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@#Hedgehog pathway regulates the physiological process of tumor cells, including proliferation, cycle, invasion and metastasis, and maintains tumorigenesis and development. With abnomal activation of Hedgehog pathway, most tumors response poorly to chemotherapy, which is mediated by Hedgehog pathway through activation of target gene and crosstalk with other pathways. Herein, Hedgehog pathway has been an important target for reversing resistance. In this study, the Hedgehog pathway and role of Hedgehog-mediated resistance in recent years are reviewed.
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Objective To determine the influence of serum complement and IgG on rituximabdependent NK cell-mediated cytotoxicity to Raji cells in vitro.Methods FcγR Ⅲ a (CD16a) polymorphism of NK cells were detected by nest-PCR. Effects of serum IgG on FcγRⅢ a expression of NK cells in vitro were analyzed by flow cytometry.The target cells(Raji cells) were stained with DIO,cultured with effector cells(NK cells) and rituximab with or without serum IgG/complement,and finally stained with propidium iodide (PI),then these cells were tested by flow cytometry and the cytotoxic index was calculated as well. Results The cytotoxic indexes of the ADCC +CDC groups were higher than those of ADCC groups, but the serum IgG groups were lower than the ADCC groups. In FcγRⅢa-158Ⅴ/Ⅴ groups, the cytotoxic indexs of the ADCC+ CDC groups,the serum IgG groups and the ADCC groups were (94.25±1.79) ％,(59.79±0.66) ％ and(69.05± 2.38) ％,respectively,and the differences among the groups were statistically significant (P＜ 0.05).In FcγRⅢ a-158Ⅴ/F groups,the cytotoxic indexs of these three groups were (66.71±5.57) ％,(18.13±2.99) ％ and (39.63±3.86) ％, respectively, and the differences among the groups were also statistically significant (P＜ 0.05).Conclusions Complement may enhance the rituximab-mediated NK cell cytotoxicity to Raji cells, whereas,serum IgG may weaken the cytotoxicity against Raji cells. It is clued up that for patients treated by tumorspecific monolonal antibody (MAb), combined infusion of fresh frozen plasma could promote its anti-tumor effect,however,MAb combined with IVIG may impair its anti-tumor effect.
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[Objective]To investigate the effects of mesenchymal stem cells (MSC) feeder layer, culture sere and freeze-thaw lysates on expansion and differentiation of cord blood CD34~+ cells in vitro. [Methods] MSC were isolated from human bone marrow and cultured until the third passage. Sera were obtained from the cultured MSC, and freeze-thaw lysates were obtained by repeated freeze-thaw procedures. Cord blood CD34~+ cells were isolated by magnetic cell separation system, and were co-cultured with the MSC feeder layer, culture sera, freeze-thaw lysates and hematopeietic growth factors (HGFs), respectively. The nucleated cells, CD34~+ cells, CD34~+CD38~- cells, CD41~+ cells and CD3~+ cells in the above culture system were detected by flow cytometry on day 6 and day 12. [Results] ①MSC feeder layer had a strong effect on nucleated cells, CD34~+,CD34~+CD38~- cells expansion. The MSC sera and freeze-thaw lysates had similar effect on cell expansion, but the effect was weaker than that of feeder layer (P<0.05). ② Both MSC sera and feeder layer inhibited cord blood CD34~+ cells differentiation toward CD3~+ cells or CD19~+ cells, and no significant differences were found between these two groups (P>0.05). ③ Both MSC sera and feeder layer promoted cord blood CD34~+ cells differentiation toward CD41~+ cells, and the effect was stronger in the feeder layer than that of the sera (P<0.05). ④ Freeze-thaw lysates had no effect on cell expansion and differentiation, and were similar with that of HGFs (P>0.05). [Conclusions] The MSC sera have positive effects on expansion of cord blood CD34~+ and CD34~+CD38~- cells, moreover they have the ability of promoting cord blood CD34~+ cells differentiation toward CD41~+ cells.
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Objective To assess whether treatment with mesenchymal stem cells (MSCs) is an effective adjunct therapy for refractory extensive chronic graft-versus-host disease (GVHD) resistant to conventional therapy. Methods 12 patients with steroid-resistant extensive chronic GVHD were treated with MSCs. One patient received one dose, 10 received two doses, and the remaining three doses. The MSCs were obtained from HI,A-identical sibling donors (n = 14), haploidentical donors (n = 2), unrelated mismatched donor (n = 1) and third-party HLA-mismatched donors (n = 7). Of the 11 patients treated with multiple infusions, 5 received cells derived from two donors. The median first dose of MSCs was 1.0 (0. 4-2. 1) × 106/kg , the median second dose was 1.2(0. 8-1.9) × 106/kg , and the third dose in one patient was 1.1 × 106/kg. Meanwhile the proportion of CD3+ ,CD4+,CD8+ ,CD19+,CD4+ CD25+ ,FOXP3+,FOXP3+CD4+ and FOXP3+ CD25+ was determined with double fluorescent-labeled antibodies and flow cytometry before and 4 weeks after the MSCs infusion. Results No patients had side-effects during or immediately after the infusions of MSCs. After a treatment course of one to three doses, 3 patients had complete response(CR), 6 showed partial response(PR) and 3 did not respond; the total effective rate was 75% (9/12). Complete resolution was seen in the involvement of skin (3/12), lung (1/3), joints (1/5), liver (3/10), oralcavity (4/12) and eye (2/7). Response rate was not related to donor HLA-match. 3 CR patients discontinued all of the immunosuppressive agents without relapse 100 to 292 days after the MSC infusion and 6 PR patients taped all immunosuppressive agents after 60 to 79 days. Mean follow-up period was 1152(795-1914) days, leukemia free survival rate was 91.7% (11/12) and the overall survival rate was 75% (9/12). The ratio of CD4/CD8 and the proportion of regulatory T cells were significantly higher than that before MSCs treatment. Conclusion Third-party MSCs were as effective as HLA-identical or haploidentical cells. This finding has practical implications and suggests that third-party cells can be prepared and stored frozen to be used for steroid-resistant extensive chronic GVHD therapy. It is concluded that MSCs may prevent the lethal cGVHD after allogeneic hematopoietic stem cell transplantation and raise the survival rate by increasing the ratio of CD4/CD8 and proportion of regulatory T cells in vivo.
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AIM: To investigate the effect of T-bet plasmid gene transfer to airway on allergen induced airway inflammation in a murine asthmatic model. METHODS: A mouse asthma model was established by sensitization with ovalbumin (OVA). Forty C57BL/6 mice were divided into 4 groups (10 mice in each group): the normal control group (group A), the asthmatic model group (group B), the pcDNA3 plasmid group (group C), and the pcDNA3-T-bet group (group D). The animals in group B, C and D were sensitized and challenged with OVA. The animals in group A were applied with normal saline. pcDNA3 plasmid at dose of 50 μg was intranasally administered at 24 h before intranasal challenges to the mice in group C, and the 50 μg pcDNA3-T-bet plasmid for the mice in group D. Bronchial alveolar lavage fluid (BALF) was collected and lung tissues were resected at 48 h after OVA challenge for later assay. RESULTS: After administration with pcDNA3-T-bet plasmid, high level of T-bet expression at 48 h was detected in the lung tissue by Western blotting. In pcDNA3-T-bet treated asthmatic models, histological evaluation revealed the significant suppression of eosinophil peribronchial and perivascular infiltration, and reduction of epithelial damage. The numbers of eosinophils, neutrophils and lymphocytes in BALF from pcDNA3-T-bet treated mice were significantly reduced compared to those in asthmatic control group (P<0.05). The level of IL-4 in BALF was significantly decreased in pcDNA3-T-bet group compared to that in asthmatic control group (P<0.05), while the level of IFN-γ in BALF was significantly increased in pcDNA3-T-bet group. No significant change of inflammation cells and cytokines in pcDNA3 plasmid group and asthmatic control group was observed (P>0.05). CONCLUSION: Intranasal pcDNA3-T-bet plasmid transfer inhibits asthmatic airway inflammation in the murine asthmatic model, suggesting a new therapeutic strategy for allergic asthma.
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BACKGROUND: Hemorrhagic cystitis (HC) is one of common complications in patients undergoing hematopoietic stem cell transplantation (HSCT). It is of great value for improvement in the HSCT outcome to describe the clinical characteristics of HC and risk factors. OBJECTIVE: To investigate the incidence of HC in children after HSCT, and to analyze its clinical characteristics and risk factors.DESIGN: Case analysis SETTING: Center of Hematopoietic Stem Cell Transplantation, Department of Pediatrics, Second Affiliated Hospital of Sun Yat-sen University.PARTICIPANTS: Experiments were performed at the Center of Hematopoietic Stem Cell Transplantation, Department of Pediatrics of Second Affiliated Hospital of Sun Yat-sen University from October 1998 to June 2004. Eighty-eight patients receiving umbilical cord blood transplantation (UCBT) and peripheral blood stem cell transplantation (PBSCT) were enrolled; 49 were males and 39 were females. The age ranged from 2 to 18 years with an average of 8.0 years. Guardians of child patients signed informed consents. The experimental procedures were approved by Medical Ethics Committee.METHODS: ①Conditioning regimens included combination of cyclophosphamide (CY, 120-200 mg/kg) with busulphan (BU, 14-20 mg/kg)-based chemotherapy and combination of CY with total body irradiation (TBI, 2-8 Gy) or total lymphoid irradiation (TLI, 2-8 Gy)-based radiotherapy. ②HC was defined according to the criteria proposed by references 7 and 8. The incidence, clinical characteristics, laboratory examination, treatment and outcome for HC were described. The association of various clinical factors including age, gender, human leucocyte antigen (HLA) typing, diseases for transplant, the type of stem cell, the type of transplantation, the occurrence of acute graft-versus-host disease (aGVHD) and cytomegalovirus (CMV) infection with the development of HC were examined.MAIN OUTCOME MEASURES: ①Incidence of HC, ②HC patient characteristics and laboratory examination, ③HC treatment and outcome, and ④risk factors analysis. RESULTS: All 88 patients were included in the final analysis. ①The incidence of HC: 16 patients (18.2%, 16/88) developed HC post-transplant with the severity graded as mild in 11 cases (68.7%) and severe in 5 cases (31.3%). ②HC patient characteristics and laboratory examination: All had hematuria and 8 cases (50.0%) had typical pollakisuria, urinary urgency, odynuria and gross hematuria; 10 cases (62.5%) had gross hematuria and 11 had proteinuria (+ to +++); Leucocytes were detected in 7 cases. ③Treatment and outcome: All patients recovered at a median of 13.5 days (range 2-53 days). ④Risk factors analysis: The incidence of HC was significantly higher in the group of ≥ 6 years old, presence of aGVHD and development of cytomegalo-virus (CMV) infection (P < 0.05-0.01). CONCLUSION: ①HC has its own clinical characteristics following HSCT in children but with good prognosis. ②The risk factors for HC are ≥ 6 years old, presence of aGVHD and CMV infection.
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BACKGROUND: Embryonic stem cells fESCs) develop from early blastular inner cell mass.Under proper condition,ESCs can maintain undifferentiated state in vitro,normal diploid nuclear type,and proliferative potential.In addition,ESCs possess multi-directional differentiation capacity and can differentiate into all sorts of cells of three germ layers. OBJECTIVE: To investigate the feasibility of directed differentiation of ESCs towards thyroid cells in vitro as well as related molecular expression. DESIGN,TIME AND SETTING: A cell-based observational experiment was performed at the Bank of Cord Blood,Second Hospital Affiliated to Sun Yat-sen University between January 2004 and December 2006.MATERIALS: Balb/c pregnant mice at pregnancy 12.5-14.5 days were used for preparation of embryonic fibroblast feeder layer.E14 mouse embryonic stem cell (ESC) strains were gifted by professor Xu from Harvard University.METHODS: Murine El4 ESCs were cultured in methylcellulose semisolid medium to form embryoid bodies (Ebs).These Ebs were transferred for further inductive culture with the stepwise addition of growth factors- thyrotropin (TSH),insulin and kalium iodidum (KI).The cultured thyroid cells of adult Kunming mice were taken as positive control.MAIN OUTCOME MEASURES: ①Cellular morphological change in the process of differentiation.② Detection of expression levels of TSHR,PAX8,TTF-2,and TIF-1 by immunofluorescence assay.③ Detection of expression levels of TSHR,PAX8,NIS,TPO,and Tg by reverse transcription-polymerase chain reaction (RT-PCR).RESULTS: The differentiated cells had clear boundary,exhibiting round,oval,shuttle-shaped,or polygon adhesive growth.On day 6 of inductive ifferentiation,the differentiated cells showed the expression of PAX8,NIS,TPO,Tg,and TSHR,the specific gene of thyroid ceils.On day 8 of inductive differentiation,the expression of TSHR,TIF-1,PAX8,and TIF-2 was detectable in the differentiated cells with morphous similar to thyroid cells.CONCLUSION: ESCs can differentiate towards thyroid cells under given inductive conditions.
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OBJECTIVE To investigate the characteristic and prognosis of nosocomial infection in children patients with malignances during deficiency stage of neutropenia. METHODS The clinical characteristic of infections and efficiency of antibiotics were analyzed retrospectively in 174 malignances children with nosocomial infection from Jan 2000 to Jun 2005. RESULTS The incidence rate of nosocomial infection in children patients with malignances during deficiency stage of neutropenia was 67.4%.Nosocomial infection was occurred mainly in the respiratory tract,oral cavity,blood,skin,and intestinal tract of patients.The main pathogens were bacteria(86.8%),and fungal infection was 13.2%.The Gram-negative bacilli were relatively sensitive to imipenem,amikacin,ceftazidine,piperacillin/tazobactam,and ticarcillin/clavulanic acid.The Gram positive cocci were sensitive to vancomycin,imipenem,meropenem,ampicillin/sulbactam,ciprofloxcin and clindamycin.The death rate due to nosocomial infection was 44.4%. CONCLUSIONS There is higher incidence of nosocomial infection in children patients with malignances during the neutropenia stage.Good nursing,intestines disinfection,the usage of granulocyte colony-stimulating factor,a reasonable usage of antibiotics and preventing the fungal infection are good to control nosocomial infection in children malignances.
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In this study,IL-4 and IL-12 levels in plasma and peripheral blood mononuclear cells(PBMC) as well as serum IgE level were prospectively assessed with double-antibody sandwich ELISA technique in children with asthma during the attack group (30 cases)and the interval group(12 cases).The results observed revealed that serum IgE level and IL-4 level in plasma and PBMC after PHA and LPS provocation during both of the attack stage and the interval stage were found to be evidently higher than those of the normal control group (P＜0.01).Otherwise,IL-12 level during both stages was much lower than that of the normal control(P＜0.01).In other hand,there were found to have a significant difference in all these 3 data between attack and interval stages.Thus, the conclusion indicates that there might be an imbalance of IL-4,IL-12 and IgE level in children with asthma during both of the attack and the interval stages.
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【Objective】To observe the efficacy and side effects of hematopietic stem cell transplantation with combination of umbilical cord blood(UCB) and neonatal peripheral blood(NPB) in the treatment of β-thalassemia major.【Methods】28 mL NPB was drawn from a HLA identical neonate within 5 hours after his birth to complement stem cell of the UCB he donated for transplantation to his sibling with β-thalassemia major.Various items of hematopoiesis reconstruction were detected in UCB and NPB respectively.After conditioning with chemotherapy by using busulfan 20 mg/kg,cyclophosphamide 200 mg/kg,melphalan 90 mg/m2 and antithymocyte globulin(ATG) 90 mg/kg,the patient received the 53 mL UCB and 28 mL NPB,achieving 5.7×107/kg nucleated cells(NC),93×105/kg CFU-GM and 3.1×105/kg CD34+CD38- cells from his HLA-identical sibling.【Results】Absolute nucleated cell(ANC) reached 0.5×109/L on 14th day post transplant,and platelets reached 20×109/L on 34th day after transplant.The heterozygosity of β-654 mutation point was detected by the PCR-RDB.The sexual chromosome changed from XX pretransplant to XY posttransplant.The patient was free red blood cell transfusion from 14th day post transplant.Her hemoglobin rose progressively from 86 g/L to 110 g/L.The patient survived for 197 days free from disease after transplantation.Following up for 9 months, the donor grew and developed normally.【Conclusion】The NPB contains a lot of stem cells.The transplantation with combination of suitable NPB and UCB is an effective tactics when the UCB cells are deficient.
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Objective:To investigate the role of blockade of CD40 ligand-CD40 costimulatory pathway by anti-CD40L mAb on T lymphocytes typing and cytokines, and provide the experimental clues of inducing donor-specific T cell anergy.Methods:To monitor primary MLR, C57BL/6 H-2b spleen T cells were isolated as responder cells, and BALB/C H-2d spleen cell as stimulator cells. Anti-CD40L mAb was added to primary MLR cultures(termed anti-CD40L mAb group), and in control group(no anti-CD40L mAb). Incubated for 7 days, the cells responsiveness rates were detected by 3 H-TdR methods at the indicated time points, and supernatants were assayed for IFN-?,IL-2,IL-4,IL-10 cytokines levels by commercial ELISA kits. Day 5 MLR-cultured cells were assessed for the expressions of CD4, CD8, CD25, CD69, CD40L and CD45RA with flow cytometry(FCM).Cells proliferation for 5 days and cytokines in secondary MLR were assayed according above-mentioned method.Results:Blockade of the CD40-CD40L pathway by anti-CD40L mAb can induce the hyporesponsiveness to alloantigen in primary and secondary MLR culture. In primary MLR culture, the expressions of CD4 +T and CD8 +T cells and CD4 +CD25 +T, CD4 +CD69 +T, CD4 +CD40L +T, CD8 +CD25 +T and CD8 +CD69 +T cells in anti-CD40L mAb group were lower than that in control group ( P 0.05).The expression of CD4 +CD45RA + T in anti-CD40L mAb-cultured group was higher than that in control group( P
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AIM:To investigate the potential of hematopoietic stem cells(HSCs)derived from mouse embryonic stem cells(ESCs)to reconstruct hematopoiesis in vivo.METHODS:Using a three-step method,a mice embryonic stem cell line,E14.1 was induced into hematopoietic stem cells.The cell markers with CD34+/Sca-1+ were identified by flow cytometry analysis,then HSCs(1?109 cells/L)from third-step were injected into SCID mice for observing teratoma formation.To validate function of HSCs,colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted.RESULTS:The method of three-step differentiation,combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells(as high as 58.64%?4.20%)with more CFU-E,CFU-GM and CFU-GEMM populations.The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining.Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation,with 71.4%(5/7)successful engraftment rate.Three recipients showed that the cell population of the peripheral blood leukocytes,red blood cells and hemoglobin approached to normal index at 40 d after transplantation,but followed relative slow renew in platelet count.Survival rate in transplant group was 43%,compared to 100% mortality in control mice.Karyotyping assays confirmed the female mice with XY.CONCLUSION:The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs.HSCs derived from mouse ESCs can reconstruct hematopoiesis.
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AIM:To explore the supportive and expansive effects of aorta-gonad-mesonephros(AGM) region derived stromal cells on hematopoietic stem cells(HSC) in vitro.METHODS:The murine stromal cells were separated and cultured from AGM region of a 11 day postcoitum(dpc) mouse embryo and 6 week mouse.After identification by Wright's staining and flow cytometry,the stromal cells were co-cultured with the embryonic stem cell(ESC)-derived,cytokine-induced HSCs,and the maintenance and expansion of HSCs were evaluated by detecting CD34+,CD34+Sca-1+cells with flow cytometry.Blast colony-forming cell(BL-CFC) and high proliferative potential colony-forming cells(HPP-CFC) were determined by semi-solid medium clonal culture.RESULTS:AGM-derived and bone marrow(BM)-derived stromal cells were similar in morphology and phenotype,and had common character of stromal cells.Supported by AGM stromal cells or by BM stromal cells,more primitive progenitor cells HPP-CFC were expanded,but BL-CFC expansion was only detected in AGM-derived stromal cells.In the supporting of BM stromal cells CD34+ hematopoietic stem/progenitor cells were expanded 3-4 times,but no significant expansion in CD34+Sca-1+ cells was observed.While in the supporting of AGM stromal cells,both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded significantly from 4 to 5 times,respectively(P
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AIM:Xaf1-Saos inducible cell lines,which contain "gene switch" system were used to detect the effect of Xaf1 on tumor necrosis factor receptor(TNFR) signal pathway and to investigate the mechanism of cooperation between Xaf1 and TNF-? in inducing cell apoptosis.METHODS:Xaf1 on TNFR1 expression was measured by RT-PCR and Western blotting.The effect of NF-?B on Xaf1 induced apoptosis was detected by DNA content flow cytometry after co-transfection.DNA binding activity of NF-?B was identified by gel mobility shift assay and transcription activity of NF-?B was analyzed by luciferase assay and RT-PCR.SAPK/JNK activity was checked by SAPK/JNK assay.RESULTS:Xaf1 did not modulate TNFR1 at protein and mRNA levels.Increased NF-?B activity in cells inhibited Xaf1 induced apoptosis.Expression of Xaf1 impaired modestly TNF-? induced NF-?B DNA binding activation and transcription activation,also modestly reduced SAPK/JNK activity.CONCLUSION:Xaf1 inhibits TNFR signal pathway,partly contributing to cooperation with TNF-? to induce apoptosis.
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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.
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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.
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AIM:To direct embryonic stem cells(ESCs)into hematopoietic stem cells(HSCs)in vitro by simulating the hematogenic microenvironment in human early embryonic aorta-gonad-mesonephero(AGM)region.METHODS:Murine E14 embronic stem cell line was used for two-step differentiation.In the first step of primary differentiation,E14 ESCs were seeded into semisolid methylcellulose-based medium containing bone morphogenesis protein 4(BMP4)and vascular endothelial growth factor(VEGF)for embryoid body(EB)formation.On days 3,6,9,12 and 15,single EB cells were analyzed for Flk-1+ cells amount through flow cytometry.In the second step,single cell from EB containing most Flk-1+ cells was further co-cultured with human AGM stromal cells in non-contact system.On co-culture days of 3,6,9 and 12 days,cells were collected for cell count,flow cytometry for Sca-1+c-kit+ cells analysis,and colony forming cell assay.RESULTS:During the EB formation,BMP4+VEGF promoted Flk-1+ cell genesis on day 9 at peak pencentage value of 27.53%?2.84%,which was statistically higher than that in control group as 8.77?1.10(P
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AIM: The expression of the mouse ? 2 microglobulin (? 2 m) in NIH3T3 cells transfected with the mouse ? 2m sense and antisense RNA was detected to clarify the effect of mouse ? 2m sense and antisense RNA on the expression of MHC classⅠgene. METHODS: The mouse sense and antisense RNA, pcDNA3-? 2mSN and pcDNA3-? 2mAN, were constructed and were transfected into NIH3T3 cells by lipofectamine. RT-PCR and Western blot were used to detect the expression of ? 2m in those cells. RESULTS: The expression of the mouse ? 2m in the cells transfected with pcDNA3-? 2mSN was increased, while it was decreased in those cells transfected with pcDNA3-? 2mAN. CONCLUSION: pcDNA3 -? 2mAN can downregulate the expression of the ? 2m in NIH3T3 cells.
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The interaction between the immune system and nervous sytem affcts nervous function.There is a close relationship between the episode of epilepsy and immunity,the molecular basis of which is the network of cytokines.In order to quest new methods of contolling epilepsy by regulating the network of cytokines,the roles of several cytokines in the ocurrence and onset of epilepsy were reviewed in this article.
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AIM: To quest the relationship bwteen BDNF and seizures. METHODS: BDNF and other correlative proteins in the hippocampus of audioepileptic rat (P77PMC) have been detected by immunohistochemistry at different stages of seizures including quiet stage, preconvulsive stage, convulsive stage and postconvulsive stage. Wistar rats were served as controls. RESULTS: At quiet stage, the BDNF in the hippocampus of P77PMC were less than that of control group and stepped up after stimulation, trkB in the hippocampus of P77PMC were high at every stages, but PY20 were increased only after stimulation. NPY in the hippocampus of P77PMC were in a low level before seizures. CONCLUSION: The endogenetic BDNF may alter the excitement of P77PMC by adjusting the expression of NPY in hippocampus and activating its receptor trkB.