OBJECTIVETo clone the full-length cDNA of a gene responsible for vascular smooth muscle cell (v-SMC) proliferation in atherogenesis, and study its function.

METHODSOxidized low density lipoprotein (ox-LDL) at optimal concentration was used as the stimulant to induce v-SMC proliferation in culture medium. A cDNA subtractive library of v-SMC proliferation specific to ox-LDL stimulation was established using subtractive hybridization technique. Methods, including blotting, Northern hybridization and gene sequencing, were used to clone new gene fragments. By using full-length cDNA screening and protein expression techniques, one full-length cDNA was cloned and its function was studied.

RESULTSOne full-length cDNA was cloned. The new gene (Genbank AF 174647) expressed a 44 kDa protein, which might be associated with the activity of ox-LDL.

CONCLUSIONThe new gene cloned may be associated with SMC proliferation in atherogenesis.

Amino Acid Sequence ; Arteriosclerosis ; genetics ; Base Sequence ; Blotting, Northern ; Cell Division ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Gene Library ; Humans ; Lipoproteins, LDL ; pharmacology ; Molecular Sequence Data ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Nucleic Acid Hybridization

OBJECTIVETo study the inhibition effect of oxLDL on the uptake and clearance of intra-cellular (3)H-cholesterol in v-SMC from the human-apoA1 transgenic mice (C57BL/6) and the changes in human-apoA1 mRNA expression in v-SMC from human apoA1 transgenic mice after oxLDL stimulation.

METHODSv-SMC originally isolated from human apoA1 transgenic mice connected with a recombined mouse metallothionein-I (MT-I) promoter was used, and the effect of oxLDL on the uptake and clearance of intracellular (3)H-cholesterol was studied in v-SMC of the transgenic and control mice respectively, the study of h-apoA I mRNA expression from v-SMC of the transgenic mice were done by RT-PCR and Northern blot.

RESULTSoxLDL (30 microg/ml) strongly promoted the SMC proliferation. No difference was found in (3)H-cholesterol uptake between nSMC and trSMC, and the uptake rates of both kinds of SMC rose 100% after oxLDL stimulation. The efflux rates of (3)H-cholesterol in trSMC were much higher than those of nSMC (40% - 50%). After oxLDL stimulation, the clearance rates fell by 28% and 10%, respectively, for nSMC and trSMC. The result of RT-PCR and Northern blot showed that h-apoA1 gene expression was markedly increased by the stimulation of oxLDL.

CONCLUSIONExpression of the h-apoA1 gene in C57BL/6 mice enables them to reduce the accumulation of cholesterol in v-SMC. The trSMC can alleviate the harmful effect of oxLDL due to the increase of h-apoA1 expression.

Animals ; Apolipoprotein A-I ; genetics ; metabolism ; Blotting, Northern ; Cell Division ; drug effects ; Cells, Cultured ; Cholesterol ; pharmacokinetics ; Dose-Response Relationship, Drug ; Gene Expression ; Humans ; Lipoproteins, LDL ; pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVETo evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.

METHODSNanoparticle-DNA complex was prepared with Poly-dl-lactic-co-glycolic acid (PLGA) bearing anti-sense monocyte chemotactic protein-1 (A-MCP-1), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 microg), 6 with A-MCP-1 (200 microg) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.

RESULTSThe package efficiency of the nanoparticle-DNA complex was 0.9%, release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300 nm. SMC genomic DNA PCR showed that A-MCP-1 gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-1 mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.

CONCLUSIONNanoparticle can act as a vector to transfect specific gene.

Animals ; Chemokine CCL2 ; biosynthesis ; genetics ; DNA, Antisense ; biosynthesis ; genetics ; Drug Carriers ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Lactic Acid ; Nanotechnology ; Particle Size ; Polyglycolic Acid ; Polymers ; Rabbits ; Transfection

Cryopreservation impairs sperm quality and functions, including motility and DNA integrity. Antioxidant additives in sperm freezing media have previously brought improvements in postthawed sperm quality. Green tea extract (GTE) is widely considered as an excellent antioxidant, and its beneficial role has been proven in other human cells. This study aims to evaluate the GTE as a potential additive in cryopreservation media of human spermatozoa. In part one, the semen of 20 normozoospermic men was used to optimize the concentration of GTE that maintains sperm motility and DNA integrity against oxidative stress, induced by hydrogen peroxide (H