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Detection of fusion gene in cell-free DNA of a gastric synovial sarcoma 被引量:5
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作者 Shinpei Ogino Hirotaka Konishi +10 位作者 Daisuke Ichikawa Junichi Hamada Katsutoshi Shoda Tomohiro Arita Shuhei Komatsu Atsushi Shiozaki Kazuma Okamoto Sanae Yamazaki Satoru Yasukawa Eiichi Konishi Eigo Otsuji 《World Journal of Gastroenterology》 SCIE CAS 2018年第8期949-956,共8页
Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we des... Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA(cf DNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction(PCR) and direct sequencing. A locked nucleic acid(LNA) probe specificto the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfD NA, while not detected in postoperative plasma cfD NA. This technique will be useful for monitoring translocation-derived diseases such as SS. 展开更多
关键词 fusion gene GASTRIC SYNOVIAL SARCOMA PLASMA cell free DNA
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Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap/GFP Fusion Protein in Mammal Cells
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作者 SONGYi-shu SONGZhi-yu +4 位作者 LIHong-jun WuYin BAOYong-li TANDa-peng LIYu-xin 《Chemical Research in Chinese Universities》 SCIE CAS CSCD 2005年第3期298-300,共3页
s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of t... s-Lap is a new gene sequence from pig retinal pigment epithelial(RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B_~16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. ~s-Lap/GFP fusion protein can be expressed in CHO and B_~16 cells with a high rate expression in the nuclei. 展开更多
关键词 s-Lap gene fusion protein Mammal cell EXPRESSION LOCALIZATION
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Detection of TMPRSS2:ERG fusion gene in circulating prostate cancer cells
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作者 Xueying Mao Greg Shaw +9 位作者 Sharon Y. James Patricia Purkis Sakunthala C. Kudahetti Theodora Tsigani Saname Kia Bryan D. Young R. Tim D. Oliver Dan Berney David M. Prowse Yong-Jie Lu 《Asian Journal of Andrology》 SCIE CAS CSCD 2008年第3期467-473,共7页
Aim: To investigate the existence of TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) from prostate cancer patients and its potential in monitoring tumor metastasis. Methods- We analyzed the frequency of T... Aim: To investigate the existence of TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) from prostate cancer patients and its potential in monitoring tumor metastasis. Methods- We analyzed the frequency of TMPRSS2: ERG and TMPRSS2:ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies, and TMPRSS2:ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using reverse transcription polymerase chain reaction (RT-PCR). Fluorescence in situ hybridization (FISH) was applied to analyze the genomic truncation of ERG, which is the result of TMPRSS2:ERG fusion in 10 of the 15 CTC samples. Results: TMPRSS2: ERG transcripts were found in 44% of our samples, but we did not detect expression of TMPRSS2:ETV1. Using FISH analysis we detected chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2:ERG fusion at the primary site. However, TMPRSS2:ERG transcripts were not detected in any of the 15 CTC samples, including the 10 cases analyzed by FISH. Conclusion: Although further study is required to address the association between TMPRSS2:ERG fusion and prostate cancer metastasis, detection of genomic truncation of the ERG gene by FISH analysis could be useful for monitoring the appearance of CTC and the potential for prostate cancer metastasis. 展开更多
关键词 TMPRSS2:ERG fusion gene prostate cancer METASTASIS circulating tumor cells fluorescence in situ hybridization polymerase chain reaction
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An Efficient Electro-Competent Cells Generation Method of Xanthomonas campestris pv. campestris: Its Application for Plasmid Transformation and Gene Replacement 被引量:1
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作者 Xiuli Wang Daning Zheng Rubing Liang 《Advances in Microbiology》 2016年第2期79-87,共9页
A simple and rapid method to prepare efficient electro-competent cells of Xanthomonas campestris pv. campestris was generated, with up to 100-fold transformation efficiencies over the existing procedures. The overnigh... A simple and rapid method to prepare efficient electro-competent cells of Xanthomonas campestris pv. campestris was generated, with up to 100-fold transformation efficiencies over the existing procedures. The overnight cultures were treated with sucrose solution and micro-centrifuged at room temperature;the entire electro-competent cells generation process can be completed in 15 minutes. It overcomes the complication and time-consuming shortcomings of the traditional conjugation or electro-transformation methods in this strain. Both the replicative plasmids and non-replicative plasmids could be transformed or integrated efficiently using this method. And the DNA concentration, cells growth stage, field strength and recovery time all had influences on the transformation efficiency. In the optimal conditions, the transformation efficiency for the replicative plasmids was 10<sup>9</sup> transformants per microgram DNA, and for non-replicative plasmids was 150 transformants per microgram DNA. Further with the homology sequences, two chromosomal target genes were deleted efficiently and the knockout strains were obtained easily. 展开更多
关键词 XANTHOMONAS Electro-Competent cells Electro-transformation gene Replacement INTEGRATION
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Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells 被引量:4
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作者 WEI-DONG JI JIA-KUN CHEN JIA-CHUN LU ZHONG-LIANG WU FEI YI SU-MEI FENG 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2006年第4期277-284,共8页
Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and ... Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared wkh that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P〈0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in pl6 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens. 展开更多
关键词 Crystalline nickel sulfide Human bronchial epithelial cell line Malignant transformation P16 gene FHIT gene
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Molecular Tissue Engineering: Applications for Modulation of Mesenchymal Stem Cells Proliferation by Transforming Growth Factor β_1 Gene Transfer 被引量:3
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作者 郭晓东 杜靖远 +3 位作者 郑启新 刘勇 段德宇 吴永超 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2001年第4期314-317,共4页
The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basi... The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF β 1 gene at different doses was transduced into the rat bone marrow derived MSCs to examine the effects of TGF β 1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectamine mediated 1 μg TGF β 1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine=1μg/3μl), flow cytometry and immunohistochemical analyses revealed a significant increase in the 3 H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF β 1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases. 展开更多
关键词 articular cartilage defect repair tissue engineering gene transfer mesenchymal stem cells transforming growth factor β 1 molecular tissue engineering
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Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene 被引量:1
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作者 游洪波 《Journal of Wuhan University of Technology(Materials Science)》 SCIE EI CAS 2011年第4期634-640,共7页
The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells (PSCs) into chondrocytes was studied. KLD-12 was synthesized by so... The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells (PSCs) into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-β3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group (P〈0.05). Quantitative real-time PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group (P〈0.01). ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group's PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated (P〈0.01). The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, which provided a fresh approach to cartilage tissue engineering. 展开更多
关键词 precartilaginous stem cells tissue engineering SCAFFOLD gene self-assembled peptide transforming growth factor
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Experimental Study of Plasmid TGF-β1 DNA Gene Transfer with Lipofectamine into Rabbit Corneal Epithelial Cells In Vitro
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作者 黄琼 胡燕华 +1 位作者 姜发纲 陈宏 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2002年第1期62-65,共4页
To investigate whether the TGF β1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF β1 in the cultured corneal epit... To investigate whether the TGF β1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF β1 in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMTGF β1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF β1 protein expression specific for pMAMTGF β1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23.37 %. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF β1 on the mechanism of physiology and pathology concerned with corneal epithelial cells. 展开更多
关键词 CORNEA epithelial cells gene transfer transforming growth factor beta
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Osteogenic Potential of Cultured Bone Marrow Stromal Cells Transfected with Transforming Growth Factor β_1 Gene in vitro
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作者 易诚青 郑启新 +1 位作者 郭晓东 刘勇 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2001年第2期130-133,共4页
To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β ... To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β 1 and Lipofectamine Reagent in vitro . The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type Ⅰ propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type Ⅰ expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF β 1 gene could promote the osteogenic potential of cultured BMSCs. 展开更多
关键词 transforming growth factor β 1 gene transfection bone marrow stromal cells osteogenic potential
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THE EFFECT OF db-cAMP ON THE GENE EXPRESSION OF CALMODULIN AND CYTOSKELETON IN THE TRANSFORMED CELLS
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作者 柳惠图 王端顺 +4 位作者 张鸿卿 薛绍白 游劲松 杜春英 王晓良 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 1992年第1期25-34,共10页
We have demonstrated that the distribution of microtubules (MT), mlcrofilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c- fos enhanced in the transformed C3H10T1/2 c... We have demonstrated that the distribution of microtubules (MT), mlcrofilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c- fos enhanced in the transformed C3H10T1/2 cells. After treatment with 1 mM db-cAMP for 1 hour and 2 hours, there was an early and repldly reduced in gene expression of Calmodulin and c-fos respectively. After db-cAMP treatment for 4 -5 days, the number of capping cells of ConA binding decreased significantly and the cell surface microvllll decreased as well. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed In G1 phase, we have found that the db- cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, mlcrofilaments and fibronectln were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the Inhibition of proliferation, alteration, of phenotype and reco- very of cytoskeleton is transformed cells after treatment with db-cAMP are related to the Inhibition of gene expression of Calmodulin. 展开更多
关键词 THE EFFECT OF db-cAMP ON THE gene EXPRESSION OF CALMODULIN AND CYTOSKELETON IN THE TRANSFORMED cellS gene FIGURE
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DIFFERENCES IN EXPRESSION AND REGULATION BETWEEN TRANSFORMED CELLS OF THE HUMAN GASTRIC CARCINOMA ONCOGENE Ha-ras AND THE UNTRANSFORMED PARENT CELLS
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作者 韩复生 刘淑萍 +5 位作者 宋建国 袁艳华 张维 施华 邓国仁 刘培楠 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 1989年第1期20-25,共6页
An Ha-ras oncogene was isolated from a cell line of gastric carcinoma called BGE-823 in order to elucidate genetic control and the influence of DNA sequences. The oncogene was cloned and identified as a single nucleot... An Ha-ras oncogene was isolated from a cell line of gastric carcinoma called BGE-823 in order to elucidate genetic control and the influence of DNA sequences. The oncogene was cloned and identified as a single nucleotide substitution of thymine for guanine in the 12th codon through the sequencing of its first axon. We compared the differences of expression and regulation between the transformed Ha-ras cells and untransformed parent cells. Data indicated that the expression of Ha-ras in the transformed cells was five-fold higher than in the untransformed cells and that the Ha-ras gene in the former was hypersensitive toward DNase I. In addition, a nuclear protein of 35 kilodaltons bound strongly to the 2.5 Kb fragment located upstream of the 6.6 Kb Ha-ras gene and contained a CC rich region. These results suggest that there might be another mechanism of activation for the ras gene besides point mutation. 展开更多
关键词 gene DIFFERENCES IN EXPRESSION AND REGULATION BETWEEN TRANSFORMED cellS OF THE HUMAN GASTRIC CARCINOMA ONCOgene Ha-ras AND THE UNTRANSFORMED PARENT cellS
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Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells
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作者 苏立 际运贞 +1 位作者 张晓刚 余强 《South China Journal of Cardiology》 CAS 2005年第1期11-15,共5页
Objectives To construct a recombinant plasmid carrying enhanced green fluore- scent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expre- ssion in rat mesenchymal stem cells... Objectives To construct a recombinant plasmid carrying enhanced green fluore- scent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expre- ssion in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP- C1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluoresce- nce microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection. Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease. 展开更多
关键词 Vascular endothelial growth factor Enhanced green fluorescent protein fusion protein Mesenchymal stem cells gene expression
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SUPPRESSION OF MALIGNANT PHENOTYPE OF A TRANSFORMED MOUSE MAMMARY EPITHELIAL CELL LINE (11A1)BY TUMOR SUPPRESSOR GENE RB
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作者 王冬梅 李申德 +1 位作者 崔惠云 杨晓洁 《Chinese Medical Sciences Journal》 CAS CSCD 1997年第2期76-79,共4页
A malignant transformed mammary epithelial cell line (11A1) was transfected with liposome encapsulated eukaryotic expression plasmid pCMV-neo-RB, yielding 4 constant clones which have obvious pheno-typic reversion cha... A malignant transformed mammary epithelial cell line (11A1) was transfected with liposome encapsulated eukaryotic expression plasmid pCMV-neo-RB, yielding 4 constant clones which have obvious pheno-typic reversion changes, and named 11A1-R1-R4 respectively. Further experiments showed that the 11A1-R1 behaved like normal epithelial cells in both morphological and biological characteristics, with decreased clonogenicity in solid argar medium as well as decreased tumorigenicity. Northern blot hybridization showed increased expression of RB gene and decreased expression of c-myc gene in 11A1-R1, 11A1-R2 cells compared to 11A1 cells. This was an ideal phenotypic reversion model for epithelial transformed cell line and demonstrated that the RB gene can reexpress and suppress malignant phenotype in RB inactive cells. 展开更多
关键词 RB gene phenotypic reversion transformed cell line
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Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system 被引量:12
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作者 DengXY WeiYZ 《Cell Research》 SCIE CAS CSCD 2001年第2期156-160,共5页
After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resist... After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us. 展开更多
关键词 CINNAMATES Anti-Bacterial Agents Arachis hypogaea cell Culture Techniques CHIMERA COTYLEDON Drug Resistance gene Expression Regulation Plant genetic Engineering Hygromycin B Osmosis Plants genetically Modified Plasmids Regeneration Research Support Non-U.S. Gov't Seeds transformation genetic
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Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer 被引量:3
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作者 Zhang, Mang-Li Lu, Sen +1 位作者 Zhou, Lin Zheng, Shu-Sen 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2008年第5期533-538,共6页
BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role ... BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of pancreatic cancer. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the ECT2 gene in pancreatic cancer. METHODS: Four cell lines (PANC-1, Colo357, T3M-4 and PancTu I) and pancreatic ductal adenocarcinoma (PDAC) tissues were used for mRNA detection. After restriction isoschizomer endonucleases (Msp I/Hpa II) were used to digest the DNA sequence (5'-CCGG-3'), PCR was made to amplify the product. And RT-PCR was applied to determine the expression of the gene. RESULTS: The mRNA expression of the ECT2 gene was higher in pancreatic tumor tissue than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the ECT2 gene were almost identical in normal, tumor pancreatic tissues, and the 4 PDAC cell lines. Some of the 5'-CCGG-3' areas in the upstream region of ECT2 were methylated, while others were unmethylated. CONCLUSIONS: The oncogene ECT2 is overexpressed in pancreatic tumor tissues as verified by RT-PCR detection. The methylation status of DNA in promoter areas is involved in the gene expression, along with other factors, in pancreatic cancer. 展开更多
关键词 pancreatic neoplasms epithelial cell transforming sequence 2 gene epigenesis genetic
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Expression, purification and bioactivity of human augmenter of liver regeneration 被引量:2
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作者 Yang-De Zhang Jian Zhou +4 位作者 Jin-Feng Zhao Jian Peng Xiao-Dong Liu Xin-Sheng Liu Ze-Ming Jia 《World Journal of Gastroenterology》 SCIE CAS CSCD 2006年第27期4401-4405,共5页
AIM: To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity. METHODS: hALRcDNA clone was obtained from plasmid pGEM-T... AIM: To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity. METHODS: hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase (GST) sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS: The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups (P 〈 0.01).CONCLUSION: Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application. 展开更多
关键词 Human augmenter of liver regeneration gene recombination EXPRESSION PURIFICATION fusion protein transformation Biological activity
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Transplantation of human hepatocytes into tolerized genetically immunocompetent rats 被引量:23
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作者 EdwinC.Ouyang CatherineH.Wu +2 位作者 CherieWalton KittichaiPromrat GeorgeY.Wu 《World Journal of Gastroenterology》 SCIE CAS CSCD 2001年第3期324-330,共7页
AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human... AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes. 展开更多
关键词 ALBUMINS Animals cell Line Transformed Disease Models Animal Female gene Expression Graft Survival Hepatitis HEPATOBLASTOMA Hepatocytes Humans Immune Tolerance IMMUNOCOMPETENCE Liver Liver Neoplasms Lymphocyte Culture Test Mixed Microscopy Confocal Pregnancy RNA Messenger RATS Rats Sprague-Dawley Research Support Non-U.S. Gov't Research Support U.S. Gov't P.H.S.
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Fusions of Dendritic Cells and C6 Cells Transfected with TGF-β1 Antisense in Treatment of Intracranial Gliomas
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作者 金贵善 刘福生 +2 位作者 柴奇 王建交 厉俊华 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2007年第2期113-118,共6页
Objective: To investigate the immunotherapy efficacy of fusion cells (dendritic-C6anti-TGF-β1 cells) in the treatment of intraeranial gliomas. Methods: Dendritic cells were isolated from rat bone-marrow precursor... Objective: To investigate the immunotherapy efficacy of fusion cells (dendritic-C6anti-TGF-β1 cells) in the treatment of intraeranial gliomas. Methods: Dendritic cells were isolated from rat bone-marrow precursors stimulated in vitro with granuloeyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4). C6anti-TGF-β1 cells originally from C6 cell line of a rat glioblastoma were transfected with plasmid of TGF-β1 anti-sense gene. Fusions of dendritic cells and C6anti-TGF-β1 cells were prepared by polyethylene glycol (PEG). The DC/C6anti-TGF-β1 fusion cells were observed and confirmed by fight microscopy and scanning electron microscopy. Experimental rats were divided into three groups at random: C6 cells (Ⅰ), dendritic-C6anti-TGF-β1 fusion cells and C6 cells (Ⅱ) and IMDM medium only (Ⅲ). The cells were injected into right parietal lobe region of the rat with stereotaxic technique. Histology, tumor necrosis and survival time were evaluated. Results: Compared with the rats that received C6 cells (survival median time was less than 20 days, tumor region was seen in all fields of observed), the rats injected with dendritic-C6anti-TGF-β1 fusion cells and C6 cells got a more prolonged life span (more than 59 days), as well as less tumor region (5.01%-6.2%). There was no tumor necrosis, but some glias were seen in surroundings. All rats were survived and no necrosis was observed in negative control group. Statistical analysis showed that group Ⅱ had significant difference compared with group Ⅰ. Conclusions: Dendritic-C6anti-TGF-β1 fusion cells could prolong the life span of rats, providing a strategy to achieve an antitumor response against tumors in the central nervous system. 展开更多
关键词 Giioma Immunotherapy Dendritic cell TGF-Β1 Anti-sense gene fusion cells
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Inhibiting Mechanism of Baculovirus p35 Gene to Apoptosis
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作者 Li Xiaofeng Qi Yipengt +1 位作者 Lin Hong Li Yan(College of Life Sciences, Wuhan University, Wuhan 43OO72, China) 《Wuhan University Journal of Natural Sciences》 EI CAS 1998年第2期243-246,共4页
We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant t... We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35 gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNA electrophoresis, Sf9-35 cells were found to resist apoptosis induced by infection of vAcΔp35 deleting p35 gene and actinomycin D treatment; And it was also found that apoptosis induced by viral infection and actinomycin D treatment can only be delayed, but can not be stopped in Sf9-35. 展开更多
关键词 APOPTOSIS transformed cells p35 gene BACULOVIRUS
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Effects of chimeric intron on the epithelial-mesenchymal transformation of non-small cell lung cancer PC-9
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作者 Liang Liao Guo-Hui Yang 《Journal of Hainan Medical University》 2021年第2期1-5,共5页
Objective:To investigate the effects of Intron on the EMT capability of non-small cell lung cancer cell line PC-9.Methods:Firstly,using the psiCHECK-2 plasmid as a basic framework to construct the recombinant plasmid ... Objective:To investigate the effects of Intron on the EMT capability of non-small cell lung cancer cell line PC-9.Methods:Firstly,using the psiCHECK-2 plasmid as a basic framework to construct the recombinant plasmid of psiCHECK-2-Intron dual-luciferase reporter gene;secondly,the psiCHECK-2-Intron and psiCHECK-2 were transfected into PC-9 cells respectively.The migration and invasion abilities of PC-9 cells were analyzed by Matrigel assay.The expression changes of EMT related hallmarks,including N-cadherin,β-catenin and snail,were detected by qRT-PCR and Western Blotting.Results:Compared with the control group,the migration and invasion abilities of PC-9 cells in Intron group significantly decreased(p<0.001).The expression of N-cadherin,β-catenin and snail also down-regulated(p<0.001).Conclusion:The introns could inhibit the EMT of PC-9 cells. 展开更多
关键词 Chimeric intron PC-9 cell line Dual-fluorescent protein reporter gene Non-small cell lung cancer(NSCLC) Epithelial-mesenchymal transformation(EMT)
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