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1.
Journal of Experimental Hematology ; (6): 1531-1537, 2018.
Article in Chinese | WPRIM | ID: wpr-689902

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of exosomes derived from miR-486 gene-modified umbilical cord mesenchymal stem cells (UC-MSCs) on biological characteristics of rat cardiomyocytes.</p><p><b>METHODS</b>The human umbilical cord mesenchymal stem cells (UC-MSCs) were isolated and cultured, then the immunophenotypes and ability of osteogenic and adipogenic differentiation of UC-MSC were identified. The structure of exosomes was observed by electron microscopy; the effect of exosomes on cell migration was detected by transwell cell migration test; the miR-486 high expression of UC-MSC was mediated by using recombinant adenovirus vector, moreover the UC-MSC with high expression of miR-486 were identified by qPT-PCR. The exosomes were isolated from cell culture supernatant by ultracentrifugation and the miR-486 expression level of UC MSC exosomes was detected by qRT-PCR. The effect of exosomes on the proliferation of cardiomyocytes was evaluated by Dye670 marking. The HO-induced cardiomyocyte apoptosis model was established, and the effect of exosomes on apoptosis of cardiomyocytes was detected by flow cytometry with Annexin V/PI double staining.</p><p><b>RESULTS</b>The exosomes derived from UC-MSCs had the diameter between 40-100 nm and double membrane stracture. The recombinant adenovirus could effectively mediate the expression of miR-486 in UC-MSC, and the expression level of miR-486 in exosomes of miR-486-modified UC-MSC significantly increased. The exosomes with miR-486 high expression possessed the pro-proliferation and pro-migration effects on cardiomyocytes, moreover the preventive effect on apoptosis of cardiomyocytes.</p><p><b>CONCLUSION</b>The exosomes derived from UC-MSC and accompamied by high expression of miR-486 can promote the proliferation and migration of cardio myocytes, yet can prevent the apoptosis of cardiomyocytes.</p>

2.
Military Medical Sciences ; (12): 130-137, 2018.
Article in Chinese | WPRIM | ID: wpr-694331

ABSTRACT

Objective To investigate the protective role and underlying mechanism of human dental pulp stem cells (DPSCs)-derived exosomes against lipopolysaccharide ( LPS) induced acute lung injury ( ALI) in pulmonary alveolar macrophage(PAM) cells of rats.Methods DPSCs were cultured in the complete culture medium , and their supernatants at passage 6 were collected after serum-free medium treatment for 24 hours.Exosomes were extracted and purified with ultracentrifugation .Rat PAM NR8383 was cultured in 12-well plate and treated with LPS of 1μg/ml alone or together with exosomes.The supernatants were then collected at 0, 6, 12 and 24 h respectively after treatment .Inflammatory cytokine levels of tumor necrosis factor-α(TNF-α)and interleukins (IL-1βand IL-6) in the supernatant were measured by ELISA assay and the expression and phosphorylation level of MAPK (p44/42), NF-κB and IκBαin cell lysates were detected with Western-blotting.Results Compared with control group , the content of TNF-α,IL-1βand IL-6 increased significantly in LPS group (P<0.05), which indicated that the inflammatory cell model was induced successfully .The levels of TNF-αand IL-1βwere obviously attenuated after a high doses of exosomes treatment (P<0.05), and the expression of IL-6 was markedly suppressed after low and high doses of exosomes treatment (P<0.05), compared with the group of LPS treatment alone.The phosphorylation of NF-κB, IκBαand p44/42 was significantly inhibited after treatment with the DPSCs-derived exosomes.Conclusion DPSCs-derived exosomes may have a potential protective effect on LPS-induced ALI, and the underlying mechanism is that the activity of MAPK (p44/42) and NF-κB/IκBαpathways are eliminated by DPSCs-derived exosomes.

3.
Journal of Experimental Hematology ; (6): 1283-1288, 2017.
Article in Chinese | WPRIM | ID: wpr-301735

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect and mechanism of miR-486 on glycometabolism of hematopoietic cells.</p><p><b>METHODS</b>qRT-PCR was applied to detect the expression of miR-486 or Sirt1 on TF-1 cells under hypoxia. Lentivirus was used to mediate the overexpression or inhibition of miR-486 on TF-1 cells and qRT-PCR was used to detect the expressions of Sirt1, glucose transporter 1(Glut1) and glucose transporter 4(Glut4). Lentivirus-mediated Sirt1-shRNA transduction was used to knockdown Sirt1 expression which was detected by qRT-PCR and Western blot. The expressions of Glut1 and Glut4 were determined by qRT-PCR.</p><p><b>RESULTS</b>Hypoxia promoted the expression of miR-486 and inhibited the expression of Sirt1. MiR-486 overexpression could inhibit the expression of Sirt1 and promote the expressions of Glut1 and Glut4, whereas miR-486 silencing upregulated the sirt1 expression and inhibited the expressions of Glut1 and Glut4. And inhibition of Sirt1 expression increased the expressions of Glut1 and Glut4.</p><p><b>CONCLUSION</b>MiR-486 can regulate the glycometabolism of hematopoietic cells by targeting Sirt1.</p>

4.
Article in Chinese | WPRIM | ID: wpr-271885

ABSTRACT

<p><b>OBJECTIVE</b>To clarify the roles of SPK pathway in the regulation of proliferation, survival and glucose consume of human erythroleukemia TF-1 cells.</p><p><b>METHODS</b>The interfering in SPK expression of TF-1 cells was performed using leutivirus vector-mediated shRNA, the interference efficacy of SPK in TF-1 cells was detected by RT-qPCR and Western blot, the viability of TF-1 cell proliferation was detected by using CCK-8 method, the apoptosis of TF-1 cells was determined by flow cytmetry with Annexin V staining.</p><p><b>RESULTS</b>Hypoxia up-regulated the expression of HIF-1α, HIF-2α, and SPK in TF-1 cells. SPK treatment resulted in reduced proliferation and induced apoptosis in TF-1 cells. Furthermore, knockdown of the SPK significantly reduced utilization and consumption of glucose.</p><p><b>CONCLUSION</b>The SPK is key signalling molecule involved in regulation of hypoxia-induced proliferation and glucose metabolism in TF-1 cells, and plays an important rote in proliferation and energy metabolism of leukemia cells.</p>

5.
Journal of Experimental Hematology ; (6): 1793-1800, 2016.
Article in Chinese | WPRIM | ID: wpr-332609

ABSTRACT

<p><b>OBJECTIVE</b>To construct a recombinant lentiviral expression vectors carrying MEG3 and to evaluate its effects on XG-7 cell apoptosis.</p><p><b>METHODS</b>A full-length genomic fragment of human MEG3 was cloned from the pcDNA3.0-MEG3 packaging plasmid and was amplified by PCR. New restriction sites were introduced to be blunted with T4 DNA Ligase. The sequence of the amplified segments was sub-cloned into lentivirus expression vector pCDH-EF1-MCS-T2A-copGFP.The recombined lentiviral expression vector was transfected into 293T cells. FACS was used to detect the effect of MEG3 on XG-7 cell apoptosis after being infected by optimized MOI.</p><p><b>RESULTS</b>The recombined lentiviral expression vector pCDH-EF1-MEG3-copGFP was constructed successfully. The results showed that pCDH-EF1-MEG3-copGFP could increase the mRNA expression of MEG3 dramatically, its transfection efficiency was more than 90%. The apoptosis rate in XG-7 cells (26.8±2.8%) was very significantly higher than that of the control group (P<0.01).</p><p><b>CONCLUSION</b>The recombined lentiviral LncRNA expression vector targeting MEG3, pCDH-EF1-MEG3-copGFP, has been successfully constructed, the pCDH-EF1-MEG3-copGFP can induce the cell apoptosis in human myeloma cell lines. This study set up a basis to further explore the relationship between human myeloma cells and LncRNA-MEG3 gene.</p>

6.
Article in Chinese | WPRIM | ID: wpr-264957

ABSTRACT

The objective of this study was to explore the effects of microRNA-17-92 on the biological characteristics of K562 cells. The expression of miR-17-92 in K562 cells transfected with miRNA-17-92 mimic was detected by real time PCR. The effect of microRNA-17-92 on K562 cell proliferation was detected by CCK-8 method. Apoptosis of K562 cells was detected by Annexin V-PI labeling. Cell cycle distribution was determined by using flow cytometry. Western blot was performed to determine the protein levels of Crk. The results indicated that the transfection with miR-17-92 mimic increased expression of mature miR-17-92 in K562 cells. Compared with control group, cell proliferation and cell amount in S-phase of miR-17-92 mimic transfected group significantly increased, cell apoptosis decreased. The expression of signal connector protein Crk was greatly up-regulated in miR-17-92-mimic-transfected K562 cells. It is concluded that miR-17-92 can promote proliferation, inhibit apoptosis and regulate the cell cycle of K562 cells.


Subject(s)
Humans , Apoptosis , Cell Cycle , Cell Proliferation , Gene Expression Regulation, Leukemic , HL-60 Cells , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Genetics , Metabolism , MicroRNAs , Genetics , Transfection
7.
Article in Chinese | WPRIM | ID: wpr-349685

ABSTRACT

This study was aimed to investigate the growth-promoting activity of thrombin on mesenchymal stem cells (MSC) and its mechanisms. Human bone marrow MSC were cultured in serum-free medium supplemented with graded concentrations of thrombin, and the proliferation status of MSC was detected by MTT test. The expression levels of protease-activated receptors (PAR) and c-MYC gene were detected by PCR. Activated Akt signaling pathway was revealed by Western blot, and specific inhibitors of the signaling pathways were used to confirm the effects. The results showed that thrombin stimulated MSC proliferation in a dose-dependent manner; the minimal concentration of thrombin for stimulating MSC growth was 0.5 U/ml, and the promoting effect reached its maximum when thrombin at a dose of 8 U/ml was employed. PCR results showed that MSC expressed the two types of PAR1 and PAR2. After PAR1 was blocked with a specific inhibitor SCH79797, the growth-promoting effect of thrombin was inhibited, while this phenomenon was not observed when MSC were exposed to FSLLRY-NH2, a specific inhibitor for PAR2. Further experiments showed that after exposure to thrombin, the AKT signaling pathway in MSC was promptly activated, and c-MYC expression was greatly up-regulated. Meanwhile, when LY294002, a specific AKT inhibitor, was added into the culture medium, the up-regulation of c-MYC expression was reduced, accompanied by the low rate of MSC growth. It is concluded that thrombin can stimulate MSC proliferation by eliciting PAR1-mediated AKT activation and subsequent up-regulation of c-MYC expression.


Subject(s)
Humans , Bone Marrow Cells , Cell Biology , Cell Proliferation , Cells, Cultured , Mesenchymal Stem Cells , Cell Biology , Receptors, Thrombin , Metabolism , Signal Transduction , Thrombin , Pharmacology
8.
Journal of Experimental Hematology ; (6): 1540-1544, 2014.
Article in Chinese | WPRIM | ID: wpr-340462

ABSTRACT

This study was aimed to explore the effect of VX-680, an aurora inhibitor, on proliferation and apoptosis of K562, KCL22 cell lines and CD34⁺ cells from chronic myeloid leukemia (CML) patients in vitro. The proliferation of K562 and KCL22 cell was detected by CCK-8 method. Apoptosis of cells was detected by Annexin V-PI labeling and flow cytometry. The colony forming ability of bone marrow CD34⁺ cells derived from CML patients and donors was determined by the colony forming test. The results showed that the treatment of K562, KCL22 and CML CD34⁺ cells with VX-680 of 20-100 nmol/L for 3 days could obviously inhibit the cell proliferation in a concentration-dependent manner (P < 0.01). VX-680 treatment significantly induced apoptosis of K562 and KCL22 cells. Compared to bone marrow CD34⁺ cells derived from the healthy donors, the colony forming ability of CML CD34⁺ cells derived from bone marrow of CML patients was remarkably reduced (P < 0.01). It is concluded that VX-680, an aurora inhibitor, can inhibit the proliferation and induce apoptosis of CML cells in vitro.


Subject(s)
Humans , Apoptosis , Aurora Kinase A , Cell Line, Tumor , Cell Proliferation , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Pathology , Piperazines , Pharmacology , Protein Kinase Inhibitors , Pharmacology
9.
Article in Chinese | WPRIM | ID: wpr-263376

ABSTRACT

The aim of this study was to investigate if transfusion of mesenchymal stem cells (MSC) could exhibit beneficial effects on rheumatoid arthritis. Human bone marrow MSC were intraperitoneally injected into Wistar rats with collagen-induced arthritis at a dose of 10(7) on the next day (preventive group) or 2 weeks (treatment group) after collagen II induction, once a week for 2 weeks (preventive group) or 4 weeks (treatment group). The control group was given normal saline (NS) at corresponding time. The symptom scorings were documented weekly from the second week of the induction. On week 6, the hind joints of the rats were pathologically examined and the activation status of splenocytes was analyzed by flow cytometry. The results showed that all the rats developed arthritis and subsequent joint abnormality. On the sixth week, symptom scores of the rats that received MSC preventive (9.5 ± 0.5) or therapeutic (9.4 ± 0.6) infusions had no significant difference between each other, but were significantly greater than those of the NS controls (7.6 ± 0.6, P < 0.05). Consistently, pathological examination on the involved knees showed that the synovitis and arthritis scorings of MSC treated rats were greatly elevated compared with NS controls. Furthermore, the ratios of CD86(+) cells in the spleens of MSC prevention, MSC treatment and NS control groups were (4.16 ± 1.48), (4.06 ± 1.97) and (4.15 ± 2.04) respectively, while those of CD11b/c(+)CD86(+) cells were (1.04 ± 0.68), (0.95 ± 0.56) and (0.98 ± 0.44), all of which were significantly higher than those of healthy controls [(0.97 ± 0.18) and (0.30 ± 0.17), P < 0.05 for both parameters]. It is concluded that MSC infusion has little beneficial effects on collagen-induced arthritis in rats, conversely, MSC therapy aggravated the damage of the involved joints, its underlying mechanisms need to be further investigated.


Subject(s)
Animals , Humans , Male , Rats , Arthritis, Experimental , Pathology , Therapeutics , Bone Marrow Cells , Cells, Cultured , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Cell Biology , Rats, Wistar , Transplantation, Heterologous , Treatment Outcome
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