Animals ; Atherosclerosis ; etiology ; genetics ; pathology ; China ; Coronary Disease ; pathology ; Genes, p53 ; Humans ; Point Mutation ; Risk Factors

Animals ; Atherosclerosis ; blood ; drug therapy ; pathology ; Dehydroepiandrosterone ; blood ; physiology ; therapeutic use ; Dehydroepiandrosterone Sulfate ; blood ; therapeutic use ; Hormone Replacement Therapy ; Humans

Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Fluorouracil ; pharmacology ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; physiology ; Neoplasm Proteins ; biosynthesis ; genetics ; physiology ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

Breast Neoplasms ; classification ; genetics ; metabolism ; pathology ; Chromosome Aberrations ; Female ; Gene Deletion ; Humans ; Phyllodes Tumor ; classification ; genetics ; metabolism ; pathology ; Twist-Related Protein 1 ; metabolism ; Wnt Signaling Pathway

Animals ; Cell Differentiation ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Endothelial Cells ; pathology ; physiology ; Humans ; Muscle, Smooth, Vascular ; pathology ; physiology ; Receptors, CXCR4 ; metabolism ; Stem Cells ; metabolism ; pathology ; physiology ; Vascular Diseases ; metabolism ; pathology ; physiopathology

OBJECTIVETo identify and select smooth muscle progenitor cells from mouse bone marrow mesenchyme stem cell population and to characterize smooth muscle progenitor cells in peripheral blood.

METHODSRecombinant expression vector with the promoter of sm22alpha was constructed to have an enhancement type green fluorescent protein expression plasmid (EGFP-1). The construct was transfected into mouse bone marrow mesenchyme stem cells using Lipofectamine 2000. Morphological assessment was performed and the expressions of myocardin at protein and mRNA levels by fluorescence microscope and RT-PCR were evaluated at 3, 5, 7, and 10 d targeting CD34 positive bone mesenchyme stem cells.

RESULTSThe transfection efficiency of the positive control group was 70% +/- 1.5% (P > 0.05). Expected green fluorescent proteins expressed at 3rd day. The numbers of green fluorescent cells in experimental groups increased with the time and reached the peak at the 7th day, and declined thereafter. The shapes of the green fluorescent cells were also different from each others. The positive ratios of green fluorescent cells at different time points: 3 d: 7% +/- 0.13%, 5 d: 10% +/- 0.32%, 7 d: 20% +/- 0.26%, 10 d: 12% +/- 0.18%, P < 0.05. Myocardin mRNA expression roughly correlated with green fluorescent expressions. CD34 was expressed on the 5th day in transfected bone mesenchyme stem cells. The CD34 positive ratio was 5.2% +/- 0.21% (P > 0.05).

CONCLUSIONSThere are smooth muscle progenitor cells among mouse bone marrow mesenchyme stem cell population. Smooth muscle progenitor cells can be selected using a Psm22alpha-EGFP-1 recombinant expression approach.

Animals ; Antigens, CD34 ; immunology ; Bone Marrow Cells ; cytology ; immunology ; Cell Separation ; methods ; Cell Shape ; Green Fluorescent Proteins ; Mesenchymal Stromal Cells ; cytology ; immunology ; Mice ; Microfilament Proteins ; genetics ; Microscopy, Fluorescence ; Muscle Proteins ; genetics ; Myocytes, Smooth Muscle ; cytology ; Nuclear Proteins ; genetics ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Recombinant Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics ; metabolism ; Transfection

Cells, Cultured ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Homocysteine ; pharmacology ; Humans ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Monocytes ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology

Animals ; Aorta ; pathology ; Atherosclerosis ; blood ; etiology ; metabolism ; Chemokine CCL2 ; metabolism ; Cholesterol ; blood ; Cholesterol, Dietary ; administration & dosage ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Dehydroepiandrosterone ; pharmacology ; Diet, Atherogenic ; Immunohistochemistry ; Rabbits ; Random Allocation ; Triglycerides ; blood ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo explore the mechanism of anisodamine in treating infectious shock through studying effect of anisodamine on endotoxin lipopolysaccharide (LPS) induced expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in vascular endothelial cells (EC).

METHODSHuman umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. In order to evaluate a possible contribution of the nuclear factor-kappa B (NF-kappa B) pathway on the transductive effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B binding oligonucleotides.

RESULTSLPS could significantly strengthen the expression of HUVEC PAI-1 protein and TF activity and its mRNA, this effect of LPS could be markedly weakened after adding Anisodamine dose-dependently. Anisodamine could also completely block the LPS induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC.

CONCLUSIONThe possible mechanism of anisodamine in treating infectious shock may be through antagonizing LPS induced HUVEC TF and PAI-1 expression, and the antagonism might be, at least partially, transduced by path of NF-kappa B.

Cells, Cultured ; Culture Media, Conditioned ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Humans ; NF-kappa B ; metabolism ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Solanaceous Alkaloids ; pharmacology ; Thromboplastin ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo investigate the expression profiles of myocardin gene during the differentiation of bone marrow-derived mesenchymal stem cell to smooth muscle cells in the conditional medium combined with a high concentration of fetal bovine serum (FBS).

METHODSMarrow-derived mesenchymal stem cells were isolated and purified from mouse femoral bone and shinbones using differential adherent methods. Cells at the third passage were induced by 20% FBS in conditioned medium, conditioned medium alone, 20% FBS or 10% FBS alone respectively. Mouse aortic smooth muscle cells were cultured as the positive control. Levels of mRNA and protein expression of myocardin and several smooth muscle cells marker genes were determined by immunofluorescence, RT-PCR and Western blot before and 3, 7, 10, 14 d after the induction. The presence of smooth muscle myofilaments was detected by using transmission electron microscope.

RESULTSNaive bone marrow-derived mesenchymal stem cells displayed multiple morphological forms including fusiform, polygon, oval, and micro-spherical, as compared to the single macro-spindle form after the induction. Typical appearance of peak valley was displayed on the 21st day after induction. At the same time, the expression of smooth muscle marker genes was reinforced along with an up-regulation of myocardin expression. Immunofluorescence study showed that the cells expressing myocardin and smooth muscle marker genes such as alpha-SMA and SM-MHC increased. Fluorescence domain of myocardin translocated from cytoplasm to nucleus and the amounts of double positive cells for myocardin with alpha-SMA or SM-MHC also increased. RT-PCR confirmed that the mRNA expression of myocardin increased gradually and remained stabilized after achieving its peak on the 7th day after induction. The expression of smooth muscle marker genes, alpha-SMA and SM22alpha, remained stable on the 10th day of induction. It was also confirmed by Western blot that the protein expression of both myocardin and alpha-SMA were markedly increased during the induction. Finally, transmission electron microscopy revealed the presence of myofilament on the 21st day after induction.

CONCLUSIONSBone marrow-derived mesenchymal stem cells can be effectively induced into smooth muscle-like cells by conditioned medium combined with 20% FBS. Myocardin plays an important role in the differentiation process of bone marrow-derived mesenchymal stem cells to the peripheral smooth muscle cells.

Animals ; Bone Marrow Cells ; cytology ; physiology ; Cattle ; Cell Differentiation ; physiology ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics ; metabolism ; physiology ; Up-Regulation