AIM:Targeting of focal adhesion kinase (FAK) gene,we aim to construct FAK shRNA lentiviral vector and to identify its function on the growth of leukemic cells.METHODS:FAK shRNA was chemically synthesized,and inserted into a GFP-lentiviral plasmid through molecular biology methods.After packaged and concentrated,the lentiviral-FAK-shRNA-vector was transduced into a human leukemic cell line.FAK gene expression was detected by reverse transcriptional PCR and Western blotting.Cell apoptosis was measured by Annexin V labeling.RESULTS:The results showed that FAK shRNA was successfully inserted into the lentival vector,and the infection efficiency varied from 10% to 25%.Compared to the control vector (lentival vector without FAK shRNA),FAK shRNA inhibited the expression of FAK mRNA and protein by 40% and 70%,respectively.Moreover,the results of apoptosis experiment showed that the percentages of Annexin V+ cells in control vector group and FAK shRNA group were (4.19 ? 0.36) % and (8.48 ? 0.58) % respectively,the difference was statistically significant (P

Objective:To investigate the effects of sensitized sera on bone marrow transplantation and clarify the role of antibody in the process of rejection.Methods:Two hundred microlitres sensitized sera or non-sensitized sera were injected into normal BALB/c one day before transplantation.Ten millions (1?107) bone marrow cells from C57BL/6 were transfused to the recipients after lethal irradiation.The donor-reactive antibodies in recipients before transplantation were measured by complement-dependent cytotoxicity (CDC) method.Moreover,the survival analysis and engraftment evaluation of the recipients were carried post transplantation.Results:The CDC results showed that donor-reactive antibodies existed in the recipients which had received sensitized sera transfusion.Eighty percent (80%) of the recipients received sensitized sera transfusion died of bone marrow failure about 10 days post transplantation,while the recipients received non-sensitized sera transfusion were long-term alive.Furthermore,the hematopoietic recovery and percentage of donor chimera analysis declined along with time in the sensitized sera transfusion recipients,and there were significant differences compared with those in the non-sensitized sera transfusion recipients (P

The study was aimed to investigate the strategy of transfusion of allogeneic hematopoietic stem/progenitor cells (HS/PC) into marrow cavity of mouse model in sensitized transplantation. A sensitized BALB/c mouse model was established by repeated transfusion of allogeneic spleen cells. The normal BALB/c mice were used as non-sensitized controls. The non-sensitized or sensitized recipients were transplanted by transfusion of allogeneic HS/PCs into bone marrow cavity. The survival rate and hematopoietic recovery were monitored. Moreover, non-sensitized and sensitized sera were obtained and incubated with allogeneic HS/PC respectively, the percentage of dead cells was calculated using complement-dependent cytotoxicity (CDC) tests. The results showed that non-sensitized recipients got long-term survival after the transfusion of HS/PC into marrow cavity, and the hematopoietic recovery increased along with time. However, among the sensitized recipients, one mouse died of anesthetic accident, the other 9 mice (9/10) died within 2 weeks after the transfusion of HS/PC in marrow cavity, and the hematopoietic recovery declined along with time. Histopathologic analysis demonstrated that the sensitized recipients died of bone marrow failure. The results of CDC tests showed that the percentage of dead cells in non-sensitized and sensitized group was 7.80 ± 1.93% and 50.80 ± 3.12%, respectively, and the differences were statistically significant (p < 0.05), indicating sensitized sera were capable of impairing allogeneic HS/PC. It is concluded that the strategy of the marrow cavity transfusion of HS/PC can not enhance engraftment of allogeneic donor cells in sensitized recipients.
Animals ; Bone Marrow ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transplantation, Homologous

OBJECTIVETo establish a murine model of sensitization, and investigate the effect and mechanism of sensitization on allogeneic donor bone marrow cells (BMCs).

METHODSSensitized BALB/c mice were established by transfusions of allogeneic splenocytes. The donor reactive antibodies were detected by binding and complement-dependent cytotoxicity assays. After irradiation, 1 × 10(7) BMCs of C57BL/6 donor mice were injected into non-sensitized or sensitized BALB/c recipient mice. The distribution pattern of donor BMCs in peripheral blood, spleen and bone marrow of recipient mice were analyzed at different time points (2 h, 12 h and 48 h) post transplantation. Hematopoietic recovery post transplantation was assessed, and survival was monitored. Moreover, sera and splenocytes derived from non-sensitized or sensitized recipients were incubated with allogeneic BMCs in vitro, and the cytotoxic indexes were calculated in the immune experiments.

RESULTSThe binding and complement-dependent cytotoxicity assays showed that a high level of donor reactive antibodies was presented in sensitized sera. Compared with the non-sensitized recipients, the homing assay showed significantly decreased distributions of allogeneic donor BMCs in peripheral blood, spleen and femur of sensitized recipients. Non-sensitized recipients survived long term after irradiation, while all the sensitized recipients died within 12-15 days. Fourteen days post transplantation, the white blood cells and BMCs of non-sensitized recipients were (3240 ± 300) × 10(6)/L and (396 ± 27) × 10(6)/femur, respectively; while the white blood cells and BMCs of sensitized recipients were (320 ± 80) × 10(6)/L and (6 ± 2) × 10(6)/femur, respectively; the differences were statistically significant between this two groups (P < 0.05). Seven days post transplantation, the percentage of donor cells in bone marrow of non-sensitized and sensitized recipients was (48.07 ± 4.70)% and (0.77 ± 0.11)%, respectively, and the differences were statistically significant (P < 0.05). Furthermore, the white blood cells and BMCs following transplantation decreased along with time in sensitized recipients. The immune experiments of complement-dependent cytotoxicity reaction, cytotoxic T lymphocytes reaction and antibody-dependent cellular cytotoxicity showed the cytotoxic indexes were higher in sensitized group than the non-sensitized group.

CONCLUSIONA sensitized model was established by transfusions of allogeneic spleen cells. Allogeneic donor BMCs were rejected in sensitized recipients, and its mechanism might be through immune impairment pathways.

Animals ; Bone Marrow Cells ; Bone Marrow Transplantation ; Disease Models, Animal ; Graft Rejection ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Tissue Donors ; Transplantation, Homologous

The aim of this study was to separate and amplify CD4(+)CD25(+)Treg cells from splenocytes of sensitized mice. The percentage of CD4(+)CD25(+)Treg cells was detected by flow cytometry in sensitized and normal control mice. CD4(+)T, CD4(+)CD25(+)Treg and CD4(+)CD25(-) T cells were isolated from mouse splenocytes by MACS. CD4(+)CD25(+)Treg cells were expanded in vitro cultures in addition of CD3/CD28 MACSiBead and IL-2. The activity of cells was detected with 0.4 trypan blue staining. The purity of cells after sorting, the main surface marker and the level of Foxp3 were detected by flow cytometry. The results showed that CD4(+)CD25(+)Treg cell proportion was higher in sensitized mice than normal control mice (P < 0.05). The average purity of CD4(+)CD25(+)Treg cells was 87. The activity of these cells was more than 97, and the expression of Foxp3 in these cells was high. The amplification multiples achieved 42 times after 2 weeks in vitro. The percentage of CD4(+)CD25(+) regulatory T cells was 85.32, and the expression of Foxp3 decreased from (76.92 ± 1.72) to (75.33 ± 2.11) (P > 0.05). It is concluded that the sorting of CD4(+)CD25(+)Treg cells is isolated successfully by MACS without affecting the vitality of target cells. The amplification of CD4(+)CD25(+)Treg cells is successful in vitro. Expression of surface markers and Foxp3 gene does not obviously change after amplification, so that to establish a practical method to recover and enlarge the amount of CD4(+)CD25(+)Treg cells in good condition.
Animals ; CD4 Antigens ; biosynthesis ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Immunomagnetic Separation ; methods ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lymphocyte Count ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; cytology

Adenosine triphosphate(ATP)production in cardio-myocytes in healthy heart depends primarily on the oxidative phosphorylation of fatty acids and,to a lesser extent,on the oxi-dation or glycolysis of glucose.Patients with heart failure devel-op severe disturbances in energy metabolism,including disturb-ances in substrate uptake and utilization,oxidative phosphoryla-tion,and ATP shuttling,resulting in inadequate cardiac energy supply.By intervening in energy metabolism pathways,cardiac efficiency can be increased,energy production deficits can be reduced,and cardiac function can be improved in patients with heart failure.We hence have reviewed and outlined the mecha-nisms and therapeutic implications of metabolic changes during heart failure.

This study was purposed to compare the effect of 3 different cell components for expanding CD4(+) CD25(+) Treg in vitro, and identify their immunosuppressive function. CD4(+) T cells, CD4(+) CD25(-)T cells and CD4(+) CD25(+)T cells were isolated from mouse splenocytes by MACS and then expanded in vitro. Phenotype of the T cell lines and expression of the FOXP3 was determined by flow cytometry. The inhibitory effect of expanded CD4(+) CD25(+) T cells on CD4(+) CD25(-)T cells was tested by MLR method. The results showed that the Treg cells from all the three groups were expanded significantly after culture for 2 weeks. In the CD4(+) T cells group, the proliferation rate was (77.8 ± 5.32) folds with a percentage of Treg cells increasing from (6.61 ± 1.00)% to (15.33 ± 1.31)%. The proliferation rate in the CD4(+) CD25(-) T cells group was (95.20 ± 7.67) folds, with the percentage of CD4(+) CD25(+) T cells raising from (0.37 ± 0.13)% to (9.84 ± 0.98)%. The proliferation rate in the CD4(+) CD25(+) T cells group was (41.20 ± 6.92) folds, the proportion of Treg cells decreased from (86.75 ± 1.25)% to (85.32 ± 1.62)%, and the expression of Foxp3 decreased from (76.92 ± 1.72)% to (75.33 ± 2.11)% during the culture, there were not significant differences in the cell purity and the expression of Foxp3, compared with pre-amplification. The inhibitory test showed that the expanded CD4(+) CD25(+) T cells could inhibit the proliferation of CD4(+) CD25(-) T cells in vitro in a cell dose-dependent manner. It is concluded that the amplification of CD4(+) CD25(+) Treg cells is successful in vitro, especially in the CD4(+) CD25(+) T cells group, the cell purity and Foxp3 gene is not obviously changes after amplification.
Animals ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Interleukin-2 Receptor alpha Subunit ; immunology ; Lymphocyte Culture Test, Mixed ; Male ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes, Regulatory ; cytology ; immunology

This study was aimed to investigate the effects of focal adhesion kinase (FAK) gene silence on leukemia cell growth, leukemogenesis and efficacy of chemotherapy drug. Vector containing lentiviral-FAK-shRNA was constructed and transfected into BCR/ABL-BaF3 leukemic cells, the cell growth and apoptosis were detected in vitro. The effect of FAK shRNA on leukemogenesis was studied in a murine model with leukemia. The apoptosis of leukemia cells and survival of leukemic mice treated by FAK shRNA combined with drug STI571 were monitored. The results showed that FAK gene expression was knocked down by lentiviral-FAK-shRNA. FAK gene silencing inhibited leukemia cell growth in vitro. The apoptosis test results showed that the percentages of Annexin V(+) cells in vector control group and FAK shRNA group were (3.46 ± 0.56)% and (7.3 ± 0.79)%, respectively, and the difference was statistically significant (p < 0.05). The mice in vector control group died at day 21 to 27, while the mice in FAK shRNA group died between day 52 and 60, and the difference was statistically significant (p < 0.05). Moreover, FAK gene silence combined with drug STI571 could enhance the apoptosis of leukemia cells and prolong survival time of leukemic mice. It is concluded that FAK gene silence inhibits leukemogenesis and promotes efficacy of chemotherapy drug on leukemia cells, indicating FAK gene silence may be considered as a new therapeutic strategy for leukemia.
Animals ; Focal Adhesion Kinase 1 ; genetics ; Gene Silencing ; Genetic Vectors ; Leukemia, Experimental ; genetics ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo evaluate the effect of sensitized sera on engraftment of hematopoietic stem cells (HSCs) and its mechanism.

METHODSBone marrow cells (BMCs) from C57BL/6 mice were incubated with sensitized sera (group I) or normal sera (group II), and then washed and transplanted into irradiated BALB/c mice. The survival analysis and engraftment evaluation of the recipients were observed. The incubated BMCs were bound with goat anti mouse IgG for and labeled with Annexin V for apoptosis detection.

RESULTSSeven out of ten recipient mice in group I died of bone marrow failure at day 10 after transplantation, while all of those in group II were long-term survived. Engraftment assay showed recipients blood count and BMCs progressively decreased along with time passing in group I; in addition, the chimeric percentage of donor cells progressively decreased as well. The percentage of BMCs binding with goat anti mouse IgG in group I or group II were (90.3 +/- 5.1)% and (5.2 +/- 2.4)%, respectively, and the difference was statistically significant (P < 0.01). However, no significant difference was found in the apoptosis detection between the two groups (P > 0.05).

CONCLUSIONThe engraftment capacity of HSCs is significantly impaired by sensitized sera, the antibodies in sensitized sera may bind to antigens expressed on HSCs but do not induce apoptosis.

Animals ; Annexin A5 ; metabolism ; Apoptosis ; Bone Marrow Cells ; metabolism ; pathology ; Cells, Cultured ; Hematopoietic Stem Cell Transplantation ; Immune Sera ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

OBJECTIVETo investigate the effect of ginsenoside Rg1 on the influence of neprilysin expression induced by LPS in BT325 cell line.

METHODMTT was used to measure the levels of the survival rate of BT325 cultured with ginsenoside Rg1 and LPS in different concentrations. The expression of NEP was measured by RT-PCR.

RESULTThe survival rate of BT325 was obviously inhibited by LPS, and the expression of NEP was decreased. The survival rate of BT325 was obviously raised by ginsenoside Rg1, and the expression of NEP was increased.

CONCLUSIONLPS can cause cell damage, and decrease the expression of NEP. Ginsenoside Rg1 can exert neuroprotective properties which protects BT325 cell line from the cell toxicity of LPS.

Cell Line, Tumor ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Ginsenosides ; isolation & purification ; pharmacology ; Glioma ; metabolism ; pathology ; Humans ; Lipopolysaccharides ; Neprilysin ; metabolism ; Neuroprotective Agents ; pharmacology ; Panax ; chemistry ; Plants, Medicinal ; chemistry