Objective To explore the transfection efficiency of primary lymphocytes from human peripheral blood by different methods to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using Ficoll-Hypaque.Cell viability was detected by Trypan blue staining.Suspending lymphocytes were sucked out and were incubated in 24-well plate after cultured in 6-well plate for 2 h.Activated lymphocytes were transfected by electroporation with plasmid(PEGFP-N1).Resting or activated lymphocytes were transfected by lentivirus vector(LVGFP) single infection or repeated infection,respectively.Green fluorescence protein (GFP) was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry at different time points after infection.At the same time,the effectiveness of lentivirus infection was compared under different conditions.Results Purity of mononuclear cells isolated by Ficoll-Hypaque was 95 ％ and its viability was over 95 ％.The percentage of lymphocytes obtained with a uniform shape was 90 ％-95 ％.Scattered fluorescence was observed by electroporation under the conditions of voltage 2 100 V,pulse width 10 ms,pulse number 1 for lymphocyte,while fluorescent became weaker over time and no green fluorescent was observed after transfection for 72 h.After resting lymphocytes were infected once for 48 h by lentivirus vector,green fluorescent was not found and positive cells were less than 1％.1％-5 ％ of activated lymphocytes could express GFP after single lentivirus infection and the expression levels were enhanced with concentration increasing,while 5 ％-10 ％ of activated lymphocytes showed strong green fluorescent by repeated lentivirus infection.In contrast with electroporation,the fluorescent with lentivirus infection was stronger over time.Conclusion Repeated lentivirus infection could efficiently transfect exogenous genes into activated lymphocytes for stable expression.

Background Acute retinal ischemia anoxic injury is common in eye disorders,such as acute glaucoma,central retinal artery occlusion and ischemic optic neuropathy,etc.This will cause retinal ischemia anoxic injury and induce retinal ganglion cells (RGCs) death in addition.Endogenous cannabinoid (CB) and its receptors are involved in the central nervous system injury,ischemia,inflammation,and poisoning and other physiological and pathological process.Objective This study was to investigate the effect of CB on RGCs damage induced by oxygen-glucose deprivation (OGD).Methods The eyeballs were obtained from 6-week-old normal C57BL/6J mice to prepare retinal frozen sectionsand the expression and distribution of cannabinoid receptors (CB1R and CB2R) in RGCs was detected by immunofluorescence staining.The eyeballs of ten newborn C57BL/6J mice (postnatal 0-3 days) were obtained after immersed by 75％ alcohol and the retinas were isolated in preeooling DMEM for the primary culture of RGCs.The cells were identified by detecting the expression of Brn3a,a marker of RGCs,with immunofluorescence staining.Then the cells cultured for 14 days were divided into normal control group (in complete culture medium+95％ air+5％ CO2) and OGD group (in glucose-free medium+95％ N2 +4％ CO2 + 1％ O2) for 20 hours.The mitochondrial damage and RGCs morphology changed were evaluated by JC-1 staining to observe the mitochondrial membrane potential change.SR141716A (CB1R antagonist,1 μmol/L),SR144528 (CB2R antagonist,1 μmol/L) and 5 or 10 μmol/L WIN 55212-2 (CB1R and CB2R agonist) were added,and the survival rate of RGCs was assayed MTT.Results CBR was positively expressed in various layers of normal mouse retinas.The cells in the normal control group showed uniform size and polygon in shape with the long and thin axons,and the expression of Brn-3a was seen in the cells.However,in the OGD group,cell shrinkage and fragments were found and most of the axons disappeared.The expression of Brn-3a was evidently weakened.The fluorescence intensity of JC-1 was evidently weakened in the OGD group compared with the normal control group,showing the reduce of mitochondrial membrane potential.MTT assay showed that the survival rate of RGCs was (100.00± 13.87)％,which was significantly higher than (89.52-± 18.16)％ in the normal control group (q =8.065,P =0.008).The mean survival rates of RGCs were (116.63±22.21)％ and (112.61 ±19.02)％ in the cells treated by SR141716A and SR144528,and that in the normal cells was (89.52 ± 18.16)％ in the OGD group,with significant differences between SR141716A-or SR144528-treated cells and normal cells (q =29.780,17.391;both at P＜ 0.01).Conclusions Hypoxia and glucose-free up-regulate the expression of CB and activate CB pathway.Inhibition of activation CBR process has a neuroprotection effect under the Hypoxia and glucose-free condition.

Objective To investigate the effects of B lymphocyte-induced maturation protein-1 ( Blimp-1) on the number and function of splenic lymphocytes.Methods The mice with defective Blimp-1 in T cells were generated by cross-breeding B6.Blimp-1flox/flox mice with B6.Lck-Cre mice.The mononuclear lymphocytes isolated from spleen of T cell conditional Blimp-1 knockout (Blimp-1CKO) mice and wild type ( WT) C57/B6 mice were comparatively analyzed.Alterations of CD4+T and CD8+T cell subsets, the secre-tion of cytokines as well as the expression of C-C chemokine receptor type 7 ( CCR7 ) and Sphingosine-1-phosphate receptor 1 (S1P1) in mice from the two groups were analyzed by flow cytometry.The changes of CD19+B cell subsets were also detected.Results Compared with WT mice, the total numbers of mononu-clear cells, T and B lymphocytes were all significantly increased in Blimp-1CKO mice ( P<0.05) .The ab-solute numbers of CD4+T, CD8+T and CD19+CD5+CD1d+B cells in mice form Blimp-1CKO group were higher than those of the control group (P<0.05), however, no significant differences with the percentages of these cell populations were observed between two groups.Higher numbers and percentages of CD19+CD5+B cells were detected in mice from Blimp-1CKO group (P<0.01).The Blimp-1CKO mice showed increased secretion of IFN-γ, TNF-α, IL-17 and IL-2, but decreased expression of CCR7 on CD8+T cells as com-pared with WT mice (P<0.05).No significant differences with the changes of S1P1 were found between the two groups.Conclusion Blimp-1 played an important role in the maintenance of number, phenotype and function of T cells.Furthermore, not only T cells but also B cell subsets in mice were affected by the dele-tion of Blimp-1 in T cells.

Objective To assess the functional role of TH 17 cells in allogeneic hematopoietic stem cell transplantation (allyHSCT).Methods Bone marrow monocytes and splenic T cells were enriched from C57/BL6 donors.Recipient Balb/c mice were irradiated with 7.5 Gy total body irradiation (TBI) and injected with 5 105 splenic T cells and 5 106 bone marrow monocytes.Survival was monitored daily,clinical graft-versus-host disease (GVHD) was assayed three times a week,and detailed histopathologic analyses of lung were performed at day six after Allo-HSCT.Flow cytometry analysis was performed using CD3-FITC,CD4-PE,CD45-PerCP-CyS.5 monoclonal antibodies.Cells were stained for intracellular cytokines using mouse TH 1/TH2/TH 17 cytokine kit.Results All the experimental animals showed GVHD manifestations on the day 6 after transplantation.Animals from BMT and HF groups were scarified and histological analysis of lung was performed.Absence of TH 17 cells induced severe pathologic pulmonary lesions.The histopathology of the lung tissue was characterized by disorganization,epithelia cell damage,interstitial fibroplasias,and monocytes infiltration.The proportion of TH1 and TH 17 in BMT group was (5.53 ± 0.11 ) ％ and ( 1.04 ± 0.34)％ respectively,both significantly different from that in HF group.The levels of IL-17A and IFN-γin BMT group were (2.81 ±0.19) and (42.97 ± 0.23) pg/mL respectively.IL-17A could not be detected in HF group,yet the level of IFN-y was only (9.89 ± 0.51 ) pg/mL.IL-10 in both HF and BMT groups was not detectable.Conclusion Lung is on target of aGVHD.IL-17A may play a key role in the lung injury after transplantation.

To investigate the effects of estrogen (E2) on telomerase activity and its mechanism in human breast cancer cells, estrogen receptor positive MCF-7 cells were treated with different concentrations of E2. Telomerase activity was measured by using TRAP-ELISA method, the cell cycle phases analyzed by using flow cytometry, and the expression of Cyclin D1 detected by using immunohistochemistry method. The results showed that telomerase activity levels were increased in MCF-7 cells treated with 10(-8) mol/L E2 during the observed period (P < 0.05), and E2 increased telomerase activity levels in a dose-dependent manner(10(-10)-10(-8) mol/L); Simultaneously, the cell cycle phases of MCF-7 cells treated with 10(-8) mol/L E2 were changed significantly: G0/G1 phase decreased from 60.52% to 50.93%. S phase increased from 29.03% to 30.83%; However, the expression of Cyclin D1 was decreased. It was concluded that estrogen can upregulate telomerase activity of MCF-7 cells, and the effect can be blocked by antiestrogen tamoxifen. Its mechanism may be closely associated with modulation of cell cycle phases.
Breast Neoplasms ; chemistry ; enzymology ; pathology ; Cell Cycle ; Cyclin D1 ; analysis ; Estrogens ; pharmacology ; Female ; Humans ; Receptors, Estrogen ; analysis ; Telomerase ; genetics ; metabolism ; Tumor Cells, Cultured

To investigate the correlation between the activity of kinases in the growth factor signal transduction pathway and the development of resistance of breast cancer to tamoxifen, reporter gene regulated by the regulating fragment of CCD1 was transfected into the MCF-7 cells, and the influence of tamoxifen on the reporter gene expression was examined under different conditions of TPA treatment. Our results showed that the reporter gene expression was inhibited by tamoxifen and promoted by TPA. Furthermore, tamoxifen exerts an agonist effect on the reporter gene expression when the cells was treated by TPA previously for 12 h. It is concluded that TPA could induce estrogen-like effect of tamoxifen on estrogen receptor positive breast cancer cells and it may be one of the mechanisms responsible for the development of tamoxifen resistance.

To investigate the correlation between the activity of kinases in the growth factor signal transduction pathway and the development of resistance of breast cancer to tamoxifen, reporter gene regulated by the regulating fragment of CCD1 was transfected into the MCF-7 cells, and the influence of tamoxifen on the reporter gene expression was examined under different conditions of TPA treatment. Our results showed that the reporter gene expression was inhibited by tamoxifen and promoted by TPA. Furthermore, tamoxifen exerts an agonist effect on the reporter gene expression when the cells was treated by TPA previously for 12 h. It is concluded that TPA could induce estrogen-like effect of tamoxifen on estrogen receptor positive breast cancer cells and it may be one of the mechanisms responsible for the development of tamoxifen resistance.

CD5-positive diffuse large B-cell lymphoma (CD5+DLBCL) is a special type of DLBCL, which is characterized with later clinical staging, high-risk of relapse in extranodular tissues like bone marrow and central nervous system (CNS).Combined chemotherapy including rituximab and salvage autotransplantation/ allotransplantation can not significantly improve the prognosis. This article reviews the clinicopathological features, the possible pathogenesis, treatment status and dilemma in order to get the better understanding of CD5+DLBCL and avail the early diagnosis and individualized treatment.

Objective: To investigate the efficacy and safety of IA regimen which contains idarubicin (IDA) 8 mg/m², 10 mg/m² or 12 mg/m² as induction chemotherapy for adult patients with de-novo acute myeloid leukemia (AML) . Methods: A total of 1 215 newly diagnosed adult AML patients, ranging from May 2011 to March 2015 in the First Affiliated Hospital of Soochow University and other 36 clinical blood centers in China were enrolled in the multicenter, single-blind, non-randomized, clinical controlled study. To compare the response rate of complete remission (CR) , adverse events between different dose idarubicin combined with cytarabine (100 mg/m²) as induction chemotherapy in newly diagnosed patients of adult AML. Results: Of 1 207 evaluable AML patients were assigned to this analysis of CR rate. The CR rates of IDA 8 mg/m² group, IDA 10 mg/m² group and IDA 12 mg/m² group were 73.6% (215/292) , 84.1% (662/787) and 86.7% (111/128) , respectively (P<0.001) . After adjusted for age, blast ratio of bone marrow, FAB classification and risk stratification, the odds ratios (95% CI) of IDA 10 mg/m² group and IDA 12 mg/m² group were 0.49 (0.34-0.70) and 0.36 (0.18-0.71) , as compared with the IDA 8 mg/m² group (P<0.001, P=0.003) . In the intermediate and favorable groups, CR rates was 76.5% (163/213) , 86.9% (506/582) and 86.1% (68/79) in different doses of IDA (P=0.007) . Interestingly, IA regimen with IDA 10 mg/m² was the only beneficial factor affecting CR in this group after adjusted for age, blast ratio of bone marrow and FAB classification[OR=0.47 (95% CI 0.31-0.71) , P<0.001]. CR rates in adverse group was 50.0% (18/36) , 60.6% (43/71) and 81.8% (18/22) respectively (P=0.089) . However, the odds ratios (95% CI) of IDA 12 mg/m² when compared with the IDA 8 mg/m² was 0.22 (0.06-0.80) , after adjusted for age, blast ratio of bone marrow and FAB classification. The median time (days) of neutrophil count less than 0.5×10⁹/L in IDA 8 mg/m² group, IDA 10 mg/m² group and IDA 12 mg/m² group were 14 (11-18) , 15 (11-20) and 18 (14-22) , respectively (P=0.012) and of platelet count lower than 20×10⁹/L were 14 (7-17) , 15 (11-20) and 17 (15-21) , respectively (P=0.001) . The incidences of lung infection in the three groups were 9.8%, 13.5% and 25.2%, respectively (P<0.001) . Conclusions For young adult patients (aged 18-60 years) with AML in China, intensifying induction therapy with idarubicin 10 mg/m² is clinically superior to IDA 8 mg/m² and IDA 12 mg/m² in favorable intermediate AML subgroup. However, idarubicin 12 mg/m² is more suitable to adverse AML subgroup.

Antiviral Agents ; chemistry ; metabolism ; Crystallography, X-Ray ; Ligands ; Protein Binding ; Protein Structure, Tertiary ; RNA Helicases ; chemistry ; classification ; metabolism ; Static Electricity ; Viral Proteins ; chemistry ; classification ; metabolism ; Zika Virus ; enzymology