Biomedical subjects

Yan-Bin Song

Publications and source records attributed to Yan-Bin Song.

11 recordsLinked to original sources

PubMed2006

[Research of the spermatozoal gene expression with gene microarrays].

OBJECTIVE: To perform the detection of spermatozoal gene expression in order to accelerate the study of spermatozoal molecular biology. METHODS: To collect the healthy adults sperm and lymphocytes respectively, and then to extract the total RNAs from them by RNeasy mini kit (QIAGEN) or Trizol reagent. Corresponding cDNAs were produced, digested, ligated, finally labeled with Cy3 (sperm) and CyS (lymphocyte) in the course of RD amplifying reactions. Hybridization with self-made microarrays contained 560 probes was carried out after the labeled cDNAs pured by PCR Product Purification Kit. RESULTS: Among the 560 probes, 72 genes were up-regulated, 321 genes were down-regulated, the others had no different expression. Furthermore, genes associated with replication, transcription, translation and regulative functions were non-different expression or down-regulated, and those belonged to the spermatogenesis associated, sperm associated antigen were up-regulated, but those involved in the glycolysis were up-regulated, in the oxidative phosphorylation were down-regulated. CONCLUSION: It had successfully confirmed that there were a plenty of genes expressed in sperm, furthermore the genes expressed were accorded to spermatozoal functions and characteristics.

Adult↗

PubMed2005

Growth inhibition of K562 cells by cyclin E gene-specific small interfering RNA.

OBJECTIVE: To study the inhibitory effect of small interference RNA(siRNA) of cyclin E gene on the growth of K562 cells. METHODS: siRNA targeting the 940 bp site of the cyclin E mRNA were designed and generated by PCR amplification. The PCR products containing U6 promoter and the siRNA were then transfected into K562 cells via Lipofectamine2000. The cells transfected with non-functional siRNA served as the negative control group and those only treated with serum-free RPMI1640 as the blank control group. Cell counting, reverse transcriptase (RT)-PCR and flow cytometry were employed to evaluate the effect of RNA interference. RESULTS: Compared with the negative and blank control groups, the viable cell count in the interference group was decreased by approximately 80%, the ratio of G(1)-phase cells increased by nearly 30%, and growth arrest was observed. Cyclin E mRNA expression in the cells of the interference group was significantly lowered by about 70%; as compared with that of the negative and blank control groups, whereas the latter two groups had similar expression levels. CONCLUSION: RNA interference induces obvious inhibition of cyclin E gene expression, which consequently affects the proliferation of K562 cells.

Cell Proliferation↗

PubMed2005

[Cloning of human obesity gene and its expression in E. coli].

OBJECTIVE: To clone the obesity gene of Chinese and express human leptin in E.coli. METHOD: The obesity gene was amplified from the total RNA isolated from cultured human adipocytes of Chinese by reverse transcriptional PCR, inserted into TA-vector and cloned into the expression plasmid pBV220 after sequence identification. RESULTS: DNA sequencing confirmed that the isolated obesity gene was identical to the previously reported sequence. The recombinant plasmid pBV220-OB was constructed and leptin successfully expressed in E.coli. CONCLUSION: Successful cloning and expression of human obesity gene in E.coli may facilitate further research of the mechanism of fat metabolism and adipocyte differentiation.

Adipocytes↗

PubMed2005

[Construction and preliminary identification of subtracted cDNA library of leukemia cell line K562].

OBJECTIVE: Subtractive hybridization technology is a common method to screen and clone differentially expressed genes. This study was to construct subtracted cDNA library of leukemia cell line K562, and screen for differentially expressed genes. METHODS: cDNA fragments of K562 cells (tester), prepared by restriction display (RD), were subtracted with the Sau3A I-digested cDNA fragments of normal lymphocytes (driver). The subtracted cDNA fragments were re-amplified, and cloned into pMD18-T vectors. Positive clones were selected by blue-white screening. The inserts in plasmid were amplified by polymerase chain reaction (PCR), and some of which were sequenced. RESULTS: The subtracted library contained 360 positive clones with cDNA fragments distributed mainly from 200 to 800 bp. The 50 randomly sequenced clones were derived from 42 known genes. CONCLUSION: Specific subtracted cDNA library of K562 cells was successfully constructed with reliable quality, and may be used to further screen and clone differentially expressed genes of K562 cells.

Cloning, Molecular↗

PubMed2003

[Analysis of gene expression patterns of leukemia K562 cells after cytochalasin B treatment].

BACKGROUND & OBJECTIVE: The advanced technique of DNA microarray makes it possible to monitor the expression of thousands of genes simultaneously in one hybridization experiment. This technique accelerates demonstration of anti-tumor drug mechanisms and discovery of new drug targets. This study was designed to investigate the differential gene expression of K562 cells after cytochalasin B treatment using cDNA microarray. METHODS: Restriction display polymerase chain reaction (RD-PCR) products of 277 human genes were spotted on a glass slide in microarray. K562 cells grew in RPMI 1640 medium with 10 microg/ml cytochalasin B. After 24 hours, the total RNA was isolated from K562 cells, and mRNA was purified. Both mRNA from the treated K562 cells and the controlled K562 cells were reversely transcribed into cDNA and labeled with two different fluorescence dyes: Cy5 or Cy3, using a method of restriction digestion and PCR labeling (RD-PCR). The probes were hybridized to the cDNA microarrays. After high-stringent washing,the cDNA microarray was scanned for the fluorescent signals and showed difference between the two cells. RESULTS: Among the 277 target genes, 18 down-regulated genes were identified after cytochalasin B treatment. CONCLUSION: There is a consistent tendency toward lower-expressed genes in partial K562 cells after cytochalasin B treatment. Most down-regulated genes were correlated with cell proliferation, signal transduction, and transcription factor.

Cytochalasin B↗

PubMed2002

[Cloning of an expressed sequence tag with restriction display polymerase chain reaction].

OBJECTIVE: To isolate gene fragments from SH-SY5Y cells by way of restriction display polymerase chain reaction (RD-PCR). METHODS: Total mRNA was extracted from SH-SY5Y cells followed by synthesis of the single-strand cDNA with Oligo (dT18) as the anchored primer, and the second strand was synthesized by nick translation. The double strands were cleft with restriction enzyme Sau3A I and the fragments ligated with a universal adapter to be amplified with the universal primers and selected primers. The products were then ligated into the pMD18-T vector and sequenced. RESULTS: One of the sequenced clones was retrieved in the National Center for Biotechnology Information (NCBI) databases with Blast program. The results showed that the sequence possessed great similarity to one fragment of the 17th chromosome in the genome. Sequence analysis with GenScan software indicated that the EST might be one section of an unknown gene. CONCLUSION: RD-PCR provides simple and efficient approach for isolating EST from cells, and cDNA clone sequencing combined with bioinformatics analysis may be helpful in identifying new genes.

Base Sequence↗

PubMed2002

[Cloning E. coli mRNAs with poly (A) tails by restriction digest polymerase chain reaction].

OBJECTIVE: To investigate the polyadenylation at the 3' terminal of the mRNAs in E.coli. METHODS: mRNAs of E.coli was enriched from total RNA with oligo (dT)-cellulose, and reverse transcription was performed using oligo(dT)18 as primer prior to synthesis of double strands cDNA which was digested with Sau 3A I to produce multiple gene fragments that were then ligated with adapters. Restriction digest-polymerase chain reaction (RD-PCR) was employed to divide the fragments into 10 groups using 10 different combinations of the 4 primers, and the products were cloned into T-vectors. RESULTS: More than 100 gene fragments were cloned, 30 of which were sequenced. CONCLUSION: Polyadenylation of E.coli mRNA is not a biochemical curiosity, and very likely, it is a general attribute of the mRNAs of bacteria.

Base Sequence↗

PubMed2002

[Construction and identification of cDNA library of E.coli mRNA with poly(A) tracts].

OBJECTIVE: To construct and identify the cDNA library of E.coli mRNA with poly(A) tracts. METHODS: The cDNA library of E.coli was constructed by restriction display-PCR (RD-PCR) technique, followed by sequencing and bioinformatics analyses. RESULTS: cDNA library of E.coli mRNA with poly(A) tracts was successfully constructed, and 66 gene fragments were sequenced. CONCLUSION: The constructed cDNA library of E.coli mRNA with poly(A) tracts contains a low rate of repetition and is of high quality.

Cloning, Molecular↗

PubMed2002

[Proliferation and denucleation of K562 cells induced by cytochalasin B].

OBJECTIVE: To observe the effect of cytochalasin B on the denucleation of K562 cells. METHODS: K562 cells were grown in RPMI 1640 medium supplemented with 10% calf serum and 4 microL cytochalasin B (CB). Denucleation was induced in the cultured cells by CB, and the cells were examined by phase contrast microscopy and Giemsa staining respectively. RESULTS: The denucleation of K562 cells induced by CB was clearly observed, and the cell proliferation was obviously inhibited. CONCLUSION: The denucleation efficiency of K562 cells is positively correlated with CB concentrations and the duration of CB treatment. CB at low doses may inhibit the cell proliferation and at high doses causes cell death.

Cell Division↗

PubMed2001

Cloning and sequence analysis of HIV-1 gene fragments isolated by restriction digest polymerase chain reaction method.

OBJECTIVE: To clone and analyze the HIV-1gene fragments isolated by restriction digest polymerase chain reaction (RD-PCR). METHOD: All the HIV-1 gene fragments were divided into 10 subgroups and amplified by RD-PCR. The PCR products of each subgroup were purified and cloned into the T-vectors, then identified rapidly. The plasmids were extracted from positive clones and the target gene fragments were amplified and sequenced. RESULTS: Sequences analysis showed that all the fragments amplified were HIV gene. CONCLUSION: A method for cloning and identifying multiple fragments has been improved and used for restriction fragments.

Journal Article↗

PubMed2000

DNA Microarray Chips Made on Surface of Ceramic Slides.

As a novel technology, DNA microarray chips are being used in gene detections. To make DNA chips capable of multiple probing, ceramic slides, instead of silicon wafer or glass slides, were used as the substrate upon which DNA microarray are deposited. Our study indicates that the ceramic DNA microarray chips can be applied to multiple hybridization and detection procedures, showing high specificity. The advantages in using ceramic substrates are discussed.

Journal Article↗