To explore the feasibility of real-time quantitative PCR (QRT-PCR) for selecting cell strains which overexpress a certain transgene, expression level of RbAp46 was detected in transfected cell strains by using optimal real-time PCR with SYBR Green I. Meanwhile, semi-quantitative RT-PCR and Western blot were performed to compare with the QRT-PCR. The results showed that values of RbAp46(N) were 2064.42 +/- 253.47, 860.94 +/- 291.07, 234.456 +/- 31.08, 18.17 +/- 5.14 and 1.46 +/- 0.54 in K562/RbAp46, K562/CMV, SHG44/RbAp46 monoclone, SHG44/RbAp46 multiclone and SHG44/CMV, respectively. The results were consistent with that determined by semi-quantitative RT-PCR and Western blot. It is concluded that QRT-PCR provides a highly efficient and reproducible method for selection of transfected cell subclones at different level of transgene expression.
Blotting, Western ; Carrier Proteins ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; Nuclear Proteins ; genetics ; metabolism ; Organic Chemicals ; chemistry ; RNA, Neoplasm ; metabolism ; Retinoblastoma-Binding Protein 7 ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Transfection ; Transgenes ; genetics

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

Acute Disease ; Blast Crisis ; genetics ; Genes, Retinoblastoma ; Genes, Wilms Tumor ; Humans ; Leukemia ; genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Polymerase Chain Reaction ; U937 Cells

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

OBJECTIVETo study the DNA methylation status of WT1 gene promoter region in hematologic malignancy cell lines and its correlation with WT1 gene expression.

METHODS1. RT-PCR and methylation-specific PCR were performed for detecting WT1 gene expression and DNA methylation status in its promoter region in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines. 2. Treatment of U937 cells with 5-aza-CdR, a demethylation inducing agent and the changes in WT1 gene expression level and its promoter region methylation status were determined.

RESULTS1. HL-60, K562, KG-1, NB4 and SHI-1 cells showed higher levels while 8226, Jurkat, Raji, U266 and U937 cells showed extremely low levels of WT1 expression. DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cells. 2. The WT1 gene expression in U937 was enhanced after treatment with 5-aza-CdR accompanied with the decrease of methylated and the increase of unmethylated levels in its promoter region.

CONCLUSIONModulation of the DNA methylation status in WT1 promoter region is one of the epigenetic mechanisms for regulating its expression.

Cell Line, Tumor ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Hematologic Neoplasms ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; WT1 Proteins ; genetics

OBJECTIVETo investigate retinoblastoma (Rb) associated protein 46 (RbAp46) gene expression levels in bone marrow (BM) cells of leukemia patients.

METHODSReal-time quantitative reverse polymerase chain reaction (QRT-PCR) method was used for detecting RbAp46 expression levels in BM cells of 140 patients with acute leukemia (AL), 13 with chronic myelogenous leukemia in chronic phase (CML-CP), 7 with CML in blast crisis (CML-BC) and 32 with non-leukemic disorders.

RESULTSThe M-Estimators of RbAp46 were higher in 98 newly diagnosed ALs and 5 relapsed ALs than in 28 ALs in complete remission (CR) and 32 non-leukemic controls (178.23 and 213.65 vs 85.89 and 88.08, respectively). No statistic difference was found between the CR group and control group, or between the newly diagnosed group and relapsed group. The M-Estimators of RbAp46 in patients with CML-CP was 58.27, similar to that in control, but much lower than that in CML-BC (173.24). Among 98 newly diagnosed ALs, the M-Estimators of RbAp46 in M(3) and M(4) were the lowest in all of the subtypes. Furthermore, the RbAp46 expression levels were not correlated with the expression of the fusion genes of bcr/abl, PML-RARalpha, and multidrug resistant gene (mdr1), but were positively correlated with Wilms' tumor gene (WT1) expression levels and negatively with AML1/ETO fusion gene expression.

CONCLUSIONRbAp46 expression levels in ALs and CML-BC were strikingly higher than that in non-leukemias and CML-CP, and might participate in leukemogenesis.

Adolescent ; Adult ; Aged ; Carrier Proteins ; genetics ; metabolism ; Child ; Female ; Gene Expression ; Humans ; Leukemia ; genetics ; metabolism ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; metabolism ; Retinoblastoma-Binding Protein 7 ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVETo develop a human ovarian carcinoma SKOV3 model in severe combined immunodeficiency (SCID) mouse and to study its biologic characteristics.

METHODSHuman ovarian carcinoma SKOV3 cells were injected intraperitoneally into female SCID mouse to establish a transplantation model of human ovarian carcinoma. The biological characteristics, metastasis and morphology of transplanted tumors were studied.

RESULTAll tumors grew progressively with no sign of regression. The tumor cells spread around the peritoneal cavity and mainly on the diaphragm, mesentery, peritoneum and around the liver, which was confirmed by histopathology. The morphology, growth pattern and CA125 secretion of primary culture of transplanted cells remained as same as those of ovarian carcinoma cell line SKOV3.

CONCLUSIONAn intraperitoneal transplantation model of human ovarian carcinoma SKOV3 in SCID mice has been developed successfully, which can simulate the biological behavior of peritoneal metastasis of human ovarian carcinoma.

Animals ; Disease Models, Animal ; Female ; Humans ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Ovarian Neoplasms ; pathology ; ultrastructure ; Peritoneal Neoplasms ; secondary ; Transplantation, Heterologous

Objective To analyze the forensic characteristics of 24 psychiatric patients who died of long-term use of psychotropic drugs.Methods Cases of sudden death of psychiatric patients from2011 to 2016 were collected.The forensic characteristics of these sudden deaths were retrospectively analyzed by systematic investigation plus results of autopsy and toxics (drugs).Results Among the 24psychiatric patients who died of long-term use of psychotropic substances, the ratio of male to female was 1.7∶1, and the average age was (59.0±8.8) years.Fifteen patients had clear disease durations (14.4±8.2) years, and other the nine were known to have disease durations of over 3 years.The death time of 62.5％of patients was the daytime in working days, and 91.7％of the patients died at home.Most patients complained different degrees of physical discomfort before death.Patients with schizophrenia (20 cases) were the most common, followed by depression (4 cases).All patients had the history of taking psychotropic drugs, with clozapine and chlorpromazine being the mostly detected ones.The causes of death were mainly circulatory diseases (15 cases), with the most common being myocarditis (11 cases) followed by pneumonia (4 cases).Conclusion Critical attention should be paid to the risk of antipsychotics-induced sudden unexpected deaths for psychiatric patients, particularly for those with schizophrenia.

Objective To explore the effect and indication of splenectomy in liver transplantation.Methods From January 2001 to April 2006,260 patients underwent piggyback orthotopic liver transplantation(PBOLT),and 28 patients had undergone combined PBOLT and splenectomy(splenectomy group).These patients were compared to 56 randomly selected non-splenectomy patients from the same transplant period,meaningly two controls were se- lected for every non-spleneetomy case.Two groups were analyzed with respect to rate of infection and survival rate, as well as biopsy-proven acute allograft rejection within 30 days after transplantation.Results Rate of infection in the splenectomy group was higher than that in the non-splenectomy patients(85.7% vs 55.4%,P

Objective Constructing physiological-psycho-social three dimensional aging index system, and scale to provide a basis for evaluating the effects of human aging and anti-aging measures. Methods Evaluation, modification and determination of the second and third level indexed by Delphi and principal component analysis and other methods. The weight of each index was weighted by the analytic hierarchy process, and the reliability and validity were calculated on the basis of the preliminary scale. A large sample of empirical studied to determine the aging of different age groups standardized score distribution table. The best model for judging functional age was sought, based on a variety of biostatistical models. Results The aging scale was constructed by three levels, the first level included 3 indicators, the second level had 10 indicators, the third level included 51 indicators, and adopted by expert certification the national society. 3 184 valid questionnaires were obtained through epidemiological investigation, the average total score of aging was (46.93±11.07) points, and the scores of aging were positively correlated with age(r=0.785, P<0.001). The average scores of aging in each dimension were different and the difference was statistically significant (all P<0.001), and each dimension had significant difference with age. The growth model (R2=0.635) was selected, the curve of the relationship between the score of aging and age was fitted. Conclusions The three-dimensional human aging scale was scientific, feasible, and has good reliability and validity. The empirical results show that the comprehensive score of aging increase with age, and show certain characteristics and laws on the curve.