The ex vivo expansion characteristic of endothelial progenitor cells

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34⁺ and CD34^- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34⁺ and CD34^-,total MNC led to a significant increase in the expansion of CD34⁺ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34⁺ and AC133⁺cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34⁺ and CD34^- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.     

EP2A promotes human CD34+ cell homing in vitro but not proliferation

AIM: To investigate the role of prostaglandin E₂ receptor 2 agonist (EP₂A) in proliferation and homing of human CD34⁺ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34⁺ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34⁺ cells and BMMSC were divided into 4 groups, and treated with PGE₂ (as positive control), DMSO (as negative control), EP₂A and EP₂A+prostaglandin E₂ receptor 2 antagonist (EP₂AA), respectively. After exposed to the reagents, human CD34⁺ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34⁺ cells in EP₂A group were not higher than those in negative control group. Furthermore, the proportion of human CD34⁺ cells treated with EP₂A in G₂/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34⁺ cells exposed to EP₂A, but the protein expression of CXCR4 in human CD34⁺ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP₂A promotes human CD34⁺ cell homing in vitro but not proliferation.     

Donor age affects confluent EPCs on phenotypic transition,proliferation and migration of smooth muscle cells

AIM: To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells (SMCs). METHODS: Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 (containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1×10⁵ units/L of penicillin and streptomycin, respectively). EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding. Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats. Vascular SMCs were cultured by tissue explant method and identified by α-SM-actin immunofluorescence. In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower chamber. The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE). The protein expression of α-SM-actin and osteopontin was detected by Western blotting. [³H]-TdR incorporation assay was used to determine the proliferation. SMC migration was analyzed by scratch wound healing assay. RESULTS: Compared with P3 group, α-SM-actin expression in P4 group significantly decreased and osteopontin protein expression obviously increased, whereas no significant change was found in P4YE group. Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs. Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phenotype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION: Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs proliferation and migration. Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.     

Role of CCL17 and CCL22 expression in dendritic cells in maternal-fetal immune tolerance

AIM: To determine the expression of CCL17 and CCL22 in dendritic cells (DC) from human decidua and endometria. METHODS: The decidua were collected from normal pregnant women undergoing induced abortion and recurrent spontaneous abortion (RSA) women undergoing early abortion.The endometria were cllected from non-pregnant women undergoing abdominal hysterectomy.The mononuclear cells in the decidua and endometria were isolated. DC were induced by GM-CSF and IL-4, cultured in vitro and identified. The expression of CCL17 and CCL22 in DC at mRNA and protein levels was analyzed by real-time PCR and ELISA. RESULTS: The mRNA levels of CCL17 and CCL22 in decidual DC in normal pregnancy group were 3.04?0.40 and 1.83?0.24, respectively, significantly higher than those in endometrial DC in non-pregnancy group (0.85?0.24 and 0.31?0.08, respectively, P<0.01) and those in decidual DC in RSA group (1.65?0.14 and 0.96?0.09,respectively,P<0.01). Decidual DC continually and strongly secreted CCL17 and CCL22. The levels of CCL17 and CCL22 in normal pregnancy group were significantly higher than those in non-pregnancy group and RSA group at the same culture time point (P<0.01). CONCLUSION: The expression of CCL17 and CCL22 in decidual DC in pregnant woman increases. This may attract more CD4⁺CD25⁺ regulatory T cells to decidua and play an important role in the establishment of maternal-fetal immune tolerance.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

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.     

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.     

Detection and clinical significance of circulating tumor cells in peripheral blood of nasopharyngeal cancer patients with modified immunomagnetic enrichment and fluorescent immunocytochemistry

AIM: To investigate the clinical significance of the new method of modified immunomagnetic enrichment of tumor cells in combination with fluorescent immunocytochemistry to detect circulating tumor cells (CTCs) in peripheral blood samples of patients with nasopharyngeal cancer (NPC). METHODS: Peripheral blood samples were obtained from 76 histology-proven patients with NPC before the initial therapy. After isolation of the mononuclear cells, the CTCs expressing cytokeratin (CK)8/18 in the blood samples were detected by the method of immunomagnetic enrichment in combination with fluorescent immunocytochemistry. The magnetic beads covalent binding with epithelial cell adhesion molecule (EpCAM) antibody were used to enrich the tumor cells which expressed EpCAM. After median following-up for 25 months, the effects of CTCs and other prognostic factors on patient prognosis were thoroughly investigated. RESULTS: None of the positive CK8/18 cells was detected in 20 normal blood samples. The CTCs were detected in 82.9% of the patients (P<0.01). Relapse patients had significantly higher number of median CK8/18⁺ CTCs than the patients without relapse (P<0.01). No association of viral capsid antigen (VCA)-IgA (P>0.05) was observed between the patients with and without relapse. Relapse-free survival rates were lower when the number of peripheral blood CK8/18⁺ CTCs was more than 3. The 2-year relapse-free survival rates were 100%, 100%, 100%, 94.1%, 71.4%, 53.3% and 44.4%(P<0.01) when the numbers of peripheral blood CK8/18⁺ CTCs were 0, 1, 2, 3, 4, 5, 6 before treatment, respectively. Overall survival rates were lower when the number of peripheral blood CK8/18⁺ CTCs was more than 5, but the difference was not significant. The 2-year overall survival rates were 100%, 100%, 100%, 100%, 100%, 100%, 80% and 77.8% (P>0.05) when the numbers of peripheral blood CK8/18⁺ CTCs were 0, 1, 2, 3, 4, 5, 6 before treatment,respectively. The VCA-IgA titer could not predict survivals. Cox multivariate analysis also showed the same results. CONCLUSION: Peripheral blood CK8/18⁺ CTCs are a prognostic factor for initial treatment of NPC.     

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.     

The effect of mifepristone on natural killer subsets of peripheral blood and decidua in early pregnancy

AIM: To investigate the effect of mifepristone on natural killer(NK) subpopulations of peripheral blood and decidua in early pregnancy. METHODS: Flow cytometry was used to detect the expression of CD56 and CD16 on lymphocytes of decidua and peripheral blood in early pregnancy, mifepristone-treated pregnancy. RESULTS: The percentages of different natural killer subsets in peripheral blood between early pregnancy and mifepristone-treated pregnancy were almost identical. The serum levels of estradiol and progesterone in mifepristone-treated pregnancy were slightly higher than in early pregnancy. The percentage of decidual CD56⁺NK cell in early pregnancy was significantly higher than in mifepristone-treated pregnancy. The CD56⁺NK cells were predominant lymphocyte population of decidua in mifepristone-treated pregnancy, but in which the CD56⁺CD16⁺ and CD16⁺NK cells were major lymphocyte subpopulations of peripheral blood. CONCLUSION: Mifepristone acted principally on feto-maternal interface, it blocked the proliferation and differentiation of decidual CD56⁺NK cells and induced embryo immune rejection.     

The vascular endothelial progenitor cells and angiogenesis in ischemic cardiovascular diseases

The vascular endothelial progenitor cells are a population of functional endothelial precursors in circulating blood, which are derived from bone marrow or cord blood. CD34⁺, Flk-1⁺ and ACl33⁺ are their molecular markers. In this review, the functional characterization of vascular endothelial progenitor cells is introduced and the relationship between vascular endothelial progenitor cells and angiogenesis in is chemic cardiovascular diseases is discussed. These data may offer a foundation for the development of therapeutic angiogenesis for the prevention and treatment of ischemic cardiovascular diseases by transplantation of vascular endothelial progenitor cells.     

Effects of Ca2+on the releases of ET-1, NO and PGI2 from bovine coronary artery endothelial cells during hypoxia

AIM: To explore the mechanism of ET-1, NO and PGI² release from coronary artery endothelial cells(CAEC) induced by acute hypoxia. METHODS: Bovine coronary artery endothelial cells were cultured and [⁴⁵ Ca²⁺] was used to investigate the difference of calcium uptake between normoxia group and hypoxia group(3% O₂). The contents of ET-1, NO and PGI² in media of normoxia group, hypoxia group and hypoxia + verapamil group were measured 24 h after hypoxia. RESULTS: [ ⁴⁵ Ca²⁺] uptake by CAEC in hypoxia group was 1.9 times more than normoxia group(P< 0.01). Hypoxia + verapamil group released more PGI², ET-1 and less NO than hypoxia group(P< 0.05). CONCLUSION: Changes of ET-1, NO and PGI² releases during hypoxia may be caused by the inflow of Ca²⁺ into coronary artery endothelial cells.     

Effect of heterogenic corneal stroma transplantation on peripheral T cells of rats

AIM: To Compare immunogenicity of three kinds of heterogenic corneal stroma. METHODS: 36 SD rats were randomized into 4 groups, each group consisting of 9 rats. Group 1 was control group. Three kinds of heterogenic corneal stroma: porcine, rabbit and chicken corneal stroma were heterotopically transplanted to subcutaneous layer of 27 (group 2-4) SD rats, respectively. The expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ on peripheral T cells was identified and analyzed by dual fluorescence flow cytometry at 7, 14, 28 days after operation. RESULTS: Compared with control group, the expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ was no significant change in porcine corneal stroma group(P＞0.05), the expression of CD4⁺ was increased in rabbit corneal stroma (P＜0.05), CD4⁺, CD4⁺ CD71⁺ markedly higher in the chicken corneal stroma (P＜0.01) at 7 days after operation. CONCLUSION: The immunogenicity of porcine stroma is the lowest in three kinds of heterogenic corneal stroma (chicken, rabbit and porcine).