Aim To study on the mitochondrial mass change in mouse thymocyte apoptosis.Method Control and Dexamethasone(DEX) groups were set;at 6h,we studied mitochondrial mass changes by NAO and Mitotracker Green(MG) staining flowcytometry and detected mitochondrial membrane potential change with DiOC_6(3) staining flowcytometry.We also used Annexin V-PE/MG double staining flocytometry to examine the mitochondrial changes in apoptosis progress.Results NAO staining results showed that 1 ?mol?L~(-1) DEX stimulation reduced the cardiolipin content of thymocyte mitochondria(P

Objective To generate hemogenic endothelial cells(HECs)from human induced pluripotent stem cells (hiPSCs)in vitro in order to learn more about the mechanism by which the vascular niche affects HECs production and self -renewal.Methods hiPSCs with reporter gene runx1c were differentiated to hematopoietic cells by spinEB method.The CD34 positive cells were sorted by magnetic-activated cell sorting(MACS)at day 10 after hematopoietic differentiation. Afterwards,these CD34 positive cells were co-cultured with DLL4 overexpressed vascular niche cells VeraVec to further differentiate to HECs.The HECs derived from the hiPSCs were characterized by FACS.Results We first established an hiPSCs single cell culture method for spinEB differentiation.Single cell cultured hiPSCs with reporter gene runx 1c were differentiated to form embryonic bodies(EBs)by spinEB method.The HECs were enriched from the day 10.Meanwhile, we cultured the E4ORF1 transfected human umbilical vein endothelial cell(HUVEC)line(VeraVec)and examined the expression of NOTCH signaling pathway related genes.According to the results, VeraVec had a high expression level of NOTCH ligand DLL4 at both mRNA and protein levels.And the CD34 positive HECs were co-cultured with DLL4 overexpressed VeraVec cells,which promoted the expression of tdTomato during hematopoitic differentiation and increased HSCs production.Conclusion A method of inducing hiPSCs differentiation by spinEB has been established, which can enrich HECs.This model can be applied to study the mechanism by which the vascular niche promotes hematopoietic differentiation from hPSCs.The generated functional HSCs are of great social and military values for HSCs transplantation and battlefield radiation injury treatment.

This study was aimed to investigate the immunological effect of modified dendritic cells (DC) which inducing cytotoxic T cells (CTL) against lymphoma cells. The DC were isolated from the lymph node and peripheral blood of patients with diffuse large B cell lymphoma (DLBCL). DC were transfected with recombinant adenovirus vector carrying human p53 gene (rAd-p53-DC). The expression of p53 gene was detected by flow cytometry. Western-blot was used to detect the expression of P53. ELISA was used to detect IL-12 level in supernatant. The mixed lymphocyte reaction (MLR) was used to detect the proliferative ability of auto-lymphocyte stimulated by DC. The lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTL. The results indicates that the expressions of DC surface molecule (except for CD1a) such as CD83, CD80, CD86 and HLA-DR were significantly higher in experiment group than that in control group and blank control group. The secretion of IL-12 in supernatant was higher in experiment group than that in control group. The autologous T lymphocyte proliferation and cytotoxic activity against the same kind of DLBL-cells increased in experiment group as compared with control group and blank control group (P < 0.05). The ability to stimulate T lymphocyte proliferation increased with the rising of the ratio of DC and T lymphocyte. However, there was statistically significant difference between rAd-p53-DC derived from Lymph node and peripheral blood (P < 0.05). It is concluded that rAd-p53-transfected DC can induce CTL response in vitro against lymphoma cells.
Adenoviridae ; Cell Line, Tumor ; Dendritic Cells ; cytology ; immunology ; Genes, p53 ; Genetic Vectors ; Humans ; Lymphocyte Activation ; Lymphocyte Culture Test, Mixed ; Lymphoma, Large B-Cell, Diffuse ; blood ; immunology ; Transfection

OBJECTIVETo explore the mechanism of occupational medicamentosa-like dermatitis (OMDT) induced by trichloroethylene (TCE) and some immunity indexes in workers occupationally exposed to TCE.

METHODSThe blood samples from 8 cases with medicamentosa-like dermatitis in 1st, 2nd, 3rd, 4th and 5th weeks after admitting to hospital were examined for liver function, immunoglobulin and some complement indexes. Thirty nine workers occupationally exposed to TCE were investigated for urinary TCE and some immuno-complement indexes. The TCE concentrations of air in workplaces were monitored.

RESULTSC3d-CIC and C3 of patients before admission were (92.86 ± 44.80) mg/L and 0.91 ± 0.19 mg/L, respectively. C3d-CIC and C3 of patients before discharge were (52.41 ± 17.75) mg/L and (1.14 ± 0.22) mg/L, respectively. There were significant differences between admission and discharge (P < 0.05). The average TCE concentration in 4 workplaces was (351.96 ± 36.72) mg/m(3), which was higher than the occupational exposure limits (OELs). The number of workers exposed to the TCE concentration-time weighted and TCA in urine over OELs were 28.21% and 56.41% of total subjects, respectively. The serum IgG and CIC levels of patients before discharge were (10.03 ± 1.21) mg/L and 103.50 ± 29.17 mU/L, which were significantly lower than those (17.21 ± 1.85) mg/L and (227.46 ± 111.67) mU/L of patients before admission (P < 0.01).

CONCLUSIONThe type II and III hypersensitivity may be associated with OMDT and the organ injure induced by TCE.

Adolescent ; Adult ; Complement System Proteins ; immunology ; Dermatitis, Occupational ; immunology ; Female ; Humans ; Male ; Occupational Exposure ; Trichloroethylene ; toxicity ; Young Adult

OBJECTIVETo establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function.

METHODSThe genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR.

RESULTSThe standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains.

CONCLUSIONSReal-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.

Animals ; Gene Rearrangement, T-Lymphocyte ; Lymphocyte Count ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell ; genetics ; metabolism ; T-Lymphocytes ; cytology ; Thymus Gland ; cytology ; immunology

Objective To understand the pathogenic distribution and epidemiological trend of hand-foot-and-mouth disease (HFMD),and provide evidence for the prevention and control of HFMD.Methods Children who were diagnosed with HFMD in a hospital between January and December 2015 were investigated,real time fluorescence PCR was used to detect enterovirus universal type EV,enterovirus 71 (EV71),and Coxsackievirus A16 (CoxA16) in specimens from children with HFMD.Positive rates and distribution of various types of EV among children of different months,genders,age groups,and infection types were analyzed.Results A total of 837 throat swab specimens from HFMD children were collected in 2015,380 (45.40％) of which were EV positive specimens.Virus typing showed that 110 (28.95 ％),7 (1.84 ％),6(1.58 ％),and 257(67.63 ％) were positive specimens for EV71,CoxA16,EV71 + CoxA16,and other types of EV.HFMD had a high prevalence since April,reached a peak in May-June,and remained high incidence in July-December.Positive rates of EV in children of different months were statistically different (P＜0.05).The age of onset was mainly in children under 3 years.Positive rates of EV and constitute ratios of different types of EV in children of different age groups were all statistically different (all P＜0.05).The positive rate of EV in severe HFMD cases was higher than common cases (65.34％ vs 27.06％,P＜0.001).The proportion of severe cases in children with EV71 infection and other types of EV infection were 90.00％ and 60.70％ respectively;children with EV71 + CoxA16 double infection were all severe cases.Constitute of EV types in children with different infection types was statistically different(P＜0.001).Conclusion In 2015,EV infection in hospitalized children with HFMD in this hospital was mainly caused by other types of EV (nonEV71 and non-CoxA16),the high prevalence season,high-risk population under 3 years of age,and severe cases should be paid high attention,prevention and treatment should be performed well.

Objective To evaluate the cytotoxicity of natural killer(NK)-92 cell lines against various human hepatocellular carcinoma cells(HCCs)and to explore the potential mechanism.Methods We established a culture method of NK-92 cell lines in vitro.Lactate debydrogenase(LDH)cytotoxicity assays and cytokine release assays were performed to determine whether NK-92 cell lines could recognize and kill HCCs in vitro.At the same time,Nu/Nu mices were housed. Subcutaneous(sc)xenografts HepG2 models of human hepatocellular carcinoma were established.1×107NK-92 cells were intravenously(iv)injected through the tail vein on days 2,9,16,23 while the control group was injected with PBS in the same way.Tumor size, tumor volume, tumor mass and mouse survival status were closely observed in experimental and control groups.Mice were euthanized when tumor-bearing time reached 28 days.Xenograft tissues were taken for general observation.Sections were cut and processed for HE staining and immunofluorescence staining.The expression of glypican-3(GPC3)protein in xenografts tissue was clearly defined.Results NK-92 cell lines that were chronically cultured in vitro and maintained typical phenotypic characteristics of NK cells with good cellular activity.Enhanced cytotoxicity and IFN-γ production of NK-92 cell lines were identified by LDH and ELISA,indicating that NK-92 cell lines could recognize and kill different kinds of HCCs.In addition,NK-92 cell lines efficiently suppressed the growth of HCC xenografts in vivo.Tumor volume in experimental group was significantly reduced compared with control group and there was low a GPC 3 expression in experimental group through immunofluorescence and immunohistochemistry results, pointing to the possibility that the cytotoxicity of NK cells was correlated with GPC3 +HCCs.Conclusion NK cells provide a promising means of therapeutic intervention for HCCs.NK-92 cell lines could eliminate HCC cells in vitro and in vivo.The cytotoxicity of NK-92 cell lines may work by killing the GPC3-positive cells in the liver cancer tissue.In addition to the anti-tumor effect, NK cells also have cytotoxicity on pathogens such as bacteria and viruses.

OBJECTIVETo study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway.

METHODSInhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points.

RESULTSThe sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01).

CONCLUSIONCHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

Apoptosis ; drug effects ; Cycloheximide ; pharmacology ; HL-60 Cells ; Humans ; Membrane Potentials ; Mitochondria ; physiology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

OBJECTIVETo evaluate the advantage and disadvantage of laser-assisted liposuction compared with conventional liposuction.

METHODSTen swines were devided into three groups, the laser liposuction group, conventional liposuction group and control group. We compared the two surgical groups with the following aspects: ecchymosi, edema, lipocrit study, hemoglobin studies and blood biochemical changes, etc.

RESULTSThere is a benefit of laser-assisted liposuction in the following aspects such as ecchymosi, edema, lipocrit and postoperative complications. The two aspects, that is, ecchymosi and lipocrit study was statistically significant. Hemoglobin change was not statistically significant between the two groups. Laser-assisted liposuction did not lead to dysfunction of organs such as liver and kidney.

CONCLUSIONSLaser-assisted liposuction can significantly decrease the blood lose, ecchymosis and edema compared with conventional liposuction, and it did not give rise to dysfunction of organs.

Adipose Tissue ; surgery ; Animals ; Female ; Lasers ; Lipectomy ; instrumentation ; methods ; Male ; Models, Animal ; Swine ; Treatment Outcome

OBJECTIVETo construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human prostate-specific antigen promoter and investigate its targeted inhibition effects in androgen-responsive prostate cancer cells LNCaP.

METHODSTo clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the pGensil-1 plasmid containing U6 promoter and EGFP. U6 promoter was replaced by hPSA promoter. Then, the recombinant EGFP-hPSA-siCXCR4 fragment was sub-cloned into pLXSN, which was evaluated by restriction enzyme. The pLXSN-EGFP-hPSA-siCXCR4 was transfected into PA317 cells with Lipofectamine 2000. The virus obtained from transfected PA317 cells was transfected into PC-3m, LNCaP and MCF-7 cells, respectively. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western blot. The invasion ability of prostate carcinoma cells was detected by Transwell experiment.

RESULTSThe recombinant pLXSN-hPSA-siCXCR4 was successfully constructed. The expression of CXCR4 mRNA and protein in LNCaP cells was blocked by pLXSN-hPSA-siCXCR4. The expression inhibition rate was (81.53 +/- 10.22)% at mRNA level detected by semi-quantitive RT-PCR and (90.52 +/- 9.31)% at protein level detected by Western blot, respectively, in LNCaP cells at 48 h. The expression of CXCR4 mRNA and protein was effectively inhibited by sequence-specific hPSA-siCXCR4 in LNCaP cells, but not in PC-3m and MCF-7 cells. The results of Transwell experiment showed that the number of cells in down-pore of micro-membrane was 139.9 +/- 14. 2 in the treated group, significantly less in comparison with 348.4 +/- 36. 4 in the controlled group (P < 0.05). However, the number of PC-3m and MCF-7 cells in down-pore of micro-membrane was not significantly different among the control and treated groups (P > 0.05).

CONCLUSIONThe downstream siRNA controlled by hPSA promoter in retrovirus system can be expressed selectively in androgen-responsive prostate carcinoma cells, showing an apparent targeting character. RNAi targeted to CXCR4 driven by hPSA promoter has a potential value in gene therapy of androgen-responsive prostate cancer.

Animals ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Male ; Mice ; NIH 3T3 Cells ; Neoplasm Invasiveness ; Plasmids ; Promoter Regions, Genetic ; Prostate-Specific Antigen ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, CXCR4 ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; Transfection