A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.
This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries. On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer. The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detectedin vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.
The pathogenesis of hyperoxia lung injury and the mechanism of amygdalin on type 2 alveolar epithelial cells (AEC2) isolated from premature rat lungsin vitro were investigated. AEC2 were obtained by primary culture from 20-days fetal rat lung and hyperoxia-exposed cell model was established. Cell proliferating viability was examined by MTT assay after treatment of amygdalin at various concentrations. DNA content and the proliferating cell nuclear antigen (PCNA) protein expression of AEC2 were measured by using flow cytometry and immunocytochemistry respectively after 24h of hyperoxia exposure or amygdalin treatment. The results showed that hyperoxia inhibited the proliferation and decreased PCNA protein expression in AEC2 of premature ratin vitro. Amygdalin at the concentration range of 50–200 μmol/L stimulated the proliferation of AEC2 in a dose-dependent manner, however, 400 μmol/L amygdalin inhibited the proliferation of AEC2. Amygdalin at the concentration of 200 μmol/L played its best role in facilitating proliferation of AEC2sin vitro and could partially ameliorated the changes of proliferation in hyperoxia exposed AEC2 of premature rat. It has been suggested that hyperoxia inhibited the proliferation of AEC2s of premature rat, which may contribute to hyperoxia lung injury. Amygdalin may play partial protective role in hyperoxia-induced lung injury.
To explore the molecular mechanisms of sodium butyrate working on SKM-1 cell proliferation/differentiation and to study its synergistic effect with all-trans retinoic acid (ATRA), SKM-1 cells were grown in the absence or presence of sodium butyrate and/or ATRA. The percentage of viable cells was determined by trypan blue exclusion. Differentiation was dtermined by nitroblue tetrazolium (NBT) reduction and cell surface adhesion molecules was analyzed by FACS. Cell cycle distribution was examined after DNA staining by propidium iodide. D-type cyclins, cdks and P21 mRNA were studied by reverse transcription-polymerase chain reaction. Our results showed that sodiun butyrate and/or ATRA blocked cells mainly in the G0/G1 phase of the cell cycle. ATRA inhibited the mRNA expression of CDK6, CDK4, cyclinD3 and cyclinD1. Sodium butyrate inhibited the mRNA expression of CDK2, cyclinD2 and cyclinD1. ATRA and sodium butyrate inhibited the mRNA expression of CDK6, CDK4, CDK2, cyclinD1, cyclinD2 and cyclinD3. Both ATRA and/or sodium butyrate stimulated p21 expression at the mRNA levels Our results suggest that the effect of sodium butyrate on cell proliferation/differentiation might be linked to its ability to induce expression of p21 mRNA and inhibit the cyclin-cdk complexes. Our observations support the notion that the sodium butyrate works synergistically with ATRA.
The protective effect of carvedilol on abnormality of L-type calcium current induced by oxygen free radical in single guinea pig ventricular myocytes was studied. Whole-cell patch clamp technique was used to study the effect of H2O2 (0.5 mmol/L) on L-type calcium current in single guinea pig ventricular myocytes and the action of pretreatment with carvedilol (0.5 μmol/L). 0.5 μmol/L carvedilol had no significant effect onICa.L and its channel dynamics. In the presence of 0.5 mmol/L H2O2, peak current ofICa.L was reduced significantly (P<0.001), theI-V curve ofICa.L was shifted upward, steady-state activation curve and steady-state deactivation curve ofICa.L were shifted left and recovery time ofICa.L was delayed significantly (P<0.001). 0.5 μmol/L carvedilol significantly alleviated the inhibitory effect of H2O2 onICa.L as compared with that in H2O2. It was concluded that carvedilol could alleviate the abnormality of L-type calcium current induced by oxygen free radical in cardiomyocytes. It shows partly the possible mechanism of the special availability of carvedilol in chronig heart failure.