Objective To investigate the antitumour effects of transfected gut-enriched Krüppellike factor(GKLF) on human gastric carcinoma cell line SGC-7901 in vitro and in vivo. Methods The expression of GKLF mRNA and protein in human gastric carcinoma cell line SGC-7901 were detected before and after transfection by real-time fluorescence quantitative PCR and Western blot,respectively. Proliferation and invasion in SGC-7901 were measured respectively by MTT assay, flow cytometry, colony formation assay and cell invasion assay after transfected with GKLF. The growth of xenograft was observed, the microvessel density(MVD) of xenograft tissue was determined by immunohistochemistry. Results The GKLF mRNA and protein in SGC-7901 were overexpressed after transfected with GKLF(P＜0.05). The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups (P＜0.05). Transfected with GKLF caused part of the G0/G1 arrest, decreased clone formation rate and the invasion ability (P＜0.05). The growth speed of xenograft in SGC7901-pcDNA3.1-GKLF group was lower, the weight and MVD of xenograft tissue in SGC7901-pcDNA3. 1-GKLF group were less (P＜ 0. 05).Conclusion Transfected with GKLF maysuppress proliferation and invasion in human gastric carcinoma cell line SGC-7901, inhibit the growth and the angiogenesis of xenograft in nude mice.

BACKGROUND: Pearl has the tranquilizing effect, and it can be applied in the treatment of hypertension. However, there is little report on the prevention and cure effect and mechanism of hydrolyzed pearl liquid on hypertension. OBJECTIVE: To observe the effect of hydrolysis of Hepu pearl (hydrolyzed Nanzhu fluid) on the biological behavior and secretion of human microvascular endothelial cells. METHODS: Human microvascular endothelial cells were cultured and passaged. There were four groups, and the microvascular endothelial cells were incubated in the 200 μL culture medium containing nothing (control group), and 120, 60 and 30 mg/L hydrolysis of Nanzhu fluid. The cell proliferation and migration was detected by cell conuting kit-8 assay and Transwell assay respectively; the cell cycle distribution was tested by flow cytometry; the cell apoptosis was assayed by TUNEL method; the secretion of nitric oxide and reactive oxygen species was tested by nitrale reduetase and chemical fluorescence method, respectively. RESULTS AND CONCLUSION: Compared with the control group, hydrolysis of Nanzhu fluid significantly promoted the proliferation of microvascular endothelial cells in a manner-dependent manner (P ＜ 0.05), suggesting the optimal concentration was 120 mg/L. Compared with the control group, the percentage of cells in S and G2 phase was significantly increased, and the percentage of cells in the G1 phase was significantly reduced in the hydrolysis of Nanzhu fluid group (P＜0.05), indicating hydrolysis of Nanzhu fluid could promote cell cycle progression. Apoptotic cells with green-stained nucleus were invisible in both groups. The number of cell migration in the hydrolysis of Nanzhu fluid group was significantly more than that in the control group (P ＜ 0.05). Compared with the control group, there was a significant increase in the nitric oxide secretion, and a significant decrease in the production of reactive oxygen species in the hydrolysis of Nanzhu fluid group (P＜0.05). To conclude, hydrolysis of Nanzhu fluid can promote the proliferation and migration of human microvascular endothelial cells in vitro, and has the function of promoting the secretion of nitric oxide and inhibiting reactive oxygen species secretion, implying its positive role in the protection of endothelial cell function.

OBJECTIVETo study the potential effects of exogenous WT1 gene isoform on the differentiation of leukemia cell line NB4 and its possible molecular mechanisms.

METHODSThe recombinant eukaryotic expression vector (pCB6 + /WTA) containing full-length human WT1 isoform (WTA: -17AA/ -KTS) cDNA and the blank pCB6 + vector were transfected into leukemia cell line NB4 by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Cell morphology, NBT reduction and CD11b antigen expression in NB4 cells were assayed to evaluate cell differentiation. Expression of PML/RARalpha, p21 and c-myc genes was determined by semi-quantitative RT-PCR after transfection.

RESULTSCompared with NB4/WTA cells, NB4 and NB4/CMV (NB4 cells transfected with pCB6 + vector) cells had higher morphological differentiation rates and higher CD11b expression levels after exposure to ATRA for 48 hours. The percentage of NBT reduction in NB4/WTA cells was lower than that in control groups. The difference in NBT reduction rate between NB4/WTA and control cells was gradually increased after treated with ATRA for three days. The expression levels of PML/RARalpha, p21 and c-myc genes in NB4/WTA cells were notably increased.

CONCLUSIONOverexpression of exogenous WTA gene could partially inhibit the differentiation of NB4 cells by up-regulating the expression of PML/RARalpha, p21 and c-myc genes.

Cell Cycle ; Cell Differentiation ; genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA, Messenger ; genetics ; Transfection ; WT1 Proteins ; genetics ; metabolism

In order to study the potential effects of exogenous WT1 gene isoform on apoptosis in leukemia cell line NB4 and its possible molecular mechanisms, the eukaryotic expression recombinant vector (pCB6(+)/WTA) containing full-length human WT1 isoform (WTA: -17aa/-KTS) cDNA and the vacant vector-alone were introduced into the leukemia cell line NB4 respectively by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Binding of Annexin V were tested by flow cytometry and agarose gel electrophoresis to verify whether exogenous WTA could induce apoptosis of NB4 cells. Expressions of p21, p53, bcl-2, bcl-XL and c-myc genes were determined by semi-quantitative RT-PCR after introducing recombinant vectors into the NB4 cells. The results showed that in exposure to As(2)O(3) at 0.8 micromol/L for 48 hours, the NB4/WTA cells exhibited the morphological hallmarks of apoptosis, the marked DNA ladder shown by gel electrophoresis, and the enhanced apoptosis rate marked by Annexin V. RT-PCR showed an increase in p21 and c-myc genes expression, a decrease in bcl-2 and a relative constant expression of p53, bcl-XL in NB4/WTA cells. It is concluded that the introduction and expression of exogenous WTA gene can lead to apoptosis of NB4/WTA cells by down-regulating the Bcl-2 gene expression and up-regulating the p21 and c-myc genes expression.
Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; WT1 Proteins ; genetics ; metabolism ; physiology ; bcl-X Protein ; genetics

BACKGROUND: Inadequate sources of islet cells mean that islet cell transplantation for diabetes cannot meet the clinical demand.Therefore,in vitro induction of pancreatic stem cells to differentiate into islets has become a focus of research. OBJECTIVE:To study the effect of Tripterygium wilfordii polysaccharides on the differentiation of pancreatic stem cells from islets in mice, so as to explore the effect of traditional Chinese medicine on the differentiation of pancreatic stem cells into pancreatic beta cells. METHODS:Tripterygium wilfordii polysaccharide was used to induce the differentiation of purified mouse pancreatic stem cells into islets in vitro.The islet-like cell clusters then underwent morphologic observation, dithizone (DTZ) staining, and western blot analysis. RESULTS AND CONCLUSION: Cell morphology, cell growth characteristics and immunocytochemical staining showed that mouse pancreatic stem cells were obtained.They were induced by Tripterygium wilfordii polysaccharide into spherical islet-like structures, which had a spindly pedicle connected with the bottom of the culture flask, and were DTZ-stained to iron red. Western blot assay detected β-cytokine proteins in the islet-like cell clusters. These findings confirm that mouse pancreatic stem cells can be induced to differentiate into islet-like cell clusters containing β cells in vitro by Tripterygium wilfordii polysaccharide.

OBJECTIVETo investigate Wilms' tumor gene (WT1) expression levels in bone marrow (BM) of acute leukemia patients (ALs).

METHODSA real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and internal reference GAPDH expression levels in BM of 108 ALs and 23 non-leukemia controls by Light Cycler.

RESULTSThe median expression levels of WT1 in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those in 23 ALs in complete remission (CR) and 23 non-leukemic controls (75.10 and 89.56 vs 2.07 and 1.51 respectively). No statistic differences was found between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, median WT1 expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias (M(5)), but there was no statistic differences among the M(1), M(2), M(3) and ALL subtypes. Furthermore the WT1 levels were not correlated to peripheral WBC counts, BM blast percentage and multidrug resistant gene (mdr1) expression at presentation, but correlated to chromosome karyotypes. Dynamic analysis of WT1 levels of 2 patients on treatment showed that WT1 expression levels predicted relapse.

CONCLUSIONWT1 expression levels in ALs were strikingly higher than that in non-leukemias. WT1 can be a marker for detecting MRD and evaluating therapy efficacy in leukemias.

Acute Disease ; Adolescent ; Adult ; Aged ; Bone Marrow Cells ; metabolism ; Child ; Female ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Leukemia ; blood ; genetics ; Leukemia, Monocytic, Acute ; blood ; genetics ; Leukemia, Myeloid ; blood ; genetics ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; WT1 Proteins ; genetics ; Young Adult

OBJECTIVEIn order to develop plans for effective intervention measures, prevalence and health-seeking behavior related to reproductive tract infection among floating married women of childbearing age in Fengtai district in Beijing were studied.

METHODSCross-sectional study was carried out. Two thousand and sixty-nine eligible women were randomly selected from strata based on their home provinces. From June to July 2001, the subjects were given face-to-face interview at the Fengtai family planning clinic in Beijing using standard questionnaire followed by gynecologic examination and laboratory tests.

RESULTSThirty point three percent of the subjects were found to have reproductive tract infections (RTI) by laboratory tests. Prevalence rates of bacterial vaginosis, candida and trichomonas vaginitis were 22.2%, 4.9% and 2.1% respectively. Prevalence rates of chlamydia, gonorrhea, condyloma acuminatum and syphilis were 2.2%, 1.6%, 0.5% and 0.2% respectively. Of these infected women, only 43.1% (270/626) were symptomatic, and 61.5% (166/270) of these women with symptoms had sought treatment.

CONCLUSIONCompared to other results in the literature, we found a relatively high prevalence of RTI in our study population. Only a small proportion of these infected women were symptomatic but only few of them sought treatment. We suggested that the provision of more family planning service and promotion of RTI knowledge to the floating women of childbearing age.

Adolescent ; Adult ; China ; epidemiology ; Cross-Sectional Studies ; Female ; Humans ; Infection ; epidemiology ; Middle Aged ; Prevalence ; Surveys and Questionnaires ; Travel ; Trichomonas Vaginitis ; epidemiology ; Urban Health ; Vaginitis ; epidemiology ; microbiology ; Vaginosis, Bacterial ; epidemiology ; Women's Health Services

OBJECTIVETo explore the mechanism of action of RbAp46 gene on leukemic cells.

METHODSK562 leukemic cells and SHG44 glioma cells were transfected with eukaryotic expression vector carrying full-length cDNA of RbAp46 driven by the cytomegalovirus promoter mediated by lipofectamine transfection reagent. Empty vector were transfected at the same time as control. G418-resistant colonies were selected after 3 weeks culturing. Series genes were amplified using RT-PCR. Growth curve and colony formation assays were performed.

RESULTSThe number of K562/RbAp46 and K562/CMV cells were (90.00 +/- 8.40) x 10(4) and (119.58 +/- 9.87) x 10(4), respectively after 4 days growth, and SHG44/RbAp46 and SHG44/CMV cells were (89.13 +/- 4.88) x 10(4) and (149.42 +/- 10.83) x 10(4), respectively after 5 days growth. Seven-day yields of K562/RbAp46 and K562/CMV cell colonies were 131.67 +/- 15.57 and 250.33 +/- 26.31, respectively (P < 0.01), while those of SHG44/RbAp46 and SHG44/CMV cells were 50.78 +/- 6.77 and 206.67 +/- 37.18, respectively (P < 0.01). The fraction of K562/RbAp46 and K562/CMV cells in S phase was (48.88 +/- 4.35)% and (62.78 +/- 4.78)% (P < 0.01), and in G(0)/G(1) phase was (29.10 +/- 4.14)% and (22.40 +/- 2.43)%, respectively (P < 0.05), and that of SHG44/RbAp46 and SHG44/CMV cells in G(0)/G(1) phase was 65.6% and 48.8%, and in S phase was 22.6% and 38.4%, respectively. Insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1) gene was induced to express only in the RbAp46-over expressing K562 cells and was not in SHG44 cells.

CONCLUSIONA regulatory pathway between RbAp46 and IGFBP-rP1 genes might exit in K562 leukemic cells.

Carrier Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Insulin-Like Growth Factor Binding Proteins ; genetics ; K562 Cells ; Nuclear Proteins ; genetics ; Retinoblastoma-Binding Protein 7 ; Transfection

The purpose of this study was to investigate the effect of simvastatin (SIM) on proliferation and apoptosis of acute monocytic leukemia cell line SHI-1 and its mechanism. Experiments were divided into control and test groups (5 µmol/L, 10 µmol/L, 20 µmol/L SIM groups). The growth inhibitory rate of SHI-1 cells was detected using methyl thiazolyl tetrazolium (MTT) method. The cell cycle distribution and apoptotic rate were measured by using flow cytometry. The expression of BCL-2, caspase-3 mRNA were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of BCL-2, caspase-3 protein levels were analyzed by Western blot. The results demonstrated that SIM inhibited the growth of SHI-1 cells in time- and does-dependent manners. Cell cycle analysis showed that SHI-1 cells significantly arrested in S phase (p < 0.05) after treating with SIM for 48 hours, as compared with control group. 5 µmol/L SIM in test group significantly blocked cell cycle progression, but can not induce apoptosis. The expressions of BCL-2 mRNA and protein were down-regulated and caspase-3 mRNA and protein were up-regulated along with the increase of SIM concentration (p < 0.05). It is concluded that SIM is able to inhibit proliferation and induce apoptosis of SHI-1 cells, the mechanism may be associated with downregulating the expression of apoptosis-related gene BCL-2, upregulating the expression of caspase-3.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Simvastatin ; pharmacology

OBJECTIVETo study the expression of annexin II (AnnII) and the fibrinolytic activity in NB4 cells and their alterations in the presence of arsenic trioxide (ATO) and daunorubicin (DNR).

METHODSLeukemia cell line NB4 was treated with ATO or DNR for 24 ∼ 72 h. Cell surface expression of AnnII and its mRNA were analysed by flow cytometry and real time PCR, respectively, the fibrinolytic activity by chromogenic assay.

RESULTSCompared with other acute leukemia cell lines, the expression of AnnII on untreated NB4 cells was relatively higher. The AnnII positive cell rates on NB4, HL-60, K562, and A3 cells were (94.5 ± 1.6)%, (40.1 ± 2.1)%, (36.3 ± 1.5)% and (11.8 ± 2.5)%, respectively. The fibrinolytic activity of NB4 cells was the greatest with a A value of 0.68 ± 0.02. The fibrinolytic activity of NB4 cells was obviously decreased by ATO, DNR or monoclonal antibody against AnnII, being decreased by 60.4%, 35.8% and 26.0% of the pretreatment level, respectively. The expressions of AnnII and its mRNA in NB4 cells were decreased dramatically after ATO and DNR treated for 48 h. Annexin II positive cells rate were (55.46 ± 4.72)% and (27.00 ± 6.18)%, respectively.

CONCLUSIONNB4 cells have strong ability to enhance the catalytic efficiency of the t-PA-dependent plasminogen activation and AnnII on the cell membrane contributes to this activity. Its high fibrinolytic activity can be corrected by ATO and DNR through down-regulating AnnII.

Annexin A2 ; Apoptosis ; Daunorubicin ; HL-60 Cells ; Humans ; Leukemia ; metabolism