Biomedical subjects

Wen-ling Zheng

Publications and source records attributed to Wen-ling Zheng.

At least 19 recordsLinked to original sources

PubMed2006

[Screening differentially expressed genes in denucleated K562 cells with restriction display technique].

OBJECTIVE: To screen differentially expressed genes in cytochalasin B (CB)-induced denucleated K562 cells by restriction display (RD) technique. METHODS: The total RNA was isolated and purified from K562 cells before and after CB (10 mug/ml) treatment. The mRNA from both treated and untreated K562 cells were reversely transcribed into cDNA, and the differentially expressed genes were separated using RD technique combined with polyacrylamide gel electrophoresis and sliver staining, followed by cloning, sequencing and homology analysis against GenBank database of these genes. RESULTS: Seven differentially expressed genes were identified in CB-treated cells including aquaporin 1 (AQP1) gene, which was verified to be up-regulated after CB treatment by RT-PCR. CONCLUSION: AQP1 gene might be in close association with the regulation of denucleation processes and CB-induced proliferation inhibition of K562 cells.

Aquaporin 1↗

PubMed2006

[Human cytomegalovirus induces apoptosis of ECV304 endothelial-like cells].

OBJECTIVE: To investigate the mechanisms for the cytopathic effect (CPE) of human cytomegalovirus (HCMV) in ECV304 endothelial-like cells. METHODS: PCR and indirect immunofluorescence were used to detect HCMV infection by examining immediate-early (IE) gene and protein expression of the virus in ECV304 cells. Phase-contrast and electron microscopies were performed to observe the morphological changes of the infected and uninfected cells, and DNA ladder analysis and flow cytometry were carried out to study HCMV-induced cell apoptosis. RESULTS: In HCMV-infected ECV304 cells, cytopathic effects were first observed at approximately 72 h post-infection. The cells with CPE changes exhibited detachment from the monolayer, cell rounding and shrinkage. The expression of the IE gene was detected. Chromatin condensation and nuclear fragmentation along with dramatic changes of the mitochondria were observed by electron microscopy at 96 h post-infection. Cellular DNA fragmentation was observed in the infected cells, which had cells apoptotic rates of 4.1% and 45.7% at 96 h and 144 h post-infection, respectively. CONCLUSION: HCMV can induce apoptosis of ECV304 endothelial-like cells.

Antigens, Viral↗

PubMed2006

[Morphological changes of ECV304 cells infected by herpes simplex virus type 2].

OBJECTIVE: To observe the pathological changes and morphological alterations of ECV304 cells after the infection by herpes simplex virus type 2 (HSV-2) in vitro. METHODS: Passaged ECV304 cells were infected with HSV-2, TCID50 and morphological changes were observed by optical microscopy and tissue staining. RESULTS: One day after HSV-2 infection, swelling, rounding, and increase of thickened cytoplasmic granules occurred in the ECV304 cells, and on day 2, cell fusion was observed with weakened nuclear staining. CONCLUSION: ECV304 cells mostly undergo necrosis after HSV-2 infection without obvious evidence of cell apoptosis.

Cells, Cultured↗

PubMed2006

[Preparation of small interfering RNA expression cassette based on PCR technique].

OBJECTIVE: To modify the current PCR-based method for rapid and efficient preparation of small interfering RNA (siRNA) expression cassette to improve the efficiency of RNA interference. METHODS: The U6 promoter sequence was amplified by PCR using the genomic DNA of K562 cells as the template, and cloned into pMD18-T vector which served as the template for further PCR amplification with the primers on the plasmid. The amplified product was directly used as the template for preparing siRNA expression cassette. The siRNA expression cassette targeting p53 gene was amplified, verified by sequencing, and transfected into SH-SY5Y cells. After a 48-hour transfection, the cells were harvested and the total RNA was for RT-PCR for evaluating the effect of RNA interference. RESULTS: The sequencing result confirmed the correct U6 promoter sequence cloned from K562 cells. After transfection of SH-SY5Y cells for 48 h with siRNA expression cassette, the p53 gene expression was inhibited at the mRNA level in comparison with the control cells as demonstrated by RT-PCR detection. CONCLUSION: The siRNA expression cassette prepared using the established method described hereby can be well applicable in RNA interference research.

Gene Silencing↗

PubMed2006

[Effects of microRNA miR-181a on gene expression profiles of K562 cells].

OBJECTIVE: To investigate the effect of microRNA on the gene expression profile of human leukemia K562 cells using microarray technique. METHODS: miR-181a RNA duplexes were designed and synthesized according to the mature sequence of miR-181a. Forty-eight hours after transfection of in vitro cultured K562 cells using Oligofectamine, gene expression profiles of the cells were studied and analyzed using Agilent Human 1A Oligo microarray. RESULTS: Totalling 228 differentially expressed genes were identified from the 20,173 screened genes, including 59 up-regulated ones (consisting of metabolism-associated genes, tumor suppressor genes, signal transduction-associated genes, immunity and defense-associated genes etc), and 169 down-regulated ones (consisting of oncogenes, DNA-binding and transcription genes, metabolism-associated genes, signal transduction-associated genes, cell cycle and development-associated genes etc.) in the transfected K562 cells as compared with the control K562 cells. Changes in expressions of CTCF, ZAP70, SEMA4C and RALA were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. CONCLUSIONS: miR-181a transfection for 48 h induces gene expression profile changes in K562 cells, indicating the functionality of the miR-181a. These differentially expressed genes are related to the functions of the microRNA, and may also be the basis of the regulation model of posttranscriptional gene silencing. These findings provide an evidence for further study of the machineries and functions of the microRNA in mammalian cells.

Gene Expression Profiling↗

PubMed2006

[Phenotypic analysis of luxS gene deletion mutants and its application in virulence regulation research in group B Streptococcus].

OBJECTIVE: To explore the mechanism of virulence regulation in group B streptococcus (BGS) by studying LuxS-related AI-2 quorum-sensing pathway in GBS. METHODS: luxS gene deletion mutants of GBS (Delta lusX) were characterized by reverse transcription (RT)-PCR, colony immunoblot analysis, growth curve measurement, and cAMP determination. Functional analysis of luxS in the deletion mutants was conducted by bioluminescence assay. RESULTS: Genetic analysis results showed that the luxS deletion in the mutant 515-Delta lusX caused upregulation of scpB gene expression. Phenotypic analysis revealed that, in comparison with the wild strain, 515-Delta lusX mutant grew slowly in DCM media but quickly in THY media. An approximately two-fold decrement in bioluminescence was detected in the luxS deletion mutants as compared with the wild strain. CONCLUSION: This study confirms the importance of LuxS molecule in the AI-2 quorum-sensing pathway in GBS and provides new insights into the virulence regulation mechanism of GBS.

Bacterial Proteins↗

PubMed2005

[c-myc gene silencing in K562 cells with RNA interference].

OBJECTIVE: To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells. METHODS: siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting. RESULTS: Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate. CONCLUSION: RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.

Apoptosis↗

PubMed2005

[Research of restriction display technique in cDNA microarrays preparation for detecting of HCV].

The cDNA microarrays for HCV detection was prepared. With the restriction display technique (RD), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and amplified by RD-PCR. We separated the differential genes through polyacrylamide gel electrophoresis and sliver staining. Single bands were isolated which were cut out from the polyacrylamide gel. The third-round PCR could be performed by using the single bands as PCR template. The RD-PCR fragments were purified and cloned into the pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting PCR products to the surface of amido modified glass slides by the robotics. We validated the detection of microarray by the hybridization and the results of sequence analysis. A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced, which were the specific gene fragments of HCV. These fragments could be further used as probes in the microarray preparations. From the results of hybridization and sequence date analysis, the specificity, sensitivity, accuracy, reproducibility and linearity in detecting HCV RNA were satisfactory. RD technique is of great value in obtaining a large number of size-comparable gene probes, which provide a swift protocol in generating probes for the preparation of microarrays, and the optimized microarray is sensitive and effective in clinical diagnosis of HCV.

DNA Probes↗

PubMed2005

[Expression and identification of F1 antigen of Y.pestis in Saccharomyces cerevisiae].

AIM: To construct the recombinant vector containing F1 gene caf1 of Y.pestis and express it in Saccharomyces cerevisiae. METHODS: F1 gene caf1 of Y.pestis was inserted into pHSS6-mTn-3xHA/lacZ plasmid to construct recombinant vector pHSS6-mTn-3xHA/lacZ-caf1. The recombinant plasmid was linearized with Not I and then transformed into yeast cells by acetate lithium (LiAc) method. Positive recombinants were selected with uracil-lack medium. The expressed F1 antigen was identified by SDS-PAGE and Western blot. RESULTS: SDS-PAGE and Western blot analysis showed that F1 antigen was expressed in Saccharomyces cerevisiae. CONCLUSION: The recombinant vector pHSS6-mTn-3xHA/lacZ-caf1 has been constructed and expressed successfully in Saccharomyces cerevisiae. These results lay the foundation for preparing gene vaccine of Y.pestis which could be taken via alimentary tract pathway.

Antigens, Bacterial↗

PubMed2005

[Comparison of two amine-modified chemical platforms for DNA microarray preparation].

OBJECTIVE: To study two amine modification procedures for DNA microarray preparation based on polymeric coatings. METHODS: One of the proposed approaches utilized poly-amine coating of silanized slides activated by 1,4-phenylene diisothiocyanate, and the other employed acrylic acid-co-acrylamide copolymer and 1-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide as the coating agents and activator, respectively. The modified slides were used for preparing lambda phage DNA microarrays, whose properties were analyzed by hybridization. RESULTS: Formation of dendrimeric structure and polymer was observed on the surface of the slides. The signal spots in uniform, steady and regular shape, in comparison with the commercial CMT-GAPS slides, indicated successful manufacture of the microarrays. CONCLUSION: The two platforms are suitable for microarray preparation, and the method of acrylic acid-co-acrylamide copolymer modification is more preferred.

Acrylamides↗

PubMed2005

A novel sample labeling method with restriction display PCR for 60-mer oligonucleotide microarray.

OBJECTIVE: To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray. METHODS: Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases. RESULTS: Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes. CONCLUSION: Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.

Humans↗

PubMed2005

[Application of green fluorescent protein gene in study of adipocyte differentiation].

OBJECTIVE: To establish a model for dynamic detection of adipocyte differentiation in vivo by green fluorescent protein (GFP). METHODS: The promoter of peroxisome proliferators-activated receptor gamma 2 (PPARgamma2) gene was amplified by PCR from the genomic DNA of human preadipocytes, inserted into the eukaryotic expression vector pEGFP-1 and transferred into NIH3T3 fibroblasts and preadipocytes which were induced to differentiate into adipocyte. RESULTS: Upon induction with insulin, dexamethasone and 3-isobutyl-1-methyl xanthine, the transformed human preadipocytes differentiated into mature adipocytes and expressed GFP, but NIH3T3 fibroblasts could not differentiate into adipocytes or express GFP. CONCLUSION: Transformation of human preadipocytes with the recombinant of PPARgamma2 promoter with GFP gene can be employed to dynamically detect the differentiation of adipocytes in vivo.

Adipocytes↗

PubMed2005

[Detection of quorum-sensing pathway and construction of LuxS gene deletion mutants of group B Streptococcus].

OBJECTIVE: To detect the possible presence of AI-2 quorum-sensing pathway and construct group B Streptococcus (GBS) mutants with deletion of LuxS gene related to quorum-sensing pathway. METHOD: V. harveyi BB170 was employed as the reporter strain to detect AI-2 pathway in GBS, and identification of LuxS homologous gene in GBS type V strain 2603 was performed by software-based analysis. LuxS gene deletion mutant Delta LusX was then constructed in GBS by means of allelic exchange and verified by Southern hybridization analysis. RESULTS: A component in the secretions of GBS could induce bioluminescence activity in the reporter strain, suggesting the presence of AI-2 quorum-sensing pathway in GBS. luxS homologous gene was detected in GBS and LuxS gene deletion mutants was successfully constructed in GBS Ia 515 and V 2603 strains. CONCLUSION: This study establishes a bacterial model for studying the role of LuxS molecule in AI-2 quorum-sensing pathway in GBS and provides new insights into virulence regulation mechanism of GBS.

Bacterial Adhesion↗

PubMed2004

DNA microarray probe preparation by gel isolation nested PCR.

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

Bacteriophage lambda↗

PubMed2004

[Amplification and cloning of the N gene of SARS-associated coronavirus].

OBJECTIVE: To amplify and clone the N gene of severe acute respiratory syndrome-associated coronavirus. METHOD: Using primer Premier 5.0 software, two pairs of nested PCR primers were designed to amplify the N gene. After purification, the amplified products were cloned into pMD18-T vectors, and the positive clones with the inserted fragments were identified by sequence analysis. RESULTS: The amplified products was about 1 375 bp in length, and sequence analysis demonstrated that the N gene fragments had been successfully inserted into pMD18-T vectors. CONCLUSION: The successful amplification and cloning of N gene facilitates further investigation of the expression of the N protein and study of its structure and functions.

Base Sequence↗

PubMed2004

[Preparation of the microarray for detecting hepatitis D virus].

OBJECTIVE: To prepare the microarray for detection of hepatitis D virus (HDV). METHOD: Several pairs of specific PCR primers were designed according to the conserved region of HDV genome. The DNA microarray were prepared by blotting the PCR products onto the surface of glass slides with the use of robotics, and restriction display PCR (RD-PCR) was employed to label the samples. RESULT: Sequences analysis showed that the products of PCR amplification were the specific gene fragments of HDV. Hybridization signals on the gene chip demonstrated good specificity and sensitivity of the microarray for HDV detection. CONCLUSION: Microarray-based clinical HDV detection can be sensitive and effective.

Base Sequence↗

PubMed2004

[DNA microarray for the detection of Yersinia pesits].

OBJECTIVE: To develop a DNA microarray technique for fast diagnosis of plague. METHODS: Restriction display polymerase chain reaction (RD-PCR) and hybridization with fluorescently labeled cy5 were employed for detecting the sample DNA of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica. RESULTS: The prepared microarray is capable of distinguishing Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica in the same genus. CONCLUSION: The constructed DNA microarray is an effective diagnostic tool for the detection of Yersinia pestis.

Fluorescence↗

PubMed2004

Design and preparation of oligonucleotide microarray for vaccinia virus detection.

OBJECTIVE: To study the preparation of oligonucleotide microarray for detecting vaccinia virus. METHODS: Oligonucleotide probes were designed and synthesized according to the specific genes of vaccinia virus. Sample DNA of the virus and the negative control sample were obtained and labeled by restriction display technique, followed by hybridization to the oligonucleotide microarray and scanned by Agilent scanner. RESULTS: Strong hybridization signals were detected from the viral DNA hybridized with the microarray, but were absent in the negative sample when positive probes were not used. CONCLUSION: Distinct differences in the hybridization signals between the virus sample and negative sample and between the samples obtained in different phase of infection demonstrate high specificity and sensitivity of the microarray for vaccinia virus detection.

Base Sequence↗