Animals ; Chromatin Assembly and Disassembly ; DNA Methylation ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic ; Histones ; metabolism ; Humans ; Methylation ; MicroRNAs ; genetics ; Neoplasms ; genetics

Animals ; Fluorescence ; Fluorescence Recovery After Photobleaching ; Fluorescence Resonance Energy Transfer ; Fluorometry ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Neoplasms, Experimental ; pathology ; Pathology

Animals ; Humans ; MicroRNAs ; physiology ; RNA, Small Interfering ; physiology ; RNA, Untranslated ; physiology

Animals ; Fluorescence ; Humans ; Microscopy, Fluorescence ; Nanotechnology ; Neoplasms ; diagnosis ; Photons ; Quantum Dots ; Staining and Labeling ; instrumentation ; methods

OBJECTIVETo observe the effect of small interfering RNA (siRNA)-induced MAPK p42 silencing on the survival of HeLa cells.

METHODSTwo siRNAs targeting at the MAPK p42 gene and one random siRNA were synthesized respectively by Silencer siRNA Construction Kit and transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) in the transfected HeLa cells was analyzed by Western blotting and immunohistochemistry, and the morphology of cells were observed with electron microscope. TUNEL assay and Annexin V/PI staining were employed for detecting the cell apoptosis.

RESULTSThe expression of p42(MAPK) in the HeLa cells was remarkably suppressed after transfection with the two siRNAs, reduced by about 2.5 and 3.2 folds respectively in comparison with the negative control. Chromatin margination in the cell nuclei were observed in the transfected cells, and TUNEL assay and Annexin V/PI staining further confirmed the occurrence of cell apoptosis.

CONCLUSIONIn vitro MAPK p42 siRNA-1 and siRNA-2 transfection can specifically silence the gene expression and induce apoptosis of HeLa cells.

Apoptosis ; physiology ; Gene Silencing ; physiology ; HeLa Cells ; Humans ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the effect of early intervention by pravastatin with two different dosage on inflammatory factors and endothelial vasodilator function in patients with unstable angina (UA).

METHODS108 patients with UA were investigated consecutively and divided randomly into three groups (group 20 mg, n = 37; group 10 mg, n = 37; group control, n = 34). Blood samples were examined at admission and 4, 8 weeks after the therapy of pravastatin. Fourty patients of UA were chosen from those three groups (15, 15 and 10 cases respectively). The endothelium-dependent vasodilation and the function of vascular endothelium of them were measured. In the dosage of 20 mg pravastatin group non-endothelium-dependent vasodilation in brachial artery was also tested by ultrasound before and 8 weeks after the therapy. Cardiac events were followed up for 2 months.

RESULTS(1) The use of pravastatin in early admission period of UA could significantly reduce inflammatory factors and improve vascular endothelium function, which was more obviously in the group of 20 mg/d than in group of 10 mg/d. These benefits occurred in 4th week and more obviously in 8th week after the therapy. (2) The lipid lowering therapy in the early stage of admission (24 - 48 h) resulted in the reduction of cardiac events in the hospital.

CONCLUSIONThe use of pravastatin 20 mg/d seems better than that of 10 mg/d in all the fields as above in early admission period of UA patients.

Adult ; Aged ; Angina, Unstable ; drug therapy ; Anticholesteremic Agents ; administration & dosage ; therapeutic use ; Female ; Follow-Up Studies ; Humans ; Inpatients ; Male ; Middle Aged ; Pravastatin ; administration & dosage ; therapeutic use ; Prospective Studies

To investigate the possible mechanism of apoptosis induced by indole-3-acetic acid (IAA) combined with horseradish peroxidase in leukemia cell line K562, cell proliferation and apoptosis of K562 cell were examined by MTT assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively; the activity of superoxide dismutase (SOD) and the quantitative change of MDA were measured by biochemical method; changes of free radical were determined by 2, 7-dichlorofluorescin diacetate (DCFH-DA) probe with confocal microscopy. The results showed that of MTT assay and TUNEL indicated that IAA/HRP could significantly inhibit cell proliferation (P < 0.05) and induce apoptosis of K562 cell (P < 0.01), at the same time a positive correlation was found between apoptosis rate and IAA concentration (r = 0.971, P < 0.01). The activity of SOD and the quantitative of MDA increased, accompanied with a rise in IAA concentration. Results detected by DCFH-DA probe indicated that the fluorescence intensity of intracellular free radical increased, as compared with control, and a positive correlation was found. It is concluded that IAA/HRP can inhibit proliferation of K562 cells and induce K562 cell apoptosis, its mechanism may be related with the increase of intracellular free radical due to the effects of IAA/HRP.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Drug Synergism ; Horseradish Peroxidase ; pharmacology ; Humans ; In Situ Nick-End Labeling ; Indoleacetic Acids ; pharmacology ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; pathology ; Microscopy, Confocal ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Time Factors

To prepare carboxyl terminus truncated human papillomavirus type 58L1 protein, and study on its in vitro bioactivity. PCR was used to amplify carboxyl terminus truncated HPV 58L1 gene, the product was inserted into the PUCMT cloning vector, preparing recombinant PFastBacHTb containing carboxyl terminus truncated HPV58L1 gene. Further more, the recombinant plasmid PfastbacHTb was used to transform DH10Bac cells, constructing recombinant Baculovirus, then the recombinant virus was successfully used to infect Sf-9 insect cells. After incubating at 27 degrees C for 72 hours, the infected cells were collected and total cellular proteins were extracted. The target protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The interested protein was purified by ProBond purification system. The purified interested protein was identified to self-assemble into VLPs by Transmission electron microscope, and induce murine erythrocyte hemagglutination, indicating that the given proteins had the conformation of VLPs, collecting, HPV58L1 proteins with carboxyl terminus truncation could be efficiently expressed in baculovirus Sf-9 cells expression system, it has identical in vitro bioactivity to the wild type HPV58L1, The present study is fundmental for preparing HPV58L1 prophylactic vaccine.
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Cloning, Molecular ; Humans ; Mice ; Mice, Inbred C57BL ; Papillomaviridae ; genetics ; Papillomavirus Vaccines ; immunology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Vaccines, Synthetic ; immunology ; Viral Proteins ; biosynthesis ; genetics ; Virion ; immunology

OBJECTIVETo screen for siRNAs that inhibit the expression of p42(MAPK) in HeLa cell line.

METHODSThree p42(MAPK) siRNAs and one random siRNA were synthesized using Silencer siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTT and flow cytometry.

RESULTSTwo siRNAs (siRNA-2 and siRNA-3) among three tested were identified to be able to downregulate the p42(MAPK) expression. A concurrent growth retardation of HeLa cell line was observed in comparison with the control.

CONCLUSIONInhibition of p42(MAPK) expression with siRNA technique can inhibit the proliferation of HeLa cells.

Cell Cycle ; Cell Proliferation ; Cell Survival ; Flow Cytometry ; HeLa Cells ; Humans ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection