Effects of 5-Bromotetrandrine and Daunorubicin on the Expression of Survivin In K562/AO2 Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4947-4947
Author(s):  
Bao-An Chen ◽  
Xiao-hui Cai ◽  
Jun Wang ◽  
Chong Gao ◽  
Jia-hua Ding ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mtt Assay ◽  
Conflicts Of Interest ◽  
Acquired Resistance ◽  
Leukemic Cells ◽  
Control Group ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Typical Experiment

Abstract Abstract 4947 Objective: The aim of this study was to investigate the expression of survivin and the apoptosis induced by DNR and BrTet in the leukemic cells K562/A02. Methods: In a typical experiment, the K562/AO2 cells were treated with daunorubicin (DNR), 5-bromotetrandrine (BrTet), or DNR and BrTet for 48 hours, and the cells treated without any drugs were used as control group. Cell proliferation was analyzed by MTT assay. Cells apoptosis and the concentration of DNR within the cells were measured by Flow cytometry (FCM). The expressions of mRNA and protein of survivin were determined by semi-quantitative reverse transcription PCR (RT-PCR) and Western blot, respectively. Results: The results of MTT assay indicated that DNR and BrTet were both able to inhibit the proliferation of K562/AO2 cells in dose-dependent manner. The fresh evidence from flow cytometry showed that a higher apoptosis rate could be induced and a higer concentration of DNR could be detected in K562/AO2 cells by DNR and BrTet as compared with those by DNR or BrTet in the same concentrations(P<0.01). RT-PCR revealed that the expression of survivin mRNA, a higer expression in K562/AO2 cells with acquired resistance to adriamycin than that in parent K-562 cells, decreased in the DNR and BrTet group (P<0.05), but there was no obvious change in other groups(P>0.05). Western bolt demonstrated that the expression of survivin protein was much lower in the DNR and BrTet group(P<0.05). Conclusion: BrTet could increase the concentration of DNR and reverse the multidrug resistance(MDR) in the K562/AO2 cells. Survivin may play an important role in apoptosis induced by DNR. Survivin could be a target for the treatment of MDR in haematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Oxymatrine Induces Apoptosis in Human Myeloma Cells By Cell Cycle Arrest and Activation of Caspase-Dependent Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5727-5727
Author(s):  
Wenjun Wu ◽  
Cai Wu ◽  
Fuming Zi ◽  
Yi Li ◽  
Li Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Growth Inhibition ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Conflicts Of Interest ◽  
Dependent Manner ◽  
Rt Pcr

Abstract Background : Multiple myeloma (MM) is a B cell malignant hematologic cancer. Despite the introduction of new drugs and improvement of chemotherapy, MM is still an incurable disease. Oxymatrine (OMT), the active ingredients of traditional Chinese herbal medicine sophora, has been reported to have antitumor activity. This study was to estimate the therapeutic efficacy of OMT in MM. Methods: The growth inhibition of myeloma cell lines (RPMI8226, U266, ARP-1) or primary cells by OMT was assessed by MTT assay. Apoptosis of MM cells was examined by annexin V-FITC using flow cytometry analysis. DNA content was analyzed by flow cytometry. RT-PCR and western-blot analysis were used to assess the expression of Bcl-2 family proteins and the IAP family proteins. Western blotting was also used to elucidate the signaling pathway that may mediate OMT-induced apoptosis of MM cells. Results: OMT treatment resulted in cell growth inhibition and apoptosis in primary MM cells and all tested MM cell lines in a dose-dependent manner (P <0.05). To elucidate OMT -induced MM cell apoptosis, MM cell lines were treated with or without OMT for 24h and assessed for caspase activation and signaling pathway by Western blotting. The results showed the cleavage of PARP, caspase-3, and caspase-9, and p-AKT were down-regulated after OMT treatment. The mRNA expression of survivin and HIAP by RT-PCR was down-regulated. OMT treatment at 5mM for 48h resulted in increased G-phase cells and decreased S-phase cells in MM cell lines (P <0.05). Cell cycle repressor P21 protein was up-regulated while CDK4, CDK6 and CyclinD1 expression was down-regulated. Our finding also showed a synergistic anti-MM activity of OMT and dexamethasone or adriamycin at a low does (CI<1). In addition, LC3-II expression was significantly increased both in RPMI8226 and U266 cells after treatment with OMT. However, treatment with different doses of OMT and 5 mM autophagy inhibitor 3-MA, significant increased cell apoptosis (P <0.05). Conclusion: Our findings demonstrate the anti-MM activity of OMT and indicate that OMT alone or together with other MM chemotherapeutics may be a prospective treatment for MM. Disclosures No relevant conflicts of interest to declare.

Here Is the Sub-Title:Apoptosis on Multiple Myeloma U266 Cell by Phosphorylation c-Jun of Brucine

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5112-5112
Author(s):  
Yanping Ma ◽  
Zhihua Li ◽  
Yihua Wang ◽  
Jing Feng
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Expression Change ◽  
U266 Cell ◽  
Apoptotic Effect

Abstract Abstract 5112 Objective: To investigate the Mechanism of the apoptotic effect of brucine on human multiple myeloma. Method: U266 cells (5×104) were plated in the presence or absence of the brucine (0, 0.05, 0.1, 0.2, 0.4 mg/ml) in 96 well culture plates for 24–72 h. The anti-proliferative response of brucine was assessed by MTT assay. The analysis of cell cycle of U266 cell with or without brucine was mesured by flow cytometry. The expression change of c-Jun after joining brucine, brucine and JNK specific inhibitor SP600125 was detected using RT-PCR. Results The apoptotic effect of brucine show a dose and time dependent manner. Cell cycle analysis using flow cytometry revealed accumulation of cells at sub-G0/G1 phase. The apoptosis rate separately were (4.137±0.01)%, (10.55±0.03)%, (12.31±0.04)%, (27.67±0.08)%, (29.67±0.09)% (p<0.01). Detecting c-Jun gene expression respectively after joining brucine, brucine and JNK specific inhibitor SP600125 by RT-PCR. The gray scale values were (0.7961±0.007),(0.4683±0.003). Conclusions Within the 0.4mg/ml concentration of brucine can induce apoptosis in U266 cells. Brucine by JNK signaling pathway through phosphorylation of c-Jun induced apoptosis in U266 cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Study on Biocompatibility of Porous nHA/PLGA Composite Scaffolds for Tissue Engineering with Rabbit ChondrocytesIn Vitro

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Lei Chen ◽  
Wei-Min Zhu ◽  
Zhi-Qiang Fei ◽  
Jie-Lin Chen ◽  
Jian-Yi Xiong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Flow Cytometry ◽  
Mtt Assay ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Control Group ◽  
Dependent Manner ◽  
Plga Scaffold ◽  
Significant Difference

Objective. To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA) composite and evaluate its feasibility as a scaffold for cartilage tissue engineering.Methods. Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffoldin vitroand the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM). The cell cycle profile was analyzed by flow cytometry.Results. The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P>0.05).Conclusion. The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

Maturation by Toll Like Receptor Ligand R848 Improves the Uptake of Apoptotic Leukemic Cells by Monocyte Derived Dendritic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2075-2075
Author(s):  
Willemijn van den Ancker ◽  
Theresia M Westers ◽  
Hetty J Bontkes ◽  
Tanja D de Gruijl ◽  
Gert J Ossenkoppele ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Toll Like Receptor ◽  
Cytokine Cocktail ◽  
Migratory Capacity

Abstract Abstract 2075 Poster Board II-52 Introduction. Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g. monocyte derived DC (MoDC) loaded with leukemia associated antigens (LAA). Many sources of LAA, whether or not combined with various adjuvants and different methods of loading of antigen onto DC, have been used in an attempt to optimize anti-tumor responses. However, the optimal source of tumor antigen combined with adjuvants remains unclear. Leukemic cells harboring all unknown and known potential LAA are an attractive antigen source. Here we explore the use of leukemic blast derived apoptotic cells and lysates loaded onto MoDC combined with various maturation stimuli, such as the clinically applicable toll like receptor 7/8 ligand (TLR-L) R848 combined with conventional cytokine cocktail consisting of IL1β, IL6, TNFα and PGE2. Material & Methods. CD14+ monocytes isolated from peripheral blood of healthy donors were differentiated into MoDC with GM-CSF and IL-4 in serum free medium. Leukemic blasts were labeled with CFSE, followed by induction of apoptosis in serum deprived medium with heat shock (2h 42°C) or preparation of lysate by 3 freeze/thaw cycles. Apoptosis was confirmed by flow cytometry using Syto(62) and 7AAD. Subsequently, the CFSE-labeled apoptotic blasts and lysates were incubated in various ratios with immunofluorescently labeled MoDC in the presence of TLR-L (Poly-(I:C), LPS, PGN, Flagellin or R848) and/or the cytokine cocktail in different time frames. Uptake was defined by flow cytometry as percentage of CFSE positive MoDC. MoDC were analyzed for expression of co-stimulatory molecules, the chemokine receptor CCR7 and maturation-associated antigens. Migratory capacity towards CCL19 was measured in a transwell system. Mixed leukocyte reaction was performed to study T cell stimulation and IL12 secretion was quantified after CD40 ligation. Results. Apoptotic cells or lysates were taken up by MoDC in a dose dependent manner, up to 26% (12-49%) and 17% (median; range: 3-47%), respectively. Of all tested TLR-L, R848 most effectively enhanced the uptake of apoptotic cells, but not of lysates. Addition of the cytokine cocktail resulted in a decreased uptake by MoDC of both apoptotic cells and lysate (n=7), but most effectively induced phenotypic DC maturation as determined by up-regulation of co-stimulatory molecules, enhanced migratory capacity and enhanced ability to stimulate T cells. Both R848 and cytokine cocktail stimulated MoDC were able to produce IL-12 (n=6). To combine the enhanced uptake induced by R848 and the optimal maturation of MoDC by the cytokine cocktail, we co-incubated blasts and MoDC in the presence of R848 for 24h followed by a 48hr incubation with cytokine cocktail (n=9). The resulting uptake was superior to the cytokine cocktail alone, and migratory and co-stimulatory capacity was improved as compared to R848 alone. Conclusion. The TLR-L R848 significantly improved the uptake of leukemic blasts by MoDC. Combination of R848 and cytokines resulted in sufficient maturation induction and subsequent T cell priming. These data may add significantly to the development of new strategies for optimization of vaccine preparation in AML. Disclosures: No relevant conflicts of interest to declare.

Biological Effects of p53 Mutation W248 and H175 on MCF-7 Cells

2013 ◽  
Vol 790 ◽  
pp. 550-554
Author(s):  
Xiang Yu Zhou ◽  
Ya Jun Liu ◽  
Dan Li
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Tumor Cell ◽  
Mtt Assay ◽  
Biological Effects ◽  
Scratch Test ◽  
Control Group ◽  
Tumor Cell Migration ◽  
Mcf 7

Objective: p53, a tumor suppressor gene, is one of the hotspots in the world of the biomedical field. Mutation of p53 gene, which is found in approximately 50% of human cancers, is a key event in carcinogenesis. This project aims to investigate the new characteristics of two p53 mutants, p53-W248 and p53-H175, in MCF-7 cells, so as to provide the experimental basis for understanding the functional alternations of mutant p53. Methods: In this study, MCF-7 cells transfected with p53-H175 or p53-W248 plasmids were used as experimental group and the MCF-7 cells transfected wild type p53 plasmid were used as control group. Then the biological effects at the cellular level were investigated using 3-(4.5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis and cell scratch test. Results: MTT assay showed that p53-W248 might promote cell proliferation in MCF-7 cells. The results of flow cytometry indicated that no significant effect on cell cycle progression and cell apoptosis by p53-H175 or p53-W248 in cells. The cell scratch test showed that p53-H175 could increase the ability of cell migration. Conclusion: p53-H175 could lead to the promotion of tumor cell migration, while p53-W248 may promote tumor cell proliferation. p53-H175 and p53-W248 might have acquired some new characteristics of oncogenes.

scholarly journals Restoration of Retinoid Sensitivity by MDR1 Ribozymes in Retinoic Acid–Resistant Myeloid Leukemic Cells

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2452-2458 ◽  
Author(s):  
Hiromichi Matsushita ◽  
Masahiro Kizaki ◽  
Hiroyuki Kobayashi ◽  
Hironori Ueno ◽  
Akihiro Muto ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cellular Proliferation ◽  
Therapeutic Potential ◽  
Acquired Resistance ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
P Glycoprotein ◽  
All Trans Retinoic Acid ◽  
Mdr1 Mrna

Complete remission is achieved in a high proportion of patients with acute promyelocytic leukemia (APL) after all-trans retinoic acid (RA) treatment, but most patients relapse and develop RA-resistant APL. We have previously reported that both RA-resistant HL-60 (HL-60R) and APL cells express P-glycoprotein and MDR1 transcripts; and these cells differentiate to mature granulocytes after culture with RA and P-glycoprotein antagonist. Ribozymes have been shown to be able to intercept a target RNA by catalytic activity. To address the role of MDR1 in overcoming RA-resistance in APL cells, we investigated the biologic effects of ribozymes against the MDR1 transcript in HL-60R cells. These ribozymes efficiently cleaved MDR1 mRNA at a specific site in vitro. The 196 MDR1 ribozyme was cloned into an expression vector, and stably transfected (HL-60R/196Rz) cells were obtained. Expression of MDR1 transcripts was decreased in HL-60R/196Rz cells compared with parental HL-60R and empty vector-transfected (HL-60R/neo) cells. Interestingly, RA inhibited cellular proliferation and induced differentiation of HL-60R/196Rz cells in a dose-dependent manner, suggesting reversal of drug resistance in HL-60R cells by the MDR1 ribozyme. These data are direct evidence that P-glycoprotein/MDR1 is responsible in part for acquired resistance to RA in myeloid leukemic cells. The MDR1 ribozyme may be a useful tool for investigating the biology of retinoid resistance and may have therapeutic potential for patients with RA-resistant APL.

scholarly journals The Role of 5-HT on Proplatelet Formation and Thrombopoietin Production

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-14
Author(s):  
Mo Yang ◽  
Enyu Liang ◽  
Jieyu Ye ◽  
Beng H Chong ◽  
Liang Li
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Traumatic Stress ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Mice Model ◽  
Synthesis Inhibitor ◽  
Rhodamine Phalloidin ◽  
Proplatelet Formation ◽  
Group 5

Background: Our previous work confirmed that serotonin (5-HT) promotes the proliferation of hemopoietic stem cells and megakaryocytes (Yang M et al, Stem Cells, 2007; 2014). However, the mechanisms remain indefinite. Methods: Q-PCR, Flow Cytometry, Western Blot, or Immunofluorescence microscope were used in the receptor and TPO study. MTT/CCK-8, Proplatelet assay, and Flow Cytometry were also used in cell proliferation and apoptosis study. The relationship between 5-HT and TPO was studied in a traumatic stress mice model. Results: In-vitro study, there was a stimulating effect of 5-HT on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum-free medium with either 5-HT (200nM) or TPO (100 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (12.3 ± 5.0)% vs. Control (6.2 ± 3.5)%, P=0.025; TPO (15.6 ± 2.5)% vs. Control, P=0.04; n=4). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to the TPO group. 5-HT2A, 2B, 2C receptors were detected in the surface of megakaryocytes. The effect of 5-HT on proplatelet formation in MK cells was via 5-HT2 receptors and this effect was reduced by 5-HT2 receptor inhibitor ketanserin. 5-HT acted on cytoskeleton reorganization in MKs via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, the polymerization actin level was higher in the 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced. Our data also demonstrated that ERK1/2 was activated in MKs treated with 5-HT for 30 minutes. In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control in this model. Conclusions: This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. 5-HT could promote the proplatelet formation from megakaryocytes. The second is the indirect effect by promoting the production of TPO, which is a paracrine secretion to influence thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

224 A GROWTH OF HUMAN BG-1 CANCER CELLS EXPRESSING ESTROGEN RECEPTORS WAS ENHANCED BY SYNTHETIC PYRETHROIDS, LAMBDA-CYHALOTHRIN AND CYPERMETHRIN, VIA AN ESTROGEN RECEPTOR-DEPENDENT SIGNALING PATHWAY

2015 ◽  
Vol 27 (1) ◽  
pp. 201 ◽  
Author(s):  
C.-W. Kim ◽  
R.-E. Go ◽  
K.-C. Choi
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Mtt Assay ◽  
Ovarian Cancer Cells ◽  
Transcriptional Level ◽  
Synthetic Pyrethroids ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Pyrethroid Insecticides ◽  
Lambda Cyhalothrin

Synthetic pyrethroids (SP) are the most common pesticides in recent use, which are used as indoor pest control. The widespread use of SPs has resulted in extensive exposure to wildlife and human. Recently some SPs are suspected as endocrine disrupting chemicals (EDC) and have been assessed for their potential estrogenicity by various assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LCT) and cypermethrin (CP), the most commonly used pyrethroid insecticides in Korea, using BG-1 ovarian cancer cells expressing oestrogen receptors (ER). To evaluate the estrogenic activities of two SPs, LCT and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT–PCR). In MTT assay, LCT (10–6 M) and CP (10–5 M) significantly induced the growth of BG-1 cancer cells, 1.61 ± 0.1 and 1.45 ± 0.06 times, respectively, as 17β-oestradiol (E2, 10–9 M, 2.73 ± 0.25 times) did. LCT or CP-induced cell growth was reversed to a control level (DMSO) by addition of ICI 182 720 (10–8 M), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT–PCR results showed that transcriptional level of ERα expression was significantly down-regulated by LCT and CP as in case of E2. Taken together, these results indicate that LCT and CP may possess estrogenic potentials to stimulate ovarian cancer cells expressing ERs via an ER-dependent manner, and these collective results confirm the carcinogenicity of these SP, LCT and CP, in ER-positive cells or tissues.

scholarly journals Heat stress combined with lipopolysaccharide alter the activity and superficial molecules of peripheral monocytes

2019 ◽  
Vol 33 ◽  
pp. 205873841982889
Author(s):  
Jiajing Luo ◽  
Yi Chen ◽  
Chengjia Ding ◽  
Jialing Qiu ◽  
Yulan Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Heat Stress ◽  
Western Blot ◽  
Inflammatory Mediators ◽  
Peripheral Blood ◽  
Heat Stroke ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α

The purpose of this study was to focus on the underlying relationship between the hyperactivity for the peripheral monocytes and heat stroke by investigating the inflammatory oxidative activity of and the expression of superficial molecules. Peripheral blood samples were collected from 10 healthy adult volunteers. Human blood monocytes were isolated by density gradient centrifugation and sequent adherent culture. The objectives were divided into four groups: 43°C heat stress combined with lipopolysaccharide (LPS) group, 43°C heat stress group, LPS group, and control group. There were 10 cases in each group. An enzyme-linked immunosorbent assay (ELISA) test was used to measure the concentrations of supernatant inflammatory mediators (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10)). After loaded by 2,7-Dichlorodi-hydrofluorescein-diacetate (DCFHDA) fluorescent probe, intracellular reactive oxygen species (ROS) levels were determined by a flow cytometry. After fluorescent microspheres incubation, the phagocytosis of monocytes was observed under a fluorescent microscope. Respectively, the flow cytometry and Western blot were used to evaluate the level of triggering receptor expressed on myeloid cells-1 (TREM-1) and Toll-like receptor-4 (TLR-4) on the monocytes. Furthermore, the mRNA expression of TREM-1 and TLR-4 was detected by real-time polymerase chain reaction (RT-PCR). The heat stress combined with LPS stimulation promoted the peripheral monocytes to produce inflammatory mediators (TNF-α, IL-1β, and IL-10) and release ROS. Otherwise, such complex strike significantly suppressed the phagocytic activity of monocytes in peripheral blood. Moreover, the expression of TREM-1, TLR-4 and CD86 was measured by the flow cytometry on peripheral monocytes which were respectively promoted by the union of heat stress and LPS. The results of Western blot and RT-PCR demonstrated the similar kinetics on these superficial molecules (TREM-1, TLR-4, and CD86) stimulated by the combination of heat stress and LPS. The underlying mechanism of the dysfunction for the peripheral monocytes may be related to the abnormal expression of superficial molecules TREM-1, TLR-4, and CD86 on the monocytes induced by heat stress and LPS.

Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4099-4099
Author(s):  
Zhenhua Qiao ◽  
Xiujuan Zhao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Hematopoietic Cell ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

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