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.     

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.     

Glucocorticoids enhance differentiation of central memory CD8+ T-lymphocytes in vitro

AIM:To investigate the effect of glucocorticoids, a kind of traditional immunosuppressive drug, on expanding central memory CD8⁺ T cells (CD8⁺ T_CM) and to provide a novel method of generating CD8⁺ T_CM for adoptive immunotherapy.METHODS:Healthy human peripheral blood mononuclear cells were isolated by density gradient centri-fugation. Naïve CD8⁺ T cells were further isolated with MACS microbeads and flow cytometry. After activated by cytokines, the cells were divided into experimental group and control group. Glucocorticoids at different concentrations and an identical volume of PBS were added. The phenotypic characteristics of T_CM in different groups were measured by flow cyto-metry at separate time points. Furthermore, the effect of glucocorticoids on CD8⁺ T cell expansion was further verified using CFSE assay and Annexin V staining.RESULTS:Glucocorticoids significantly increased the proportion of CD8⁺ T_CM in vitro. Glucocorticoids sustained CD8⁺ T cell expansion. Glucocorticoids had low toxicity for CD8⁺ T cells.CONCLUSION:Glucocorticoids effectively increase the number and proportion of CD8⁺ T_CM in vitro, which provides novel insights into the generation of human CD8⁺ T_CM and reveals a novel potential clinical application of glucocorticoids for immunotherapy.     

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.     

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.     

Effect of microRNA-7 knockdown on ConA-induced acute liver injury in mice

AIM: To study the effect of microRNA-7 (miR-7) knockdown (KD) on concanavalin A (ConA)-induced acute liver injury (ALI) in mice.METHODS: Wild type (WT) mice and miR-7KD mice were received ConA (30 mg/kg) to induced acute liver injury model by intraperitoneal injection, and the morphological changes, liver weight and weight index were measured 48 h later. The pathological changes of the liver tissues were observed by HE staining. The levels of serum alanine aminotransferase (ALT), IL-4 and IFN-γ were detected by ELISA. The proportional changes of CD4⁺ T cells and the relative levels of IL-4 and IFN-γ were analyzed by flow cytometry.RESULTS: The color of the liver tissue became lighter, and the weight and weight index were changed significantly in miR-7KD mice compared with control group (P<0.05). HE staining showed that the inflammatory cell infiltration was increased in the liver of miR-7KD mice. Moreover, the level of serum ALT was significantly increased (P<0.05). The serum level of IFN-γ elevated significantly (P<0.01), while the IL-4 levels decreased significantly (P<0.01) in the serum of miR-7KD mice. Furthermore, the proportion of CD4⁺ T cells and relative IFN-γ cells increased obviously (P<0.01).CONCLUSION: miR-7 knockdown promotes the pathogenesis of the ConA-induced acute liver injury in mice.     

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.     

Study on the potential to differentiate into myocytes of the CD34+ cells

AIM:To investigate the potential of differentiatng into myocytes of the granulocyte colony-stimulating factor(G-CSF)-mobilized CD34⁺ cells. METHODS:Three hours after intraperitoneal injecction of isoprenaline(ISO)to develop acute ischemic model,rats’bone marrow hematopoietic stem cells were mobilized to the site of myocardial infarction by G-CSF.The techniques of immunohistochemisty and HE stain were used to detect the infiltration of CD34⁺ cells and the regeneration of myocytes in the infarct zones. RESULTS:24 hours after administration of ISO, a large amount of infiltrative monocytes and regenerative myocytes which were CD34 positive expression could be found in the infarct zones of the G-CSF treatment group, while majority of the infiltrative inflammatory cells in control group were neutrophils and there was no infiltrative cells and myocytes which were CD34 positive expressio, 2 weeks after administration of ISO, there were a plenty of scar in control group, but not in the G-CSF treatment group. CONCLUSION:G-CSF-mobilized CD34⁺ cells possess the potential to differentiate into myocytes and it may be used in treating acute myocardial infarction.     

CD4+CD25+FOXP3+ Tregs and HBV-specific CTLs in peripheral blood from chronic hepatitis B patients

AIM: To investigate the roles of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) and HBV-specific cytotoxic T-lymphocytes (CTLs) in peripheral blood from the patients with chronic hepatitis B (CHB).METHODS: Peripheral blood mononuclear cells from 28 patients with CHB and 15 healthy controls were analyzed for Treg frequency using flow cytometry and for HBV-specific CTLs using enzyme-linked immunospot assay (ELISPOT).The clinical data of HBV-infected patients were considered.RESULTS: The frequency of CD4⁺CD25⁺FOXP3⁺Tregs was higher in the patients with CHB than that in the patients of healthy controls (3.14%±0.97% vs 1.95%±0.68%, P<0.05), and a positive correlation was found between Tregs and the DNA levels of HBV (r=0.831, P<0.01).HBV-specific CTLs were detected by ELISPOT in CHB patients and a negative correlation was observed between Tregs and CTLs (r=-0.540, P<0.01).CONCLUSION: Peripheral blood CD4⁺CD25⁺FOXP3⁺ Tregs in CHB patients are increased and closely correlated with the DNA replication of HBV and CTLs, suggesting that the clearance of HBV can be influenced by the inhibition of cellular immunoreaction through Tregs.     

Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.     

Effects of miR-155 over-expression on expression of inflammatory factors and indolamine 2, 3-dioxygenase in microglial BV-2 cells

AIM:To explore the effect of microRNA-155(miR-155)over-expression on the expression of inflammatory factors and indolamine 2, 3- dioxygenase (IDO) in the microglial BV-2 cells. METHODS:For over-expression of miR-155, the BV-2 cells were transfected with lentiviral vector carrying mmu-miR-155. The expression of inflammatory factors was detected by cytometric bead array system (CBA). The mRNA expression of inflammatory factors and IDO was analyzed by real-time PCR. The protein levels of suppressor of cytokine signalling 1 (SOCS1), p-p38 MAPK and IDO were determined by Western blot. RESULTS:The expression of miR-155 was up-regulated in the BV-2 cells transfected with lentiviral vector carrying mmu-miR-155 compared with LPS treatment group (P<0.01). The miR-155 over-expression promoted the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and IL-10, and inhibited the secretion of IL-12. The miR-155 over-expression increased the mRNA expression of IL-6, TNF-α, IL-10 and IDO, also increased the protein levels of IDO and p-p38 MAPK, but decreased the protein expression of SOCS1 (P<0.01). LPS promoted the secretion of IL-6, TNF-α, MCP-1 and IL-12, also increased the mRNA expression of IL-6, TNF-α and IDO, meanwhile, increased the protein levels of IDO, p-p38 MAPK and SOCS1 (P<0.01). CONCLUSION:Over-expression of miR-155 promotes the secretion of related imflammatory factors and protein expression of IDO in microglial BV-2 cells mediated with SOCS1 and p38 MAPK signaling pathway.     

Methods for expansion of human CD8+ memory T cells in vitro

AIM：To investigate new methods for the expansion of human CD8+ memory T cells in vitro and provide novel means of anti-viral and anti-tumor adoptive immunotherapy. METHODS：Six kinds of stimuli, anti-CD3 antibody, anti-CD28 antibody, CD70, IL-2, IL-7 and IL-15, for the expansion of human CD8+ memory T cells in vitro were selected and arranged for their combinations, resulting in 63 kinds of stimulating combinations. Normal human CD8+ T cells were isolated and exposed to these stimuli. After 14 days of cell culture, the number and purity of CD8+ T cells, and the percentages of CD8+ central memory T cells (TCM) and CD8+ effector memory T cells (TEM) were detected. The expansion folds of CD8+ T cells, CD8+ TCM and CD8+ TEM were calculated and the relatively better stimulating combination was determined. RESULTS：The combination of anti-CD3 antibodies, IL-2 and IL-7 was a better method for the expansion of human CD8+ T cells, CD8+ TCM and CD8+ TEM in vitro, and the expansion folds were 13.19, 13.28 and 15.27, respectively. CONCLUSION：The combination of anti-CD3 antibodies, IL-2 and IL-7 is the relatively better method for the expansion of human CD8+ memory T cells in vitro.     

Effects of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children

AIM: To investigate the effect of glucocorticoid inhalation on the levels of CD4+CD25+ regulatory T cells in peripheral blood of asthmatic children. METHODS: Glucocorticoid inhalator was inhaled by 70 children with attack asthma. The levels of CD4+CD25+Tr in peripheral blood of asthmatic children were tested by flow cytometry (FCM). RESULTS: The CD4+CD25+Tr levels in peripheral blood of asthmatic children were (5.62%±1.29%) and (7.05%±1.61%) before and after of regulated glucocorticoid inhalation, respectively (P<0.01). The Tr levels were (7.56%±1.88%), (7.09%±1.23%) and (6.11%±1.96%) in the complete control group, part control group and poor control group, respectively (P<0.05). The Tr level in formal treatment group (7.05%±1.61%) was higher than that in irregular treatment group (5.91%±1.76%), P<0.01. CONCLUSION: The level of CD4+CD25+Tr is remarkable increased by regulated glucocorticoid inhalation, and the level of Tr can reflect the effects of glucocorticoid inhalation.     

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.     

Effects of PFOA exposure on inflammatory mediators IL-4, IFN-γ and glucocorticoid receptor in asthmatic mice

AIM: To investigate the effects of perfluorooctanoic acid (PFOA) exposure on the changes of asthmatic mouse airway inflammation, inflammatory mediators interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum, and glucocorticoid receptor (GR) expression in the lung tissue.METHODS: BALB/c mice (n=30) were randomly divided into 5 groups:normal control (C) group, asthma (A) group, asthma+low-dose PFOA (AP10) group, asthma+ mode-rate-dose PFOA (AP50) group and asthma+high-dose PFOA (AP100) group. Asthma model and PFOA exposure model of mice were established according to the grouping. The animals were sacrificed and their lungs were collected for HE staining, transmission electron microscopy, Western blot and immunohistochemical staining. ELISA was applied to detect the levels of IL-4 and IFN-γ in the serum.RESULTS: HE staining of the lungs showed that the asthmatic mice, compared with the normal control mice, had obvious mucus secretion around the airways and infiltration of inflammatory cells around airways and blood vessels, and the effects were much more marked in AP groups. Ultrastructural alteration of the lung tissues in the asthmatic mice were indicated by transmission electron microscopy. Compared with C group, the results of ELISA in A group and AP groups proved that IL-4 in the serum was increased and IFN-γ was decreased significantly (P<0.05). Compare with A group, IL-4 was significantly increased and IFN-γ was decreased in AP100 group (P<0.05), and no difference of those between AP10 group and AP50 group was found. The results of Western blot indicated that GR protein expression in the asthmatic mice were decreased compare with the normal mice (P<0.05), and no difference of that among A group and AP groups was observed. Immunohistochemical staining manifested that GR protein was mainly located in the cytoplasm of bronchial columnar epithelial cells, airway smooth muscle cells and vascular smooth muscle cells.CONCLUSION: Acute airway PFOA exposure in asthmatic mice dose-dependently exacebates lung inflammation by inducing Th2 type immune responses, promotes infiltration of inflammatory cells and mucus secretion around the airways and blood vessels, and destroys the ultrastructure of the lung tissues.     

Imperatorin increased sensitivity of lung cancer CD133+ cell subsets to gefitinib by down-regulating c-met expression

AIM: To investigate the role of imperatorin in reversing the resistance of the PC9 CD133⁺ cell subsets to gefitinib. METHODS: MTT assay was performed to evaluate the viability of PC9 cells treated with imperatorin and gefitinib. The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot. The percentage of CD133⁺ cell subsets population and the apoptotic rate of the PC9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry. RESULTS: The sensitivity of the PC9 CD133⁺ cell subsets to gefitinib was significantly lower than that of the PC9 CD133^- cell subsets. Treatment with gefitinib alone significantly inhibited the protein levels of EGFR/PI3K/AKT in the PC9 CD133^- cell subsets but not the PC9 CD133⁺ cell subsets. Treatment with gefitinib alone increased the percentage of CD133⁺ cell subsets population in the PC9 cells. However, combination of gefitinib with imperatorin significantly inhibited the enrichment of CD133⁺ cell subsets population. Imperatorin down-regulated c-met expression, suggesting the c-met was the target of imperatorin in the PC9 CD133⁺ cell subsets. The results of MTT assay, Western blot analysis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC9 CD133⁺ cell subsets.CONCLUSION: Imperatorin increases the sensitivity of lung cancer CD133⁺ cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib.     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

Relationship of CTCs with CD4+/CD8+, NLR,TTV and tumor stage in patients with primary hepatocellular carcinoma

AIM: To explore the relationship of circulating tumor cells (CTCs) with CD4⁺/CD8⁺, neutrophil-to-lymphocyte ratio (NLR), total tumor volume (TTV) and tumor stage in the patients with primary hepatocellular carcinoma.METHODS: We selected 80 cases of histologically diagnosed primary hepatocellular carcinoma in the study. The method of CanPatrol^TM was used, which was developed by SurExam biotechnology company to identify CTCs in the blood. CD4⁺ T cells and CD8⁺ T cells were counted by flow cytometry. The patients were divided into 2 groups according to the levels of CD4⁺/CD8⁺ ratio, NLR and TTV. The patients were also divided into Ⅰ＋ Ⅱstage group and Ⅲ＋ Ⅳ stage group according to the seventh edition of TMN staging criteria.RESULTS: The numbers of peripheral blood CTCs and mesenchymal CTCs in high CD4⁺/CD8⁺ ratio group were significantly lower than those in low CD4⁺/CD8⁺ ratio group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in high TTV group were significantly higher than those in low TTV group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in I＋ II stage group were significantly lower than those in III＋ IV stage group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs, hybrid CTCs and epithelial CTCs between high NLR group and low NLR group had no statistical difference.CONCLUSION: CTCs exist in the peripheral blood of the patients with primary hepatocellular carcinoma. The peripheral blood CTCs have significant correlations with TTV, tumor stage and T-cell immunity.The worse cell immune function, the larger TTV and the later tumor stage a patient has, the more the peripheral blood CTCs are.     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.     

Bone marrow-derived mesenchymal stem cells regulate the function of Th17/ Treg in peripheral blood of severe asthmatic children

AIM：To investigate the regulatory function of bone marrow-derived mesenchymal stem cells (MSCs) on T helper 17 cells (Th17) and regulatory T cells (Treg) in peripheral blood of severe asthmatic children. METHODS：MSCs were isolated, cultured and identified in vitro. MSCs digested with mitomycin were cocultured with T lymphocytes (TLC) at different ratios (1∶1, 1∶2, 1∶10 and 1∶20) from severe asthmatic children for 72 h. The proliferation of TLC was measured by CCK-8 method. In the coculture system of the 1∶2 ratio and the single TLC system, the supernatant levels of interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) were measured by ELISA. The mRNA expression of retinoic acid-related orphan nuclear receptor C (RORC) and forkhead box protein 3 (Foxp3) in TLC was detected by qRT-PCR. RESULTS：After cocultured with MSCs, the proliferation of TLC decreased significantly in a dose-dependent manner (P<0.05). It also showed decreases in IL-17 (3 799±441 vs 4 890 ±373, P<0.05) and RORC mRNA level (1.21±0.14 vs 3.85±0.48, P<0.05), while an increase in TGF-β level (209±32 vs 117±26, P<0.05) was observed. No influence on the mRNA expression of Foxp3 was found (P>0.05). CONCLUSION：MSCs suppresses Th17 polarization of naive peripheral blood CD4+ T cells and matures Th17 cells secreting IL-17, which may effectively revise Th17/Treg imbalance of asthma.