer were tested by MTT.Results sMICA was (205.36±71.27)ng/L in breast cancer patients and 81.6 % samples were detected.There were positive correlations between sMICA levels with breast cancer stages.There were no difference of NK cells percentage between breast cancer and healthy person.The cytotoxicity of NK cells and expressions of NKG2D were obviously lower in breast cancer with sMICA(+) than in healthy person,but CD158b was higher in healthy person.After cultured with sMICA,NK cells cytotoxicity decreased from(76.2±6.7)% to(48.4±4.1)% and the expression of NKG2D reduced from(92.5±7.1)% to (62.5±6.4)%,but the the expression of CD158b increased from(10.6±3.2)% to (43.6±3.4)%.IL-15 up regulated the expression of NKG2D and NK cells cytotoxicity,but decreased the expression of CD158b by co-culturation with IL-15 and sMICA in sMICA+ patients with breast cancer.Conclusion sMICA reduced the expression of NKG2D and increased the expression of Kill,which lead to the down regulation of NK cells cytotoxicity.IL-15 can reverse this effect.
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Objective To observe the effect of Jiaweibugan decoction on c-jun mRNA expression of sciatic nerve in experimental di-abetic rat and explore the preventive and therapeutic effects of Jiaweibugan decoction on peripheral neuropathy in experimental diabetic rats. Methods The diabetic rat model, was established by streptozotocin (STZ). The rots were killed on the 4th or 8th week from the beginning of treatment, and c-jun mRNA of sciatic nerve was detected by reverse-transcriptase polymerase chain reaction (RT-PCR). Serum SOD was determined by xanthine oxidase method. Results The results of RT-PCR showed that c-jun mRNA expression of the model group on the 4th or 8th week was higher than that of the normal control group (P <0.05) , while those in model rats treated by Jiaweibugan decoction were markedly lower than those in non-treated model rats(P <0.05). The results showed that SOD of the model group on the 4th or 8th week was lower than that of the normal control group (P < 0.05) , while those in model rats treated by Jiaweibugan decoction were markedly higher than those in non-treated model rats(P < 0.05). Conclusion Jiaweibugan decoction has a preventive and therapeutic effect on peripheral neuropathy in experimental diabetic rats.
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BACKGROUND: Hematopoietic stem cells have an ability of multi-directional differentiation and can be differentiated into megakayocytes in the presence of suitable hematopoietic growth factors and lineage special growth factors.Inducing of CD34+ stem cells to megakaryocytes in vitro involves many changes in gene expression, especially the signal transduction genes.OBJECTIVE: To study the expression of signal transduction genes after differentiation from stem cells into megakaryocytes.DESIGN: Open experiment.SETTING: Department of Tumor Immunology, Fujian Provincial Tumor Hospital.MATERIALS: 60-145 mL of cord blood was collected from normal labor fetuses under sterile conditions by using the blood-bag collective method. Patients and their relatives provided the confirmed consent. Reagents and equipments were detailed as follows: VarioMACS segregation apparatus, CD34+ Isolation Kit, SCGM serum-free medium, CD monoclonal antibodies, cytokines, human genome Oligo Set Version 2.1 from the CapitalBio Corporation, and LuxScan 10K/A bi-passage laser scanning apparatus. The researchers obtained consent from the local ethic committee.METHODS: This experiment was carried out in the Department of Tumor Immuonlogy, Fujian Provincial Tumor Hospital and the CaptialBio Corporation. from July 2005 to June 2006. RNA was extracted separately from CD34+ cells purified from cord blood and CD41+ cells induced from the same cord blood. The cDNA probes were synthesized by reverse transcription and purified. CD34+ cells before culturing and CD41+ cells after culturing were labeled with Cy3-dCTP and Cy5-dCTP respectively. Labeled DNA was dissolved in 30 μL hybridization fluid and hybridized at 42 ℃ over night MAIN OUTCOME MEASURES: The differential gene expression between haematopoietic stem cells and megakaryocytes involving cell signal transduction were detected by using DNA microarray analysis. According to the gene chip results,17 differentially expressed genes were selected for further analysis by RT-PCR.RESULTS: In this experiment, 3 522 differentially expressed genes were screened, including 1705 up-regulated genes and 1 817 down-regulated genes. In the 3 522 differentially expressed genes, there were 343 genes involved in cell signaling, 150 genes involved in transcription regulation, 21 genes involved in cell differentiation. The expression of the CD61 gene increased 369.83 fold, the expression of the CD41 gene increased 27.38 fold, and the expression of PF4 gene increased 24.06 folds; The expression of mitogen-activated protein kinases (MAPKs), G protein-coupled receptors (GPCRs) and members of RAS oncogene family increased mostly. The expression of the genes involved in STAT pathways, both SOCS1 and JAK2 were up-regulated, but STAT5A was down-regulated. As determined by the gene chip results, 17 differentially expressed genes were selected for further analysis by RT-PCR. GAPDH was used as the internal control, and RT-PCR results were in agreement and confirmed the finding from the gene chip expression results.CONCLUSION: Thrombopoeitin (TPO) and other hematopoietic growth factors may enhance the proliferation and differentiation of megakaryocytes derived from cord blood stem cells by GPCRs-Ras-MAPK pathway.