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Objective To investigate the heterogeneity of immunomodulatory function of exosomes secreted from human umbilical cord-derived mesenchymal stem cells(hUC-MSC).Methods Five different hUC-MSCs lines were isolated and cultured.Exosomes were isolated from the supernatant.The expression of specific surface markers CD9,CD63,CD81 and CD44 was detected by flow cytometry.The protein concentration of hUC-MSCs exosomes(hUC-MSCs-ex)was evaluated by the BCA assay.Concanavalin A and rhIL-2 stimulated umbilical cord blood mononuclear cells (UBMCs) from healthy donor were co-cultured at different concentrations of hUC-MSCs-ex from different cell lines for 72 h.The growth rate of UBMCs was detected by MTT assay.ELISA was used to test the levels of IFN-γ and TNF-α of the supernatants.The UBMCs co-cultured with hUC-MSCs were co-cultured with K562 cells at the ratio of 5∶1.The cytotoxic activity was calculated by MTT assay.Results hUC-MSCs-ex expressed CD9,CD63,CD81and CD44.Different hUC-MSCs lines had different regulating activities on the proliferation,secretion and killing ability of UBMCs.Conclusion hUC-MSCs-ex has heterogeneity of immunomodulatory function on UBMCs.
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Objective To evaluate the efficacy and adverse effect of valproic acid (VPA) in combination with low dose chemotherapy on intermediate and high-risk myelodysplastic syndrome. Methods A total of 41 patients with intermediate (34) and high-risk (7) myelodysplastic syndrome were retrospectively analyzed. Among them, 19 patients received low dose chemotherapy regimen and 22 received low dose chemotherapy plus VPA.Low dose chemotherapy regimen included: homoharringtonine,1-2 mg·m-2·d-1 intravenously,14-28 d; clarubicin,5-7 mg·m-2·-1 intravenously,1-8 d,15-23 d;cytarabine 15 mg/m2 subcutaneously once every 12 h, 14-21 d; and subcutaneously use of granulocyte colony-stimulating factor 200 μg·m-2·d -1 when neutrophil deficiency.The outcome and adverse effect were recorded after the treatment. Results The overall response rate in the low dose chemotherapy regimen group was 47.4% (9/19), 6 patients (31.6%) achieved complete response (CR). The overall response rate in the VPA group was 77.2% (17/22), 9 patients (40.9%) achieved CR. The overall response rate of the low dose chemotherapy in combination with VPA group was significantly higher than that in the low dose chemotherapy group (P＜0.05) while no difference was found in CR rate. The adverse effect of the low dose chemotherapy in combination with VPA regimen was tolerated. Conclusion With acceptable adverse effect, the low dose chemotherapy in combination with VPA regimen is effective for the treatment of intermediate and high-risk myelodysplastic syndrome. Long-term outcome needs further investigation.
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OBJECTIVE: To investigate the effect of high glucose on the expression of c-met in human kidney fibroblasts in vitro, and to explore the regulation of Radix Astragali. METHODS: A cell culture system of human kidney fibroblasts was developed in vitro. The human kidney fibroblasts were divided into normal control group, high glucose group and mannitol group. Expressions of c-met and transforming growth factor-beta1 (TGF-beta1) mRNAs were detected by reverse transcription polymerase chain reaction (RT-PCR) and the expressions of c-met protein were analyzed by Western blot method after 6-, 12-, 24-, 48- and 96-hour culture. The human kidney fibroblasts were also cultured with 10% Radix Astragali containing serum; the expressions of c-met mRNA and protein were detected after 24- and 48-hour culture. RESULTS: Compared with the normal control group, expression of c-met mRNA in the high glucose group was significantly increased after 12-hour culture (P<0.05), arriving at the peak after 24-hour culture (P<0.01). The level of TGF-beta1 mRNA was higher in the high glucose group than that in the normal control group after 24-hour culture (P<0.05), arriving at the peak after 96-hour culture (P<0.01). Forty-eight hours after treating with 10% Radix Astragali containing serum, the levels of c-met mRNA and protein in fibroblasts were increased, and were higher than those in the high glucose group (P<0.01, P<0.05). CONCLUSION: High glucose can induce the expressions of c-met mRNA and protein in earlier period, and then inhibit the expressions. Radix Astragali can up-regulate the expressions of c-met mRNA and protein of human kidney fibroblasts, which may be one of its action mechanisms in delaying the progression of diabetic nephropathy.
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Objective To examine the expression of hepatocyte growth factor(HGF) receptor (c-met) and investigate the changes in activity of the HGF/c-met system in human kidney fibroblast by high glucose. Methods HGF and c-met mRNA levels in fibroblast induced by high glucose were detected by RT-PCR. C-met protein was examined by Western blotting. At the same time, the expression of TGF-? and c-met in the exogenous HGF and treating with anti-c-met antibody in vitro were measured. Results Extremely rapid induction of HGF and c-met mRNA was observed at the first six hours by high glucose. On the other hand, both c-met mRNA and c-met protein were markedly increased. HGF (50 ng/ml) induced the expression of c-met ( P
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Objective To observe high glucose induced expression of hepatocyte growth factor (HGF) and c met in human kidney fibroblast. Methods The effects of glucose concentrations on expression of HGF, c met and plasminogen activator inhibitor (PAI) 1 in cultured human kidney fibroblasts were observed by RT PCR. In the same system, the effect of exogenous HGF on the expression of PAI 1 was investigated. Results Human kidney fibroblasts cultured in high glucose concentration (25 mmol/L) showed higher HGF and c met expressions in the early stage and then manifested a gradient decrease of HGF and c met expressions, but PAI 1 expression was gradiently increased. Exogenous HGF resulted in inhibiting PAI 1 expression. Conclusion HGF is a potential anti fibrogenic factor and activates matrix degradation pathways in diabetic kidney by reducing PAI 1 expression.
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The gene expression of both the mouse plasmocytoma (SP2/0) and the hybrid cells crossed with rat nucleated erythroblasts were detected by in situ hybridization technique using the probes of mouse ?-globin gene and 7 oncogenes (v-Ki-ras, v-H-ras, v-sis, erb-B, v-abl, v-fos, c-myc). After plasmic amplification, DNA was isolated by alkali lysis, purified and recovered, the DNA containing gene fragments were labelled with ~(32)P to become high activity ~(32)P-cDNA probes through nick translation, and the labelled probes were used to detect the gene transcripts in cellular level. The results indicated that: (1) no mouse ?-globin gene transcripts could be detected in the cytoplasm of SP2/0 cells, as well as in hybrid cells within 72 hours after cell fusion, but transcript signals could be observed in hybrid cells from 4th to 26th passages. (2) Active expression of multioncogenes in SP2/0 cells was demonstrated, all the 7 oneogenes tested, except v-sis, were expressed more strongly. On the other hand, the expression of oncogenes in hybrid cells was found to be dramatically decreased, among them, the oncogenes of c-myc and Ki-ras been suppressed completely. After long term of passages (26th subcultures), the expression of c-myc and Ki-ras was still lower than that of SP2/o ceils although in some cases other oncogenes increased in their expression levels. These results confirmed that the multistep carcinogenesis involved multi-oncogenes expression and that the decancerization of tumor cells may be due to the suppression of multi-oncogenes activity as well as to activate the expression of differentiation genes.