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Objective To explore the culture method of mass amplification for tumor-infiltrating lymphocytes (TILs) from malignant pleural/ascites in vitro, and identify the function and molecular phenotype of these amplified cells. Methods The pleural/ascites fluid was extracted under aseptic conditions, and lymphocytes were isolated by density gradient centrifugation. Then TILs were amplified by the program based on combined IFN-γ, OKT3 and IL-2, and the cell morphology and growth rate were recorded. The molecular phenotypes of the amplified lymphocytes were analyzed by Flow cytometry, and the killing ability against tumor cells was detected by CCK-8 assay. Results In this culture program, TILs remained in good condition until the 26th day, and the proliferation rate began to decrease on the 30th day. The proportions of CD4-CD8+ and CD8+CD56+ T cells gradually increased as cell culture time extended while the proportions of CD4+CD25+ T cells decreased gradually. Unlike the proportions prior to amplification, the proportions of SLAMF7, CD45RO, PD-1 and granzyme B positive cells in T lymphocyte subpopulation were significantly increased, meanwhile, the expression of exhausted T-cell marker CD57 was also gradually increased. The cytotoxicity of amplified CD8+ T cells from TILs was significantly stronger than that from PBMC, and the cytotoxicity reached the peak at the effect-target ratio of 10:1 and was significantly different among tumor cell types. Conclusion A culture program for TILs amplification from cancerous thoracic/ascites is established. The method is simple and efficient. The effector cells are mainly CD8+ T lymphocytes with active phenotype.
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Humanos , Linfócitos T CD8-Positivos , Linfócitos do Interstício Tumoral , Ascite/metabolismo , FenótipoRESUMO
OBJECTIVES@#To construct a prognosis risk model based on long noncoding RNAs (lncRNAs) related to cuproptosis and to evaluate its application in assessing prognosis risk of bladder cancer patients.@*METHODS@#RNA sequence data and clinical data of bladder cancer patients were downloaded from the Cancer Genome Atlas database. The correlation between lncRNAs related to cuproptosis and bladder cancer prognosis was analyzed with Pearson correlation analysis, univariate Cox regression, Lasso regression, and multivariate Cox regression. Then a cuproptosis-related lncRNA prognostic risk scoring equation was constructed. Patients were divided into high-risk and low-risk groups based on the median risk score, and the immune cell abundance between the two groups were compared. The accuracy of the risk scoring equation was evaluated using Kaplan-Meier survival curves, and the application of the risk scoring equation in predicting 1, 3 and 5-year survival rates was evaluated using receiver operating characteristic (ROC) curves. Univariate and multivariate Cox regression were used to screen for prognostic factors related to bladder cancer patients, and a prognostic risk assessment nomogram was constructed, the accuracy of which was evaluated with calibration curves.@*RESULTS@#A prognostic risk scoring equation for bladder cancer patients was constructed based on nine cuproptosis-related lncRNAs. Immune infiltration analysis showed that the abundances of M0 macrophages, M1 macrophages, M2 macrophages, resting mast cells and neutrophils in the high-risk group were significantly higher than those in the low-risk group, while the abundances of CD8+ T cells, helper T cells, regulatory T cells and plasma cells in the low-risk group were significantly higher than those in the high-risk group (all P<0.05). Kaplan-Meier survival curve analysis showed that the total survival and progression-free survival of the low-risk group were longer than those of the high-risk group (both P<0.01). Univariate and multivariate Cox analysis showed that the risk score, age and tumor stage were independent factors for patient prognosis. The ROC curve analysis showed that the area under the curve (AUC) of the risk score in predicting 1, 3 and 5-year survival was 0.716, 0.697 and 0.717, respectively. When combined with age and tumor stage, the AUC for predicting 1-year prognosis increased to 0.725. The prognostic risk assessment nomogram for bladder cancer patients constructed based on patient age, tumor stage, and risk score had a prediction value that was consistent with the actual value.@*CONCLUSIONS@#A bladder cancer patient prognosis risk assessment model based on cuproptosis-related lncRNA has been successfully constructed in this study. The model can predict the prognosis of bladder cancer patients and their immune infiltration status, which may also provide a reference for tumor immunotherapy.
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Humanos , Linfócitos T CD8-Positivos , Prognóstico , RNA Longo não Codificante/genética , Bexiga Urinária , Neoplasias da Bexiga Urinária/genética , Cobre , ApoptoseRESUMO
Objective The objective of this study was to investigate the imaging findings of 18 F-FDG PET-CT in patients with multiple myeloma(MM).Methods Fifteen patients with multiple myeloma were enrolled in our hospital from January 2014 to October 2016.The clinical data of these patients were retrospectively analyzed to obtain the range of SUVmax values by 18F-FDG PET-CT imaging performance.Results Fifteen MM patients with 18F-FDG PET-CT showed a total number of lesions 202,the number of lesions 176 in PET and the number of lesions 171 in CT.The SUVmax range of 18F-FDG PET-CT imaging in the diagnosis of MM was 1.41 ～ 16.28.Fourteen cases of 15 MM patients were found varying degrees of bone destruction;one case showed a slightly increased bone density and also slightly increased FDG metabolic activity.Bone destruction was more performance for the chisel-like,osteolytic,swelling and flaky bone destruction.The osteogenesis lesions were rare.Conclusion 18F-FDG PET-CT imaging can clearly show the imaging features of MM by combining functional metabolic and anatomical information,and provide more clues for clinical diagnosis,which can improve the diagnostic accuracy of MM.
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Objective To study the expression and clinical significance of miR-183-5p, TβRⅠ and TβRⅡ in esophageal squamous cell carcinoma (ESCC). Methods The mRNA and protein expression of miR-183-5p, TβRⅠ and TβRⅡ were examined in ESCC cell lines ECA-109, TE-1, normal esophageal epithelial cells, tumor tissues and tumor-free tissues from 72 ESCC patients. Their clinical significance and the relationship between miR-183-5p and the latter two were analyzed. The effects of miR-183-5p on the expression of TβRⅠand TβRⅡ in ECA-109 cells and the cell functions of ECA-109 were also investigated. Results Compared with the normal esophageal epithelia cells, ESCC cell lines TE-1 and ECA-109 were statistically characterized by a high expression of miR-183-5p (all P<0.05) and low expression of TβRⅠand TβRⅡ(all P<0.05). The expression of miR-183-5p in ESCC tissues was higher than that in adjacent normal tissues, while the expressions of TβRⅠ and TβRⅡ were lower (all P< 0.05). The expression of miR-183-5p was closely related to sex, tumor differentiation, tumor staging, distant metastasis, lymphatic metastasis, and tumor location (all P<0.05). TβRⅠlevel was associated with sex, lymph node metastasis and tumor size (all P<0.05). Experimental data showed the negative correlation between the expression of miR-183-5p and TβRⅠin ESCC tissues (r= -0.521, P< 0.05). Over expression of miR-183-5p significantly inhibited the expression of TβRⅠ in ECA-109 cells (P< 0.05) and promoted the growth, invasion and metastasis of ECA-109 cells (P< 0.05). Low expression of miR-183-5p significantly promoted the expression of TβRⅠ in ECA-109 cells (P< 0.05), and suppressed the growth, invasion and metastasis of ECA-109 cells (P< 0.05). There was no significant change in the expression of TβRⅡ in the transfection experiments. Conclusion MiR-183-5p is closely related to the abnormal expression of TβRⅠ, which may exert an important role in the progression of lymphatic metastasis.
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Objective To investigate the expression and clinical significance of UL16 binding protein 3 (ULBP3) in human esophageal squamous cell carcinoma (ESCC) and its correlation with nature killer (NK) cells.Methods The relative expression of ULBP3 in the ESCC tissues and corresponding carcinoma adjacent tissues of 40 patients was detected by realtime-poly merase chain reaction (PCR),immunohistochemical staining and Western blot methods.The percentage of NK cells in peripheral blood of same patients was examined by flow cytometry.The correlation between ULBP3 and the percentage of NK cells was analyzed with Pearson method.Results The expression of ULBP3 at mRNA level in the tumor tissues ([4.96 ±-6.11]×10-3) was significantly higher than that of corresponding carcinoma adjacent tissues ([1.64 ± 2.96]× 10-3,t =3.656,P＜ 0.01).The immunohistochemical staining results indicated that the positive rate of ULBP3 in the tumor tissues was 60％ (24/40),however that of corresponding carcinoma adjacent tissues was only 32.5％(13/40,t=3.921,P＜0.01).The Western blot results indicated that the expression of ULBP3 at protein level in the tumor tissues was significantly higher than that in the corresponding carcinoma adjacent tissues.The relative expresssion ULBP3 at mRNA level in carcinoma tissues of ESCC patients with lymph node metastasis and at TNM stage Ⅲ was higher than that of ESCC patients without metastasis and at TNM stage Ⅰand Ⅱ (t=4.839,4.192,P＜0.05).There was no significant correlation between the expression and ages,gender,location of tumors and the differentiation degree of tumor (P＞0.05).At early and mid stage of the tumor,the expression of ULBP3 at mRNA level was positively correlated with the percentage of NK cells in peripheral blood (r=0.5233,P＜0.05),however there was no correlation at advanced stage.Conclusion ULBP3 was highly expressed in ESCC and may be involved in the immune regulation of NK cells.
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Objective To explore the role of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) in the tubulointerstitial lesions of immune-mediated nephrotoxic nephritis (NTN) and the intervention regulation by anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb). Methods WKY rats were randomly divided into control,NTN and PsL-EGFmAb-treated groups. The mrs in NTN group were injected with 1 ml nephrotoxic rabbit serum per kilogram of rat body weight; the ones in PsL-EGFmAb-treated group were injected with 2 mg PsL-EGFmAb per kilogram of rat body weight simultaneously and 2 h later after nephrotoxic rabbit serum injection; and those in control group were injected with equal volume of 0.9% saline. Renal function and pathology were observed at day 4, 7 and 14 after the induction of NTN. Distribution of DC-SIGN + dendritic cells (DCs) in renal tissues was measured by immunofluorescence. Real-time PCR was performed to examine the expression of P-selectin,RANTES, TNF-α, IL-10, IFN-γ and IL-4. Expression of MHC Ⅱ , CD80 and DC-SIGN on dendritic cells was analyzed by flow cytometry. Transendothelial migration was used to detect the ability of DCs migration. DCs ability to activate T cells was determined by mixed lymphocyte reaction (MLR). ELISA was used to detect the concentration of IFN-γ and IL-4 in the supernatant of MLR. Results At day 4, immature DC-SIGN+ DCs infiltrated the rat renal tubulointerstium of NTN group, matured at day 14, and enhanced the ability to migrate and activate T cells. The distribution of DC-SIGN + DCs was significantly related to the form of crescent, tubulointerstial lesions and renal function. In addition, expression of chemokine RANTES and proinflammatory cytokine TNF-α continuously augmented since day 4, while anti-inflammatory eytokine IL-10 decreased after markedly increased at day 4. At day 14, IFN-γ/IL-4 mRNA increased, which was obviously related to DCs maturation. The intervention of PsL-EGFmAb supressed the expression of DC-SIGN and CD80 on DCs, depressed DCs maturation, migration and ability to activate T cells,down-regulated proinflammatory cytokines and up-regulated anti-inflammatory cytokines in kidney,and thus regulated Th1/Th2 bias. At the same time, kidneys showed the decrease of crescents,improvement of tnbulointerstium damage and renal function. Conclusions DC-SIGN may mediate DCs tubulointerstitial infiltration. It may be also a potent regulator of local immune reaction imbalance and pathology of tubulointerstium. PsL-EGFmAb may depress DCs migration and downregulate DCs maturation and function through DC-SIGN, and thus having a role in prevention and treatment.
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In order to investigate the effect of T-bet on malignant cells, we selected SGC-7901, a kind of human gastric carcinoma cell line, and used gene clone technique and adeno-associated virus (AAV) packing technology, thus obtaining a recombinant rAAV-eGFP-T-bet and T-bet gene-transfected SGC-7901 cells. Then the function of T-bet gene-infected SGC-7901 cells was researched by detecting the levels of IFN-gamma and T-bet production. The results showed: (1) It was verified that rAAV-T-bet's packing was completed; (2) After SGC-7901 cells was transfected by rAAV-eGFP-T-bet, a green fluorescence was found in about 30%-40% SGC-7901s, and the gene of 1670 bp (T-bet) and 388 bp (IFN-gamma) were generated from SGC-7901s cells; (3) The proteins of IFN-gamma and T-bet secreted by SGC-7901 cells were also detected. These reveal that SGC-7901 cell is efficiently infected by rAAV encoding T-bet, which can induce transfected cells to secret IFN-gamma. It may be useful in the researches on cancer immune therapy of transfecting T-bet gene.