OBJECTIVETo investigate the effect of MTS1 gene beta promoter transcriptional activation in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and identify the fragment with transcriptional activation.

METHODSSeven pGL3 recombinant plasmids with the same 3'-end transcriptional start site but the different 5'sequences were constructed by gene recombinant technique and transfected into Jurkat cell line which is biallelic deletion of MTS1 gene by transient transfection. Luciferase report gene was detected to observe beta promoter transcriptional activation.

RESULTSSeven pGL3 recombinant plasmids containing different fragments of beta promoter were obtained, all of them showed transcriptional activation in Jurkat cell line. Among them, the 0.38 kb fragment cut by SacII-SacI is fundamental in transcription.

CONCLUSIONMTS1 gene beta promoter can be activated in Jurkat cell line.

Genes, p16 ; Humans ; Jurkat Cells ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; Transfection

OBJECTIVETo explore the effects of vascular endothelial growth factor (VEGF) antisense phosphorothioated oligodeoxynucleotide (AS-ODN) on the expression of VEGF in human leukemic cell lines (HL-60 and K562 cells).

METHODSThe levels of VEGF mRNA and protein in leukemic cells incubated with VEGF AS-ODN were measured by RT-PCR, immunohistochemistry assay and ELISA. MTT test was used to examine the influence of the culture supernatant (CS) of VEGF AS-ODN treated leukemic cells on the proliferation of human umbilical vein endothelial cells (ECV304).

RESULTSAfter leukemic cells were treated with different concentrations (2.5 approximately 15.0 micro mol/L) of VEGF AS-ODN for 24 h, VEGF mRNA level in the cells decreased remarkably in a concentration dependent manner, no change was found in the VEGF missense ODN treated cells (MS-ODN). When the leukemic cells were treated with 5 micro mol/L VEGF AS-ODN for 24 h, VEGF protein level decreased greatly both in the cells and in the CS; and the proliferation stimulating effect of the treated CS on the ECV304 cells reduced. Meanwhile, there was no obvious change in VEGF protein and its effect in the VEGF MS-ODN treated group.

CONCLUSIONVEGF AS-ODN could inhibit VEGF expression in human leukemic cell lines in vitro.

Enzyme-Linked Immunosorbent Assay ; HL-60 Cells ; Humans ; Immunohistochemistry ; K562 Cells ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics

OBJECTIVESTo 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.

METHODSSTAT5 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.

RESULTSConfocal 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.

CONCLUSIONSSTAT5 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.

Cell Proliferation ; Cyclin D1 ; genetics ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Expression ; Humans ; K562 Cells ; Liposomes ; Microscopy, Confocal ; Oligodeoxyribonucleotides, Antisense ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; STAT5 Transcription Factor ; genetics ; physiology ; Transfection ; bcl-X Protein ; genetics

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

OBJECTIVETo observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.

METHODSThe recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.

RESULTSInfection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.

CONCLUSIONThere is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.

Adenoviridae ; genetics ; Centrosome ; metabolism ; Genes, p53 ; genetics ; Genetic Vectors ; Humans ; K562 Cells ; Transfection ; Tubulin ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo select the optimal pregnancy time window of embryonic pig skin precursor tissue for xenotransplantation and study its ability in wound repair.

METHODSSkin precursor tissues were obtained from pig fetus of fetal age of 35, 42, 56, 70 days, and were minced into microskin and transplanted to dorsal wounds of BALB/c nude mice, then they were covered with residual skin after plastic surgery of patients or adult pig skin (white). The characteristics of growth and development were observed after transplantation. Pathological examination was performed on 6 and 12 post operation weeks respectively to observe the tissue structure and tumorigenicity.

RESULTSSkin precursor tissues from fetal pig survived and developed after transplantation, and the microskin fused. New tissue area from skin precursor tissues with fetal age of 42 days was (47 +/- 6) mm2, which was higher than that of 35 days (18 +/- 8 mm2), 56 days (31 +/- 12 mm2), 70 days (20 +/- 8 mm2, P < 0.05). The skin precursor developed into "intact skin" with hair, sebaceous glands and sweat glands, and melanocytes were also detected in epidermis. The newly-grown skin tissue included epidermal and dermal layer, and obvious dermal papillae. Teratoma was not found after transplantation in skin precursor tissue with fetal age of 56, 70 days.

CONCLUSIONFetal pig skin precursor tissue with fetal age of 56 days can be used to repair wound as xenotransplantation.

Animals ; Fetal Tissue Transplantation ; Fetus ; Gestational Age ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Skin Transplantation ; Swine ; Transplantation, Heterologous ; Wound Healing

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONSHnRNP 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.

CCAAT-Enhancer-Binding Protein-alpha ; genetics ; Cell Differentiation ; genetics ; Gene Expression Regulation ; Heterogeneous-Nuclear Ribonucleoproteins ; genetics ; Humans ; K562 Cells ; RNA ; genetics ; Translating

BACKGROUNDEpidemiologic studies have shown an independent and definite association between obstructive sleep apnea (OSA) and hypertension. This study aimed to define the association between daytime blood pressure and severity of OSA in Chinese population in mainland of China.

METHODSTwenty university hospital sleep centers in mainland of China were invited by the Chinese Medical Association (CMA) to participate in this epidemiologic study and 2297 consecutive patients (aged 18 - 85 years; 1981 males and 316 females) referred to these twenty sleep centers for evaluation of OSA between January 2004 and April 2006 were prospectively enrolled. Nocturnal polysomnography was performed in each patient, and disease severity was assessed based on the apneahypopnea index (AHI). These patients were classfied into four groups: nonapneic control (control, n = 257) with AHI < or = 5 episodes/hour; mild sleep apnea (mild, n = 402) with AHI > 5 and < or = 15 episodes/hour; moderate sleep apnea (moderate, n = 460) with AHI > 15 and < or = 30 episodes/hour and severe sleep apnea (severe, n = 1178) with AHI > 30 episodes/hour. Daytime blood pressure measurements were performed under standardized conditions in each patient at 10 a.m. in office on the day of referring to sleep centers for getting average value. All the patients were requested to quit medications related to blood pressure for three days before the day of assessing.

RESULTSBoth daytime systolic blood pressure and diastolic blood pressure values were significantly related to AHI positively (r = 0.201 and 0.276, respectively; both P values < 0.001) and to nadir nocturnal oxygen saturation negatively (r = -0.215 and -0.277, respectively; both P values < 0.001), which were the parameters of OSA severity. In two special designed mean plots, means of daytime systolic and diastolic blood pressure increased gradually with increasing AHI. Beyond AHI of 61 - 65, this increasing trend reached a plateau.

CONCLUSIONSThe results showed that OSA severity was associated with daytime blood pressure until AHI of 61 - 65, providing evidence for early OSA management, especially in OSA patients with concomitant hypertension.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Blood Pressure ; physiology ; China ; Female ; Humans ; Male ; Middle Aged ; Sleep Apnea, Obstructive ; pathology ; Young Adult

BACKGROUNDThe nocturnal nondipping and elevated morning blood pressure (BP) in patients with obstructive sleep apnea syndrome (OSAS) have not yet been well investigated in Chinese patients. This study aimed to describe the BP profile, and to elucidate the relationships between daytime BP and nighttime BP, and between evening BP and morning BP in patients with OSAS.

METHODSTwenty teaching hospital sleep centers in China were organized by the Chinese Medical Association to participate in this study and 2297 patients were recruited between January 2004 and April 2006. BP assessments were made at four time points (daytime, evening, nighttime and morning) and polysomnography (PSG) was performed and subjects were classified into four groups by their apnea-hypopnea index (AHI): control, n = 213 with AHI < 5; mild, n = 420 with AHI ≥ 5 and < 15; moderate, n = 460 with AHI ≥ 15 and < 30; and severe, n = 1204 with AHI ≥ 30. SPSS 11.5 software package was used for statistical analysis and figure drawing.

RESULTSAll the average daytime, nighttime, evening and morning BPs were positively correlated with AHI and negatively correlated with nadir nocturnal oxygen saturation. The ratios of nighttime/daytime and morning/evening average BP were positively correlated with AHI. The ratio of nighttime/daytime systolic BP became a "reversed BP dipping" pattern until the classification reached severe, while the ratio of nighttime/daytime diastolic BP became reversed at moderate. Similarly, the ratio of morning/evening diastolic BP becomes reversed even at mild.

CONCLUSIONSOSAS may result in higher BP levels at all four time points. The ratios of nighttime/daytime and morning/evening BP increase with increased AHI. The increasing of diastolic BP, which is inclined to rise more quickly, is not parallel with increasing systolic BP.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Anthropometry ; Blood Pressure ; physiology ; Female ; Humans ; Hypertension ; physiopathology ; Male ; Middle Aged ; Sleep Apnea, Obstructive ; physiopathology ; Young Adult