Study on oral absorption mechanisms of puerarin in nanocrystals self-stabilized pickering emulsion / 中国中药杂志

Nanocrystals self-stabilized Pickering emulsion(NSSPE) is a new kind of emulsion where only nanocrystals of poorly soluble drugs are used as stabilizers. Our previous study showed that NSSPE with Ligusticum chuanxiong oil as the main oil phase can significantly promote oral absorption of puerarin. The present study aimed to explore its absorption mechanism in oral administration. The in vitro dissolution test was carried out to study the effect of NSSPE on release of puerarin. The effects and mechanism of NSSPE on uptake and transport of puerarin across Caco-2 cell were investigated. The results showed that the drug release rate of NSSPE was similar to that of nanocrystals, with their cumulative dissolution of puerarin not affected by pH of releasing mediums, both significantly higher than that of crude material. The uptake of puerarin in NSSPE was concentration-dependent and significantly higher than that of solution or surfactant stabilized emulsion. Genistein and indomethacin, inhibitors of lipid rafts/caveolin, could significantly reduce the uptake of puerarin in NSSPE. Compared with solution, NSSPE and surfactants stabilized emulsion obviously increased transport rate K_a and apparent permeability coefficient P_(app) of puerarin in AP → BL direction, but there was no significant difference in BL → AP direction. It could be inferred that there were both passive and active transport mechanisms, as well as lipid raft/caveolin mediated endocytosis for absorption of NSSPE. The promoted oral absorption of puerarin in NSSPE was mainly related to the existing nanocrystal form which could promote dissolution, puerarin as well as Ligusticum chuanxiong oil which could promote drug transmembrane transport and inhibit drug efflux. It is the unique structure and composition of the compound NSSPE that promoted the oral absorption of puerarin.

Clinical Significance of TF and VEGF Expressions on Peripheral CD14 Positive Monocytes in Patients with Diffuse Large B Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical significance of tissue factor (TF) and vascular endothelial growth factor (VEGF) expression on peripheral blood CD14 positive monocytes in patients with diffuse large B cell lymphoma (DLBCL).@*METHODS@#The expressions of TF and VEGF on peripheral CD14 monocytes in 41 patients with DLBCL (DLBCL group) before chemotherapy and after 4 chemotherapeutic courses, and in 20 healthy subjects (control group) were detected by flow cytometry respectively, meanwhile, the relationship of the expression of TF and VEGF with international prognostic indexes (IPI) and short-term effects were analysed.@*RESULTS@#The expression levels of TF and VEGF on peripheral CD14 monocytes in DLBCL group were significantly higher than those in control group (P0.05), the survival of patients in group with low expression of TF and VEGF was superior to that in group with high expression of TF and VEGF (P<0.05).@*CONCLUSION@#The paripheral blood CD14 monocytes in DLBCL patients highly express the TF and VEGF, which relate with IPI, therapeutic efficacy and survival, thus the TF and VEGF expression levels are of reference significance for evaluating the therapeutic efficacy and prognosis of patients.

Effect of OCT4A Gene on the Biological Characteristics of K562 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis.</p><p><b>METHODS</b>Two recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR.</p><p><b>RESULTS</b>The OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05).</p><p><b>CONCLUSION</b>Two lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.</p>

Construction of A Lentiviral Vector Carrying CUEDC1 Gene and Its Effect on the Proliferation and Colony-formating Ability of MOLT-4 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct a lentiviral vector carrying human CUEDC1 gene, to establish leukemic cell line MOLT-4 stably expressing recombinant plasmid, to analyze the expression of CUEDC1 in MOLT-4 cells and to investigate its effect on the proliferation of MOLT-4 cells.</p><p><b>METHODS</b>The CUEDC1 gene was amplified by RT-PCR, and then was subcloned into the lentiviral vector pCDH to generate a lentiviral vector pCDH-CUEDC1. Recombinant lentivirus was generated by co-transfection of 3 plasmids, and transfected into MOLT-4 cells. The Real-time PCR and Western blot were respectively applied to detect the expression of CUEDC1 mRNA and protein, the CCK-8 and colony formation assay were used to evaluate the effect of CUEDC1 on proliferation of MOLT-4 cells.</p><p><b>RESULTS</b>The recombinant lentiviral vector pCDH-CUEDC1 had been constructed successfully. After infection of MOLT-4 cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of CUEDC1 and protein. The CCK-8 detection and colony formation assay showed that exogenous CUEDC1 could significantly promote cell growth and the colony formation of MOLT-4 cells.</p><p><b>CONCLUSION</b>The recombinant lentiviral vector carrying human CUEDC1 has been successfully constructed, exogenous CUEDC1 can significantly promote cell growth and the colony formation of MOLT-4 cells.</p>

Inducing Effect of Akt Kinase Inhibitor MK2206 on Apoptosis in U937 Cells and RS4;11 Cells, and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was purposed to investigate the effect of Akt kinase inhibitor MK2206 on proliferation and apoptosis of U937 cells and RS4;11 cells, and to explore its possible mechanism.</p><p><b>METHODS</b>U937 and RS4;11 cells were cultured with different concentrations of MK2206 for 24 h and 48 h, and cell growth curve was analyzed by CCK-8; cell apoptosis was analyzed by Annexin V/7-AAD double labeling; cell cycle changes were analyzed by flow cytometry. The BAX, BCL-2, XIAP, CDK1, caspase-3 mRNA expressions were determined by real time PCR.</p><p><b>RESULTS</b>MK2206 significantly inhibited the growth of U937 and RS4;11 cells in a time-and dose-dependent manner, and the IC50 values of U937 cells for 24 h and 48 h were (0.48±0.15) µmol/L and (0.09±0.01) µmol/L respectively, while IC50 values of RS4;11 cells for 24 h and 48 h were (0.91±0.02) µmol/L and (0.68±0.11) µmol/L respectively. U937 were cultured with 0.5 µmol/L MK2206 and RS4;11 cells were cultured with 1.0 µmol/L MK2206 for 24 h and 48 h, and the both apoptosis rates were higher for 24 h or 48 h than that in control group (P<0.05), meanwhile the apoptosis rates for 48 h were higher than 24 h. The results of cell cycle detection showed that the both cells were arrested in G2/M phase compared with control group. The real time PCR assay revealed that the expressions of BAX, caspase-3 mRNA in cells treated with MK2206 were increased, while BCL-2, XIAP, CDK1 were reduced compared with control group.</p><p><b>CONCLUSION</b>MK2206 can inhibit proliferation and induce apoptosis of U937 and RS4;11 cells, and the both cells are arrested in G2/M phase. The mechanism of promoting apoptosis may be related with up-regulating BAX, caspase-3 and down-regulating BCL-2, XIAP, meanwhile the cell cycle arrested in G2/M phase may be associated with down-regulating CDK1.</p>

Effect of Metformin on Proliferation, Differentiation and Apoptosis of THP-1 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of metformin on proliferation, differentiation and apoptosis of THP-1 cells and explore its possible mechanism.</p><p><b>MEHODS</b>THP-1 cells were cultured with different concentrations of metformin for 24 h and 48 h. The cell proliferation was evaluated by CCK-8, the cell apoptosis was analyzed by Annexin V/7-AAD double labeling, the expression of CD14 and CD11b (surface differentiation antigens on THP-1 cells) was evaluated by flow cytometry, the BCL-XL, BAX, BIM and caspase-3 mRNA expressions of THP-1 cells were detected by real time quantitative PCR.</p><p><b>RESULTS</b>Metformin could significantly inhibit the growth of THP-1 cells in a time- and dose- dependent manner. After treated with 20 mmol/L metformin for 24 h, the expressions of CD14 and CD11b in THP-1 cells didn't change much (P>0.05), the early apoptosis rates in exprimental and control groups were (2.02±0.85)% and (4.46±1.33)% respectively, the late apoptosis rates in experimental and control groups were (1.43±0.83)% and (3.31±0.59)% respectively. In process of inducing effect of 20 mmol/L metformin on THP-1 cells, the expressions of BCL-XL and BIM did not significantly changed, while the expressions of BAX and caspase-3 significantly increased (P<0.01).</p><p><b>CONCLUSION</b>Metformin can effectively inhibit proliferation and induce apoptosis of THP-1 cells. However, it has no significant effect on differentiation of THP-1 cells, its mechanism inducing apoptosis maybe related with up-regulating BAX and caspase-3.</p>

Expression of NANOG gene in acute lymphoblastic leukemia cells and construction of lentiviral vector carrying NANOG specific shRNA / 中国实验血液学杂志

The aim of this study was to detect the expression of NANOG gene in acute lymphoblastic leukemia (ALL) cells, and to construct the lentiviral vector carrying NANOG specific shRNA. The expression of NANOG was detected by RT-PCR and Western blot in MOLT-4, CCRF-HSB2, Jurkat cells and bone marrow cells from 15 patients with ALL in our hospital. The lentiviral vector carrying NANOG specific shRNA was constructed. After infection of MOLT-4 cells with the lentivirus constructs, GFP (+) cells were harvested by flow cytometry. The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot. The results showed that the expression of NANOG mRNA and protein was detected in MOLT-4, CCRF-HSB2 cells and 33.3% samples of bone marrow from patients with ALL. The sequencing results demonstrated that the mRNAs amplified from these leukemic cells showed higher homology to NANOGP8 than NANOG1. The lentiviral vector pLB-shNANOG-1, pLB-shNANOG-2 and pLB-shcontrol were constructed. The viral particles were harvested and concentrated by ultracentrifugation. The virus titers were (1.83-3.12) ×10(8) IU/ml. After infection of MOLT-4 cells with the lentivirus, flow cytometry detection indicated that the GFP(+) cells were harvested by real-time quantitative PCR and Western blot, the assays showed that the 2 designed shRNA could significantly down-regulate expression of NANOG gene and protein. It is concluded that NANOGP8 is expressed in various types of ALL cells and in 33.3% of marrow cell samples obtained from ALL patients. After infection with the lentivirus constructs, MOLT-4 cells which stably down-regulate the expression of NANOG mRNA are obtained.

Construction of lentiviral vector carrying human OCT4A gene and establishment of leukemic cell line K562 stably expressing OCT4A / 中国实验血液学杂志

This study was purposed to construct a lentiviral vector carrying human OCT4A gene and green fluorescent protein (GFP) , and infect the leukemic cell line K562, observe the expression of OCT4A in K562 cells. According to the sequence of OCT4A mRNA which was found in GenBank, the special primer sequences were synthesized. The OCT4A gene was amplified by RT-PCR, and then cloned into the pCR-Blunt vector. The OCT4A DNA fragment was subcloned into the lentiviral vector pLVX-IRES-ZsGreen1 which was restricted by EcoR1 to generate a lentiviral vector pLVX-OCT4A-ZsGreen1. The sequence of the recombinant plasmid was identified by DNA sequencing. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT cells using lipofectamine 2000 and transfected into K562 cells. Real-time PCR and Western blot were applied to detect the expression of OCT4A mRNA and protein, CCK-8 and colony formation assay were performed to evaluate the effects of OCT4A on proliferation of K562 cells. The results showed that the recombinant lentiviral vector pLVX-OCT4A-ZsGreen1 was successfully constructed. The virus titers were (1.43 ± 0.25) × 10(8) U/ml. After infection of K562 cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of OCT4A gene and protein according the real-time PCR and Western blot detection results. CCK-8 and colony formation assay showed that exogenous OCT4A gene could significantly promote cell growth and the colony formation of K562 cells. It is concluded that the recombinant lentiviral vector pLVX-OCT4A- ZsGreen1 carrying human OCT4A gene is successfully constructed; K562 cells which stably up-regulates the expression of OCT4A mRNA are obtained, the results of this study provide fundamental basis for further study on mechanism of OCT4A in human leukemia development.

Effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside on proliferation, differentiation and apoptosis in U937 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on proliferation, differentiation and apoptosis of U937 cells and explore its possible mechanism.</p><p><b>METHODS</b>U937 cells were cultured with different concentrations of AICAR for 24 h and 48 h. Cell proliferation was evaluated. Cell growth curve was analyzed by CCK-8; cell apoptosis was analyzed by cell morphology, Annexin V/7-AAD double labeling. The differentiation of U937 cells was evaluated by expression of CD11b. The Bcl-xL, Bax, Bim, caspase-3 mRNA expressions of U937 cells were determined by real time PCR.</p><p><b>RESULTS</b>AICAR significantly inhibited the growth of U937 cells in a time-and dose-dependent manner, with a 24 h IC50 value of 1.1 mmol/L and 48 h of 0.9 mmol/L. 1.0 mmol/L AICAR didn't induce differentiation of U937 cells with the increase of CD11b expression for 24 h (P > 0.05). The U937 cells apoptosis was confirmed by cell morphology and Annexin V/7-AAD labeling. AICAR induced apoptosis of U937 cells and the apoptosis rate was (6.81 ± 1.16)% at 1 mmol/L AICAR higher than control group (2.74 ± 0.32)% without AICAR for 24 h treatment (P < 0.05). The real time PCR assay revealed that as compared with control group, the expression of Bim and caspase-3 mRNA were increased, while Bcl-xL and Bax were unchanged on the AICAR treatment.</p><p><b>CONCLUSION</b>AICAR can effectively inhibit proliferation and induce apoptosis of U937 cells. However, it has no significant effect on differentiation of U937 cells. The mechanism may be related with up-regulating Bim and Caspase-3.</p>

Construction of shRNA expression vector targeting AATF and establishment of stably transfected U937 cells / 中国实验血液学杂志

This study was aimed to construct the targeting AATF shRNA eukaryotic expression vector and establish the stably transfected U937 cell lines. The sequence of AATF mRNA was obtained from GenBank. After excluding homology, three plasmid expression vectors coding shRNA targeting 228 ∼ 249, 303 ∼ 324 and 443 ∼ 464 of AATF gene sequence were synthesized. Two terminals of shRNA carried BamHI and HindIII restriction sites. The selected nucleotides were cloned into the plasmid pSilencer 3.1-H1 neo respectively, and the resultant recombinant plasmids were named as pSA-1, pSA-2, pSA-3. The sequences of the recombinant plasmids were identified by DNA sequencing. The recombinant plasmids were transfected into the cell line U937 by electroporation with Neon(TM) Transfection System. The transfected cells were persistently screened under G418 (500 mg/L), and isolated with a limited dilution for 8 weeks. The inhibition of AATF mRNA and protein expression was respectively detected by RT-PCR and Western blot. The results indicated that RNAi eukaryotic expression vectors targeting AATF had correct reading frame and nucleotide sequence. Real-time PCR revealed that AATF shRNA effectively silenced mRNA expression of AATF. Western blot analysis found that AATF shRNA obviously suppressed protein expression of AATF (P < 0.05). It is concluded that the shRNA eukaryotic expression vector has been successfully constructed which can inhibit the expression of AATF, and the establishment of stably transfected U937 cell lines provide a original route for exploring the mechanism of AATF in human Leukemia further.

Effects of NANOG gene down-regulation on the apoptosis of T-cell acute lymphoblastic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore gene expression of NANOG in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells.</p><p><b>METHODS</b>Real-time PCR (RT-PCR) and Western blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells (Annexin V⁺/7-AAD⁻) and late apoptotic cells (Annexin V⁺/7-AAD⁺) were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected.</p><p><b>RESULTS</b>Both NANOG gene and protein expression was positive in MOLT-4 and CCRF-HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-sh control were successfully constructed, as evidenced by the viral titers (1.83-3.12)× 10⁸ IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1 and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1[(8.06 ± 1.61)%]and shNANOG-2[(5.67 ± 1.59)%]were significantly increased as compared to that of MOLT-4 group[(1.13 ± 0.40)%]or sh-control [(1.15±0.49)%](P<0.05). However, no statistical difference among them was observed for late apoptotic cells (P>0.05). The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG-2 group was augmented as compared to that of MOLT-4 group or sh-control (P<0.05). Reversely, a significant down-regulation of Bcl-2 gene expression was observed (P<0.05).</p><p><b>CONCLUSION</b>NANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.</p>

Expression of interleukin-22 and relative CD4(+) T cell subsets in patients with immune thrombocytopenia / 中国实验血液学杂志

The aim of this study was to detect the expression of interleukin-22 (IL-22) and relative CD4(+) T cell subsets in untreated adult patients with immune thrombocytopenia (ITP), and to explore their roles in the pathogenesis of ITP. Fourty adult active ITP patients were enrolled in this study and 40 healthy subjects were taken as control. Plasma IL-22 level was quantified by ELISA. The percentages of Th1, Th17 and Th22 cells in peripheral blood were determined by flow cytometry. The Pearson correlation test was used to evaluate correlations between IL-22 level and Th1 cells, Th17 cells and Th22 cell percentages. The results showed that the plasma IL-22 levels in untreated ITP patients [(364.12 ± 94.22) pg/ml] were significantly higher than that in healthy controls (P < 0.001). The percentages of both Th1 [(18.92 ± 6.03)%] and Th22 [(2.28 ± 0.51)%] cells in ITP patients were elevated as compared to healthy controls (P < 0.05). Elevated plasma concentration of IL-22 positively correlated to the percentages of Th1 (r = 0.42, P = 0.022) and Th22 (r = 0.40, P = 0.030) cells in untreated ITP patients. The percentage of Th17 cells was not significantly different between untreated patients and normal controls, and there was no statistical correlation between the IL-22 level and the percentage of Th17 cells in active ITP patients. It is concluded that elevated IL-22 level correlates to Th1 and Th22 cells percentage, which may play a synergistic effect in the immunopathogenesis of ITP, while Thl7 cells may not be associated with the occurrence of active ITP.