Abstract:The transforming growth factor (TGF)-beta/Smad signaling pathway is thought to play a major role in keloid formation. This study showed that the expression of inhibitory Smad7 was significantly down regulated in keloid compared with normal scar (P < 0.05) and normal skin (P < 0.05), however, no significant difference of Smad2 , 3 and the phosphorylation of Smad2,3. But the mechanism of reduced expression of Smad7 is unclear. Sp1 binding sites were found in Smad7 promotor by bioinformatics system analysis, then, the expression of TIEG1 mRNA and proteins in keloids, in normal skin and in normal scars tissues and fibroblasts were investigated. Dermal fibroblasts were obtained from biopsies of keloids, normal scars and normal skin. Fibroblasts were cultured in vitro. The expression of TIEG1 mRNA was analysed by RT-PCR and protein expression was determined by Western blot analysis. The results demonstrated increased mRNA and protein expressions of TIEG1 in keloid tissue and fibroblasts as compared to normal scar tissues and fibroblasts (P < 0.05) and normal skin tissues and fibroblasts (P < 0.05). It suggested that TIEG1 might play a significant role to the decreased expression of the inhibitory Smad7 in keloid. Further investigation is necessary to illuminate the mechanism of TIEG1 regulating Smad7.

Abstract:Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of the physiological response to hypoxia. To study the regulation mechanism of HIF-1α in proliferation and differentiation of stem cells, two small interference RNA expression vectors of HIF-1α were constructed, and transfected into the fetal liver stromal cell lines (FLSCs) stably by lentiviral system. The efficiency of virus transfection was identified by expression of green fluorescence protein(GFP) analyzed by fluorescence microscope, then the high GFP expression FLSCs were sorted by fluorescence-activated cell sorting (FACS) according to strong GFP expression. Analysis of efficiency of RNA interfering on HIF-1α was detected by real time-PCR and Western-blot. The HIF-1α gene expression at mRNA level of FLSCs transfected by lentivirus plasmids pSicoR-HIF-1α-1 and pSicoR-HIF-1α-2 are 18.8% and 25.5% of the FLSCs transfected by the control lentivirus under 20% O₂; 21.2% and 29.3% under hypoxia. The HIF-1α protein were down regulated by siRNA. The pSicoR-HIF1 showed higher interfering efficiency than pSicoR-HIF2. The expression of SDF-1α gene in HIF-1α silencing FLSCs were detected by RT-PCR, fluorescent immunocytochemistry analysis and ELISA. The mRNA and protein of SDF-1α gene were down regulated after silencing of HIF-1α. Therefore, the regulation of SDF-1α gene under hypoxia could be activated by HIF-1α, that plays an important role in the influence on the proliferation and differentiation of stem cell.

Abstract:The LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and recombined with plasmid pUCP18 reversely. The recombinant pUCP18/lasR^antisense was verified with restriction analysis, PCR and sequence and was transformed in Pseudomonas aeruginosus. The biological effect of pUCP18/lasR^antisense was detected by RT-PCR, NAD method and the assay of pyocyanin. The air tubes of rats were infected by pUCP18/lasR^antisense strain and then carried on histopathologic slide check. Expected full length LasR fragment (721bp) can be extended from Pseudomonas aeruginosus gene with PCR technology. And it is consistent with LasR gene of Pseudomonas aeruginosa covered in GenBank (NO. NC_002516). The recombinant plasmid was constructed and transformed into Pseudomonas aeruginosus sucessfully. Compared with the rats which were infected by standard strain, the bronchitis of the rats which were infected by pUCP18/lasR^antisense strain was obviously eased. It can be concluded that the antisensenucleic acid of LasR gene can depress the virulence of Pseudomonas aeruginosus and reveal a new target site for treatment.

Abstract:Several methods, including PepTag® Assay, RT-PCR, Western blot, oil red staining and HPLC, were used to explore the role of PKC in lipid-accumulation mediated by adipophilin in THP-1 macrophage treated with PKC activator PMA and inhibitor Calphostin C. 100 nmol/L PMA activated cytomembrane PKC activity ((0.2514±0.0154) U/ml), also synergistically enhanced the expressions of PKCα, PPARγand adipophilin and the lipid-accumulation in the presence of oxLDL. Together with oxLDL, PMA stimulated the ratio of intracellular CE/TC to (69.8±9.5) %. 300 nmol/L Calphostin C inhibited cytomembrane PKC activity((0.0927±0.0056) U/ml) of lipid-loaded THP-1 macrophage, reduced intracellular lipid droplets and the ratio of intracellular CE/TC ((40.1±9.1) %). Calphostin C downregulated the PKC activity and the expressions of PKCα, PPARγ and adipophilin in a dose-dependent manner. 400 nmol/L Calphostin C ultimately reverse the effect induced by 50 mg/L oxLDL. In conclusion, the changes of PKC activity can have effects on the lipid-accumulation mediated by adipophilin, PPARγ may play an important role in the regulation mechanism.

Abstract:In order to investigate the ability of PEG-PEI copolymers as gene carriers for delivery of VEGF165. A series of PEG-PEI copolymers with different PEG grafting was prepared and the cytotoxicity was evaluated. Simultaneously，the VEGF165 gene segment with HindⅢ and BamHⅠ site was obtained by PCR, which was cloned into pEGFP-C1. PEG-PEI/ pEGFP-VEGF165 complexes were formed by self-assembly and transfected HUVEc. Transfection efficiency was evaluated by measuring the percentage of cells expressing green fluorecensce protein. The VEGF expression was detected by ELISA, RT-PCR, and the effect of transfection on growth of endothelial cell was evaluated by MTT. The results suggested that the formation of PEG-PEI copolymers could help to reduce the cytotoxicity of PEI. After transfection, the strong expression of green fluorescence protein was observed by fluorescence microscopy. The transfection efficiency was influenced by the number of PEG side chains and N/P ratio. Of all copolymers tested, the transfection efficiency of PEG-PEI(5-25-1) at N/P = 30 reached a maximum, which was much higher than that of PEI. The expression of VEGF protein and mRNA increased significantly, and HUVEc proliferation was accelerated after transfection.These results indicates PEG-PEI copolymers can be used as effective gene carriers for delivery of pEGFP-VEGF165 gene.

Abstract:Arsenic is highly effective in treating acute promyelocytic leukemia (APL), especially for relapsed patients. However, the treatment is highly affected by the resistance of the drug by patients, while the arsenic-resistance mechanism has not been well studied. A genome-wide screen was performed against a pool of 4 757 Saccharomyces cerevisiae mutants, each with one different gene individually deleted, to isolate genes that may mediate cellular resistance to arsenic. A one-step selection method was used. An aliquot of the pooled yeast library was plated on YPD agar plates supplemented with 3 mmol/L sodium arsenite. The genomic DNAs of the arsenic resistant strains were separately extracted, and amplified by PCR to get DNA fragments with UPTAG. The corresponding deleted genes were identified by comparing the PCR-amplified sequences with the UPTAG sequences from the Saccharomyces Genome Deletion Project. Mutations were identified in 104 genes/ORFs showing resistance to arsenic as compared to the wild type strain. To rule out the possibility that the resistant phenotype of these mutants is a result of arsenic-induced mutation during the screening process, the individual deletion strains from the mutant collection were picked up and tested individually for arsenic resistance using the spot assay. Of the 104 mutants identified in the screen, all exhibited significantly more resistance than the wild-type cells. Among the verified strains, 32 mutants turned out to have stronger phenotype that is resistant to 5 mmol/L arsenite. Five of the 32 mutants (FPS1, TMA20, UPF3, YAL066W, YOR309C) showed resistance to 7 mmol/L arsenite. The phenotype data were mapped onto the regulatory network. Bioinformatic studies of the genes revealed four neighborhoods, including mRNA catabolism, response to stress, histone acetylation, and protein synthesis and catabolism.

Abstract:To screen small molecule peptide specific binding to non-small cell lung cancer cell (A549) using the “one-bead one-peptide” combinatorial library. Twenty-nine positive beads binding to A549 cell were totally obtained after primary screening. Consensus peptide sequence of -NGXG- was identified by amino acid sequencing in ten beads. Peptide cNGQGEQc was re-synthesized on beads and further studied for its cell specificity, alanine scanning and site-directed deletion. The results showed that cNGQGEQc is specific for cell attachment to non-small cell lung cancer cells including A549, Calu-1 and H178, but not to other tumor cell lines. Both motif of -NGXG- and the length of six peptides are very important for A549 adhesion. Peptide cNGQGEQc labeled with FITC can specifically bind to A549 cell. In a blocking assay with anti-integrin antibodies(α1～6, αv/β1～5), cell adhesion of A549 to peptide beads was obviously inhibited by integrin α3 combining with any β subunits. Results suggested that small molecule peptide cNGQGEQc can bind specifically to non-small cell lung cancer cell A549 via integrin α3 on cell surface.

Abstract:Two sets of primers were designed according to the sequences of xynA and xynB from Aspergillus sulphureus, and the DNA fragments composed of 574 bp and 594 bp were amplified by polymerase chain reaction (PCR), respectively. The two PCR products respectively digested with EcoRⅠ/BamHⅠ and BglⅡ/HindⅢ were ligated into multiple cloning sites of pET-28a(+). The resulting plasmid is pET-xynAB, in which xylanase A and B are ligated by a 7-amino acid peptide (GlyGlyGlySerGlyGlyGly). E. coli BL21 transformed with pET-xynAB was induced by IPTG, and a special protein band about 50 ku was detected by SDS-PAGE. The protein purified with Ni-NTA column was denatured by 8 mol/L urea and dialyzed for refolding. The recombinant xylanase showed optimal activity at 50℃ and pH 4.4. The enzyme retained above 75% of its activity at the range of pH 2.4～5.4. The xylanase displayed about 50% retained acitivity after incubating at 80℃ for 30 min. Various metal ions have different effects on activity of the recombinant xylanase.

Abstract:Small interfering RNAs (siRNAs) can efficiently inhibit gene expression by sequence-specific RNA interference (RNAi). A common 5′ leader sequence exists in the genomic RNA and all subgenomic RNAs of SARS-CoV, and is well conserved among various SARS-CoV strains, thus providing a preferable target for RNAi of SARS-CoV replication. Here efficient depletion of the SARS-CoV mRNAs by either a synthetic siRNA or DNA vector-derived short hairpin RNAs (shRNAs) targeting the leader sequence in mammalian cell lines were reported. The siRNA or shRNAs efficiently suppressed the expression of an EGFP reporter gene which contains the leader sequence at the 5′ end. Both the siRNA and shRNAs efficiently knocked down the levels of leader-containing transcripts of three SARS-CoV genes encoding the spike protein, membrane protein and nucleocapsid protein were demonstrated. The results suggest that RNAi targeting the leader sequence is a potential efficient strategy for anti-SARS-CoV therapy.

Abstract:After analyzing the coding sequence (CDS) of chicken prolactin gene by DNA sequencing, one point mutation of C1607T in exon2 and two mutations of C5749T and T5821C in exon5 were discovered. Although none of the mutations changes amino acid encoded, they have different codon frequencies in the five populations. The three mutations in 370 individuals of the five populations by single-strand conformation polymorphism (SSCP) were screened, and seven haplotypes in the CDS of prolactin were discovered. Moreover, the seven haplotypes show different high frequency value, which was defined by the number of high frequency codon of the haplotype. Analyzing the laying performance of the five breeds, the positive correlation between the laying performance and high frequencies value wea found. After detecting the expression of the prolactin by enzyme linked immunosorbent assay (ELISA), it was found that the higher number of high frequency condon will lead to the more quantity of prolactin. The results demonstrate that the codon frequency is a factor which influence the laying performance, and synonymous mutation might influence phenotype by changing codon frequency.

Abstract:Researches for susceptibility of complex diseases need large-scale SNPs genotyping across many individuals. It leads to a requirement for a simple, cost effective, automatic and high-throughput genotyping method. Herein, a novel high-throughput SNP genotyping method using magnetic particles (MPs) in situ PCR and universal tags was described. PCR products were directly amplified by MPs in situ PCR and interrogated by hybridization with wild and mutant tagged probes and a pair of dual-color (Cy3, Cy5) universal detectors to determine SNP, and then genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured detectors on an unmodified clean glass slide. Morever, a pair of given dual-color universal detectors can be applied to any SNP loci by hybridization with allele specific tag probes. In this study, this new method have been applied to the analyze the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism in 96 different samples. The fluorescent ratios (match/mismatch signal) of homozygous samples were over 4.5, whereas heterozygous samples had ratios near to 1.0. The genotyping results were additionally validated by sequencing. By using universal tags and in situ magnetic particle PCR, this simple and cost-effective approach can be widely used in high-throughput SNP genotyping.

Abstract:With Saccharomyces cerevisiae cell surface engineering, combining with enzyme-linked immunosorbent assay (ELISA), a new method has been, which is named as ‘yeast-ELISA’ detection. Here, the recombination yeast cell MT8-1 surface display expressing antigen HPSα6 (Human proteasome subunit alpha 6), and its monoclonal antibody 3D7D12D4 (hybridoma cell line No) was used as study object. The fitful yeast cell concentration of this method is 0.50 A₆₀₀, and it was used to detect monoclonal antibody and its valance (1∶5×10⁵). The specificity and sensitivity of the method was demonstrated by crossing test and interdiction test. The results indicated that ‘yeast-ELISA’ could detect monoclonal antibody directly, and the valance of which is concordance with the result of the proteantigen indirect ELISA. And also this method could be used to antiserum detection, which also has good result. It demonstrates that the method of the ‘yeast-ELISA’ detection was established, which is the supply and application of Saccharomyces cerevisiae cell surface engineering.