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Objective To investigate the effect of hyperoside on proliferation and killing activity of NK cells against pancreatic cancer PANC1 cells in vitro,and explore its potential mechanism.Methods Peripheral blood mononuclear cells of healthy donors were isolated,NK cells were induced with medium contained with IL-2 and different concentrations of hyperoside (0.3,1.6,8,40 and 200 μg/ml) for 12 days.Cell viability was observed by trypan blue staining.Phenotype and perforin,granzyme B expression of NK cells were detected by flow cytometry.Killing activity of NK cells against PANC1 cells were analyzed with lactate dehydrogenase (LDH) releasing method.Results The proportion of NK cells in control group and experimental group treated with different concentration of hyperoside both reached about 80％,respectively.The proliferation of CDs-CD56 + NK cells treated by hyperoside at 0.3,1.6 and 8 μg/ml was (93.76 ±8.77),(106.67 ± 12.35) and (118.50 ± 11.51) times,respectively,which were significantly higher than (73.70 ± 9.43) times of the control group.The expressions of perforin in NK cells treated with hyperoside at 1.6,8 and 40 μg/ml were significantly higher than those of the control group [(82.34 ± 2.90) ％,(89.15 ±3.54) ％,(81.78 ± 2.81)％ vs (72.93 ± 2.06)％].The expressions of granzyme B in NK cells treated with hyperoside at 1.6 and 8 μg/ml were significantly higher than those of the control group [(87.30 ± 1.70) ％,(92.16 ±3.05)％ vs (82.35 ±2.73)％].The killing activity of NK cells against PANC1 cells treated by hyperoside at 1.6 and 8 μg/ml was significantly higher than those of the control group [(63.18 ± 3.77)％,(65.34 ± 4.97) ％ vs (52.16 ± 5.48) ％].The differences were statistically significant (all P ＜ 0.05).Conclusions Hyperoside could promote the proliferation of NK cells at certain concentrations and maybe enhance the killing effect against pancreatic cancer PANC1 cells through up-regulating the expression of perforin and granzyme B in NK cells.
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Objective:To investigate the effect of 2-deoxy-D-glucose (2-DG) on hunmanγδT cells on the expression of CCR5 and killing function in vitro.Methods:UsingγδT medium to cultivate peripheral blood mononuclear cell( PBMCs) in vitro.After co-cultured with various concentrations of 2-DG for 48 h,the expression of CCR5 and killing activities of γδT cells for each group were detected by flow cytometry and CCK-8 methods.Results: 2-DG could not promote the growth of γδT cells with the increase in concentration from 0 μmol/L to 1.0 μmol/L and decreased thereafter.The certain concentration ( 0-2.0 μmol/L ) of 2-DG could upregulate the expression of CCR5 in dose dependent manner.Besides,at 0.5μmol/L and 1.0μmol/L of 2-DG could increase the ex-pression of CD107a and perforin and have no effect on the granzyme B.Conclusion: Human γδT cells isolated from peripheral blood treated with 2-DG could promote the expression of CCR5 and increase the killing activities at certain concentration in vitro.
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Objective:To investigate the mechanisms of TWS119 induced CCR5 expression in hunman γδT cells. Methods:After treatment with various concentrations of TWS119 for 48h, the expression of CCR5 in γδT cells were detected by flow cytometry. The p-STAT3 and GAPDH expression were examined by Western blot analysis. Results: TWS119 could upregulate the expression of CCR5 in dose dependent manner. Western blot analysis revealed that TWS119 inhibit phosphorylation of STAT3,but had no significant impact on GAPDH. In addition, pretreatment of γδT cells with 0. 5 μmol/L STAT3 specific phosphorylation inhibitor Stattic could upregulate the expression of CCR5 and enhance the TWS119 induced CCR5 expression. Conclusion: TWS119 could upregulate CCR5 expression of γδT cells by inhibiting STAT3 phosphorylation in vitro.
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Objective:To investigate the anti-tumor effect of Hyperoside.Methods: Human γδT cells were amplified by isopentenyl pyrophosphate from peripheral blood cells.The proliferation capacity of γδT cells was measured with CCK-8 assay after treated with different concentrations of Hyperoside.Cytotoxicity of γδT cells was detected with LDH assay , and the expression of granzyme,perforin CD107a and IFN-γonγδT cells were measured by flow cytometry before and after treatment.Results: Hyperoside could significantly stimulate the proliferation of γδT cells at the concentration of 3.13-12.5 μg/ml.Cytotoxicity and expression of granzyme,perforin and IFN-γofγδT cells were increased after treatment.Conclusion:Hyperoside could enhance cytotoxicity of humanγδT cells through up-regulation of granzyme ,perforin CD107 a and IFN-γexpression.
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Objective:To investigate the effect of glycogen synthase kinase-3β inhibitor TWS119 on hunman γδT cells growth and phenotypic characteristics. Methods:Using γδT medium to cultivate human peripheral blood γδT cells in vitro. After co-cultured with different concentrations of glycogen synthase kinase-3β( GSK-3β) inhibitor 4, 6-disubstituted pyrrolopyrimidine ( TWS119 ) for indicated time,growth curve and Wnt/β-catenin activation of in each group were determined by CCK-8 and Western blot assays. The CD62L and CD45RA expression theγδT cells were detected using flow cytometry. Results:Wnt/β-catenin pathway ofγδT cells could activate by TWS119. In the first group,TWS119 could upregulate the expression of CD62L and CD45RA in dose dependent manner while inhibit the growth and ratio of γδT cells. In the second group,TWS119 could promote the growth and ratio of γδT cells with the increase in concentration from 0 μmol/L to 4. 0 μmol/L and decreased thereafter. Besides,TWS119 could promote the expression of CD62L in a dose-dependent and had no effect on the CD45RA. Conclusion: Human γδT cells isolated from peripheral blood treated with TWS119 gave rise to two subsets of CD45RA+CD62L+γδT cells and CD62L+γδT cells.
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<p><b>OBJECTIVE</b>To study the influence of heat stimulation on expression of coxsackie-adenovirus receptor (CAR) in keratinocytes (KCs) of mouse skin and the effect of CAR on production of cell growth factors by dendritic epidermal T lymphocytes (DETCs).</p><p><b>METHODS</b>(1) Twenty BALB/c mice were divided into heat stimulation group (HS) and control group (C) according to the random number table, with 10 mice in each group. Mice in group HS were inflicted with scald milder than superficial-thickness by dressing wet hot gauze, which had been soaked in 100°C hot water for 3 min, in the hair removed area on the back for 1 to 3 s, while mice in group C were sham injured by dressing a wet gauze which had been soaked in water of room temperature for 3 min in the hair removed area on the back for 1 to 3 s. Square full-thickness skin specimens measuring 2.0 cm × 2.0 cm in size were obtained from the center of the bare skin. The expression of CAR in skin tissue sections were detected by immunohistochemistry staining. The mRNA and protein expression levels of CAR in skin tissue sections were respectively determined by real-time fluorescent quantitation RT-PCR and Western blotting. (2) KCs were isolated and cultured from full-thickness skin obtained from the trunk of 2 fetal BALB/c mice, and they were divided into 2 groups according to the random number table, with 5 wells in each group. The cells in group HS and group C were respectively cultured in 42°C and 37°C, 5% CO2 incubator for 1 h, and then all the cells were cultured in 37 °, 5% CO2 incubator for 6 h. The apoptosis of the cells and their expression of CAR were detected by flow cytometer. (3) Five BALB/c mice were sacrificed, and full-thickness skin was obtained from the trunk. The DETCs were divided into 7 groups according to the random number table after being isolated and purified from the skin specimens. Cells in group C were cultured without any stimulation, and cells in the 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/L CAR groups were respectively cultured with corresponding concentration of recombinant mice CAR nutrient solutions, with 5 wells in each group. The contents of insulin-like growth factor I (IGF-I) and keratinocyte growth factor (KGF) were determined with ELISA. Data were processed with independent samples t test and one-way analysis of variance.</p><p><b>RESULTS</b>(1) The immunohistochemistry staining showed that there was mild positive staining in the skin tissue sections of mice in group C, while the positive staining was more obvious in group HS. The positive staining was mainly located in KCs, hair follicles, and sweat gland epithelial cells, while no positive staining was observed in fibroblasts. The mRNA expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.157 ± 0.027 and 0.773 ± 0.029. There was statistically significant difference between them (t = 3.052, P < 0.01). The protein expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.23 ± 0.09 and 0.89 ± 0.14. There was statistically significant difference between them (t = 2.556, P < 0.05). (2) The apoptosis rates of KCs in group C and group HS were respectively (5.7 ± 1.3)% and (7.4 ± 1.7)% (t = 0.464, P > 0.05). The expression rates of CAR in KCs in group C and group HS were respectively (48 ± 6)% and (80 ± 8)%. There was statistically significant difference between them (t = 2.585, P < 0.05). (3) The contents of IGF-Iin culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (23.1 ± 1.8), (22.5 ± 2.1), (31.2 ± 2.5), (39.7 ± 2.3), (61.8 ± 3.5), (45.1 ± 2.8), and (29.0 ± 2.0) µg/L. There was statistically significant difference among 7 groups (F = 3.414, P < 0.05). The contents of KGF in culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (131 ± 9), (217 ± 12), (355 ± 21), (563 ± 21), (535 ± 34), (292 ± 20), and (245 ± 10) ng/L. There was statistically significant difference among 7 groups (F = 5.063, P < 0.01).</p><p><b>CONCLUSIONS</b>The expression of CAR in KCs would rise after HS. The optimum CAR concentration to increase IGF-I and KGF production in DETCs is low.</p>
Subject(s)
Animals , Female , Male , Mice , Burns , Metabolism , Cells, Cultured , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Metabolism , Fibroblast Growth Factor 7 , Metabolism , Hot Temperature , Insulin-Like Growth Factor I , Metabolism , Keratinocytes , Metabolism , Mice, Inbred BALB C , Skin , Cell Biology , T-Lymphocytes , MetabolismABSTRACT
Objective To investigate the in vitro effects of quercitrin on the proliferation and the cytotoxicity of human γδT cells.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects and cultured with isopentenyl pyrophosphate and IL -2 to induce human γδT cells.The hu-manγδT cells were cultured with quercitrin at various concentrations for 48 hours.CCK-8 kits were used to analyze the in vitro proliferation and cytotoxic activities of γδT cells.Flow cytometry was performed to meas-ure the expression of granzyme B and perforin in γδT cells.The expression of p-ERK, p-Akt and Bcl-2 at protein level were detected by Western blot .Results The percentage of human γδT cells in PBMCs was in-creased from (2.96±1.83)%to (88.94±2.36)%after 10 days of culture.The quercitrin at concentrations of 10 to 80 μg/ml could promote the growth of γδT cells and up-regulate the expression of granzyme B , per-forin, p-ERK, p-Akt and Bcl-2 in a dose dependent manner .The cytolytic activities of γδT cells against co-lonic carcinoma cells ( HCT116 ) were enhanced by quercitrin .Conclusion Quercitrin could promote the proliferation of γδT cells and enhance the expression of granzyme B and perforin at certain concentrations in vitro.ERK1/2 and Akt signal transduction systems might be involved in the process .
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Objective To investigate the effect of DHA on proliferation and killing activity of γδT cells against SW1990 cells in vitro.Methods γδT cells were generated in vitro by stimulating peripheral blood mononuclear cells of healthy donors in RPMI 1640 completed medium containing IPP and IL-2 for 8 d,and then co-cultured with different concentrations of DHA for 48 h.Proliferation rates of γδT cells for each group were detected by MTT method.The perforin,granzyme B and CD107a expression in γδT cells were verified by flow cytometer.The cytotoxic activity of γδT cells against SW1990 cells were analyzed by CCK-8 kit.Results The purity of γδT cells in each group reached 75.46％ ±5.32％ after 8 d of culture.Compared with the control group,the proliferative capability of γδT cells were enhanced significantly after treated with 50-100 μmol/ml DHA for 48 h,moreover,cytotoxicity against SW1990 cells and perforin,granzyme B and CD107a expression of the γδT cells treated with DHA were higher than the control group.Conclusion DHA could enhance the antitumor activity of γδT cells,which may be associated with the upregulation of perforin and granzyme B expression in γδT cells.
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Objective To investigate the early changes of CD4 + T cells and the relevant cytokines in blood of patients with severe trauma.Methods Sixty-one consecutive patients with trauma admitted into the 97th Hospital of PLA from September 2009 to November 2011 were enrolled in this study.The exclusion criteria included:① Patients were younger than 18 or older than 75 years.②Patients received blood transfusion.③Those suffered from immune system disorders,tumors or diabetes,or recent history of virus,bacteria or parasites infections.④ Those had current or recent treatment with corticosteroids or immunosuppressive drugs.According to ISS score ≥ 16,there were 61 traumatic patients divided into mild trauma group and severe trauma group.Seventeen healthy volunteers were taken as control subjects.At admission,the peripheral venous blood samples of patients were taken to count the number of CD3+,CD4 +,CD8 + T cells by flow cytometry and to detect TNF-α,INF-γ,IL-1,IL4,IL-6 and IL-12 levels in plasma by enzyme linked immunosorbent assay,meanwhile the ratio of Th1 / Th2 type cytokine were calculated.Data were analyzed with t test and ANOVA or Kruskal-Wallis test by using SPSS version 16.0package.P value ＜ 0.05 was considered to be statistical significance.Results Compared with control group,mild trauma and severe trauma groups showed a similar decrease in number of CD3 + T cells,and severe trauma group showed a most significant decrease in number of CD4 + T cells.Severe trauma group had a lower INF-γ level compared with control group; IL-1 level in both mild trauma and severe trauma groups were lower than that in the control group; INF-γ/ IL-6 in severe group was significantly lower than that in the mild group.However,INF-γ IL-6 in mild group was higher than that in the control group.Conclusions The early stage of severe trauma exhibits a significant decrease in number of both CD3 + and CD4 +T cells,accompanied by a significant reduction in most of cytokines,and has a tendency of shift to Th2 type cytokine.Therefore,the changes of immune cells should be promptly and successively monitored after severe trauma.
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Objective To investigate the effects of hydrocortisone (HC) on proliferation and killing activity of NK cells against pancreatic cancer SW1990 cells in vitro.Methods Peripheral blood mononuclear cells of healthy people were isolated and cultured with NK cells medium containing IL-1S.When the purity of NK cells reached above 70％,different concentrations of HC (10-6,10-5,10-4,10-3 μmol/L) were added and co-cultured with NK cells for 7 days.And NK cells without HC were used as control.CD3-CD56 + NK cell numbers of each group were countered by trypan blue staining.Perforin,granzyme B and IFN-γ expression of CD3-CD56+ + NK cells were verified by flow cytometry.NK cells and SW1990 cells were co-cultured with a 20∶1effector to target ratio,then the cytotoxic activity of NK cells against SW1990 cells were analyzed by CCK-8 kit.Results After treatment with different concentration of HC for 7 days,NK cells purity of each group reached 70.72％ ～ 76.39％,and it was not significantly different with that in control group [(72.61 ± 3.76) ％].The proliferation folds of NK cells treated with 10-6,10-5,10-4,10-3 μmol/L HC were (9.13 ± 0.94),(9.67 ±1.51),(10.33±1.07),(8.40±1.47) times,respectively,while it was (4.23 ±0.82) times in control group (all P ＜0.01).The killing effects of NK cells on SW1990 cells were (58.58 ± 4.89) ％,(62.27 ± 5.63) ％,(64.02 ± 5.79) ％,(63.88 ± 3.61) ％,which were higher than that in control group [(57.46 ± 5.11) ％],moreover,the difference between NK cells of 10-4 μmol/L HC treatment group and control group was statistically significant(P ＜ 0.05).The expressions of perforins of 10-4,10-3 μmol/L HC treatment group were (96.71 ± 3.04) ％,(97.56 ± 2.18) ％,which were significantly higher than that in control group [(92.40 ±3.53)％,P ＜0.05 or 0.01].The expression of granzyme B in 10-5 μmol/L HC treatment group was (78.23 ±2.94)％,which were significantly higher than that in control group [(73.68 ±3.52) ％,P ＜0.05].The expressions of IFN-γ in 10-5,10-4,10-3 μmol/L HC treatment group were (96.61 ±2.04)％,(97.58 ± 2.17)％,(98.00 ± 1.77)％,which were significantly higher than that in control group [(92.44 ± 2.74)％,P＜0.01].Conclusions HC can promote IL-15 activated NK cells proliferation and enhance NK cells mediated killing activity against SW1990 cells with proper concentration,and up-regulation of perforin,granzyme B and IFN-γ expression may be the main mechanisms.
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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 observe the effect of pituitary adenylate cyclase activiting polypeptide (PACAP) on traumatic brain injury (TBI) and on tbeCD4+/CD8+ T cell number in blood and spleen of rats.MethodsThe male SD rats were randomly (random number) divided into sham operation group ( n =6),normal saline + TBI group ( n =6) and PACAP + TBI group ( n =6).Right parietal cortical contusion was produced by a weight-dropping method.PACAP was administered intra-cerebroventricularly in a dose of 1 μg/5 μl 20 min before TBI.Brain tissue samples were taken 24 h after trauma.The injured brain tissue of rats was examined by HE stain.The numbers of CD4+/CD8+ T cells in blood and spleen were deteced with flow cytometry.Results Edema,hemorrhage,inflammatory cell infiltration,swollen,degenerated neurons and neurons arrayed disorderly around the injured cortex in hippocampus were found under light microscope.The average numbers of CD4 + T lymphocytes counts in blood and spleen were lower ( P =0.000,P =0.005 ),and number of CD8 + T lymphocytes was higher ( P =0.01 ) in TBI rats group than those in the sham operation group.Micro-injection of PACAP into cerebroventricular attenuated the injury,significantly increased the number of CD4 + T lymphocytes in blood and spleen ( P =0.019,P =0.839),and decreased the number of CD8 + T lymphocytes.ConclusionsPretreatment with PACAP may protect against TBI via influencing periphery T cellular immune function.
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Objective To observe the local immue response and changes of angiogenic factors of tumor cells in Heps-bearing mice after mesenchymal stem cell (MSC) are administrated. And to explore the feasibility and safety of MSC for liver tumors therapy. Methods MSC were obtained through adherent culture method. Phe-notypes of MSC were analyzed by flow cytometry. MSC were labeled with DAPI in vitro. 54 Mice of 8 weeks of age with subcutaneously transplanted liver carcinomas were developed randomly. When the maximal diameters of the tumor reached 0.5 - 0.8cm, they were divided into three groups randomly: MSC group, DAPI group and NS control group. 2 × 10~6 MSC and MSC marked by DAPI were administrated into the mice right rear back tumor tissue. The survival time of the tumor-bearing mice was recorded and the mean survival time was calculated. Immunohistochemical staining was performed to count CD4~+ T cells and CD8~+ T cells in the local tumor,as well as to examine the expression of vascular endothelial growth factor ( VEGF) in tumor cells. Results In the MSC group,the mean survival time was 45 d (95%CI;33 ～56 d) ,in the NS control group, the mean survival time was 33 d ( 95%CI : 28 ～ 37 d). There was a statistical significance in the difference between them ( P < 0.05). Immunohistochemical staining results showed as follow: the number of CD4~+ T cells and CD8~+ T cells in the MSC group decreased significantly in comparison with the NS control group at early stage. The expression of VEGF also decreased obviously in comparison with the NS control group and induced tumor cells necrosis at late stage. The survival time of MSC group was prolonged. Conclusion MSC can engraft in Heps-bearing tumor tissue, and inhibit T lymphocyte cellular immunity at early stage. It can reduce the number of CD4~+ T cells and CD8~+ T cells and promote tumor growth. MSC can down regulate VEGF expression and induce tumor cells necrosis at late stage. By this way,it can prolong the survival time of Heps-bearing mice.