Adolescent ; Adult ; Child ; Humans ; MELAS Syndrome ; diagnosis ; pathology ; Magnetic Resonance Imaging ; Male ; Tomography, X-Ray Computed

OBJECTIVETo study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELF) cells.

METHODSThe stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle.

RESULTSB[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C (10, 100, 500, 1000, and 5000 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone.

CONCLUSIONSB[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.

Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; Blotting, Western ; methods ; Cell Cycle ; drug effects ; physiology ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; G1 Phase ; drug effects ; physiology ; Humans ; Lung ; cytology ; embryology ; RNA, Antisense ; genetics ; S Phase ; drug effects ; physiology ; Transfection ; methods

OBJECTIVETo study the role of cyclinD1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line (2BS) induced by silica.

METHODSRecombination vectors with sense and antisense pXJ41-cyclinD1 and pXJ41-CDK4 were constructed, and then transfected into the malignant transformed cells induced by silica, respectively. At the same time, pXJ41-neo was used as the control.

RESULTSDuring the progress of the malignant transformation of 2BS cells induced by silica, cyclinD1 and CDK4 were overexpressed. Antisense RNA suppressed cyclinD1 and CDK4 gene expression in the antisense pXJ41-cyclinD1 and pXJ41-CDK4 transfected cells. Antisense RNA led to cell cycle arrest, resulting in lengthened G1 phase (the percentages of cells in the G1 phase changed from 45.1% to 52.7% and 58.0% for cyclinD1 and CDK4 transfected cells, respectively), and eventually attenuated the increase of the proliferation of malignant transformed cells induced by silica. Compared with malignant transformed cells induced by silica, cells transfected with antisense pXJ41-cyclinD1 and pXJ41-CDK4 showed obviously reduced growth rates. On the 8th day, the suppression rates were 58.69 and 77.43% (the growth rate of malignant transformed cells induced by silica was 100%), doubling time changed from 21.0 h to 31.4 h and 21.0 h to 42.7 h, respectively, the growth capacities on soft agar of cells transfected by antisense pXJ41-cyclinD1 and pXJ41-CDK4 decreased obviously.

CONCLUSIONCyclinD1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.

Carcinogens, Environmental ; toxicity ; Cell Line ; Cell Proliferation ; Cell Transformation, Neoplastic ; chemically induced ; Cyclin D1 ; genetics ; metabolism ; physiology ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; physiology ; Humans ; Plasmids ; RNA, Antisense ; metabolism ; RNA, Messenger ; analysis ; metabolism ; Silicon Dioxide ; toxicity

OBJECTIVETo genotype the RHCE gene of Hans, Xinjiang's Uigurs and Kazakstans in China, and to compare the results of RHCE genotyping with that of RhCc/Ee phenotyping.

METHODSRHCE genes of 98 Hans with RhD positive and 230 Hans, 72 Uigurs and 18 Kazakstans with RhD/RHD negative were genotyped with PCR-sequence specific primer (SSP) technique.

RESULTSThe results of RHE/RHe genotyping from samples with RhD positive and negative were in accord with that of phenotyping. It would result in 4.44% error using C-->G polymorphism at nt48 of RHCE gene to genotype RHCE, and 4.05% failure of detection using the 109 bp insertion to detectRHCE gene in Chinese Hans. The results of RHE/RHe genotyping in unrelated 72 Uigurs and 18 Kazakstans with RhD phenotype were consistent with that of phenotyping, and false positive and false negative were not found in genotyping in Uigurs and Kazakstans tested.

CONCLUSIONThe results of RHE/RHe and RHc genotyping were correct with PCR-SSP and accordant with that of phenotyping. Using the C48G polymorphism in exon 1 of RHCE to genotype RHC gene would result in false positive resulting from RHc mutation at this locus, and using the 109 bp insertion to genotype RHC gene would result in false negative because of the absence of the 109 bp. Therefore it is necessary to genotype RHC gene using more than two polymorphic loci.

Ethnic Groups ; genetics ; Genotype ; Humans ; Phenotype ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; blood ; genetics ; Serologic Tests ; methods

OBJECTIVETo study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.

METHODSThe phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.

RESULTSThe phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.

CONCLUSIONERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; embryology ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the etiology, pathology, and clinical characteristics of cryptogenic liver diseases in order to develop a pathogenic profile for clinical diagnosis and therapeutic design.

METHODSThe data of the 566 patients diagnosed with abnormal liver function and who had undergone liver biopsy at our institute between January 2006 to March 2010 were retrospectively analyzed. The Chi-squared (x²) test was used to assess disease correlation with sex and the rank sum test was used to assess disease correlation with continuous data since all data had asymmetric distribution.

RESULTSAmong the 566 patients, abnormal liver function was attributed to alcoholic liver disease (n=175; 30.92%), drug-induced or environmentally-induced liver disease (n=101; 17.84%), hereditary and metabolic disease (n=93; 16.43%), infectious hepatitis disease (n=84; 14.84%), fatty liver disease (n=53; 9.36%), and autoimmune liver disease (n=30; 53.00%). Thirty patients had unknown etiology, despite liver biopsy analysis. Among these disease subgroups, there were distinct correlations with sex, age, and levels of alanine transaminase (ALT) and gamma-glutamyltransferase (GGT). The autoimmune liver disease group was correlated with sex (q=9.14, 7.435, 5.071, 9.529, and 12.5, respectively; P less than or equal to 0.01). The alcoholic liver disease group and autoimmune liver disease group were correlated with age (vs. genetic metabolic disease group: q=17.254 and 10.302; infectious hepatitis group: q=17.523 and 10.697); drug/environmentally-induced liver damage group: q=9.170 and 5.266); fatty liver group: q=7.118 and 4.661) (P less than or equal to 0.01). In addition, the alcoholic and autoimmune liver disease groups were correlated with GGT levels (vs. genetic metabolic disease group: q=8.003; infectious hepatitis group: q=4.793; drug/environmentally-induced liver damage group: q=4.404) (P less than or equal to 0.01).

CONCLUSIONLiver pathology is important for the diagnosis of cryptogenic liver diseases. Patient age, sex, and biochemistry index may facilitate diagnosis and treatment in the absence of pathology.

Adolescent ; Adult ; Biopsy ; Child ; Child, Preschool ; Female ; Humans ; Liver ; pathology ; Liver Diseases ; classification ; diagnosis ; pathology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo study the role of cycline dependent kinase 4 (CDK4) in the malignant transformation of human fetal lung diploid fibroblast cell (2BS) induced by silica.

METHODSRecombination vectors with antisense pXJ41-CDK4 were constructed, and then were transfected into the malignant transformed cells induced by silica. In situ hybridization and immunohistochemistry were used to analyze the expression of CDK4. Cell growth curve, doubling time, cell cycle distribution and the growth capacities on soft agar were analyzed before and after antisense CDK4 RNA was transferred into malignant transformed cells induced by silica.

RESULTSDuring the malignant transformation of 2BS cells induced by silica, CDK4 gene was overexpressed. Antisense pXJ41-CDK4 transduction suppressed CDK4 gene expression in the antisense pXJ41-CDK4 transfected cells. Antisense CDK4 RNA led to cell cycle arrest, resulting in lengthened G1 phase (the percentages of cells in the G1 phase increased from 45.1% to 58.0%), and eventually attenuated the proliferation of malignant transformed cells induced by silica. At the 8th day, the suppression rates decreased by 77.43%. The doubling time prolonged from 21.0 h to 42.7 h. The growth capacities on soft agar of cells transfected by antisense pXJ41-CDK4 were decreased.

CONCLUSIONCDK4 might play an important role in maintaining the transformed phenotype of the cancer cells.

Cell Transformation, Neoplastic ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinases ; genetics ; physiology ; Embryo, Mammalian ; Fibroblasts ; cytology ; drug effects ; Humans ; Lung ; cytology ; Proto-Oncogene Proteins ; genetics ; physiology ; RNA, Antisense ; pharmacology ; RNA, Messenger ; genetics ; Silicon Dioxide ; toxicity

BACKGROUNDTo investigate the prognostic significance and role of coagulation factor V (CFV) levels in clinical diagnostic criteria for severe hepatitis.

METHODSThe CFV level and prothrombin activity (PTA) were tested by turbidimetry for 129 times in 58 patients with severe hepatitis. Comparative studies and clinical significance of CFV and PTA were analyzed by SPSS and SDAS softwares.

RESULTS1. The levels of CFV and PTA were 15.3%+/-9.7% and 23.5%+/-10.0%, respectively, at the onset of severe hepatitis. 2. The mortality of severe hepatitis gradually increased with the gradual decrease of CFV or PTA during the most severe stage of the illness (P=0.000). 3. The levels of CFV and PTA decreased continually and rapidly in patients who died but gradually increased in survivors. The decrease or increase of PTA preceded that of CFV on the exacerbation or convalescent stage. 4. Hepatic encephalopathy occurred in 14 cases (24.14%). In 10 cases, it occurred in the terminal stage of the illness, far later than the time of the decrease of CFV. 5. The level of CFV was closely related to PTA (the correlation coefficient was 0.812), the level of CFV was almost consistent with that of PTA.

CONCLUSION1. The level of CFV is an important prognostic indicator in severe hepatitis and is more specific than PTA. 2. Simultaneous determination of CFV and PTA may be helpful in earlier and more accurate diagnosis of severe hepatitis. 3. Possible use of CFV as one of the criteria for liver transplantation in patients with severe hepatitis should be studied.

Adult ; Aged ; Diagnostic Techniques and Procedures ; Factor V ; analysis ; metabolism ; Female ; Hepatitis ; diagnosis ; metabolism ; Humans ; Male ; Middle Aged ; Nephelometry and Turbidimetry ; methods ; Prognosis ; Prothrombin ; analysis ; metabolism ; Young Adult

OBJECTIVETo study the role of E2F1/4 pathway in vitamin C reversing benzo (a) pyrene [B (a) P]-induced changes of cell cycle in human embryo lung fibroblasts (HELF) and the relationship between E2F1 and cyclin D1/CDK4.

METHODSThe stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established to detect the relationship of signaling pathway. Cells were cultured and pretreated with vitamin C before stimulation with B (a) P for 24 hours. The expression levels of cyclin D1, CDK4, E2F1 and E2F4 were determined by Western blot and the band intensity was analysed as the relative value to control by using the Gel-Pro 3.0 software. Flow Cytometric Analysis was employed to detect the distributions of cell cycle.

RESULTSB (a) P significantly elevated the expression levels of cyclin D1, CDK4, E2F1 and E2F4 in HELF cells. Vitamin C decreased the expression levels of above proteins in B (a) P-stimulated HELF cells. The expression levels of these proteins in B (a) P-treated above transfectants were lower than those in B (a) P-treated HELF cells. The expression levels of above proteins with vitamin C combined with antisense cyclin D1 were decreased as compared to those with antisense cyclin D1 alone. B (a) P increased the percentage of S phase as compared to the controls [(41.1 +/- 0.2)% vs (33.5 +/- 3.2)%, P < 0.05]. Both vitamin C [(33.2 +/- 0.6)% vs (41.1 +/- 0.2)%, P < 0.05] and antisense cyclin D1 [(31.2 +/- 1.3)% vs (41.1 +/- 0.2)%, P < 0.05] suppressed the changes of cell cycle induced by B (a) P. Vitamin C combined with antisense CDK4 markedly suppressed B (a) P-induced changes of cell cycle as compared to those with antisense CDK4 alone.

CONCLUSIONVitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.

Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; antagonists & inhibitors ; toxicity ; Cell Cycle ; drug effects ; Cyclin D1 ; metabolism ; E2F1 Transcription Factor ; metabolism ; E2F4 Transcription Factor ; metabolism ; Humans ; Lung ; cytology ; embryology ; Signal Transduction

OBJECTIVETo explore the role of telomerase in asbestos dust induced malignant transformation of human embryonic lung fibroblasts in vitro.

METHODSHuman telomerase catalytic subunit (hTERT) was transferred into human embryonic lung fibroblasts (HELF). Chrysotile dust at concentration of 2.5 microg/cm(2) was added to HELF transduced with and without hTERT (HELF-T+), respectively, and their transduced foci were separated. Biological characteristics of the cells, telomerase activity, length of telomere and cell growth curve were observed. Colony forming test was performed on soft agar to evaluate the nature of transformation.

RESULTSThe hTERT gene was transferred into HELF steadily, and HELF-T+ was established. Malignant transformation occurred in both HELF and HELF-T+ by asbestos stimulation. Asbestos dusts could induce higher rate of transformations in HELF-T+ [(2.08 +/- 1.08)/utensil] than in HELF [(1.08 +/- 0.10)/utensil], P < 0.05. Telomerase activity in both transformed malignant cells and HELF-T+ was higher, as well as the longer length of telomere in them.

CONCLUSIONRate of malignant transformation in cells with more activity of telomerase and longer length of the telomere was higher after stimulation with asbestos, indicating telomerase could play an important role in asbestos induced human cells malignant transformation.

Asbestos, Serpentine ; toxicity ; Cell Transformation, Neoplastic ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; Embryo, Mammalian ; Fibroblasts ; pathology ; Gene Transfer Techniques ; Humans ; Lung ; pathology ; Telomerase ; genetics ; metabolism