Analysis of CDSS Experimental Indexes in 333 Cases of Newly Diagnosed Acute Leukemia / 中国实验血液学杂志

OBJECTIVE@#To analyze the 5 experimental indexes of CDSS in the patients with acute leukemia (AL) so as to provide the laboratorial basis for the early diagnosis and treatment of the secondary DIC in AL.@*METHODS@#Three hundred and thirty three patients with AL were divided into 7 groups, such as AML-M1-M5, other AML and ALL. The experimental indexes and CDSS scores of all AL groups were compared and analyzed in pairs, meanwhile 100 healthy persons were taken in the control group. Clinical events such as early death in all cases were also analyzed.@*RESULTS@#The highest positive rate of Platelet was 59.76%, among the 5 experimental indexes, followed by D-D (30.93%), and the lowest APTT with only 2.70%. Compared with the control group, the differences of remaining indexes were statistically significant (P＜0.01), except APTT in group AML-M3 and FIB in the other AML groups. The score of laboratory index was (1.50±1.51) in all AL patients, and the positive rate of overt DIC ( score≥4) was 14.11% ( 47 cases). The highest score of CDSS was (3.34 ±1.71) in group AML-M3. The difference in the incidence of early death and cerebral (pulmonary) hemorrhage in DIC patients were statistically significant (P＜0.05 and P＜0.01).@*CONCLUSION@#The application of quantitative integral method of experimental indexes in CDSS is objective and feasible, which is of great significance for early diagnosis and early treatment of acute leukemia complicated with DIC.

Death caused by ST19 Klebsiella oxytoca with wcaG virulence gene in leukemic patient / 中国人兽共患病学报

We investigated the cause of a leukemia patient induced by infect in a strain of Klebsiella oxytoca with hypermucoviscosity (HMV) phenotype.Identification and drug susceptibility of the isolate were carried out with VITEK-2 compact system.HMV phenotype was detected by string-test.The major high virulence capsular serotypes (K1,K2,K5,K20,K54 and K57) and virulence factors (rmpA,wcaG,allS,kfu,aerobactin,fimH,uge,wabG and cf29a) were detected by polymerase chain reaction and DNA sequencing.Molecular typing was performed by multilocus sequence typing (MLST).Results showed that the isolates of blood and lung tissue were Klebsiella oxytoca belonged to ST 19,which were sensitive to the antibiotics used in test,expressing the HMV phenotype.The virulence gene wcaG was found,while other virulence genes and the major high virulence capsular serotypes were negative.It indicates that ST19 Klebsiella oxytoca with wcaG virulence gene is the main reason causing leukemic patient death.

Determination of N-trans-feruloyl tyramine in Lycii Fructus by HPCE / 中国药学杂志

OBJECTIVE: To establish an HPCE method for determination of N-trans-feruloyl tyramine in Lycii Fructus. METHODS: The chromatographic separation was performed on an uncoated fused silica capillary of 50 cm × 75 μm ID (40 cm of effective length). 50 mmol · L-1Na2B4O7(pH 9.0 with H3PO4) was selected as the running buffer. The applied voltage was 20 kV, and the column temperature was maintained at 25°C. The chromatogram was detected at 214 nm. RESULTS: The linearity for N-trans-feruloyl tyramine was in a range of 0.25 - 15 μg · mL-1 (r = 0.9998, n = 7). The average recovery for N-trans-feruloyl tyramine was 101.8% with RSD of 2.06%. CONCLUSION: The method is accurate, reliable, and can be used as a quality control method for N-trans-feruloyl tyramine in Lycii Fructus, which can provide basis for development and utilization of Lycii Fructus. Copyright 2012 by the Chinese Pharmaceutical Association.

Phenylhexyl isothiocyanate (PHI) regulates histone methylation and acetylation and induces apoptosis in SMMC-7721 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of PHI on histone acetylation and methylation in hepatocellular carcinoma line SMMC-7721 cells.</p><p><b>METHODS</b>Apoptosis was measured by TUNNEL assay. Histone methylation and acetylation were detected by Western blot.</p><p><b>RESULTS</b>PHI inhibited cells growth and induced apoptosis. PHI treatment resulted in increased acetylation of histone H3 and H4 , elevated level of histone H3 lysine 4 methylation, and decreased level of histone H3 lysine 9 methylation.</p><p><b>CONCLUSIONS</b>PHI can modulate both histone acetylation and methylation, which could remodel chromatin structure. PHI may be a novel anticancer drug.</p>

Modulation of histone acetylation and induction of apoptosis in SMMC-7721 cells by phenylhexyl isothiocyanate / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect of phenylhexyl isothiocyanate (PHI) on histone acetylation and apoptosis in hepatocellular carcinoma cell line (SMMC-7721) in vitro.</p><p><b>METHODS</b>The viability of SMMC-7721 cells was determined by trypan blue exclusion. Apoptotic cells were assessed by TUNEL assay. The proteins of Bcl-2, Procaspase-9, Procaspase-8, Procaspase-3, caspase-9, caspase-3, histone acetylated H3 and H4 were detected by Western blot.</p><p><b>RESULTS</b>Compared with the vehicle control, PHI at 5, 10, 20, 40 and 80 µmol/L reduced the cell viability of SMMC-7721 cells in a concentration-dependent manner. PHI induced apoptosis in SMMC-7721 cells. An increased amount of apoptotic cells was detected after 7 hours exposure to PHI at 10, 20, and 40 µmol/L, 6.9% ± 2.4%, 17.5% ± 4.2% and 54.5% ± 5.4%, respectively, while that of the vehicle control was 4.5% ± 2.3% (P < 0.05). Along with the prolongation of time and increase of dose, the expressions of bcl-2, procaspase-9, procaspase-3 were decreased, that of caspase-9 and caspase-3 was increased. In contrast, alteration of procaspase-8 was not significant at those concentrations. PHI accumulated acetylated histone H3 and H4. After 3 hours exposure to PHI at 10, 20 and 40 µmol/L, the level of histone acetylated H3 was 1.87-, 2.43-, 3.67-fold increased and histone acetylated H4 was 1.29-, 1.45-, and 2.25-fold increased, compared with that of the vehicle control. The protein of histone acetylated H3 and H4 was significantly accumulated after 7 hours exposure.</p><p><b>CONCLUSION</b>PHI is a new histone deacetylation inhibitor. It may induce accumulation of histone acetylation H3 and H4, inhibit cell growth and induce apoptosis in SMMC-7721 cells via the mitochondrial pathway.</p>

Targeted blockage of RNA binding protein E2 by decoy RNA induces the granulocytic differentiation of K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To use a decoy RNA targeted blockage of the RNA binding protein E2 (hnRNP E2) resulting in the CCAAT/enhancer-binding protein alpha (C/EBP alpha) gene's abnormal translation and investigate its effect on the granulocytic differentiation of K562 cells and the probable molecular mechanism.</p><p><b>METHODS</b>The hnRNP E2 decoy RNA expression plasmid was constructed and transfected into K562 cells with cationic liposome, and stable expression cells were obtained by G418 selection. The changes of C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR) gene expression were detected by RT-PCR and Western blot. The morphologic changes were observed after Wright-Giemsa staining. The expression of granulocytic differentiation antigens CD13 and CD15 was studied by immunocytochemistry.</p><p><b>RESULTS</b>The stably expressed pG cells were obtained. Its C/EBP alpha mRNA level remained unchanged, while 42kD-C/EBP alpha protein expression was increased by (49.7 +/- 5.5)% (P < 0.05); and G-CSFR mRNA was increased by (42.1 +/- 3.6)% (P < .05), and its protein was increased by (37.4 +/- 6.2)% (P < 0.05) compared to that in the K562 control cells. The characteristics of polymorphonuclear neutrophils appeared in pG cells and CD13 and CD15 positive cell ratios were (18.7 +/- 2.5)% and (26.3 +/- 2.9)% respectively.</p><p><b>CONCLUSIONS</b>HnRNP E2 decoy RNA can induce granulocytic differentiation of K562 cells, and G-CSF promotes this effect. The mechanisms may be that decoy RNA specifically blocks hnRNP E2, hence regulates the translation of C/ EBP alpha mRNA, restores the expression of 42kD-C/EBP alpha, and then up-regulates the expression of G-CSFR gene.</p>

Effect of STAT 5 decoy oligodeoxynudeotide on tumorigenicity of K 562 cells in nude mice / 肿瘤

Objective: As a potential gene therapeutic method, transcription factor decoy strategy has been used to block the transcription function of multi-transcription factors. This study aims to further verify the inhibitory effect of signal transducer and activator of transcription 5 (STAT 5) decoy oligodeoxynucleotides (ODN) on tumor growth in nude mice. Methods: The xenografted tumor model was established by subcutaneously injecting leukemia K 562 cells in nude mice. Liposome-ODN complex was directly injected into tumor body once daily every 3 days. The tumor growth and tumor size were measured everyday. Nude mice were sacrificed at the end of the experiment. The tumor was removed and weighed. RT-PCR and Western blotting were performed to detect mRNA and protein expression of bcl-xL, cyclin D1, and c-myc, respectively. Results: The tumorigenicity of K 562 cells was significantly inhibited by decoy ODN. The tumor weight was markedly reduced in decoy ODN group than mutant ODN group [(0.485 ± 0.178) g vs (0.928 ± 0.223) g, P < 0.05 ]. The tumor growth inhibition rate was 47.7%. RT-PCR and Western blotting showed that the mRNA and protein expression level of bcl-xL, cyclinD1, and c-myc were down-regulated. Conclusion: Transcription factor decoy strategy effectively blocks transcription of STAT 5 target genes in xenografted tumor of nude mice.

Targeted blockage of STAT5 by a decoy oligodeoxynucleotide inhibits the growth and proliferation of K562 cells / 中华血液学杂志

<p><b>OBJECTIVES</b>To investigate targeted blockage of BCR/ABL oncoprotein mediated cell transformation by STAT5 decoy oligodeoxynucleotide (ODN), its effect on the growth and proliferation inhibition of K562 cells and the related molecular mechanisms.</p><p><b>METHODS</b>STAT5 decoy ODN, designed and synthesized in vitro, was transfected into K562 cells by cationic lipid. The cell growth curve and colony formation assay were used to reflect the growth and proliferation capacity of K562 cells, RT-PCR to detect the expression of three genes downstream STAT5.</p><p><b>RESULTS</b>Confocal microscopy demonstrated that STAT5 decoy ODN was successfully transfected into K562 cells (95.2% positive cells). STAT5 decoy ODN inhibited the growth of K562 cells (inhibition rate 77.7%) and their colony formation capacity (Decoy ODN treated group 8.3% vs control group 35.7%, P < 0.05) after the treatment with STAT5 decoy ODN, the expressions of c-myc, bcl-X(L), cyclin D1 mRNA were down-regulated by 15.4%, 30.8%, 29.1%, respectively in the K562 cells.</p><p><b>CONCLUSIONS</b>STAT5 decoy ODN inhibits the growth and proliferation of K562 cells. The mechanisms may be that decoy ODN blocks the transcriptional activation potent of STAT5 and down-regulates the expression of these tumor related genes downstream STAT5.</p>

Quantification of bcr/abl mRNA in patients with chronic myeloid leukemia by using real-time quantitative fluorescence PCR with self-quenched primer / 中华检验医学杂志

Objective To establish a quantitative RT-PCR method with self-quenched fluorogenic probe for detection of bcr/abl mRNA in patients with chronic myeloid leukemia for providing a useful tool for diagnosis of CML,evaluation of therapeutic effect and monitoring of minimal residual disease(MRD). Methods bcr/abl gene from cultured K562 cells was amplified by conventional RT-PCR.The standard quantitative plasmid was constructed by A-T clone method.The self-quenched fluorogenic quantitative RT- PCR method(FQ-RT-PCR)for determination of bcr/abl mRNA was established successfully using the ABI PRISM 7000 PCR Detector.The linear range,sensitivity,stability,and repetitiveness of the method were determined.The marrow samples from 25 CML patients and 3 ALL patients were assessed.Results The sensitivity of the FQ-RT-PCR was 10 copies/?l recombined plasmid,and bcr/abl mRNA can be detected from 1 K562 cell in 10~5 normal cells.The linear range was 10~2-10~9 copies/?l recombined plasmid.The coefficient variation(CV)value was 2.1% in intra-assay and 6.1% in inter-assay.The median ber/abl mRNA expression level was 4.50?10~4 copies/?g RNA [(0.45-89.00)?10~4],5.45?10~4 copies/?g RNA [(2.95-19.30)?10~4 ],13.00?10~4 copies/?g RNA [(4.10-89.00)?10~4] and 2.35?10~4 copies/?g RNA [(0.45-5.12)?10~4] in 25 CML patients,11 patients in the incipient chronic phase,6 patients in blastic crisis,8 patients in chronic period after treatment,respectively.The bcr/abl mRNA level in blastic crisis was significantly higher than that in chronic phase(q= 3.41,P

Screening the hepatitis B virus PreS1 associated protein by the yeast two-hybrid system / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen the hepatitis B virus PreS1 associated protein from normal human liver cDNA library by the yeast two-hybrid system and explore the role of PreS1 protein in the infection of hepatitis B virus (HBV).</p><p><b>METHODS</b>PCR was preformed to amplify the PreS1 gene containing EcoRI and PstI from HBV positive serum, and the production was inserted into plasmid pAS2-1 after digesting with the former two restricted endonuclease, then the bait vector pAS2-1-PreS1 was verified by auto-sequencing assay. The PreS1-BD fusion protein expressed in the yeast cells was confirmed by western blot, after pAS2-1-PreS1 was transfected into the yeast cell AH109. Yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library grew in selective SC/-trp-leu-his-ade2 medium, and the second screening was performed with LacZ report gene. Furthermore, segregation analysis and mating experiment were done to eliminate the false positive, then the true positive clones were submitted for PCR and sequencing. The results were submitted to the BLAST notebook of World Wide Wed Site NCBI to seek homologous sequence.</p><p><b>RESULTS</b>Bait vector pAS2-1-PreS1 included the anticipated fragment of PreS1 gene. Western blot showed that pAS2-1-PreS1 could correctly express PreS1-BD fusion protein in the yeast cells. After yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library, 97 colonies grew in the selective SC/-trp-leu-his-ade2 medium, only one clone was positive and showed high homology with Homo sapiens nascent-polypeptide-associated complex alpha polypeptide.</p><p><b>CONCLUSION</b>Bait vector pAS2-1-PreS1 is successfully constructed, and nascent-polypeptide-associated complex alpha polypeptide protein expressed in hepatocyte can interact with PreS1 by the yeast two-hybrid system.</p>