The feature of clonal expansion of TCR Vβ T cells from peripheral blood in patients with acute monoblastic leukemia

AIM: To investigate the distribution and clonal expansion of TCR Vβ subfamily T cells in patients with acute monoblastic leukemia (M5). METHODS: The CDR3 of TCR Vβ 24 subfamily genes were amplified in peripheral blood mononuclear cells from 9 cases with M5 using RT-PCR and the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Vβ T cells. RESULTS: Only 1-10 Vβ subfamily T cells were identified in M5 cases. Genescan analysis showed that oligoclonal (clonal expansion) T cells were found in some Vβ subfamilies from 8 cases with M5, Vβ 2 oligoclonal T cells were identified in six cases, whereas Vβ 7- or Vβ 9 clonal expansion T cells were detected in the other two cases, respectively. In addition, except Vβ 2, Vβ 7 or Vβ 21 oligoclonal T cells could be detected in two cases, respectively. CONCLUSION: The skew distribution and clonal expansion of TCR Vβ subfamily T cells could be found in patients with M5.It may be a specific ant i-leukemia immune response with which the host T cells were activated by the leukemia-associated-antigen.The clonal expansion T cells were tendentious in Vβ 2,which may be related to the M5 leukemia cells as sociated antigen.     

The inducement,restricted usage and clonal expansion of TCR Vβ subfamily T cells related to chronic myelogenous leukemia

AIM: To investigate the anti-leukemia effect, the restricted usage and clonal expansion of TCR Vβ subfamily T cells from donor peripheral blood induced by chronic myelogenous leukemia(CML) cells, K562 cells and bcr-abl peptide, respectively. METHODS: T cells in donor's peripheral blood were stimulated with CML cells, K562 cells and bcr3-abl2 peptide and amplified by MLTC, to induce the CML specific cytotoxic T lymphocytes. The induced T cells were further analyzed for the restricted usage and clonal expansion of TCR Vβ subfamilies by using RT-PCR and genescan analysis, and the detection of specific cytotoxicity in CML by LDH release assay. RESULTS: 10-13 Vβ subfamilies were expressed in T cells from donor peripheral blood which were induced with CML cells, K562 cells and bcr-abl peptide in 1-2 weeks by MLTC. Oligoclonal T cell in Vβ16, Vβ21 and oligoclonal tendency T cells in Vβ5, Vβ13 subfamilies were identified in induced T cells, which have the ability of specific cytoxicity to CML cells and K562 cells. CONCLUSION: The anti-CML cytotocity T cells were induced by CML cells, K562 cells and bcr-abl peptide. These induced T cells with specific cytoxicity effect may come from the clonal expansion TCR Vβ subfamily T cells.     

Effects of dendritic cell stimulation on cytotoxic activity of cord blood derived cytokine-induced killer cells,natural killer cells and CD45RO expression in CIK cells

AIM: To investigate the efficacy of dendritic cells (DCs) that augments the cytotoxic activity of cytokine-induced killer (CIK) cells, natural killer (NK) cells from a same donor and the CD45RO expression on CIK cells. METHODS: The expanded killer cells were divided into two groups: group A was pre-cocultured with DCs for 6 days, group B was the control that without any stimulation. Cytotoxicity of CIK and NK cells was measured at different effect-target ratio against K562 and HL-60. CD45RO and CD45RA expression on CIK cells in different groups were detected by flow cytometry. RESULTS: Cytotoxicity of CB derived killer cells was positive correlation with effect-target ratio. The cytotoxicity of group A against HL-60 was higher than that of group B significantly. At 20∶1 effector-target ratio, the lytic activity of group A CIK, NK cells against K562 was higher than that of group B significantly, but no significant difference between them at 10∶1 effector-target ratio. The CD45RO expression on CIK cells in groups A was significantly higher than that in groups B. CONCLUSION: CIK and NK cells cocultured with DCs can augment the killer's cytotoxicity against tumor cells and promote the CD45RO expression on CIK cells.     

Restricted expansion of T cell receptor (TCR)Vβ genes in leukemic patients subjected to allogeneic hematopoeitic stem cell transplantation

AIM: To investigate restricted expansion of TCR Vβ gene repertoire in patients with leukemia following allogeneic hematopoietic stem cell transplantation. METHODS: TCR Vβ subfamily genes in peripheral blood mononuclear cells from 7 cases of leukemia was amplified using RT-PCR. RESULTS: Only two-eight fragments of Vβ genes were detected in samples from these patients, and the detected fragments are different in different patients. CONCLUSION: TCR complexes were abnormal in all patients, part of the genes were seletively expansed and part of them were suppressed after transplantation.     

Function of dendritic cells in cord blood

AIM: To explore the function of dendritic cells in cord blood.METHODS: Dendritic cell precursor subsets pDC/pDC(CD11c+CD123- /CD11c-CD123+) in cord blood and adult peripheral blood were analyzed gated by CD34-Lin- HLA-DR+ cells and the levels of IL-12p40,IL-10,IFN-γ,IL-4 in the serums were tested by ELISA.RESULTS: The level of IL-12p40 in cord blood serum was higher than that in peripheral blood.pDC1/pDC2,IL-10,IFN-γ,IL-4 in cord blood were similar to that in peripheral blood.CONCLUSION: Function development of dendritic cells in cord blood may be consummate.     

Biological characters of mesenchymal stem cells of human umbilical cord blood

AIM：To study the isolation,expansion and purification of mesenchymal stem cells (MSCs) from human umbilical cord blood (UCB),and investigate some biological identities of MSCs.METHODS：(1) MSCs of UCB,adult bone marrow (BM) and fetus BM were isolated by centrifugation with Ficoll,and the different kinds of MSCs were observed everyday.(2) Surface markers of MSCs were identified by flow cytometry.(3) The level of HGFs (TPO,SCF,FLT-3L,IL-6) secreted by different sources of MSCs was checked by ELISA method.RESULTS：(1) No difference in morphology of the colonies between UCB MSCs and BM MSCs was observed.However,the mononuclear cells needed in culture of UCB MSCs was about 3 times more than that in culture of BM MSCs.The times of UCB MSCs colony formation and confluencing were longer than that of BM in primary culture.(2) After passaged,there was no significant difference in the proliferation rates of 3 kinds of MSCs.Only 4 of 15 UCB samples contained a homogeneous population of MSCs.(3) UCB MSCs shared the same markers with BM MSCs.Neither hematopoietic marker nor immunologic recognition antigens were expressed.(4) The level of hematopoietic growth factors (HGFs) secreted by 3 kinds of MSCs was similar.CONCLUSIONS：(1) MSCs were isolated from UCB,but the amount of MSCs in UCB was smaller than that in BM,and just seldom samples of UCB contained homogeneous MSCs.(2) MSCs from UCB and BM shared the same biological characteristics,such as proliferation ability,surface markers,immunophenotypes and HGFs secretion.     

Biological characterics of human fetal cord blood mesenchymal stem cells

AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105⁺/CD166⁺ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56.32%±3.28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.     

The feature of TCR Vα repertoire and clonal expansion of T cells from peripheral blood in patients with acute monoblastic leukemia

AIM: To investigate the distribution and clonal expansion of 29 T cell receptor (TCR) Vα subfamily T cells in patients with acute monoblastic leukemia (AML-M5)．METHODS: The CDR3 of TCR Vα 29 subfamily genes were amplified in peripheral blood mononuclear cells (PBMCs) from 8 cases with AML-M5 using RT-PCR，and the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size,to evaluate clonality of the detectable TCR Vα subfamily T cells.9 normal individuals served as control.RESULTS: Most Vα subfamily genes could be detected in PBMCs from normal individuals,whereas only 1-10 subfamily T cells were identified in 8 cases with AML-M5,the highest frequently expressed Vα subfamily was Vα3 (75%),and Vα12 was the second (62.5%),15 Vα subfamilies (Vα1,4,5,7,9,14-18,20,21,26,28 and Vα29) were absent.Genescan analysis showed that clonal expanded T cells were found in T cells from 6 out of 8 AML-M5 cases.The highest frequency of clonal expanded T cells predominated in Vα12 (3 out of 5 positive samples).In PBMCs from two cases,clonal expanded Vα3 T cells were the unique detectable Vα subfamily T cells.CONCLUSION: The selected usage and clonal expansion of TCR Vα subfamily T cells from peripheral blood could be found in patients with AML-M5.It may be a specific immune response which the host T cells are activated by the M5 leukemia cells.The distribution pattern of Vα subfamily clonal expansion displays individual specificity．     

Phagocytosis of viable apoptotic cells inhibits the activation of T lymphocytes

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF_β1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF_β1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF_β1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF_β1 secretion in local site.     

Changes of serum T-PSA,F-PSA and F/T ratio in patients with prostate cancer (PCa) and its clinical significance

AIM: To evaluate the changes of serum total prostate specific antigen (T-PSA), free prostate specific antigen (F-PSA) and the ratio of F-PSA to T-PSA (F/T) in patients with prostate cancer and its clinical significance. METHODS: The concentrations of T-PSA and F-PSA in serum were measured by micropartical enzyme immunoassay (MEIA) using AxSYM System, and the F/T ratio was calculated. RESULTS: Before operation, the concentrations of T-PSA and F-PSA in patients with PCa were much higher and F/T ratio was significantly lower than that in patients with benign prostate hyperplasia (BPH). T-PSA and F-PSA levels decreased, but F/T ratio increased after operation in PCa and BPH. F/T ratio in 83.5% PCa and 6.5% BPH was less than 0.16. To diagnosis PCa, the sensitivity of F/T ratio was 83.5%, and the specificity was 86.7%. CONCLUSION: Serum T-PSA, F-PSA and F/T ratio are important parameters for the early diagnosis of prostate cancer.     

Effects of p27kip1 gene on DNA and protein synthesis in esophageal carcinoma cells

AIM: To investigate the effects of p27kip1 gene on DNA replication and protein synthesis in esophageal carcinoma cells. METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effects on DNA replication, protein synthesis and the growth of esophageal carcinoma cells were explored by means of cell growth count, [3H]-TdR, [3H]-Leucine incorporation method and flow cytometry. RESULTS: The growth of esophageal carcinoma cells was inhibited obviously. The radioactive intensity of -TdR and -Leucine in Ad-p27kip1 group decreased significantly than that in control group after EC-9706 transfection (P<0.01), while the multiplication of esophageal carcinoma cell was inhibited obviously. CONCLUSION: Ad-p27kip1 inhibited the multiplication of the esophageal carcinoma cells, which may be related to its suppressive function for DNA replication and protein synthesis.     

Apoptosis and expression of P53 mRNA and protein in mouse spermatogenic cells induced by low dose ionizing radiation

AIM:The effect of low dose radiation (LDR) with different doses of X-rays on apoptosis and its related gene P53 expression were studied in spermatogenic cells of male Kunming mouse testis. METHODS:The different kinds of spermatogenic cells were separated using density gradient centrifugation and their apoptosis was measured by flow cytometry. At meantime, P53 protein and P53 mRNA was measured with immunohistochemical SABC and in situ hybridization, respectively. RESULTS:The apoptosis in all kinds of spermatogenic cells induced by LDR had a remarkable regularity. When the doses were 0.025 and 0.05 Gy, spermatogone apoptosis was domaint. With the increase in irradiation dose (0.075-0.2 Gy), spermatocytes also showed an apoptotic change, but the apoptotic percentage of spermatogonia was significantly higher than that of spermatocytes. Moreover, the apoptosis of spermatids and spermatozoa scarely occurred after LDR. P53 protein expressed in spermatogonia and spermatocytes in varying degrees, and the former was significantly higher than that of the latter after LDR. With the increase in irradiation dose, P53 protein expression showed a upregulated tendency, but that of spermatids and spermatozoa scarcely occurred. P53 mRNA primarily expressed in spermatids and spermatocytes when the dose was 0.025 Gy. With the increase in irradiation doses (0.05-0.2 Gy), that of spermatogonia also showed an enhancement. P53 mRNA expression in spermatogonia and spermatocytes showed a remarkable dose-effect relationship. CONCLUSION:The apoptosis of spermatogenic cells was selectively induced by LDR of X-rays, which had remarkable the dose-effect and time-effect relationships. The mechanism of the selective apoptosis in spermatogenic cells by LDR is closely related to the upregulation of P53.     

Roles of Foxc2 in the development of axial skeleton

AIM: To investigate the roles of forkhead box c2( Foxc2 ) in axial skeloton. METHODS: Mice lacking Foxc2 locus were produced by targeted mutation and the developmental anomalies in axial skeleton were analyzed. RESULTS: Foxc2 protein increased markedly in Myoblasts C2C12 after treating with BMP-2, and alkaline phosphatase activity and production of osteocalcin in Myoblasts C2C12 were significantly higher than that in cell lines transfecting antisense Foxc2 sequence. Expressed median cleft palatine, dysplasia of auditory ossicles and vertebral and anteroposterior facial dysplasia were found in all 15 homozygous mouse neonates with targeted mutation of Foxc2 . CONCLUSION: These results suggest that Foxc2 has indispensable roles during skeletogenesis in mesoderm and cells derived from the neural crest.     

《中国蔬菜品种志》

AIM: To characterize the gene expression of sortilin on adipogenic and osteogenic differentiation in mesenchymal stem cells (MSCs) in vitro and explore its significance.METHODS: MSCs derived from human bone marrow were isolated and cultured in vitro, then were stimulated in osteogenic medium and adipogenic medium, respectively. Osteopontin and lipoprotein lipase were detected by RT-PCR. Sortilin expression was analyzed by semiquantitative RT-PCR. RESULTS: 1.MSCs displayed the potential of differentiation into osteoblast and adipocyte. 2.Sortilin was upregulated one day after osteogenic induction and remained upregulated for a week. The expression of sortilin was significant increased on day 3(P＜0.01). 3. No significant changes of sortilin expression was found in adipogenic differentiation (P＞0.05).CONCLUSION: Sortilin may be useful to modulate the osteogenic differentiation and may not be necessary for adipocyte commitment in MSCs. The regulation of sortilin expression may provide new protocal and strategy for the treatment of osteoporosis and osteopenic disease.     

Effects of psychological stress on in vitro expression of activated surface molecules on T cells of peripheral blood from healthy persons

AIM: To study cellular and molecular mechanism involved in increasing susceptibility of infection in psychological stress persons. METHODS: Comparative studies were performed with double staining and flow cytometry analysis on immunophenotyping and in vitro expression of early activating surface molecule CD69 in response to mitogens on T cells from peripheral blood of 20 healthy college student volunteers before and after psychological stress. A series of term final examinations was defined as psychological stress. RESULTS: Immunophenotyping analysis showed no statistically significant difference in the percentage of CD2, CD3, CD4, CD8, CD19, CD20, CD16 and CD56 positive lymphocyte populations before and after psychological stress. There was a statistically significant decrease in the in vitro expression of CD69 in response to polyclonal stimulators on the T cells from persons after psychological stress than those before psychological stress. The percentage of CD69 expression (CD69⁺CD3⁺/CD3⁺%) in response to PHA and PDB in the whole blood culture for 72 hours decreased respectively from 28.1±4.1 and 80.7±6.8 on the T cells obtained before psychological stress to 17.6±3.8 and 65.8±7.9 on those obtained after psychological stress, while there was no statistically significant difference between the CD69 expression rates without stimulators on the T cells obtained before and after psychological stress. CONCLUSIONS: The effects of psychological stress to immune system is not on the level of changing proportions of the sub-populations within peripheral blood lymphocytes. Psychological stress can decrease the activating response of T cells in healthy persons, which may be responsible for the increase of susceptibility to infection in the psychological stress persons.     

Preliminary study of IL-17 signal transduction pathway in peripheral blood mononuclear cells from lupus nephritis patients

AIM: To investigate the role of IL-17 and its signal conduction component-JNK activity in the pathogenesis of LN. METHODS: Peripheral blood mononuclear cells (PBMC) were separated and cultured from 15 cases of active lupus nephritis (LN) patients. IL-6 level was detected by ELISA, IL-6 mRNA was checked with RT-PCR, and JNK activity was measured by Western blot. RESULTS: At same IL-17 end concentrations, there was a much higher level of IL-6 in LN group than in control group (all P<0.05). IL-17 induced a significant elevation of IL-6 mRNA expression and JNK activity in PBMC from LN patients in a time- and dose-dependent manner, which could be blocked markedly by IL-17 monoclonal antibody, mIgG₂₈, and dexamethasone. Much higher IL-6 mRNA expression was observed in LN group than in control group under medium culture or IL-17-conditioned culture (all P<0.01). Under medium culture or IL-17-conditioned culture, there was much higher JNK activity of PBMC in active LN group than that in control group. There was a positive linear correlation between PBMC JNK activity and their SLEDAI or IL-6 mRNA expression level in LN patients (r1=0.638, P<0.01; r2=0.644, P<0.01). CONCLUSION: These results suggest that IL-17 may take part in the initiation and progression of LN through induction of IL-6 overexpression by PBMC,and JNK hyperactivity may be necessary in the IL-17 signal transduction.     

Localization and expression of TGF-β1,2 in fetal and adult skins

AIM:To explore the localization and expression of transforming growth factor-β_1,2 (TGF-β_1,2) and alpha-smooth muscle actin (α-ASMA) in fetal and adult skins. METHODS:Skins of 15 cases of fetuses with different gestational ages and 5 cases of adults were taken, embedded with paraffin wax, and sectioned. Immunohistochemistry method and pathological method were used to detect the expression intensity and distribution of TGF-β_1,2 and α-ASMA. RESULTS: Positive immunohistochemical signals of TGF-β_{1, 2} and α-ASMA were found in fetal and adult skins. In skins derived from young fetus, the positive signals of these three proteins were very weak. Along with the increment in gestational age, the positive cellular rates of TGF-β_1,2M and α-ASMA were elevated progressively. In elder fetal and adult skins, TGF-β_1,2 were mostly distributed in epidermal cells, endothelial cells and some fibroblasts, while α-ASMA was mainly located in myofibroblasts and sweat gland epithelial cells. CONCLUSION:The endogenous TGF-β_1,2might be involved in the cutaneous development at embryonic stage, in the cutaneous structure maintenance at adult stage, and in the wound healing after injury.