Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Establishment of method for PD-1 immunophenotype detection in TCR Vβ repertoire of CD3+, CD4+ and CD8+ T-cell subsets

AIM:To establish the method for detecting the immunophenotype of immunosuppressive receptor programmed cell death protein 1 (PD-1) in T-cell receptor (TCR) Vβ repertoire of CD3⁺, CD4⁺ and CD8⁺ T-cell subsets, therefore to evaluate the distribution of PD-1 in T-cell repertoire from human peripheral blood (PB). METHODS:The PB samples from 10 cases of healthy individuals (HI) were collected. Using multi-colored fluorescence flow cytometry, the distribution frequency of PD-1 in TCR Vβ repertoire was detected with a wide panel of anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and 24 anti-TCR Vβ repertoire (IOTest^® Beta Mark TCR Vβ Repertoire Kit, containing 8 tubes which labeled A~H, each tube is a composite antibody of FITC and PE coupling, each cocktail contains antibodies direc-ted to 3 different Vβ subfamilies) monoclonal antibodies. RESULTS:The total number of the 24 TCR Vβ repertoire detected in CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells from 10 cases of HI was consistent with the Quick Reference Card data provided by the kit. The preliminary results showed that the frequency of Vβ usage in CD3⁺, CD4⁺ and CD8⁺ T cells was different. High usage of Vβ2, Vβ3, Vβ8, Vβ9, Vβ5.1, Vβ13.1 and Vβ13.2 was found in CD3⁺ T cells, while high usage of Vβ2, Vβ3, Vβ8, Vβ5.1, Vβ9 and Vβ13.1 in CD3⁺CD4⁺ T cells, and high usage of Vβ1, Vβ2, Vβ3, Vβ9, Vβ13.1 and Vβ13.2 in CD3⁺CD8⁺ T cells were also observed. Further analysis showed that the expression of PD-1 was detected in all 24 TCR Vβ subfamilies of CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. The higher frequency of PD-1⁺ T cells was CD4⁺Vβ5.2⁺ T cells, whereas the higher frequency of PD1⁺ T cells in CD8⁺Vβ11⁺ and CD8⁺Vβ13.6⁺ T cells was detected. CONCLUSION:The method for detection of the immunosuppressive receptor PD-1 in TCR Vβ repertoire of T-cell subsets is successfully established, which provides a new method for further analysis of immunosuppressive characteristics of TCR Vβ repertoire in the patients with leukemia.     

The characteristics of TCRVα24+ NKT cells in response to in vitro stimulation

AIM: To investigate the amount and patterns of expressing CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells, and compare with that of CD3⁺ T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin (Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells and CD3⁺ T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRVα24⁺ NKT cells to CD3⁺ T cells was about (1.34±0.42)%. The expression rates of CD69 on TCRVα24⁺ NKT cells and CD3⁺T cells in response to PDB+Ion for 6 h were (96.71±1.33)% and (98.60±0.47)%, respectively, while the ratio were (11.47±2.86)% and (1.07±0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-γ on TCRVα24⁺ NKT cells stimulated with PDB+Ion for 6 h were (48.62±2.44)% and (46.65±8.91)%, respectively, which were significantly higher than that of unstimulated group [(31.57±3.31)%, (13.45±6.29)%] and that of stimulated CD3⁺ T cells, though the expression rates on stimulated CD3⁺ T cells were significantly higher than that of unstimulated CD3⁺ T cells. CONCLUSION: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-γ and IL-4 on these lymphocytes are higher than CD3⁺ T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.     

Changes of CD28, CD56 and CD57 expression on CD8+T cells in the peripheral blood of healthy elderly individuals

AIM:The expression of CD28, CD56 and CD57 on CD8⁺T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS:Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8⁺CD28⁺, CD8⁺CD56⁺ and CD8⁺CD57⁺T cells in the peripheral blood between the young and elderly groups.RESULTS:CD8⁺CD28⁺T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07±5.28 and 49.84±7.43,respectively (P＜0.05). Conversely, CD8⁺CD56⁺T cells and CD8⁺CD57⁺T cells in the peripheral blood of the elderly group were significantly higher than those in the young group, and the percentage was 6.60±2.40 vs 2.10±0.35,41.82±6.01 vs 22.89±2.80, respectively(P＜0.05).CONCLUSION:The expression of CD28, CD56 and CD57 on the CD8⁺T cells in the peripheral blood are changed significantly with aging. The decrease in CD28 expression may play an important role in the immunosenescence, while the increase in CD56 and CD57 expression seems to be a compensatory adaptation for the immune dysfunction.     

Elevation of blood Th17 cells and CD3+CD8-IL-21+ T cells in patients with cervical cancer

AIM: To investigate the role of Th17 cells in the patients with cervical cancer.METHODS: We measured the peripheral levels of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in 37 cervical cancer (CC) patients, 25 cervical intraepithelial neoplasia (CIN) patients and 18 healthy controls by flow cytometry. The percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in total CD4⁺ cells were calculated.RESULTS: Compared with controls, the patients with CC or CIN had higher proportions of Th17 cells (all P<0.01) and CD3⁺CD8^-IL-21⁺ T cells (all P<0.05). Notably, in CC patients, the increased percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells were independently associated with the clinical stage(all P<0.05), lymph node metastasis (P<0.01,P<0.05) and vasoinvasion (all P<0.01), while the elevated percentage of CD3⁺CD8^-IL-21⁺ T cells was also associated with the tumor size(P<0.01). Furthermore, the percentage of Th17 cells was positively correlated with that of CD3⁺CD8^-IL-21⁺ T cells in healthy controls and CC patients, but not in CIN patients.CONCLUSION: Our results indicates a possible role of Th17 cells in CC patients correlated with CD3⁺CD8^-IL-21⁺T cells, and the elevated percentage of circulating Th17 cells may be involved in the development and progression of cervical cancer.     

The biological characteristics of cytokine-induced killer cells

AIM:To investigate the biological characteristics of cytokine-induced killer (CIK) cellsin vitro.METHODS:The non-adhere peripheral blood monoclear cells from healthy donors were induced into CIK cells in the presence of IFN-γ, IL-1β, IL-2 and anti-CD3 antibody. LAK (lymphokine activated killer) cells were prepared as a control. The cellular phenotype were detected by FCM and immunocytochemistry and the cytotoxicity was measured by LDH release assay.RESULTS:After 2 weeks of induction, the proliferation rate of CIK cells reached a peak and the proportion of CD3⁺ population was above 95%, and then the cells growth entered to plateau phase at week 3. The proportion of CD3⁺CD56⁺ NKT subset cells was 16.5% on day 15 and it had no obvious variety between 2 and 4 weeks. Correspondingly, LAK cells grew slowly and had lower proliferation rate compared with the CIK cells (P<0.01). CIK cells showed higher specific lysis rates to BeL-7402 hepatoma cells than those of LAK cells at different effector to target ratio (P<0.01). Immunocytochemical staining showed the CIK cells highly co-expressed HLA-DR and CD54 antigens. The NKT cells were slightly bigger than CD3⁺CD56^- cells and a large quantity of pseudopodia were observed on their surface.CONCLUSION:The CIK cells have higher proliferation potency and stronger cytotoxicity to lyse tumor cells than LAK cellsin vitro.Within the span of time from 14 to 21 days, the proliferation rate and the proportion of CD3⁺CD56⁺ subset of CIK cells all reach peaks. Therefore, CIK cells in this period are suitable for clinical application.     

中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛征文通知

"中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛"将于2012年10月在西安举行,即日起征集:①研究论文摘要,②有关园艺学进展的综述文章。经审查合格的摘要和综述将分别收入"中国园艺学会2012年学术年会论文摘要集"和"庆祝《园艺学报》创刊50周年—园艺学进展论坛专辑",并于会前以     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Effect of pioglitazone on balance of regulatory and effector T cells of uremic apolipoprotein E knockout mice in vitro

AIM: To investigate the effects of pioglitazone on the quantity and function-related factors of regulatory and effector T cells (Treg and Teff ) of uremic apolipoprotein E knockout mice in vitro with or without the stimulation of atherosclerotic plaque-specific antigen oxidized low-density lipoprotein (oxLDL). METHODS: Uremic apolipoprotein E knockout mouse model was established by 2-step surgical procedure. After intervention with different concentrations (2 μmol/L and 20 μmol/L) of pioglitazone and PPARγ antagonist GW9662 (5 μmol/L) on splenocytes of uremic mice for 12 h in the presence or absence of oxLDL (2 mg/L), the levels of CD4⁺ CD25⁺ Foxp3⁺ Treg and IFNγ⁺ CD4⁺ Teff were determined by flow cytometry. The mRNA expressions of Foxp3 and IFNγ was detected by real-time fluorescent quantitative PCR. RESULTS: In vitro, oxLDL induced a Treg/Teff imbalance in splenocytes from the uremic mice. Pioglitazone upregulated the level of Treg and mRNA expression of Foxp3 in the presence of oxLDL, which was not antagonized by GW9662. Meanwhile, pioglitazone downregulated the level of Teff and mRNA expression of IFNγ in the presence or absence of oxLDL, which was reversed by GW9662. CONCLUSION: oxLDL induces a Treg/Teff imbalance in uremic apolipoprotein E knockout mice. Pioglitazone modulates the Treg/Teff imbalance probably through PPARγ-independent and-dependent mechanisms.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Detection and clinical significance of myeloid-derived suppressor cells in peripheral blood of patients with Parkinson disease

AIM: To detect the myeloid-derived suppressor cells (MDSCs) in peripheral blood from the patients with Parkinson disease (PD) and its clinical significance. METHODS: The patients (n=80) diagnosed PD from January 2016 to March 2017 in our hospital and 20 healthy volunteers were selected as the subjects. According to the Hoehn-Yahr staging, 80 PD patients were staged, of whom 22 were I, 24 were Ⅱ, 20 were Ⅲ, 14 were IV, and 0 was V. Peripheral blood (5 mL) samples from the patients with PD and the healthy volunteers were collected and the mononuclear cells were isolated. The levels of CD14⁺CD11b⁺ cells and CD14^-CD11b⁺ cells in the peripheral blood were detected by flow cytometry. The two populations of the cells were sorted by magnetic beads. The mRNA levels of arginase 1 (ARG1), interleukin-10 (IL-10) and cyclooxygenase 2 (COX-2) were detected by qPCR. The expression of surface membrane proteins CD14 and CD11b, and immunosuppressive factors ARG1, IL-10 and COX-2 was determined by Western blot and ELISA. RESULTS: No significant change of CD14⁺CD11b⁺ cells between the patients with PD and normal controls was observed, but the cells with CD14^-CD11b⁺ increased significantly in the patients with PD compared with the control people (P<0.05). The CD14^-CD11b⁺ cells in peripheral blood of the patients were related to the stage of Hoehn-Yahr. The CD14^-CD11b⁺ and CD14⁺CD11b⁺ cells showed high levels of IL-10 and COX-2, and the high level of ARG1 was only expressed in the CD14^-CD11b⁺ cells. The expression of ARG1 in the CD14^-CD11b⁺ population from PD patients was significantly different from that of CD14⁺CD11b⁺ population and normal subjects (P<0.05). CONCLUSION: The CD14^-CD11b⁺ cells and ARG1 expression level in peripheral blood of the PD patients can be used to evaluate the pathogenesis and staging. Immunosuppression may play an important role in the pathogenesis and development of PD.     

Influences of protein kinase C inhibitors on the expression of IL-2 and IFN-γ by human T-lymphocytes

AIM:To investigate the influences of protein kinase C(PKC) inhibitors on the expression of interleukin-2(IL-2) and interferon-γ(IFN-γ) byin vitro activated T-lymphocytes. METHODS:Double fluorescent staining together with flow cytometry was adopted to detect intracellular cytokines and to analyze the effects of H7 and gossypol on IL-2 and IFN-γ expression levels of T-lymphocytes stimulated with phorbol ester (PDB)+ionomycin(I) in the presence of monensin.RESULTS:The expression rates of IL-2 and IFN-γ of CD3⁺ T cells stimulated with PDB+I for 4 h were 16.64±2.04 and 25.81±3.53(x±s), respectively, which were significantly higher than that of control (1.06±0.22 and 3.12±0.77)(P＜0.05). Gossypol was able to inhibit the expression of IL-2 and IFN-γ significantly, with the expression rates of 2.08±0.12 and 9.01±1.90, respectively. At the presence of 50 μmol/L H7, the rates of IL-2⁺ and IFN-γ⁺ CD3⁺ T cells were 0.43±0.06 and 2.40±0.27, respectively. The effect of H7 was stronger than that of gossypol. CONCLUSION:PKC plays an important role in the expression of IL-2 and IFN-γ of CD3⁺T cells and its inhibitors H7 and gossypol exert significant inhibitory effect on the expression of these two cytokines. It is suggested that H7 and gossypol may have modulatory effect on T-cell-dependent specific immune responses by inhibiting PKC activity.     

Phenotype of decidual NK cells in women with hypertensive disorder complicating pregnancy

AIM: To investigate the phenotype of uterine decidual natural killer cells (dNK cells) in women with hypertensive disorder complicating pregnancy (HDCP). METHODS: All the study subjects were collected from Department of Obstetrics and Gynecology, The First Affiliated Hospital of Jinan University, Guangzhou, China. Twenty cases of singleton pregnancy who underwent caesarean section because of HDCP were selected as HDCP group, and 15 cases of singleton pregnancy received selective caesarean section because of social-psychological concerns were also randomly selected as normal control group. The decidual tissues were sampled immediately after caesarean section. The mononuclear cells were extracted from the tissues by means of mechanic grinding and gradient centrifugation. The technique of flow cytometry was used for dNK cell sorting and the expression of CD56 and CD16 on the surface of cells was also examined. RESULTS: In HDCP group and normal control group, the proportions of CD56^brightCD16^-CD3^- dNK cells were significantly higher than those of CD56^dimCD16⁺CD3^-dNK cells (P<0.01). Neither the CD56^brightCD16^-CD3^- subset nor the CD56^dimCD16⁺CD3^- subset had statistical difference between HDCP group and normal control group. No significant difference of CD16 expression on the surface of dNK cells between HDCP group and normal control group was observed (P>0.05). CONCLUSION: The phenotypes of dNK cells from the women with HDCP and from healthy pregnant women are both dominated by CD56^brightCD16^-CD3^- subset, without significant difference.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Expression of EPCAM,CD44 and CD24 in gastric cancer tissues and its clinical significance

AIM: To evaluate the relationship between epithelial cell adhesion molecule (EPCAM)/cluster of differentiation 44 (CD44)/cluster of differentiation 24 (CD24) expression and the clinicopathological characteristics/prognosis in 95 gastric cancer patients. METHODS: The expression levels of EPCAM, CD44 and CD24 were detected using the two-step method of immunohistochemistry in 95 gastric cancer patients who underwent surgical excision and were pathologically diagnosed as gastric cancer. RESULTS: There were 56 EPCAM-positive patients (58.95%), 41 CD44-positive patients (43.16%) and 56 CD24-positive patients (58.95%). Thirty patients were both EPCAM and CD44 positive (31.58%), 45 patients were both EPCAM and CD24 positive (47.37%), 32 patients were both CD44 and CD24 positive (33.68%), and 25 patients were EPCAM, CD44 and CD24 positive (26.32%). EPCAM expression was correlated with age, depth of tumor infiltration and WHO histological classification. CD44 expression was correlated with BORRMANN and WHO histological classification as well as CEA value. CD24 expression was correlated with the depth of infiltration, location of the tumor, WHO histological classification and viscera invasion. All positive expression of EPCAM, CD44 and CD24 was correlated with the depth of infiltration, location of the tumor and WHO histological classification (P<0.05). The difference of survival rate between EPCAM positive group and negative group was observed, and the CD44 positive group and negative group had the same result (P<0.05 and P<0.01, respectively). The difference of survival rate between EPCAM⁺CD44⁺CD24⁺ group and EPCAM^-CD44^-CD24^- group was statistically significant (P<0.05). The difference of survival rate between EPCAM^-CD44⁺CD24⁺ group and EPCAM^-CD44^-CD24^- group was also significant (P<0.05). CONCLUSION: The positive rates of EPCAM, CD44 and CD24 expression are high in gastric cancer tissues and these 3 proteins can be used as primary screening targets.     

Effects of thymosin α1 on the development and maturation of thymocytes

AIM: To investigate the effect of Thymosin α₁ on the development and matutation of thymocytes. METHODS: The proportion of CD4⁺CD8⁺ thymocytes and the expression of smoothened (Smo) of the hedgehog (Hh)-signaling in CD4^-CD8^-thymocytes were examined to observe the effect of thymosin α₁ on thymocyte development and matutation. RESULTS: Flowcytometric analysis showed that thymosin α₁ showed activity at a low dose of 30 μg/kg, and 30 μg/kg thymosin α₁ accelerated the replenishment and maturation of thymocytes according to the expression of Smo of the Hh-signaling in CD4^-CD8^-thymocytes, the potent negative regulator of proliferative responses. CONCLUSION: Thymosin α₁ can accelerates thymocyte development from CD4^-CD8^- to CD4⁺CD8⁺.     

Relationship between invasion of tumor-associated macrophages and phenotype and immune efficacy of tumor-infiltrating lymphocytes in advanced ovarian carcinoma

AIM:To explore the relationship between the invasion of tumor-associated macrophages(TAM) and the phenotype and immune efficacy of tumor-infiltrating lymphocytes(TIL) in advanced ovarian carcinoma. METHODS:Immunohistochemical analysis of TAM density in 175 cases of poorly-differentiated ovarian cancer tissue biopsy was performed. The cases were divided into TAM high-density(TAM^High) group and TAM low-density(TAM^Low) group according to the median of TAM density. The control group included 32 cases of benign ovarian lesions. The changes of CD8⁺ and CD25⁺ phenotypes of TIL were detected by flow cytometry analysis. TIL in the 2 groups were cultured in vitro and the conditioned-medium was collected for detecting the expression of IL-2, IL-10, TGF-β and IFN-γ by ELISA. RESULTS:The average TAM infiltration density was 62.8/high-power field(HP, ×400) in 175 cases of poorly-differentiated ovarian carcinoma, and the median was 53.3/HP. TAM^High group was 87 cases and TAM^Low group was 88 cases. A significant difference between malignant ovarian carcinoma group and control group(10.5/HP) was observed. The mean expression of CD8⁺ TIL in TAM^High group was 24%, and CD8⁺ TIL in TAM^Low group was 52%(P<0.05). The mean expression of CD25⁺ TIL in TAM^High was 48%, and CD25⁺ TIL in TAM^Low was 25%(P<0.05). The average infiltration density of CD8⁺ and CD25⁺ TIL in control group was 7%. The average infiltration density of CD8⁺ and CD25⁺ TIL in TAM^High and TAM^Low groups was significantly higher than that in control group(P<0.05). Compared with TAM^Low group, TIL destruction cytokines IL-2 and IFN-γ were significantly decreased in TAM^High group(P<0.05), while the inhibitory cytokines IL-10 and TGF-β were significantly increased(P<0.05). CONCLUSION:In high-density TAM infiltration of ovarian cancer tissues, CD25⁺ TIL type and inhibitory cytokines IL-10 and TGF-β increase, while CD8⁺ TIL type and destruction cytokines IL-2/IFN-γ decrease, suggesting that the high-density TAM has relationship with the phenotype and immune efficacy of TIL.     

High-efficient genetic transfection of CD41+,UT7, U937 and MDA-MB-435 cells with a recombined murine stem cell retroviral vector

AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadher in-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41⁺ cells and cell culture: Cells expressing CD34⁺ from cord blood were isolated. The inducement of cells expressing CD41 from CD34⁺ cells was performed by using TPO and cells CD41⁺ were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41⁺, UT7, U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1×10⁷) and EC1-4 pMSCV retroviruses (1.0×10⁸). With 8-folds dilution retroviruses, 60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells, 72.56% in U937 cells and 30.57% in CD41⁺ cells, respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41⁺ and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION:The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41⁺,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.