OBJECTIVETo observe the effect of RNA interference on CHK1 and CHK2 expression and change of G2/M phase arrest in esophageal carcinoma cells after irradiation.

METHODSFour sequences short hairhip RNA (shRNA) of each CHK1 and CHK2 genes were constructed and connected with vector of pENTR/U6 plasmid, respectively, and then transfected into Eca109 cells with lipofectamine 2000 reagent. Protein and mRNA expression of CHK1 and CHK2 genes were detected with Western blotting and RT-PCR, respectively. Cell cycling was measured by flow cytometry after 5 Gy irradiation. Cell survival rate after 5 Gy irradiation was evaluated by clonegenetic assay.

RESULTSFour shRNA vector each of CHK1 and CHK2 genes were successfully constructed and transfected into Ecal09 cells, respectively. Protein expression of CHK1 and CHK2 were obviously decreased. Their mRNA expressions were also decreased after transfected with shRNA of CHK1 and CHK2. Arrest of G2/M stage in Eca109 cells were obviously decreased only in cells transfected with CHK1 shRNA but not with CHK2 shRNA at 12 h after 5 Gy irradiation. In first progeny Eca109 cells transfected with CHK1 and CHK2 shRNA, expression of CHK1 and CHK2 protein was also decreased. The level of phosphorylated CHK2-T68 expression was decreased at 1 h after 5 Gy irradiation, and at 72 h only transfected with CHK2 shRNA but not with CHK1 shRNA. Phosphorylation level of CHK1-S345 was not increased after transfected with CHK1 or CHK2 shRNA, but arrest of G2/M stage still remained at 12 h after 5 Gy irradiation and at 72 h accordingly. The cell survival rate was decreased in Eca109 cells transfected with CHK1 or CHK2 shRNA after 5 Gy irradiation.

CONCLUSIONAfter transfected with shRNA of CHK1 or CHK2, their expressions of mRNA and protein in Ecal09 cells are markedly inhibited and this inhibition effect can be observed in their first progeny cells and at least hold for 3 days. Arrest of G2/M phase can be reduced after irradiation when teansfected with shRNA of CHK1 and the radiosensitivity of Ec109 cells can be increased.

Blotting, Western ; Cell Division ; genetics ; physiology ; radiation effects ; Cell Line, Tumor ; Cell Survival ; genetics ; physiology ; radiation effects ; Checkpoint Kinase 1 ; Checkpoint Kinase 2 ; Esophageal Neoplasms ; genetics ; pathology ; physiopathology ; G2 Phase ; genetics ; physiology ; radiation effects ; Gamma Rays ; Genetic Vectors ; Humans ; Protein Kinases ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo explore the effect of static magnetic field (SMF) on the differentiation and proliferation of rat embryonic midbrain neurons cells.

METHODSThe micromass culture of rat embryonic midbrain neurons cell was applied to study the effect of varieties of SMF (1.0, 10.0, 50.0, 100.0, 200.0 mT) and FACS.

RESULTSSMF inhibited the differentiation of the cell without affecting cell proliferation. The concentration of 50% inhibition of cell differentiation (ICD(50)) was 25 mT. The concentration of 50% inhibition of cell differentiation (IVD(50)) was 45 mT.

CONCLUSIONSThe inhibition of SMF on embryonic midbrain neurons cells may be associated with impact of protein syntheses, and lipid peroxidation.

Animals ; Cell Differentiation ; radiation effects ; Cell Division ; radiation effects ; Cells, Cultured ; Electromagnetic Fields ; adverse effects ; Embryo, Mammalian ; Female ; Lipid Peroxidation ; radiation effects ; Mesencephalon ; cytology ; radiation effects ; Neurons ; cytology ; radiation effects ; Protein Biosynthesis ; radiation effects ; Rats ; Rats, Sprague-Dawley

To evaluate the effect of Ligustrazine on the expression of VEGF in bone marrow stromal cells (BMSCs) of radiation injured mice and to explore the effect of VEGF on the recovery of hematopoiesis and the mechanism of signal transduction, the protein expression of VEGF, focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) in BMSCs were assayed by Western blot, the cell cycle and apoptosis rate of BMSCs were tested by flow cytometry. The effect of Ligustrazine on the hematopoiesis was evaluated at the same time. The results showed that the protein expression of VEGF in BMSCs was decreased significantly after irradiation and increased slowly with the time. The value in Ligustrazine-treated group almost reached normal level, but it remained lower than that in control group on day 14. The changes of phosphorylated FAK and MAPK protein expression had the same tendency. After (60)Co gamma-irradiation, the BMSCs were arrested in G0-G1 phase and apoptosis rate increased; these values recovered slowly with the time and remained higher than that in normal control group on day 14. The recovery of these values in Ligustrazine-treated group was sooner than that in irradiated control group, and they almost reached to the normal levels on day 14. It is concluded that irradiation could inhibit the expression of VEGF in BMSCs and induce apoptosis. The Ligustrazine promotes the recovery of bone marrow microenvironment probably by increasing the expression of phosphorylated FAK and MAPK in BMSCs.
Animals ; Apoptosis ; drug effects ; Bone Marrow Cells ; drug effects ; metabolism ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Mice ; Protein-Tyrosine Kinases ; analysis ; Pyrazines ; pharmacology ; Radiation Injuries, Experimental ; drug therapy ; metabolism ; Radiation-Protective Agents ; pharmacology ; Stromal Cells ; drug effects ; metabolism ; Vascular Endothelial Growth Factor A ; analysis

OBJECTIVESince most reports on bystander effect have been only concerned with radiation-induced damage, the present paper aimed at disclosing whether low dose radiation could induce a stimulatory or beneficial bystander effect.

METHODSA co-culture system containing irradiated antigen presenting cells (J774A.1) and unirradiated T lymphocytes (EL-4) was established to observe the effect of J774A.1 cells exposed to both low and high doses of X-rays on the unirradiated EL-4 cells. Incorporation of 3H-TdR was used to assess the proliferation of the EL-4 cells, expression of CD80/86 and CD48 on J774A.1 cells was measured with immunohistochemistry and flow cytometry, respectively. NO release from J774A.1 cells was estimated with nitrate reduction method.

RESULTSLow dose-irradiated J774A.1 cells could stimulate the proliferation of the unirradiated EL-4 cells while the high dose-irradiated J774A.1 cells exerted an inhibitory effect on the proliferation of the unirradiated EL-4 cells. Preliminary mechanistic studies illustrated that the differential changes in CD48 expression and NO production by the irradiated J774A.1 cells after high and low dose radiation might be important factors underlying the differential bystander effect elicited by different doses of radiation.

CONCLUSIONStimulatory bystander effect can be induced in immune cells by low dose radiation.

Animals ; Antigen-Presenting Cells ; immunology ; metabolism ; radiation effects ; Antigens, CD ; immunology ; B7-1 Antigen ; immunology ; B7-2 Antigen ; Bystander Effect ; radiation effects ; Cell Division ; immunology ; Cell Line ; Coculture Techniques ; Dose-Response Relationship, Radiation ; Membrane Glycoproteins ; immunology ; Mice ; Nitric Oxide ; biosynthesis ; T-Lymphocytes ; immunology ; X-Rays

OBJECTIVETo study the effect of panax notoginsenosides (PNS) on the proliferation of hematopoietic progenitor cells (HPC) in mice with immune-mediated aplastic anemia.

METHODSBalb/c mice model of immune-mediated aplastic anemia was established by radiation with sublethal dose of 60Co following the intravenously infusing lymphocytes of DBA/2 mice. Model mice in the treated groups were treated separately with high, middle and low dose of PNS, 3.2 mg, 1.6 mg and 0.8 mg per day respectively by intraperitoneal injection. Model mice in the control group and normal mice in the normal control group were treated with normal saline. The peripheral white blood cell (WBC) count and pathological examination of bone marrow were carried out 12 days later, the bone marrow was taken to be incubated in semi-solid culture system for observing proliferation of HPC.

RESULTSPNS could (1) increase peripheral WBC count: as compared with that in the model control, WBC in the high, middle and low dose PNS groups was raised by (34.3 +/- 2.9)%, (29.2 +/- 1.7)% and (14.5 +/- 1.6)% respectively, P < 0.01 and P < 0.05; (2) improve the bone marrow inhibition: pathological examination showed in the model group, the hematopoietic structure was destroyed and replaced by fatty tissue, while in the PNS treated groups, the structure of marrow was rather complete and filled with abundant hematopoietic cells; (3) promote the proliferation of HPC: as compared with the model group, the colony formation of CFU-GM were increased by (64.4 +/- 2.8)%, (67.3 +/- 2.4)% and (21.9 +/- 1.8)% respectively and that of CFU-E increased by (31.9 +/- 3.6)%, (20.7 +/- 2.4)% and (12.8 +/- 2.6)% respectively in the three PNS treated group (P < 0.01 and P < 0.05).

CONCLUSIONPNS could enhance hematopoiesis by promoting proliferation of CFU-GM and CFU-E progenitors so as to improve the hematopoietic function in mice of immune-mediated aplastic anemia.

Anemia, Aplastic ; blood ; etiology ; immunology ; Animals ; Cell Division ; drug effects ; Female ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred DBA ; Panax ; Radiation Injuries ; Random Allocation

OBJECTIVETo investigate the effect of SS8, a monomer extracted from Spatholobus suberectus Dunn used for invigorating the circulation of blood, on proliferation of hematopoietic progenitor cells in mice with bone marrow depression.

METHODThe method of semi-solid culture with methylcellulose of CFU-GM, CFU-E, BFU-E, CFU-Meg was adopted in bone marrow depressed mice which were treated with SS8 for a long time.

RESULTThe experimental data demonstrated that the numbers of CFU-GM, CFU-E, BFU-E, CFU-Meg in bone marrow depressed mice were raised distinctly under the control of SS8 as compared with those of contrast group. The effect of SS8 became stronger as time went on and the dosage rose.

CONCLUSIONSS8 can distinctly stimulate the proliferation of hematopoietec progenitor cells in mice with bone marrow depression in a time-and-dosagedependent manner.

Animals ; Bone Marrow Cells ; radiation effects ; Cell Division ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Erythroid Precursor Cells ; drug effects ; radiation effects ; Fabaceae ; chemistry ; Female ; Hematopoietic Stem Cells ; cytology ; drug effects ; radiation effects ; Male ; Mice ; Plants, Medicinal ; chemistry ; Whole-Body Irradiation

To determine whether gamma irradiation influences phenotype and function of human dendritic cells (DC) in vitro, dendritic cells were induced from the peripheral blood mononuclear cells of multiple sclerosis patients with RPMI 1640 medium containing recombinant human GM-CSF (rhGM-CSF, 800 U/ml) and recombinant human IL-4 (rhIL-4, 500 U/ml). Phenotypic changes were monitored by light microscopy. Lipopolysaccharide at a concentration of 5 micro g/ml was added into the cultures after 6 days of growth for DC complete maturation, and the cells were cultured for another 24 hours. The harvested DC on day 7 were divided equally into several parts. One part was used as non-irradiated DC (naive DC) while the other parts were irradiated by gamma ray at a dose of 25 Gy and 30 Gy respectively. Cell surface molecules were analyzed by flow cytometry. The capability of DC to stimulated autologous T cell proliferation were determined. The results showed that gamma irradiation reduced expression of CD86, CD80 and HLA-DR molecules on dendritic cells, especially CD86 molecules. Dendritic cells effectively stimulated autologous T cells proliferation while irradiated DC in all groups showed profound decrease of capability to promote T cells proliferation. It is concluded that gamma irradiation of dendritic cells not only influenced phenotype of DC but also altered their function as stimulator cells in mixed lymphocyte reaction.
Antigens, CD ; analysis ; B7-1 Antigen ; analysis ; B7-2 Antigen ; Cell Division ; immunology ; Dendritic Cells ; drug effects ; immunology ; radiation effects ; Flow Cytometry ; Gamma Rays ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HLA-DR Antigens ; analysis ; Humans ; Immunophenotyping ; Interleukin-4 ; pharmacology ; Membrane Glycoproteins ; analysis ; Multiple Sclerosis ; blood ; Recombinant Proteins ; pharmacology ; T-Lymphocytes ; cytology ; immunology

OBJECTIVETo study the effects of low frequency pulsed magnetic field on the proliferation and differentiation of HepG2 cells.

METHODS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetry method and ELISA assay of alpha-fetoprotein (AFP) were used to determine the cell proliferation and differentiation after the cells were exposed to pulsed magnetic fields with different frequency but the same field intensity.

RESULTSThere were no significant differences in cell proliferation between sham and treated groups exposed to the field of 80 Hz, 1.55 mT for 1, 4, 8, 12, 24 h (P > 0.05). There were also no significant differences in cell proliferation and AFP secretion between sham and treated groups exposed to 16 Hz, 1.55 mT pulsed magnetic fields for 1, 4, 8, 24 h (P > 0.05).

CONCLUSIONThere were no "window effects" found in HepG2 cells proliferation or AFP secretion at 16 Hz and 80 Hz pulsed magnetic fields.

Cell Differentiation ; radiation effects ; Cell Division ; radiation effects ; Cell Line, Tumor ; cytology ; metabolism ; radiation effects ; Electromagnetic Fields ; Humans ; alpha-Fetoproteins ; analysis

OBJECTIVETo explore the inhibitory effect of He-Ne laser irradiation on fibroblast growth of hypertrophic scars in culture.

METHODSHe-Ne laser with wavelength of 632.8 nm, power density of 50 mW/cm(2) and doses of 3 J/cm(2), 30 J/cm(2), 90 J/cm(2) and 180 J/cm(2) was used to irradiate human scar fibroblasts in culture 1, 3 and 5 times respectively, and then the cell count and cell cycle analysis were done.

RESULTSRepeated irradiation with He-Ne laser at dose of 180 J/cm(2) three and five times led to an evident decrease in total cell number compared with that of the control group and there was a significant difference (P<0.05). The cell cycle analysis showed after three and five times of irradiation with 180 J/cm(2) He-Ne laser the cell number in S-phase decreased from 51% to 20% and 14% respectively, the cell number in G(0)/G(1) phase increased from 28% to 55% and 60% respectively, and the cell percentage in Sub-G1 phase was 6.7% and 9.8% respectively.

CONCLUSIONSRepeated irradiation with 180 J/cm(2) He-Ne laser can inhibit scar fibroblasts growth in culture. It may be that He-Ne laser irradiation causes cell stagnation in G(0)/G(1) phase and apoptosis.

Cell Division ; radiation effects ; Cells, Cultured ; Cicatrix ; pathology ; Dose-Response Relationship, Radiation ; Female ; Fibroblasts ; cytology ; radiation effects ; Helium ; Humans ; Lasers ; Male ; Neon

OBJECTIVETo seek for the appropriate concentration, at which IGF-II can exerts its strong effects on postirradiation proliferation, physiological function and differentiation of the rat's osteoblast-like cells (ROB).

METHODSThe osteoblast-like cells used were isolated from the calvariae of neonatal (one-day-old) SD rats by sequential enzymatic digestion. The third passages of the cells were irradiated with gamma-ray from a (60)Co source at the doses of 100, 400, 600, and 900 cGy. The medium was changed immediately after irradiation and 5 concentrations of IGF-II, i.e., 0, 0.1, 1.0, 10.0, and 100.0 microgram/L were added. 6 days after radiation (9 days in culture), the examination, or the measurement of relative cell number, was carried out.

RESULTSRadiation inhibited the ROB, even lethally. IGF-II completely counteracted the inhibitory effects when the cells were exposed to the radiation at lower dose (100 cGy), and partially when at higher dose (400 cGy). But after the radiation at much higher dose as 900 cGy, the damages were irreversible, even with the existence of this growth factor.

CONCLUSIONSAt least a portion of effective recovery of postirradiation damages may be due to IGF-II-induced radioresistance. Incubation with IGF-II can increase radioresistance or repair of radiation-induced cells damages. However, this effect depends on the dose of radiation.

Animals ; Cell Division ; drug effects ; radiation effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Dose-Response Relationship, Radiation ; Insulin-Like Growth Factor II ; pharmacology ; Osteoblasts ; drug effects ; physiology ; radiation effects ; Radiation Tolerance ; drug effects ; Rats ; Rats, Sprague-Dawley