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Objective:To study the antitumor mechanism of W40,a monomer purified from Wedelia prostrate (Hook.et Arn.) Hemsl.Methods:The effects of W40 on the cell proliferative of GLC-82 cells were detected by MTT assay and colony formation assay.The migratory abilities of GLC-82 cells were observed by wound healing assay.Cell apoptosis was evaluated by Annexin V-FITC/PI staining analysis.The levels of apoptosis-relative proteins and cell proliferation-related proteins,such as Caspase-3,PARP,Stat3 and ERK,were detected by Western blotting.Results:MTF assay showed that W40 had a significant cytotoxic effect on non-small cell lung cancer GLC-82 cells.Colony formation assays showed that W40 significantly inhibited GLC-82 cells proliferation.The migration of GLC-82 cells was inhibited by W40 in a dose-dependent manner.Flow cytometry showed that the apoptotic rate increased gradually in a concentration-dependent manner.W40 down-regulated Stat3 as decreasing p-Stat3 and downstream proteins of Bcl-2 and Mcl-1.At the same time,W40 up-regulated the expression of pro-apoptotic protein Bax,and increased the cleavaged Caspase-9,Caspase-3 and PARP.W40 also down-regulated BRAF / MAPK / ERK signal pathway as decreasing p-BRAF,p-MEK and p-ERK.Conclusions:W40 induced apoptosis by inhibiting BRAF / MAPK / ERK and Stat3 signaling pathways.
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Objective To investigate the protective effects on the renal allografts from brain dead (BD) donor rats pretreated with bone marrow mesenchymal stem cells (MSCs).Method Three groups [normal transplant group (G1).BD transplant group (G2),and MSCs pretreated + BD transplant group (G3)] were set up.Male F344 rats served as donors and male Lewis rats as recipients.In G1,kidneys from F344 donor rats were implanted into Lewis recipients.In G2,kidneys from F344 BD donor rats were engrafted into Lewis recipients.In G3,after BD was established in F344 rats,MSCs were given intravenously to the rats.The kidneys harvested 6 h later were transplanted to Lewis recipients.Cyclosporine was intromuscularly given daily to the recipient rats for 10 days.Right kidneys were resected from recipients on day 10.Creatinine level was examined on day 14,21,28,and 35.Renal allografts harvested on day 35 were pathologically detected.The irnmunochemistry expression of interleukin (IL)-1β and tumor necrotic factor (TNF)-α in renal allograft tissue was tested.Result Serum creatinine levels in G2 were remarkably higher than those in G1 and G3 (P＜0.01) on day 14,21,28,and 35 postoperatively.The creatinine levels on the above mentioned time points had no statistically significant difference between G3 and G1 except on day 21.Postoperative pathological changes in G2 of both pronounced infiltration of mononuclear cells and tubular epithelia[inflammation were notably increased in renal allografts as compared with those in G1 and G3.There was no obvious difference between G1 and G3 in infiltrated mononuclear cells and tubular epithelial inflammation.Positive expression levels of both IL-1β and TNF-α in glomerular,tubular and interstitial epithelial cells were statistically enhanced in G2 as compared with those in G1 and G3 (H =7.210,P =0.027),while there was no statistically significant difference in the expression of both IL-1[β and TNF-α between G1 and G3.Conclusion Brain dead donor rats pretreated with bone marrow MSCs might reduce renal allograft injury via decreasing both inflammatory cell infiltration and IL-1β and TNF-α expression.
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Objective To compare the proteome difference between multiple myeloma cell line U266 cells treated and untreated with PS-341, to investigate the potential drug targets, and to provide theoretical evidence for clinical therapy of multiple myeloma. Methods Two-dimensional gel electrophoresis (2-DE) was performed to separate proteins from treated and untreated U266 cells with proteasome inhibitor PS-341. ImageMaster 2D Platinum software was used to analyze 2-DE image, and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to identify the differentially expressed proteins. The expression levels of differential protein BAG-2 in the 2 groups of U266 cells lines were detected by Western blot. Results The 2-DE reference pattern of treated and untreated U266 cells with PS-341 was established. A total of 31 differential proteins were identified by MALDI-TOF-MS, 27 of which were down-regulated after PS-341 treatment. The differential expression level of BAG-2 in the 2 groups of U266 cells was confirmed by Western blot. Conclusion Some down-regulated proteins may be the potential drug targets of proteasome inhibitor PS-341.
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BACKGROUND: CpG oligodeoxynucleotide (ODN) is a type of highly effective immune adjuvant with low toxicity, which has an extensive application in gene therapy for many diseases. However, the specificity for species and cells leading to low uptake by cells and degradation by nuclease blocks its clinical application. OBJECTIVE: To explore the specific delivery and its immunologic efficacy of CpG ODN targeting B lymphocytes of umbilical cord blood by CD40 ligand-receptor-mediated carrier system. DESIGN, TIME AND SETTING: An observation and control experiment was performed at the Department of Hematology, and Department of Pediatric, the First Affiliated Hospital of Jinan University from April 2004 to October 2007. MATERIALS: Fresh umbilical cord blood with heparin was obtained from healthy, natal infant. Informed consent was obtained from his parents, and the experiment was approved by the hospital Ethics Committee. METHODS: CD40 ligand (CD40L)-EDC-PLL-CpG ODN conjugated complex was prepared. Mononuclear cells (MNCs) from umbilical cord blood were co-cultured with conjugated complexes. Uptake rate, mean fluorescence intensity of FAM marked CpG ODN, expressions of MNCs, proliferations of lymphocytes and the IgG levels of culture supematants were detected by flow cytometry, fluorescence techniques, MTT assay and ELISA, respectively. MAIN OUTCOME MEASURES: The uptake rate, the mean fluorescence intensity of CpG ODN by MNCs, subgroups and proliferations of lymphocytes, and IgG levels of culture supematants. RESULTS: Compared to the pure CpG ODN group, the uptake rate of the conjugated complexes group was higher (98%), the peak level of up-taking occurred earlier, and intracellular fluorescence intensity maintained much more stable. Expressions of CD19+, CD22+, and CD20+ was increased, A value and IgG levels in supematants were all higher than that of the control group. CONCLUSION: CD40 tigand-receptor-mediated carrier system is helpful for CpG ODN delivery targeting to B lymphocyte, enhancing its immunological efficiency.
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Mesenchymal stem cells(MSCs) have became the hot spot in cell tissue engineering,cell replacement therapy,gene therapy and transplant research fields. Recent studies have shown that changes in oxygen concentrations affect many biological characteristics of MSCs. Under different oxygen concentrations,MSCs have different proliferation,differentiation,migration and chemotoxis abilities. Hypoxia is a kind of common pathophysiological status,which can promote the proliferation,apoptosis,migration and chemotoxis abilities of MSCs,while hypoxia impacts the differentiation ability depending on different cell types. The mechanism of these response might be involved in hypoxia-inducible factor-1(HIF-1) ,chemokines and their receptors,and matrix metalproteinases. Hypoxia can activate HIF-1 signaling pathway,which upgrades the expression of stromal-derived factor-1(SDF-1) ,and forms microenvironments which stem cells are adapted to and grow in. SDF-1 increases the adhesion,migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue,and promotes degradation of extracellular matrix,then enhances the migration ability of MSCs by modulating the expression of matrix metalloproteinase and its protein as well.
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To explore the teaching model and effect of emergency care simulator(ECS) teaching in "Shock"during clinical bed side teaching,fifty-six students were randomly divided into two groups, receiving model and traditional teaching model respectively. After one semester,the students’examination results and general effect were evaluated. The students’ex-amination results,clinical theory and clinical skills in ECS teaching group were significantly better than traditional teaching group.
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<p><b>OBJECTIVE</b>To evaluate the long-term outcome of 60 leukemia patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) prepared with busulphan-cyclophosphamide (BU-CTX(2)) conditioning regimen.</p><p><b>METHODS</b>From April 1994 to August 2000, 60 leukemia patients (35 male, 25 female; median age 32 years old), including 20 acute myeloid leukemia (AML, CR(1) n = 19, CR(2) n = 1), 15 acute lymphocytic leukemia (ALL, CR(1) n = 8, CR(2) n = 6, CR(3) n = 1), and 25 chronic myeloid leukemia (CML, CP(1) n = 24, CP(2) n = 1) received allogeneic hematopoietic stem cells from HLA-identical siblings (n = 53), 1-locus mismatched siblings (n = 4), or HLA-identical unrelated donor (n = 3). BU-CTX(2) was used as conditioning regimen. All patients received cyclosporine A and either methotrexate (n = 54) or methylprednisolone (n = 6) for graft-versus-host disease (GVHD) prophylaxis.</p><p><b>RESULTS</b>All 60 patients got sustained engraftment. Acute GVHD (aGVHD) occurred in 22 patients (36.7%), while the incidence of aGVHD was 48.0% for the CML, 30.0% for the AML and 26.7% for the ALL patients. Thirty-eight patients are still alive in complete remission with a median follow-up of 30 (12 approximately 84) months and 22 died. The main causes of death were aGVHD in 3, CMV-IP in 7, and relapse in 11 patients. The remaining one died of pulmonary infection. Among 11 patients who died of relapse, 8 were ALL relapsed in the early posttransplant stage. All 4 long-term survivors of ALL developed chronic GVHD. The probability of DFS at 3 year for CML, AML and ALL patients was 80.0%, 70.0% and 26.7%, respectively.</p><p><b>CONCLUSION</b>BU-CTX(2) is an effective conditioning regimen for patients with AML and CML, resulting in a low relapse and high long-term survival rate. However, it is not effective enough for patients with ALL, because of a higher incidence of relapse.</p>
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Humans , Busulfan , Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Transplantation Conditioning , Transplantation, HomologousABSTRACT
AIM: To investigate the distribution and clonal expansion of TCR V? subfamily T cells in patients with acute monoblastic leukemia (M5). METHODS:The CDR3 of TCR V? 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 V? T cells. RESULTS:Only 1-10 V? subfamily T cells were identified in M5 cases. Genescan analysis showed that oligoclonal (clonal expansion) T cells were found in some V? subfamilies from 8 cases with M5, V? 2 oligoclonal T cells were identified in six cases, whereas V? 7- or V? 9 clonal expansion T cells were detected in the other two cases, respectively. In addition, except V? 2, V? 7 or V? 21 oligoclonal T cells could be detected in two cases, respectively. CONCLUSION:The skew distribution and clonal expansion of TCR V? subfamily T cells could be found in patients with M5. It may be a specific anti-leukemia immune response with which the host T cells were activated by the leukemia-associated-antigen. The clonal expansion T cells were tendentious in V? 2, which may be related to the M5 leukemia cells associated antigen.
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AIM: To evaluate the effect and safety of Rhodamine 123 (Rh123)-mediated photodynamic treatment (PDT) on acute graft versus host disease. METHODS: An acute graft versus host disease (aGVHD) mice model was established using C57B/6 mice as donors and BALB/c mice as recipients. Mixed lymphocytic cells were cultured with Rh123 (50 nmol/L) and irradiated by argon laser 30 mW/cm2 for 3 min, then transplanted to BALB/c recipient mice mixed with donor bone marrow. Hepatopoietic recovery, aGVHD occurrence, survival time after transplantation and pathological changes were observed. In addition, CD3+CD69+ positive rates of MLC were examined by flowcytometry. RESULTS: Occurrence of aGVHD decreased, degree of pathological manifestation became milder, survival rates were higher than non PDT groups. CD3+CD69+ rates of MLC cells treated with photodynamic therapy (PDT) and cultured for 24 h significantly decreased. CONCLUSION: Rh123-mediated PDT can effectively prevent aGVHD of allogeneic bone marrow transplantation in mice.
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98%), the peak level of uptake occurred earlier, intracellular fluorescence intensity maintained much more stable. Expressions of CD19+, CD22+, CD20+ increased significantly. A_~570 values of MNCs proliferation and IgG levels in supernatant were all higher. CONCLUSION: CD40 ligand-PLL carrier system may delivery CpG ODN targeting to B lymphocytes, enhancing its immunological efficiency.
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AIM: To design,prepare and screen out functional small interfering RNA(siRNA) for specifically silencing proliferating cell nuclear antigen(PCNA) gene expression and effectively inhibiting cell proliferation on human hepatoma cell line SMMC-7721,human gastric carcinoma cell line SGC-7901 and human colorectal carcinoma cell line Caco2.METHODS: PCNA siRNA was designed based on previous studies about design guidelines and synthesized in vitro by T7 Mega short script reaction kit according to the manufacturer's instructions.After purification,the integrities of siRNA were identified through 19% denaturing polyacrylamide gel electrophoresis,and the concentrations of the generated siRNA were measured by testing the absorbance at 260 nm using a spectrophotometer.Four synthesized double-strand siRNA were transfected into three types of carcinoma cell lines by LipofectamineTM2000 reagent,respectively.WST-8 assay was employed to examine the proliferative inhibitions of these three cell lines.The subsequent alterations on PCNA mRNA and protein levels were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) and immunocytochemistry,respectively.RESULTS: 3 sequences,No.2,No.3 and No.4 PCNA siRNA showed effective inhibitions on tumor cells among the 4 candidate siRNA,and a single dose of 50 nmol/L of No.2,No.4 PCNA siRNA showed the most effective inhibitory rates as more than 62% at 48 h after transfection.50 nmol/L of No.2 and No.4 PCNA siRNA transfection caused 72% decrease of PCNA mRNA level and almost completely loss of the protein in human Caco2 cells.CONCLUSION: No.2 and No.4 PCNA siRNA have been screened out in this study,which show the capability to effectively down-regulated PCNA expression and significantly inhibit the proliferation of carcinoma cells.The optimal concentration is 50 nmol/L and satisfactory effects are achieved 48 h after transfection in vitro.