RESUMEN
OBJECTIVE: To clone the circular RNA hsa_circ_0000254 and construct its lentiviral over-expression vector. METHODS: The sequence of hsa_circ_0000254 (a total of 524 bp long) was synthesized and cloned by using pGH vector. The vector was cut by EcoR I and BamH I, and artificial hsa_circ_0000254 was obtained, then inserted in pLCDH-ciR to construct the recombinant expression vector pLCDH-circ254(C254), which was confirmed by DNA sequencing. The lentiviral expression vectors pLCDH-circ254(C254) and NC(pLCDH-ciR) were cotransfected into 293T cells by lipofectamineTM 2000(lipo2000). After transfection for 40 hours, the cells were collected and verified by PCR and sequencing. RESULTS: Restriction analysis and DNA sequencing demonstrated that the lentiviral vector pLCDH-circ254(C254) was constructed successfully, the expression efficiency increased 10000 times after transfection of cells. CONCLUSION: The successful construction of the lentiviral expression vector pLCDH-circ254(C254) results in the production of high-titer lentivirus, so as to facilitate further study of the molecular functions of hsa_circ_0000254.
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Leucemia Mieloide Aguda , Vectores Genéticos , Humanos , Lentivirus , ARN Interferente Pequeño , TransfecciónRESUMEN
OBJECTIVE: To observe the effect of panaxadiol saponin (PDS) and panaxtrol saponin (PTS) on proliferation of human bone marrow hemopoietic progenitor cells (HPC). METHODS: PDS and PTS were separated and purified from ginsenosides, and the effects on HPC were studied using in vitro hemopoietic progenitor cell colony-forming technique, by observing the proliferation of human burst forming unit-erythroid progenitor (BFU-E), colony-forming unit-erythroid (CFU-E), colony-forming unit-granulocyte/macrophage (CFU-GM) and colony-forming unit-pluripotent hemopoietic progenitor (CFU-Mix) in mice after PDS and PTS stimulation. RESULTS: Different concentration of PDS (2.5-200 micrograms/ml) could stimulate the proliferation of HPC obviously, showing increase of CFU-E, BFU-E, CFU-GM and CFU-Mix by 54.9 +/- 6.3%, 48.8 +/- 5.1%, 27.6 +/- 4.2% and 48.9 +/- 3.9% respectively, which was higher than that of the control group. While stimulated by PTS of the same concentration, the CFU-E and BFU-E was lower than that of control significantly (P < 0.05); when the terminal concentration of PTS was 200 micrograms/ml, CFU-E and BFU-E was zero respectively. In the CFU-GM culture, PTS in concentration of 12.5 micrograms/ml could cause the proliferation increased by 29.7 +/- 2.2% (P < 0.05), but in concentration of 100 micrograms/ml and 200 micrograms/ml, it showed inhibitory effect on CFU-GM, the inhibition rate being 48.6 +/- 3.9% and 100% respectively. CONCLUSION: PDS is the effective component of ginsenosides in stimulating proliferation of human bone marrow HPC. PTS is an component with inhibitory action on proliferation of CFU-E and BFU-E and its effect on CFU-GM was depending on its concentration.
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Ginsenósidos/farmacología , Células Madre Hematopoyéticas/citología , Triterpenos/farmacología , Células de la Médula Ósea/citología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides , Factor Estimulante de Colonias de Granulocitos , Hematopoyesis/efectos de los fármacos , Humanos , Panax/química , Saponinas/farmacologíaRESUMEN
OBJECTIVE: To investigate the effects of sodium copper chlorophyllin (SCC) on the proliferation, differentiation and immunomodulatory function of mesenchymal stem cells (MSCs) from mice with aplastic anemia. METHODS: A mouse model of aplastic anemia was established by exposure of BALB/c mice to sublethal doses of 5.0 Gy Co60 γ radiation, followed by transplantation of 2×10(6) lymph node cells from DBA/2 donor mice within 4 h after radiation. Aplastic anemic BALB/c mice were randomly divided into six groups: the treated groups, which received 25, 50, or 100 mg/kg/day SCC, respectively; a positive control group treated with cyclosporine A (CsA); and an untreated model control group (model group); while, the non-irradiated mice as the normal control group. SCC or CsA were administered by gastrogavage for 20 days, starting on day 4 after irradiation. Peripheral blood cells were counted and colony-forming fibroblasts (CFU-F) in the bone marrow were assayed. The ability of MSCs to form calcium nodes after culture in osteoinductive medium was also observed. The immunosuppressive effect of MSCs on T lymphocytes was analyzed by enzyme-linked immunosorbent assay and flow cytometry, to evaluate the efficacy of SCC in mice with aplastic anemia. RESULTS: Peripheral blood white cell and platelet counts were increased by medium and high SCC doses, compared with the untreated control. CFU-Fs were also increased compared with the untreated control, and the numbers of calcium nodes in MSCs in osteoinductive medium were elevated in response to SCC treatment. The percentage of Forkhead box protein 3 (FOXP3(+)) T cells was increased in T cell-MSC cocultures, and the cytokine transforming growth factor ß1 was up-regulated in SCC-treated groups. CONCLUSION: The results of this study suggest that SCC not only promotes the proliferation and differentiation of MSCs, but also improves their immunoregulatory capacity in mice with aplastic anemia.
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Anemia Aplásica/terapia , Clorofilidas/farmacología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Anemia Aplásica/sangre , Anemia Aplásica/patología , Animales , Antraquinonas/metabolismo , Biomarcadores/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Femenino , Terapia de Inmunosupresión , Recuento de Leucocitos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Recuento de Plaquetas , Linfocitos T/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the effects of Panax notoginseng saponins (PNS) on the proliferation and differentiation in NIH3T3 cells. METHODS: NIH3T3 cells were treated by various concentrations of PNS 0, 0.05, 0.10, 0.20, and 0.40 g/L. The vitality and proliferation potential of cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the alkaline phosphatase (ALP) activity was measured by p-nitrophenyl phosphate (pNPP) assay, and the mineralization formation ability was tested for the cellular differentiation toward osteoblast, as well as the expression level of phosphorylated extracellular signal-regulated kinase1/2(P-ERK1/2), extracellular signal-regulated kinase1/2 (ERK1/2) protein kinase was analyzed by Western blot with total cell lysate of NIH3T3 cells treated by PNS. RESULTS: Both MTT and pNPP assay showed that optical density (OD) values were increased in response to PNS treatment at a dose-dependent pattern. The mineralization formation ability was enhanced in PNS-treated NIH3T3 cells compared with untreated cells. Meanwhile, the expression level of P-ERK1/2 protein kinase was up-regulated in PNS-treated NIH3T3 cells, while, the expression level of ERK1/2 protein kinase revealed no obvious difference with or without PNS treated cells. CONCLUSION: PNS could pay a role to promote the proliferation and differentiation in NIH3T3 cells by means of up-regulation of P-ERK1/2 protein kinase.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Panax notoginseng/química , Saponinas/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Calcio/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/enzimología , Ratones , Células 3T3 NIH , Osteocalcina/metabolismoRESUMEN
The aim of this study was to investigate the effects of aescinate on inhibition and apoptosis of HL-60 cell line from promyelocytic leukemia. HL-60 cells at logarithm phase were treated with aescinate. Cell survival rate and cell morphology were observed, and the cell apoptosis was analyzed by Annexin V/PI-FITC double labeling and DNA electrophoresis. The results showed that HL-60 cells could be inhibited in the presence of 15-120 mg/L of aescinate for 48 hours, survival rates were (92.2+/-0.69)%-(8.2+/-0.96)%, which were significantly lower than that of non-aescinate control (99.4+/-0.31)% (all p<0.01). The apoptosis of cells could be induced by aescinate treatment at dosage of 15-60 mg/L for 24 hours, the Annexin V positive cells accounted for (12.7+/-0.58)%-(65.4+/-1.30)% which were significantly higher than that of non-aescinate control (0.57+/-0.03)% (all p<0.01). The typical DNA ladder of HL-60 cells treated with aescinate was shown on the DNA electrophoresis pattern. It is concluded that aescinate can specifically induce apoptosis of leukemic HL-60 cells, which provides an experimental evidence for treatment of leukemia with aescinate as a supplementary agent to chemotherapy.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Fitoterapia , Células HL-60 , HumanosRESUMEN
This study was aimed to explore the inhibition effect and mechanism of hedyotis diffusa wild injection (HDI) on leukemia cell line (HL-60) in vitro. The leukemia cell line HL-60 was used as target cells. The inhibitory effects of HDI on proliferation of HL-60 cells were observed by MTT assay. The positive rate of cell apoptosis and the surface marker of granulocytic differentiation (CD33 and CD15) were measured by flow cytometry. The expressions of anti-apoptosis related gene (survivin and bcl-2) were detected by RT-PCR. The results showed that the growth of HL-60 cells was inhibited by higher concentration of HDI (3.12 - 12.5 ml/L) and inhibited obviously in dose-dependent manner (p < 0.05 or p < 0.01), but not suppressed by low concentration of HDI (1.56 ml/L) in liquid culture system (p > 0.05). The FCM and DNA Ladder results showed that the phenomenon of typical apoptosis did not detected after HL-60 cells were treated with the different concentrations of HDI for 24, 48 and 72 hours respectively. After HL-60 cells were treated with HDI (1.56, 3.12, 6.25 and 12.5 ml/L) for one week, the expression level of CD15 surface marker was all enhanced obviously. When treated with HDI (6.25 ml/L) for 3 weeks, the expression levels of survivin and bcl-2 gene were also decreased obviously by 60% and 44% respectively. It is concluded that HDI can inhibit HL-60 cells in the presence of its higher concentrations. The mechanisms of HDI may induce HL-60 cells differentiation, and suppress the expression of anti-apoptosis related gene (survivin or bcl-2) to inhibit the growth of HL-60 cells.
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Antineoplásicos Fitogénicos/farmacología , Proliferación Celular/efectos de los fármacos , Células HL-60 , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , SurvivinRESUMEN
AIM: To investigate the effects of panax notoginosides (PNS) on the proliferation of human hematopoietic stem/progenitor cells, and to explore the signaling pathway of the nuclear transcription factor of the glucocorticoid receptor (GR-NTF) initiated by PNS related with the proliferation. METHODS: The human CD34+ cells and bone marrow nuclear cells were exposed to PNS at a concentration of 0, 10, 25, 50, and 100 mg/L, respectively, in semi-solid culture system to observe colony forming unite of all lineages, granulocyte, erythrocyte, and megakaryocyte (CFUGEMM, CFU-GM, CFU-E, and CFU-MK). Three lineages of human hematopoietic cell lines, including granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF- 288, and Meg-01 cells were incubated with PNS at 20 mg/L for 14 d. Meanwhile, dexamethasone (Dex) was used as a positive control. The nuclear protein of the cells was analyzed by Western blotting with monoclonal antibodies against the amino or carboxyl terminus of GR-NTF. Electrophoretic mobility shift assay performed by using the 32P-radiolabeled GR-NTF consensus oligonucleotide. RESULTS: PNS promoted the proliferation of CD34+ cells and significantly raised the colony numbers of CFU-GEMM by 34.7%+/-16.0% over the non-PNS control (P<0.01). PNS also enhanced the proliferation of CFU-GM, CFU-E, and CFU-MK by 39.3%+/- 5.7%, 33.3%+/-7.3%, and 26.2%+/-3.2%, respectively. GR-NTF protein levels of either the amino or carboxyl terminus in K562, CHRF-288, and Meg-01 treated by PNS increased by 2.4-2.8 fold and 1.3- 3.9 fold over the untreated cells. GR-NTF binding activity, initiated by either PNS or Dex, was apparently elevated to form the complex of GR-NTF with DNA as higher density bands in K562 and CHRF-288 cells, and some activity appeared as a band in HL-60 cells induced by PNS. CONCLUSION: PNS displayed the action of hematopoietic growth factor-like or synergistic efficacy to promote proliferation of human progenitor cells, may play a role in the upregulation of gene expression related to proliferation of hematopoietic cells through increasing the GR-NTF synthesis and its DNA binding activity.
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Proliferación Celular/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Receptores de Glucocorticoides/metabolismo , Saponinas/farmacología , Factores de Transcripción/metabolismo , Animales , Antígenos CD34/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Células Cultivadas , Células Madre Hematopoyéticas/citología , Humanos , Panax notoginseng/química , Receptores de Glucocorticoides/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de Transcripción/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The study was aimed to investigate the action of Panax Notoginosides (PNS, extracted from notoginseng herb) on the expression of the apoptosis-related proteins (Daxx, Fas) and transcription factors (NFkB, c-Rel) in the hematopoietic cells and to explore the mechanisms of supporting cells to survive. The colony formation of CFU-GM and CFU-E in human bone marrow was assayed in the presence of various concentrations of PNS. The viability of cells was assayed by trypan blue and the changes of cell morphology were observed with microscope. The Annexin-V positive cells were detected by FCM. Three lineages of human myeloid HL-60, erythroid K562, megakaryoid CHRF-288 and Meg-01 cells were incubated in addition of PNS (10 mg/L) for 14 days. The nuclear or cytoplasm protein of cells was extracted and analyzed by Western blot with monoclonal antibodies against Daxx, Fas or NFkB, c-Rel. The results showed: (1) the proliferation on hematopoietic progenitor cells (CFU-GM and CFU-E) and four cell lines was promoted by PNS; (2) after the four cell lines were promoted by PNS and hungered through wiping off the sera, the viability of the four cell lines was high without significant morphological change and neither the detection of Annexin-V positive cells; (3) the expression of Daxx and Fas protein could be inhibited by PNS. Western Blot showed that Daxx in four cell lines treated by PNS were 33.3-61.5% lower than that in untreated controls. The Fas protein was also descended in three cell lines of K562, CHRF-288 and Meg-01 by 33.3-71.4% respectively, while Fas protein in HL-60 cells was no detectable difference after PNS treatment. (4) The transcription factors NFkB and c-Rel protein could be increased by PNS. The NFkB, c-Rel protein were also enhanced in three cell lines of K562, CHRF-288 and Meg-01 by (2.0-2.7) and (1.5-2.3)-fold respectively, while there were also no detectable difference in HL-60 cells after PNS treatment. It is concluded that PNS inhibites the expression of Daxx and Fas proteins, may decrease the apoptosis of the hematopoietic cells. The level of NFkB and c-Rel proteins can be enhanced by PNS, which not only stimulates the proliferation of cells, but also inhibits the activity of the waterfall of caspase and apoptosis of the hematopoietic cells. PNS may treat the disease with over-apoptosis of hematopoietic cells, as aplastic anemia.
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Proteínas Reguladoras de la Apoptosis/biosíntesis , Células de la Médula Ósea/metabolismo , Ginsenósidos/farmacología , Células Madre Hematopoyéticas/metabolismo , Panax notoginseng/química , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Apoptosis , Células de la Médula Ósea/citología , Células Cultivadas , Proteínas Co-Represoras , Proteína Ligando Fas/biosíntesis , Células HL-60 , Células Madre Hematopoyéticas/citología , Humanos , Células K562 , Chaperonas Moleculares , FN-kappa B/biosíntesis , Proteínas Nucleares/biosíntesis , Receptor fas/biosíntesisRESUMEN
The object of this study was to explore the effects of Panax notoginosides (PNS) on proliferation and differentiation of human CD34(+) stem/progenitor cells. CD34(+) cells were isolated from human bone marrow by using immune beads of Dynal M- 450 system. The cells were exposed to PNS at different concentrations in both liquid and semi-solid culture for 14 days. The cells were marked with monoclonal antibodies and analyzed by flow cytometry after culture. The CFU-Mix colony formation from CD34(+) cells was assayed. The results showed that: (1) The yield of CD34(+) cells after being selected by immune beads were (1.03 +/- 0.74)% out of bone marrow nuclear cells with purity of 86% - 93%. (2) PNS (10 - 25 mg/L) stimulated the proliferation of CD34(+) cells, and raised the colony numbers of CFU-Mix obviously in vitro. PNS 25 mg/L was the optimal concentration to promote proliferation of CD34(+) cells, the increasing rate of CFU-Mix colony was (34.7 +/- 16.0)%. (3) The differentiation of CD34(+) cells was induced by exposure to PNS (25, 50 and 100 mg/L) in liquid culture for 14 days. The percentages of CD33(+) and CD15(+) cells were increased after PNS exposure, which were significantly higher than those of control (P < 0.01), however CD71(+) and G-A(+) cells were no obviously difference after PNS treatment. In conclusion, Panax notoginosides not only promote the proliferation of CD34(+) cells, but also induce the differentiation committed to granulocytes.
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Antígenos CD34/análisis , Ginsenósidos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Humanos , Antígeno Lewis X/análisis , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
To observe the effects of Panax Notoginosides (PNS) on up-regulation of AP-1 family transcription factors NF-E2, c-jun and c-fos for exploring intracellular signal pathway of PNS in hematopoietic cells, four human hematopoietic cells lines including myeloid HL-60, erythroid K562, megakaryoid CHRF-288 and Meg-01 were incubated in the presence of PNS for 14 days. The nuclear protein of cells were extracted and analyzed by Western blot with antibodies against NF-E2, c-fos and c-jun. Electrophoretic mobility shift assay (EMSA) was performed by using (32)P labeled AP-1 consensus oligonucleotide which contains binding site for NF-E2, c-jun and c-fos. The results showed that the transcription factors NF-E2, c-jun and c-fos of AP-1 family could be induced by PNS. Western blot demonstrated that the nuclear protein of both NF-E2 and c-jun in four cell lines treated by PNS were increased by 1.5-2.5- and 2.0-3.0-fold over untreated cells respectively. The c-fos protein in three cell lines of K562, CHRF-288 and Meg-01 was also elevated by 2.0-3.0-fold respectively, while c-fos protein in HL-60 cells was no detectable difference after PNS treatment. EMSA results in four cell lines indicated that AP-1 binding activity initiated by PNS was apparently elevated to form higher density band of AP-1-DNA complex. In conclusion, the intracellular transcription regulation initiated by PNS was involved in transcription factors NF-E2, c-jun and c-fos of AP-1 family members, which could play an important role in the up-regulation of genes expression related to proliferation and differentiation of hematopoietic cells.