Cloning the full-length cDNA of a gene responsible for vascular smooth muscle cell proliferation in atherogenesis and study of function / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To clone the full-length cDNA of a gene responsible for vascular smooth muscle cell (v-SMC) proliferation in atherogenesis, and study its function.</p><p><b>METHODS</b>Oxidized 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.</p><p><b>RESULTS</b>One 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.</p><p><b>CONCLUSION</b>The new gene cloned may be associated with SMC proliferation in atherogenesis.</p>

Effect of NF-kappaB on the induction of PDGF-B transcription by angiotensin II in the ECV304 cell line / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To examine the effect of angiotensin II (Ang II) on nuclear factor-kappa B (NF-kappaB) activation in human endothelial cell line ECV304 and the molecular mechanism by which Ang II activates NF-kappaB.</p><p><b>METHODS</b>ECV304 cells were transiently cotransfected with an NF-kappaB/luciferase reporter gene and inactive NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKK(alpha)), IkappaB kinase beta (IKK(beta)) mutants or vectors, respectively. The effect on NF-kappaB was detected by using an electrophoretic mobility shift assay (EMSA) and overexpression of the mutants enabled blocking of reporter gene activation induced by Ang II. With immunofluorescence and immuno-electronic microscope techniques, including confocal microscopy and gold particle labeled electronic microscopy, definite cytoplasmic-to-nuclear translocations of NF-kappaB activation were detected using subunits p50 and p65 induced by Ang II.</p><p><b>RESULTS</b>The translocation of p50 in nuclei was highly remarkable 2 hours after Ang II stimulation, and the activity was somewhat reduced 6 hours after stimulation to the 18th hour. Northern blot also showed PDGF-B mRNA increased by stimulation of Ang II for 18 hours.</p><p><b>CONCLUSION</b>Ang II is effective in stimulating NF-kappaB activation through a pathway dependent on NIK, IKK(alpha) and IKK(beta), and induces PDGF-B transcription in the endothelial cell line, ECV304.v</p>

Effect of oxLDL on the uptake and clearance rate of cholesterol in v-SMC originated from human apoA1 transgenic mice / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>v-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.</p><p><b>RESULTS</b>oxLDL (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.</p><p><b>CONCLUSION</b>Expression 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.</p>

Expression of antisense thrombin receptor gene inhibits intimal hyperplasia of rat carotid artery after balloon injury / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the mechanism of restenosis after angioplasty and to clarify the effect of thrombin and its receptor on restenosis development.</p><p><b>METHODS</b>Balloon catheter-induced injury was adopted to induce intimal hyperplasia of the carotid arteries in rats. Antisense thrombin receptor (ATR) cDNA was transfected by perfusing recombinant LXSN ATR plasmid/nanoparticle complex into the segment of the injured carotid artery.</p><p><b>RESULTS</b>PCR result showed integration of the recombined gene. Dot blot showed the expression of antisense TR mediated by recombinant LXSN ATR plasmid/nanoparticle complex in the wall of common carotid arteries of the experimental group rats, which enabled to inhibit TR gene expression and intimal hyperplasia of the injured arteries.</p><p><b>CONCLUSIONS</b>Thrombin and its receptor play an important role in the formation of neointima after the injury, which provides a potential clue in developing a new approach for prevention and treatment of restenosis after angioplasty.</p>