Synthesis of matrine derivative ZS10 and its mechanism of apoptosis induced by PI3K/AKT signaling pathway in human hepatocellular carcinoma cell line BEL-7402 / 中国药理学通报

Aim To explore the signaling pathway of matrine derivative ZS10 in inhibiting proliferation and inducing apoptosis of BEL-7402 cells. Methods ZS10 was synthesized by organic synthesis. The inhibitory effect of ZS10 on the proliferation of BEL-7402 cells was analyzed by MTT method at the time of 24 h, 48 h and 72 h, respectively, and IC50 was calculated. DAPI staining was used to observe the state of BEL-7402 cells. Clone formation method was used to observe the colony formation of BEL-7402 cells, flow cytometry was used to observe the cell cycle arrest and apoptosis of BEL7402 cells, and Western blot was used to detect the expression level of PI3K/AKT pathway and related proteins. Results MTT results showed that the IC50 was(6.62±1.11)μmol·L-1; DAPI staining showed that the cell state changed significantly with the increase of drug concentration, and the results of colony formation showed that ZS10 significantly inhibited the colony formation of BEL-7402 cells. The results of flow cytometry showed that ZS10 induced S phase arrest and cycle apoptosis of BEL-7402 cells. Western blot showed that ZS10 at the concentration of 08 μ mol·L-1 could regulate the PI3K/AKT pathway and its related proteins in a dose-dependent manner. Compared with the control group, the expression of PI3K, AKT, P-AKT and anti-apoptotic protein Bcl-2 significantly decreased, the expression of pro-apoptotic protein Bax significantly increased, the expression of Cyclin D1 and CDK2 significantly decreased, and the expression of EGFR and N-cadherin, Vimentin significantly decreased in the treatment group. The expression of E-cadherin increased. Conclusions Matrine derivative ZS10 can inhibit the growth and proliferation of hepatocellular carcinoma cell line BEL-7402.

Effect of Buyang Huanwu Tang on Axonal Regeneration of Rats After Ischemic Stroke Injury / 中国实验方剂学杂志

Objective:To investigate the effects of Buyang Huanwu Tang （BHT） on axonal regeneration and neurological rehabilitation of the rats suffering ischemic stroke （IS）. Method:A total of 180 SD rats were used to establish a middle cerebral artery infarction （MCAO） model. The animals that were successfully modeled were randomly divided into model group， BHT group （12 g·kg-1） and nimodipine group （20 mg·kg-1）， and a sham group was established， with 28 rats in each group. After seven-days intragastric administration of BHT， the animals were sacrificed. TTC staining was used to test cerebral infarction. Brain water content was measured to observe cerebral edema. Bielschowsky's silver staining and immunofluorescence were performed to observe axonal degeneration and the protein expression of neurofilament protein-200（NF-200）. Quantitative real-time polymerase chain reaction （PCR） was used to analyze the mRNA expression of repulsion oriented molecule a （RGMa）， Ras homologous enzyme （Rho）， Rho kinase （ROCK）， and collapsion response regulatory protein 2 （CRMP2）. Neurological function scores assay was used to examine neurological recovery. Result:Compared with sham group， the cerebral infarction volume and brain water content increased significantly（P<0.01）， and motor function was markablely decreased in the model group. Axonal degeneration and nerve fiber damage were obviously observed. Also， gene expression of axon growth-related protein was deviation from normal （P<0.01）. Compared with model group， the cerebral infarction rate （P<0.01）， brain water content （P<0.01） and axonal degeneration of BHT group and nimodipine group were significantly reduced. The expression of NF-200 was increased. Also， the mRNA expression of RGMa， Rho and ROCK was lower （P<0.05） while the mRNA expression of CRMP2 was higher （P<0.01）. And the neurological function was significantly improved （P<0.05）. Conclusion:BHT can promote axon regeneration after ischemic stroke injury by regulating the mRNA expression of axon growth-related protein， thereby improving nerve function.

Biological Properties of Mesenchymal Stem Cells Derived from Induced Pluripotent Stem Cells / 中国实验血液学杂志

OBJECTIVE@#To obtain induced pluripatent stem cells (iPSC) from peripheral blood mononucleated cells and further induce differentiation into mesenchymal stem cells (MSC), and to compare the biological characteristics of iPSC-derived MSC and other-derived MSC.@*METHODS@#Peripheral blood mononucleated cells were obtained and transduced with reprogramming factors by sendai virus vector. Induced differentiation of MSC was performed in 1 strain of iPSC that completed all identification, and their cell morphology and immunophenotype were identified by immu-nohistochemistry and flow cytometry. Adipogenic and osteogenic media were used to induce the adipogenic and osteogenic differentiation. The expression of immune-related transcription factors was identified by PCR to systematically elucidate the biological characteristics of iPSC-induced MSC.@*RESULTS@#After transfection with sendai virus with reprogramming factor, the fate of peripheral blood mononucleated cells was reversed, initiating the expression of stem cell characteristics, the iPSC was successfully cloned, amplified, and purified, and finally the stable proliferation of iPSC was obtained. Mesenchymal stem cells derived from iPSC, had morphology consistent with other-derived MSC, and the immunophenotypes met the standard. iPSC-MSC possessed the ability of lipogenic and osteogenic differentiation. RT-PCR showed that iPSC-MSC was high expression to PDL1, and low expression to A20; besides, the expression level of STAT3 was equal to BM-MSC; and also as to the expression level of HIF1α and UC-MSC, which was lower than BM-MSC.@*CONCLUSION@#Peripheral blood mononucleated cells successfully initiated the expression of stem cell characteristics after the transduction of sendai virus vector with reprogramming factors, and obtained multi-competent iPSC. iPSC can successfully be induced to the differentiation of MSC, and the iPSC-MSC have standard cell morphology, immunophenotype and differentiation ability. High expression of PDL1 and low expression of A20 in iPSC-MSC suggest that iPSC-derived cells have different biological characteristics in cell proliferation and immune regulation.

UPLC-Q-TOF-MS/MS Analysis of Brain and Blood Absorption Components of Buyang Huanwu Tang After Intragastric Administration / 中国实验方剂学杂志

Objective:To analyze and identify the brain and blood absorption components of rats after intragastric administration of Buyang Huanwu Tang(BYHWT). Method:The brain tissue,plasma of normal rats and the cerebral ischemia-reperfusion rats were analyzed by UPLC-Q-TOF-MS/MS.The prototype components in BYHWT were identified according to retention time,accurate relative molecular weight,primary and secondary mass spectrometry data. Result:After the administration of BYHWT,five compounds were found to enter the normal brain tissue through the blood-brain barrier and identified as calycosin-7-glucoside,albiflorin,formononetin-7-O-β-D-glucoside-6″-O-acetyl,safflower yellow A and astragaloside A;two compounds penetrated the blood-brain barrier and entered modeling brain tissue,and they were identified as calycosin-7-glucoside and formononetin-7-O-β-D-glucoside-6″-O-acetyl;seven compounds entered normal plasma and were identified as calycosin-7-glucoside,albiflorin,hydroxysafflor yellow A,et al;three compounds entered model plasma and identified as calycosin-7-O-β-D-glucoside-6″-O-acetyl,6″-O-acetyl-(6αR,11αR)-9,10-dimetho-xypterocarpan-3-O-β-D-glucoside and formononetin-7-O-β-D-glucoside-6″-O-acetyl. Conclusion:BYHWT has different pharmacological material basis in normal and cerebral ischemia-reperfusion rats.

Effects of Exosomes Derived from miR-486 Gene Modified Umbilical Cord Mesenchymal Stem Cells on Biological Characteristics of Rat Cardiomyocytes / 中国实验血液学杂志

<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>

Roles of SPK in Regulation of Hypoxia Induced Proliferation and Glucose Consume of Leukemia Cells / 中国实验血液学杂志

<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>

MiR-486 Regulates the Glycometabolism of Hematopoietic Cells by Targeting Sirt1 / 中国实验血液学杂志

<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>

Design, virtual screening, synthesis and anti-hepatitis B virus of oxime derivatives / 药学学报

A series of new oxime and oxime ethers compounds were designed and virtually screened with target using the Molecular Operating Environment (MOE) software. Twelve unreported compounds including 4 oximes and 8 oxime ethers were synthesized with benzene, toluene, methoxybenzene and chlorobenzene as initial raw materials. Structures of compounds were elucidated by 1H NMR, 13C NMR and MS. The results of bioactive screening showed that a part of compounds displayed obviously anti-HBV activities. Inhibitory activities of compounds 4B-2 in secretion of HBsAg and HBeAg were IC50 HBsAg=81.15 μmol·L-1, SIHBsAg=9.20 and IC50 HBeAg=90.66 μmol·L-1, SIHBeAg8.24, respectively. Preliminary structure-activity relationship study shows that methyl oxime ethers displayed better anti-HBV activities than the oximes.

Construction of Lentiviral Vector Over-Expressing MEG3 and Its Effect on XG-7 Cell Apoptosis / 中国实验血液学杂志

<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>

Study on recovery and its influencing factors of ferulic acid and tetramethylpyrazine in cerebral microdialysis probe / 中国中药杂志

To establish a method for detecting microdialysis recovery of tetramethylpyrazine (TMP) and ferulic acid (FA) and investigating the influencing factors, providing the basis for further in vivo microdialysis experiments. The concentration of FA and TMP in dialysates were determined by high pressure liquid chromatography ( HPLC) and probe recovery were calculated respectively. The influence of the flow rates, medium concentration, temperature and in vivo probe stability on the recovery of FA and TMP were investigated by using concentration difference method (incremental method and decrement method). The recovery obtained by incremental method were similar to by decrement method. The in vitro recovery rate of FA and TMP decreased with the increase of 1-2.5 μL min(-1), and increased obviously with the temperature of 25-42 degrees C under the same conditions. The concentration of FA and TMP had no obvious effect on the probe recovery under the same flow rate. In addition, the recovery of TMP and FA remained stable and showed similar trends under the condition of four concentration cycles, indicating that the intra day reproducibility of the concentration difference method was good. The recovery of brain microdialysis probes in vivo 8 h maintained a relatively stable, but certain differences existed between different brain microdialysis probes, demonstrating that each probe was required for recovery correction in vivo experiment. Microdialysis sampling can be used for the local brain pharmacokinetic study of FA and TMP, and retrodialysis method can be used in probe recovery of FA and TMP in vivo.

Effect of aurora inhibitor VX-680 on proliferation and apoptosis of CML cells / 中国实验血液学杂志

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.

Effect of microRNA-17-92 cluster on the biological characteristics of K562 cells and its mechanisms / 中国实验血液学杂志

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.

Promoting effect of thrombin on proliferation of bone marrow-derived mesenchymal stem cells and its mechanisms / 中国实验血液学杂志

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.

Analysis of allergen variation for 3292 patients with allergic rhinitis / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the variation of common allergens in patients with allergic rhinitis in recent 4 years in Tianjin First Center Hospital.</p><p><b>METHODS</b>The medical records of skin prick test on 3292 patients with allergic rhinitis between 2009 and 2012 were restrospectively analyzed. The changing trend of various allergens in 4 years and distribution differences were compared. The differences of the top 5 allergens in under age group, adult group and different gender group were further analyzed by SPSS 19.0 software.</p><p><b>RESULTS</b>The positive rate of dermatophagoides farinae was increasing year by year, from 45.1% in 2009 to 66.3% in 2012, and the positive rate of dermatophagoides pteronyssinus increased from 42.0% in 2009 to 58.6% in 2012, the difference was statistically significant (χ(2) value was 68.70, 41.55, all P < 0.01). The positive rate of dermatophagoides farinae and dermatophagoides pteronyssinus in adult group and male group showed significant upward trend year by year (χ(2) value was 75.85, 69.93, 274.25, 42.62, all P < 0.01), but not in adult group and female group. The positive rate of quinoa, mugwort and humulus scandens decreased year by year between 2009 and 2011(χ(2) value was 22.08, 11.64, 203.19, all P < 0.01), but increased again in 2012(χ(2) value was 21.55, 29.38, 12.40, all P < 0.01).</p><p><b>CONCLUSIONS</b>There is a tendency of change of allergens in patients with AR. This phenominon may be helpful for doctors to choose the type of skin prick liquid.</p>

Advances of research on autophagy regulation in leukemia cells / 中国实验血液学杂志

Autophagy is a conservative self-degradation system in eukaryotic cells, which involves in multiple physiologic and pathologic processes. Autophagosome is a typical characteristics of autophagic process, and its formation and degradation are the key points to control autophagy. Due to its dual characteristics to promote survival and death, to some extent, autophagy determines cell fate for survival or die. Autophagy plays important roles in cancer development, metastasis and drug-resistance. Thus targeting autophagy may provide novel strategies for treating cancer and overcoming drug resistance. With the advances of study on autophagy regulation in leukemia cells, the novel therapeutic targets and strategies to cure leukemia will be developed. This review focuses autophagy characteristics and regulation, autophagy and tumor, autophagy and leukemias as well as autophagy regulation in leukemia cells.

In vivo tracing of transplanted bone marrow mesenchymal stem cells with bioluminescence imaging / 中国实验血液学杂志

Mesenchymal stem cell (MSC)-based cell therapy has shifted into clinical trials to repair the damage of various tissues. In this setting, the survival of the transplanted cells contributes critically to the therapeutic effectiveness. To investigate the in vivo tracing of MSCs, a recombinant retroviral vector carrying firefly-luciferase reporter gene [pL (FLUC) SN] was constructed and several GPE+86 cell clones that stably expressed fluc were selected. The retroviral supernatants were collected and used to transfect MSC derived from C57 mice. The cells were then screened with G418 and the expression of the exogenous gene was identified by luciferase enzyme activity analysis. Labeled mouse MSCs (2x10(6)) were injected into skeletal muscles, and the in situ expression was noninvasively tracked by in vivo bioluminescence imaging for 1, 3 and 6 days after transplantation. The results showed that the survival rates of the grafted cells dropped sharply with time, they were 57.2+/-11.7%, 8.6+/-2.5% and 5.4+/-3.1% on day 1, 3 and 6 after transplantation, and no fluorescent signals above background were detected on day 10. It is concluded that the method described above could be used for in vivo tracing of grafted cells. Furthermore, MSCs could not survive even transplanted into the none-ischemic skeletal muscles.

SphK-1/S1P signal pathway in CML cells / 中国实验血液学杂志

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. In order to investigate the role of sphingosine kinase-1 (SphK-1)/sphingosine 1-phosphate (S1P) signal pathway in the expression of CML cells, and to explore whether P210(bcr/abl) involved is activating SphK-1/S1P signal pathwey, the expressions of SphK-1 and S1P receptor mRNA in bcr/abl positive K562 cells and bcr/abl positive primary CML cells were detected by RT-PCR, the imatinib mesylate, the specific inhibitor of P210(bcr/abl) was employed to inhibit the P210(bcr/abl) tyrosine kinases of K562 cells and CML primary cells, and then the intracellular SphK-1 activity was assayed. The results indicated that after being cultured with 2.5 micromol/L imatinib mesylate for 0.5, 2, 6, 24 and 48 hours, the intensions of inhibiting SphK-1 activity were 0.007%, 38.9%, 34.6%, 28.1% and 76.1% resepectively. SphK-1 activity in CML cells also was reduced by 2.5 micromol/L imatinib mesylate (16.8% - 41.9% decrease). It is concluded that the CML cells express SphK-1 and different S1P receptor, and P210(bcr/abl) fusion protein in CML cells can activate SphK-1.

Mesenchymal stem cells applied in therapy for acute radiation injury / 中国实验血液学杂志

Mesenchymal stem cells are a kind of non-hematopoietic adult stem cells with selfrenewal and multilineage differentiation potential, which have special biological characteristics, such as secreting hematopoietic growth factors, reconstructing hematopoietic microenvironment, low immunogenicity, and can be transfected and expressed by exogenous gene. This article summarizes the biological characteristics of MSCs and their models of application to acute radiation disease in animals.

Influence of rhG-CSF on activity of sphingosine kinase in monocytes / 中国实验血液学杂志

The aim of this research was to understand the influence of rhG-CSF on the sphingosine kinase (SphK) activity of monocytes. The peripheral blood monocytes were collected from 6 peripheral blood progenitor cell donors on the fifth day of mobilization with rhG-CSF and from 5 blood donors' buffy coats. The mRNA expressions of monocyte G-CSF receptor and SphK were tested with RT-PCR. The changes of SphK activity of monocytes were assayed after being treated with rhG-CSF. The results showed that the two kinds monocytes collected from both blood donors and peripheral blood progenitor cell donors mobilized with rhG-CSF expressed mRNA of G-CSF receptor and SphK. The SphK activity of monocytes collected from blood donors was not changed significantly after being treated with rhG-CSF (P > 0.05). The SphK activity of monocytes collected from peripheral blood progenitor cell donors transiently increased by (39.6 - 87.2)% after being treated by means of rhG-CSF (P < 0.05) without obviously dose-dependent effect. It is concluded that the SphK activity of monocytes collected from peripheral blood progenitor cell donors can be activated by rhG-CSF.

Construction of 293pT2-P210 cell line enables expression of bcr/abl to be regulated by Tet-off inducing-expression-system / 中国实验血液学杂志

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210(bcr/abl) fusion protein, which is generated by the reciprocal translocation t (9; 22), inhibits apoptosis and increase proliferation. P210(bcr/abl) plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable thoroughly to study the function of bcr/abl fusion gene and its signal regulation mechanism.