Dynamics of Proliferation and Differentiation after Transplantation of Hematopoietic Cells in Mouse / 中国实验血液学杂志

OBJECTIVE@#To observe the dynamic changes of hematopoietic reconstitution and multiple lineages differentiation at early phase after transplantation.@*METHODS@#Whole bone marrow mononuclear cells (wBMMNC, 5×10) and enriched c-Kit hematopoietic stem/progenitor cells (HSPC, 3×10) from the BM of B6-Ly5.1 mice were transplanted into lethally irradiated B6-Ly5.2 mice, the frequencies and absolute numbers of donor-derived cells (including LKS and LKS) were detected by flow cytometry. The multiple lineages differentiation of donor-derived cells was also monitored by flow cytometry. The homing and early phase proliferations of donor-derived cells were observed by two-photon microscope.@*RESULTS@#The donor-derived cells started to proliferation from 5-7 days after transplantation and reached the peak value at 2-3 weeks after wBMMNC transplantation. The donor-derived cells proliferated from 1-2 weeks and maintained until 4 weeks after c-kitHSPC transplantation. At 1 week after transplantation, the donor-derived cells mainly differentiated into myeloid cells with a few lymphoid cells production (B cells) but the production of T cells was not observed at most in wBMMNC transplanted group, while myeloid cells occupied the majority of donor-derived cells at 2-4 weeks; donor-derived cells almost totally differentiated into myeloid cells at 1-3 weeks after transplantation in c-Kit transplanted group and donor-derived B cells appeared at 4 weeks. The absolute number of donor-derived LKS and LKS cells in the BM of c-Kit transplanted group were much higher than that of wBMMNC group (P＜0.001) at 2 weeks respectively. The clustering proliferation of cKit cells at 4-5 days after transplantation was observed by two photon microscope.@*CONCLUSION@#The dynamical rate of proliferation and reconstitution of donor-derived cells are much earlier and quicker in c-Kit group than those of wBMMNC group. c-Kit cells mainly differentiate into myeloid cells within 1-3 weeks and the lymphoid cell differentiation starts at 4 weeks after transplantation. The immediate proliferation and differentiation of c-Kit cells within 1 week maybe due to the urgent needs of hematopoietic regeneration under the myeloablated hosts.

Preparation of monoclonal antibodies against Carprofen / 中国免疫学杂志

Objective:Preparation of monoclonal antibodies against Carprofen,which lays a foundation for the rapid detection of Carprofen.Methods:Via an active ester method,Carprofen was conjugated to bovine serum albumin (BSA) as immune antigen and ovalbumin (OVA) as detection antigen,respectively.Hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from the mice immunized with Carprofen-BSA.Hybridomas 51C3 secreting antibodies against Carprofen were obtained and subcloned.Results: Ascites of monoclonal antibodies (McAb) were prepared by injecting cells of hybridoma 51C3 into mice abdomen.The titer of purified McAb was 3.2×105and the McAb was IgG1 subtype.McAb was highly specific for Carprofen and the cross-reactivity (CR50%) was less than 0.04% with Ibuprofen, Ketoprofen, Naproxen, Feinuoluofen, Indoprofen, Fenbufen respectively.The sensitivity of the McAb to carprofen was 0.32 ng/ml and the IC50value was 0.882 ng/ml.The recoveries of Carprofen in the pork samples were from 81.4% to 104.4 % and coefficients of variation were from 16.3% to 25.8%.Conclusion:All results indicate that the McAb 51C3 is suitable to develop an immunoassay colloidal gold rapid dipstick test.

Investigation of the cognition and behavior on drug safety in Beijing middle school students / 北京大学学报(医学版)

Objective:To understand the cognition and behavior of drug safety in Beijing middle school students and provide advice for relevant education.Methods:A cross-sectional survey using paper questionnaires was carried out on the student body of nine Beijing middle schools.Multi-stage proportionate stratified cluster sampling was adopted to enroll participants.In addition to demographic questions,the questionnaire included 17 questions assessing the cognition and behavior of safe drug use,prioritizing questions that aligned with the health education guideline for primary and secondary school students from Chinese Ministry of Education.Descriptive statistical methods were applied using the SAS 9.2 software.Results:Of the 4 220 students investigated,2 097(49.7％) were males and 2 123(50.3％) were females.The average age was (14.3 ± 1.7) years.2 030(48.1％) students were from downtown areas,1 511(35.8％) were from urban-rural linking areas and 679(16.1％) were from rural areas.Half (51.5％) of the respondents were junior high school students,and the others were from senior high schools (34.2％) and vocational high schools (14.3％).Most of the students (89.6％) lived off campus.The awareness rate of drug safety knowledge was 74.4％,the median score of drug safety behavior was 4 points (full score was 5 points) and there was a statistically positive correlation between the two (Spearman's correlation coefficient was 0.156,P ＜0.001).Both the awareness rates and the drug safety behavior scores were statistically different among the students in different regions,different school types and different residence types (P ＜ 0.001).Multiple factors analysis demonstrated the correlation between the cognition degrees of both drug safety knowledge,behavior and the above factors.Of all the students,80.4％ agreed that any drug could have adverse drug reactions;40.5％ were aware that antibiotics couldn't kill viruses;as many as 49.6％ mistook aspirin as antibiotic;97.4％ would read drug instructions before taking them;Only 42.4％ put expired drugs into special recycling bins;49.8％ would deviate from the suggested dosage and frequency of their medication when they were sick with common diseases.Conclusion:Overall,the cognition of drug safety in Beijing middle school students is good,but problems still exist in medication adherence,the management of expired drugs and the antibiotics cognition,which need to be fixed through specific,pointed way of education.And more efforts should be made to improve the cognition in rural regions,vocational high schools and on campus students.

Prevention of aseptic loosening prothesis of alendronate in beagle:abiomechanical study / 国际外科学杂志

Objective To investigate effect of alendronate on prevention of aseptic loosening prothesis.Method Twelve beagles which had been implanted a titanium plug in femur were divided into two groups randomly,one received alendronate,and the other placebo as control.After 12 weeks,all beagles were sacrificed.specimens of femur were studied by biomechanical test.Independent sample t test was used for statistical analyses.Results The pull-out strength of the experimental group was higher than that of the control (P ＜ 0.05),and the torsion strength was also higher in the experimental group(P ＜ 0.05).Conclusion The therapy of alendronate can change the bone strength,inhibit aseptic loosening of prosthesis in beagle and could be a good drug for the prevention and treatment of aseptic loosening prothesis.

Effects of grain-sized moxibustion on expression of Abeta(1-42) in prefrontal cortex and hippocampus in double-transgenic AD mice / 中国针灸

<p><b>OBJECTIVE</b>To explore the therapeutic effect of grain-sized moxibustion at "Xinshu" (BL 15) and "Shenshu" (BL 23) on early-stage Alzheimer's disease (AD) in transgenosis AD mice.</p><p><b>METHODS</b>The genotyping of amyloid precursor protein/presenilin 1(APP/PS1I) double-transgenic AD mice were detected by PCR method. Seventeen 1.5-month female transgenic (Tg 6799) mice were randomly divided into a model group (9 cases) and a treatment group (8 cases). Nine female C57BL/6J wild-type mice with identical age and background were selected into a normal group. The treatment group was treated with grain-sized moxibustion at bilateral "Xinshu" (BI. 15) and "Shenshu" (BL 23), once a day, ten treatments were considered as one course, and total 9 courses were given. The model group and normal group were treated with stimulus such as grabbing, immobilization and non-ignited moxa cone. Morris water maze (escape latency, crossing times and dwell time in the target quadrant) was applied to evaluate the learning and memory ability. Hematoxylin-Eosin (HE) staining was used to observe morphology changes in the brains of AD mice. beta-amyloid protein 1-42 (Abeta(1-42)) in the area of prefrontal cortex and hippocampus was detected by immunohistochemical method.</p><p><b>RESULTS</b>After the treatment of grain-sized moxibustion, learning and memory ability in the treatment group was increased; compared with the model group, the escape latency was shorten, crossing times was increased, and dwell time in the target quadrant was prolonged (all P<0. 05). The crossing times and dwell time in the target quadrant in the treatment group were not significantly different from those in the normal group (both P>0.05). Compared with the normal group, the positive area and integral optical density of Abeta(1-42) in prefrontal cortex and hippocampus in the model group were increased (all P<0.01). Compared with the model group, the positive area and integral optical density of Abeta(1-42) in prefrontal cortex and hippocampus in the treatment group were reduced (P<0.01, P<0.05).</p><p><b>CONCLUSION</b>The grain-sized moxibustion at "Xinshu" (BL 15) and "Shenshu" (BL 23) could significantly improve the learning and memory ability in APP/PS1 double- transgenic AD mice, and inhibit the over expression and accumulation of Abeta(1-42).</p>

Multi-central randomized controlled study on electroacupuncture at Fenglong (ST 40) for regulating blood lipids / 中国针灸

<p><b>OBJECTIVE</b>To investigate the clinical effects of electroacupuncture (EA) at Fenglong (ST 40) on blood lipids.</p><p><b>METHODS</b>Two hundred and four patients of hyperlipidemia were randomly divided into a Fenglong group and a Xuezhikang group, 102 cases in each group. The patients in the Fenglong group were treated with electroacupuncture at Fenglong (ST 40). After arrival of qi, the needles were connected with acupoint nerve stimulator (LH 202 H type, HANS). The primary parameters of EA: for high triglycerides (TG) type, AM 50 Hz, intensity 1 mA, needle-retained time 20 min, twice per week; for high cholesterol (CHO) type, AM 100 Hz, intensity 1 mA, needle-retained time 30 min, thrice per week; for high low-density-lipoprotein (LDL-C) type, the same parameters as the high CHO type except the tolerable and comfortable intensity; for the mixing type, corresponding methods were alternatively used. The patients in the Xuezhikang group received Xuezhikang capsule orally, 2 capsules each time and twice daily, for total 11 weeks.</p><p><b>RESULTS</b>The total effective rates of the Fenglong group and the Xuezhi-kang group were 83.0% and 85.9%, respectively, with no significant difference between the two groups (P > 0.05), and there was no significant differences in the function of regulating blood lipids between the two groups (all P > 0.05). After one month follow-up survey, the total CHO, TG and LDL-C decreased and high-density-lipoprotein (HDL-C) increased, of which there was a significant difference in TG reduction (P < 0.05). There were no relapses in both groups.</p><p><b>CONCLUSION</b>EA at Fenglong (ST 40) can effectively regulate blood lipids with a better after-effect, which can be applied as a safe and effective method to replace medication for regulating blood lipids.</p>

ERK, JNK/AP-1 pathway was involved in silica-induced cell cycle changes / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the alteration of activator protein-1 (AP-1) luciferase activity in human embryo lung fibroblasts (HELF) after exposed to silica, and the role of mitogen activated protein kinase (MAPK)/AP-1 pathway on silica-induced cell cycle changes.</p><p><b>METHODS</b>After HELF cells were treated with 200 microg/ml silica, immunofluorescence assays were employed to detect the translocation and the phosphorylation level of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK), flow cytometry was used to detect the distributions of cell cycle, the dominant negative mutant of ERK, JNK and p38 were applied to detect the upstream or downstream relationship of signaling pathways.</p><p><b>RESULTS</b>After HELF-AP-1 cells were exposed to 200 microg/ml silica 6, 12, 24 h respectively, silica exposure lead to AP-1 activation in a time-dependent manner, inducing significant AP-1 activation at 6 h, reaching a maximum activation at 12 h, and having a little decrease at 24 h. After silica exposure 1 h, phosphorylation level of ERK and JNK increased mainly in cytoplasm, however, after exposure 2 h, they translocated to nucleus. The proportion of cells in G1 phases was decreased from (63.80 +/- 9.57)% to (32.23 +/- 7.22)%, and the proportion of cells in S phases was increased from (35.17 +/- 10.33)% to (66.00 +/- 8.07)% after exposed to silica 24 h. Curcumin, a chemical inhibitor of AP-1, impaired the decrease of cells in G1 phases. Furthermore we found expression of dominant-negative mutant of ERK and JNK impaired silica-induced AP-1 activation, whereas, dominant-negative mutant of p38 did not show the effect.</p><p><b>CONCLUSION</b>These result suggested that 200 microg/ml silica exposure can induce AP-1 activation, induce cell cycle changes through ERK, JNK/AP-1-dependent pathway.</p>

Effects of p53 in benzo (a) pyrene induced p21 and E2F-1 expression and cell cycle changes / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the roles of p53 in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo(a) pyrene[ B(a) P], and relationships between p53 and p21, E2F-1.</p><p><b>METHODS</b>Cells transfected with p53 siRNA plasmid (p53-H) and CMV vector (HELF/CMV) were cultured with serum-free R/MINI-1640 for 48 hours, then treated with 2 micromol/L B(a)P for 24 hours. Flow cytometry assay was used for detecting the cell cycle alteration after being exposed to B(a)P. Changes of p53 and p21 expressions were checked using Western blot assay, and the cytoplasmic and nuclear extraction was used to observe the subcellular localizations of p53 and p21. The immunofluorescence assay was used to check changes of E2F-1 expression and the distribution of E2F-1 in nuclear and cytoplasm after exposed to B (a)P. p53siRNA plasmid and the chemical inhibitor of p53 [pifithrin-alpha (PFT)] were used to observe effects of p53 in B(a)P induced cell cycle changes and the relationships of p53 and p21, E2F-1.</p><p><b>RESULTS</b>After 2 micromol/L B(a)P exposure, the ratio of G1 phase cells (71 +/- 5)% was decreased to (39 +/- 4)% (P < 0.05). p53, p21 and E2F-1 expressions were increased significantly, and over expressed proteins were mostly located in nuclear after B(a)P treatment. When p53 expression was inhibited by p53 siRNA or PFT, the decreases of G1 phase in response to B(a)P treatment still existed, and over expression of p21 induced by B(a)P was attenuated, especially in nuclear, but E2F-1 over expression was not changed significantly.</p><p><b>CONCLUSION</b>B(a)P could induce cell cycle changes through p53 independent pathways. And p53 could regulate p21 expression positively, but not E2F-1.</p>

Activated protein 1-cyclin D1/E2F 1 pathways involved in cell cycle changes induced by benzo (a) pyrene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the roles of activated protein 1 (AP-1) in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo (a) pyrene [B (a) P], and relationships between AP-1 and cyclin D1/CDK4-E2F-1/4.</p><p><b>METHODS</b>Cells transfected with AP-1 luciferase reporter plasmid (AP-H) were cultured with serum-free RPMI1640 for 48 h, and treated with 2 micromol/L B (a) P for 24 h. AP-1 relative activity was detected by luciferase assay. Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometer and Western blot assay.</p><p><b>RESULTS</b>After B (a) P was treated for 24 h, the ratio of G1 phase cells (71 +/- 2)% was decreased to (48 +/- 3)% (P < 0.05), and an increase was observed in the ratio of S phase. AP-1 activity and cyclin D1/E2F-1 expression were increased significantly, but CDK4/E2F-4 expression did not change after B (a) P treatment. When AP-1 activity was inhibited by curcumin, decreases of G1 phase in response to B (a) P treatment were blocked, and overexpression of cyclin D1/E2F-1 was attenuated, but CDK4/E2F-4 expression was not changed significantly.</p><p><b>CONCLUSION</b>AP-1 is involved in B (a) P induced cell cycle changes, and is the upstream signals of cyclin D1/E2F-1, but not CDK4/E2F-4.</p>

Study on optimization parameters of electroacupuncture at Fenglong (ST 40) for adjusting blood lipids / 中国针灸

<p><b>OBJECTIVE</b>To study the effects of different parameters (frequency, intensity, needle-retained time and treatment interval) of electroacupuncture at Fenglong (ST 40) for adjusting blood lipids, so as to find out the optimization parameter.</p><p><b>METHODS</b>Fifty-four cases meeting the criteria for hyperlipoidemia were randomly divided into 27 groups with orthogonal design L27 (3(13) ). According to the orthogonal design program they were treated with electroacupuncture at Fenglong (ST 40). Ten sessions constituted one course with a one week's interval between two course. The treatment was given for 2 courses.</p><p><b>RESULTS</b>(1) The parameters of EA at Fenglong (ST 40) for regulating blood lipids in primary and secondary orders are: frequency, needle-retained time, interval of treatment, intensity. (2) The parameters of EA at Fenglong (ST 40) for various programs in regulating various blood lipids are: for TG, frequency AM 50 Hz, needle-retained time 20 ain, intensity 1 mA, twice each week; for TC, frequency AM 100 Hz, needle-retained time 30 min, intensity 1 mA, once every other day; for LDL-C, frequency Am 100 Hz, needle-retained time 30 min, intensity tolerable and comfortable, once every other day.</p>

c-Jun NH2-terminal kinase and extracellular signal-regulated protein kinase signaling pathways in regulation of benzo(a)pyrene-induced c-Jun activation in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of mitogen activated protein kinases (MAPKs) signaling pathways in the regulation of benzo(a)pyrene (B(a)P)-induced c-Jun activation in human embryo lung fibroblasts (HELFs).</p><p><b>METHODS</b>HELFs were cultured with 2.0 micromol/L B(a)P for various time (0, 3, 6, 12, 24 h) or with various concentration of B(a)P (0.0, 0.5, 1.0, 2.0 micromol/L) for 12 h. Western blot was performed to examine the effect of B(a)P on c-Jun activation. The dominant negative mutants of p38, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) were applied to establish stable transfectant, and to detect the relationship of MAPK signal molecules and c-Jun activation in B (a) P-treated cells.</p><p><b>RESULTS</b>B(a)P treatment resulted in a marked activation of c-Jun in time-dependent manner with a peak at 12 h (the densitometric ratios of phosphorylated c-Jun Ser63, Ser73 to actin were 20.1, 15.2 times for control respectively) and in dose-dependent manner. However, there was no evident change on total c-Jun expression in B(a)P-treated HELFs. Moreover, B(a)P-induced activation of c-Jun was inhibited by stable expression of dominant negative mutants of JNK or ERK, but not by dominant negative mutant of p38.</p><p><b>CONCLUSION</b>JNK and ERK signaling pathways, but not p38 pathway regulate B(a)P-induced c-Jun activation in HELFs.</p>

Benzo (a) pyrene-induced human embryo lung cell cycle alterations through positive regulation of mitogen-activated protein kinase signal pathways / 中华预防医学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>ERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.</p>

The activation of MAPK/cyclin D1-CDK4 signaling pathway caused by quartz / 中华劳动卫生职业病杂志

<p><b>OBJECTIVES</b>To study the phosphorylation level of mitogen activated protein kinase (MAPK) in human embryonic lung fibroblasts (HELF), and the expression level of cyclin D1-CDK4 protein in S-HELF and whether the expression level of cyclin D1-CDK4 protein mediated by MAPK/AP-1 signaling pathway in S-HELF.</p><p><b>METHODS</b>Two kinds of treatment: (1) Cells were harvested after stimulation 2 h for the detection of cytokines. (2) Cells were stimulated by quartz for a long time (2 months) for transformation characters (S-HELF). The MAP kinase was detected by western blot. Cyclin D1 and CDK4 (cyclin dependent kinase 4) proteins was measured by immunocytochemistry. Flow cytometry was used to evaluate the alternation of cell cycle.</p><p><b>RESULTS</b>Crystalline quartz could cause the phosphorylation level of ERKs, p38K, and JNKs in HELF increase. However, activated levels of ERKs and p46 of JNKs increased in S-HELF, and p38K activation decreased, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Cyclin D1 and CDK4 protein expression levels increased in S-HELF as compared with parental HELF. Inhibition of ERKs activation by AG126, AP-1 by curcumin, and JNKs by SP600125 could reduced the induction of cyclin D1 and CDK4, whereas inhibition of p38K by SB203580 did not show any inhibitory effects on S-HELF.</p><p><b>CONCLUSIONS</b>The phosphorylation levels of ERK1/2, JNK1/2, and p38 increased in HELF exposed to quartz. The phosphorylation levels of ERK1/2 and JNK1 increased, but the phosphorylation level of p38 decreased in S-HELF. The expression level of cyclin D1-CDK4 protein increased in S-HELF. Overexpression of cyclin D1-CDK4 is due to the activation of ERKs, JNKs/AP-1 signaling pathway in S-HELF.</p>

ERK and JNK/AP-1 pathways involved in benzo(a)pyrene induced cell cycle changes in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the role of mitogen activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway in benzo(a)pyrene (B(a)P)-induced changes of cell cycle in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>AP-1 luciferase activity was determined by the Luciferase reporter gene assay using a luminometer. The expression levels and activity of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. The dominant negative mutant of ERK2, JNK1 and p38 were applied to detect the upstream or downstream relationship of signaling pathways.</p><p><b>RESULTS</b>B(a)P treatment resulted in a marked activation of AP-1 and its upstream MAPK, including ERK, JNK and p38 in human embryo lung fibroblasts (HELF). B(a)P exposure also led to increase the population of cells at S phase compared to control (P < 0.01) with a concomitant decline of cells at G(1) phase. B(a)P-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutant of ERK2 or JNK1, but not p38. B(a)P-induced AP-1 transactivation was inhibited by the overexpression of dominant-negative mutant of ERK2 or JNK1, but not p38. Inhibition of the activation of AP-1 by curcumin, a chemical inhibitor of AP-1, significantly inhibited the cell cycle changes in response to B(a)P treatment.</p><p><b>CONCLUSION</b>ERK and JNK, but not p38, mediated benzo(a)pyrene-induced cell cycle changes by AP-1 transactivation in HELF.</p>

Vitamin C reverses benzo (a) pyrene-induced cell cycle changes by E2F pathway / 中华预防医学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>B (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.</p><p><b>CONCLUSION</b>Vitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.</p>

Vitamin C inhibits benzoapyrene-induced cell cycle changes partly via cyclin D1/E2F pathway in human embryo lung fibroblasts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>B[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.</p><p><b>CONCLUSIONS</b>B[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.</p>

Inhibition of the pathway of benzo (a) pyrene-induced cell cycle changes by all-trans retinoic acid in lung fibroblast / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>After 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.</p><p><b>RESULT</b>After 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.</p><p><b>CONCLUSION</b>ATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.</p>