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

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

OBJECTIVETo explore the effects of excessive fluoride on the gene expression of rat osteoblasts.

METHODSRat osteoblasts were treated with 2.0 mmol/L of sodium fluoride (NaF) for two weeks in vitro, and difference in the gene expression between the NaF-treated and normal osteoblasts was compared with mRNA differential display (DD-PCR) technique.

RESULTSAmong the six differentially expressed gene fragments which had been cloned, expression of the ribosomal protein L5 gene, ATPase Na(+)K(+) transporting beta polypeptide 3 gene, karyopherin alpha 2 gene and cis-Golgi body p28 gene was lower and expression of ubiquitous-conjugating enzyme E2D 3 gene and a newly-discovered gene fragment in this study showed up-regulated in the NaF-treated osteoblasts of the rats.

CONCLUSIONSExpression of genes changed in the osteoblasts after treatment with fluoride for two weeks and most of them associated with synthesis, transportation and processing of protein. It suggested that excessive fluoride could affect the protein synthesis in osteoblasts by changing the expression of the related genes. A novel gene related to excessive fluoride exposure was also found.

Animals ; Base Sequence ; Cells, Cultured ; Cloning, Molecular ; DNA, Complementary ; genetics ; Fetus ; Gene Expression Profiling ; Molecular Sequence Data ; Osteoblasts ; cytology ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Sequence Analysis, DNA ; Skull ; pathology ; Sodium Fluoride ; toxicity

OBJECTIVETo investigate the effects of excessive intake of fluoride on the expression of type II collagen gene and types and morphological change of collagen fiber in the bone tissues of rats.

METHODSA rat model with fluorosis was established by adding 221 mg/L of sodium fluoride (NaF) to drinking water for the rats for 15 days, 30 days and two months, respectively. Type II collagen alpha1 (II) cDNA probe was prepared, and cDNA-mRNA in-situ hybridization was employed to detect change in expression of type II collagen mRNA in the bone tissues of rats with excessive intake of fluoride (221 mg/L NaF). Picrosirius-polarization method was used to observe types of collagen and morphology of collagen fiber in the bone tissues.

RESULTSChondroblasts were found in the femur and other bone tissues of the rats after exposure to fluoride. cDNA-mRNA in-situ hybridization showed that expression of type II collagen gene could be observed in the cytoplasm of chondrocytic lacuna and chondrified bone tissues. mRNA in collagen of chondrocytes of the rib cartilage reached the peak level 15 days after exposure to fluoride, and decreased gradually one month and two months after exposure. Polychromatic type II collagen, breakage of collagen fiber, disorder array and reduced content of type II collagen could be found in the bone tissues with picrosirius-polarization method.

CONCLUSIONSExcessive intake of fluoride could lead to changes in types and structure of collagen (cross-linkage) of bone tissues, which caused expression of type II collagen gene in the chondrified bone tissues and enhanced its expression in the rib cartilage tissues.

Animals ; Bone Diseases ; metabolism ; pathology ; Chondrocytes ; metabolism ; Collagen Type II ; biosynthesis ; genetics ; Fluoride Poisoning ; genetics ; metabolism ; pathology ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo investigate the reverse effect of all-trans retinoic acid (ATRA) on Benzo (a) pyrene (B (a) P)-induced cyclin D1, CDK4, E2F-1 and E2F-4 expression and cell cycle progression in human embryo lung fibroblast (HELF).

METHODSAfter HELF cells was treated with ATRA, they were exposed to 2 micromol/L of B (a) P. Western blotting was employed to detect protein expression level; the RNA transfection techniques was used to investigate ATRA-induced signal pathway; flow cytometry was used to detect cell cycle progression.

RESULTAfter treatment with 2 micromol/L B (a) P for 24 h, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF; the expression of E2F-4 and CDK4 were not changed markedly; pretreatment with 0.1 micromol/L ATRA for 24 h could efficiently decrease B (a) P-induced overexpression of cyclin D1 and E2F-1; stimulation to antisense cyclin D1 or antisense CDK4 by B (a) P could significantly impair E2F-1 up-regulation; pretreatment with ATRA, cells with antisense cyclin D1 or antisense CDK4 showed a less decrease in B (a) P-induced overexpression of E2F-1 compared to similarly treated control cells; flow cytometry analysis showed B (a) P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B (a) P-induced cell cycle progression by an accumulation of cells in the G(1) phase.

CONCLUSIONATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Lung ; cytology ; metabolism ; Signal Transduction ; drug effects ; Tretinoin ; pharmacology

OBJECTIVETo investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]P.

METHODSHuman embryo lung fibroblasts (HELFs) were treated with 2 micromol/L or 100 micromol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs). Cyclin D1, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle.

RESULTSAfter B[a]P treatment, the proportion of the first gap (G1) phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 micromol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs (A-D1 and A-K4) and T-HELFs (T-A-D1 and T-A-K4). After 2 micromol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4.

CONCLUSIONSCyclin D1/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 micromol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 micromol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.

Benzo(a)pyrene ; pharmacology ; Cell Cycle ; drug effects ; Cell Line ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F4 Transcription Factor ; metabolism ; Fibroblasts ; drug effects ; enzymology ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; enzymology ; metabolism

OBJECTIVETo study the role of cyclin D1 in malignant transformation of human embryonic lung diploid fibroblasts (HELF) induced by quartz.

METHODSpXJ41-cyclin D1 expressing sense and antisense cyclin D1 RNA were transinfected into malignant transformed HELF induced by quartz with DNA recombination and gene transduction. The expression of cyclin D1 was detected with hybridization in situ and immunohistochemistry methods to analyze changes in cell growth, double multiplication time, distribution of cell cycles, colony forming ability on soft agar, etc., before and after cyclin D1 transduction.

RESULTSDuring the process of malignant transformation of HELF induced by quartz, cyclin D1 gene was overexpressed. Antisense pXJ41-cyclin D1 RNA could suppress the growth and proliferation of malignant transformed cells induced by quartz. Growth speed of antisense pXJ41-cyclin D1 transinfected cells decreased by 58.69% on the 8th day in culture, as compared to malignant transformed cells induced by quartz, and its double multiplication time prolonged from 21.0 h to 31.4 h. Antisense cyclin D1 RNA led to cell cycle arrest, resulting in lengthened G1 phase (proportion of cells in phase G1 increased to 52.7% from 45.1% and that of cells in phase S decreased to 33.1% from 40.3%). Colony forming rate reduced significantly and size of colony became smaller.

CONCLUSIONSAbnormal expression of cyclin D1 in cells related to their malignant transformation induced by quartz. Highly expressed cyclin D1 could play an important role in maintaining the transformed phenotype of malignant cells.

Cell Transformation, Neoplastic ; Cells, Cultured ; Cyclin D1 ; biosynthesis ; genetics ; Embryo, Mammalian ; Fibroblasts ; cytology ; Humans ; Lung ; cytology ; Quartz ; toxicity

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

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

The present study aimed at investigating the effects of chronic multiple stress on learning and memory functions of rats. Adult male Wistar rats were randomly divided into stressed and control groups. Rats in the stressed group were irregularly and alternately exposed to the situation of vertical revolution, sleep deprivation, noise stimulation, and night illumination 6 h per day for 6 weeks to prepare a chronic multiple stressed model. Learning and memory performance of rats was measured by using Morris water maze first and Y-maze afterwards. Neurons in the dentate gyrus(DG), CA3 and CA1 regions of the hippocampus were stained by using Cresyl violet method and counted. The results showed that: (1) After chronic multiple stress, compared with the control rats, the escape latency to the hidden platform in Morris water maze was significantly shortened in stressed rats. In stressed and control groups, the escape latency periods were (15.89+/-9.15) s and (27.30+/-12.51) s, respectively, indicating that spatial memory of the stressed rats was stronger than that of the control ones. In brightness-darkness discrimination learning in the Y- maze, the correct trials and correct percentage of entering safe arm was remarkably increased in the stressed rats, the correct rates of stressed and control groups were (79.01+/-1.23)% and (66.12+/-1.61)%, respectively, indicating that brightness-darkness discrimination learning ability of the stressed rats was better than that of the control ones. (2) After chronic multiple stress, nerve cell density in DG, CA1 and CA3 of the hippocampus in stressed rats was higher than that of the control group, the cell densities in DG, CA1 and CA3 of the stressed and the control group were (223.78+/-26.52), (112.07+/-14.23) and (105.55+/-18.12) as well as (199.13+/-15.36), (92.89+/-13.69), and (89.02+/-15.77) respectively. These results suggest that the chronic multiple stress may enhance the capability of spatial memory and brightness-darkness discrimination learning of rats. Possible reasons for the chronic multiple stress-induced learning and memory enhancement of rats were also discussed.
Animals ; Hippocampus ; physiology ; Learning ; physiology ; Male ; Maze Learning ; Memory ; physiology ; Neuronal Plasticity ; physiology ; Rats ; Rats, Sprague-Dawley ; Spatial Behavior ; physiology ; Stress, Physiological ; physiopathology