To analyze the relation of matrix metalloproteinase-2(MMP-2) and Fibronection (FN) mRNA expression with metastasis of breast cancer and elucidate the role of MMP-2 and FN in breast cancer metastasis.Methods The expres...To analyze the relation of matrix metalloproteinase-2(MMP-2) and Fibronection (FN) mRNA expression with metastasis of breast cancer and elucidate the role of MMP-2 and FN in breast cancer metastasis.Methods The expression of MMP-2 and FN mRNA in breast cancer cell lines was detected by fluorescence-quantitative RT-PCR.The expression of MMP-2 and FN protein was detected by Western blots.Results The expression of MMP-2 and FN mRNA was down-regulated in high metastatic cell lines MDA-MB-231,MDA-MB-435,but up-regulated in low metastatic cell lines MDA-453,T47D,SK-BR-3 and non-metastatic cell line MCF-7,ZR-75-30.The protein expression of MMP-2 and FN was up-regulated in high mestastic cell lines,and down-regulated in low metastatic cell lines.Conclusion The mRNA and protein expression of MMP-2 and FN was related with breast cancer metastasis.The mRNA expression of MMP-2 and FN is feed-back regulated with protein expression.6 refs,4 figs,2 tabs.展开更多
AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a ...AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.展开更多
In order to investigate the roles of MTA2 in the pathogenesis of ovarian epithelial cancer, the expression of MTA2 in 4 ovarian cell lines were detected by semi-quantitative RT-PCR and Western-blot assays. MTA2 expres...In order to investigate the roles of MTA2 in the pathogenesis of ovarian epithelial cancer, the expression of MTA2 in 4 ovarian cell lines were detected by semi-quantitative RT-PCR and Western-blot assays. MTA2 expression in normal, borderline, benign and malignant epithelial ovarian tissues was immunohistochemically examined. The expression of MTA2 mRNA and protein was detected in all of 4 cell lines of ovarian epithelial cancer. The expression of MTA2 mRNA and protein was higher in strong migration cell lines than in weak migration ones. In borderline and ma lignant ovarian tissues tested, MTA2 staining was dramatically stronger than in normal and benign tissues （P〈0. 01）. The expression levels in malignant ovarian tissues were significantly higher than that in borderline epithelial ovarian tissues （P〈0.01）. The expression of MTA2 was correlated with clinical stage, histopathological grade and lymph node metastasis. It was concluded that the high expression of MTA2 was associated with more aggressive behaviors of epithelial ovarian cancer. MTA2 provides a novel indicator of ovarian cancer.展开更多
OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA ...OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2. CONCLUSIONS: Our data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.展开更多
文摘To analyze the relation of matrix metalloproteinase-2(MMP-2) and Fibronection (FN) mRNA expression with metastasis of breast cancer and elucidate the role of MMP-2 and FN in breast cancer metastasis.Methods The expression of MMP-2 and FN mRNA in breast cancer cell lines was detected by fluorescence-quantitative RT-PCR.The expression of MMP-2 and FN protein was detected by Western blots.Results The expression of MMP-2 and FN mRNA was down-regulated in high metastatic cell lines MDA-MB-231,MDA-MB-435,but up-regulated in low metastatic cell lines MDA-453,T47D,SK-BR-3 and non-metastatic cell line MCF-7,ZR-75-30.The protein expression of MMP-2 and FN was up-regulated in high mestastic cell lines,and down-regulated in low metastatic cell lines.Conclusion The mRNA and protein expression of MMP-2 and FN was related with breast cancer metastasis.The mRNA expression of MMP-2 and FN is feed-back regulated with protein expression.6 refs,4 figs,2 tabs.
基金the"Hundred Talents"Program of Shanghai Municipal Government,No.98BR018
文摘AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.
文摘In order to investigate the roles of MTA2 in the pathogenesis of ovarian epithelial cancer, the expression of MTA2 in 4 ovarian cell lines were detected by semi-quantitative RT-PCR and Western-blot assays. MTA2 expression in normal, borderline, benign and malignant epithelial ovarian tissues was immunohistochemically examined. The expression of MTA2 mRNA and protein was detected in all of 4 cell lines of ovarian epithelial cancer. The expression of MTA2 mRNA and protein was higher in strong migration cell lines than in weak migration ones. In borderline and ma lignant ovarian tissues tested, MTA2 staining was dramatically stronger than in normal and benign tissues （P〈0. 01）. The expression levels in malignant ovarian tissues were significantly higher than that in borderline epithelial ovarian tissues （P〈0.01）. The expression of MTA2 was correlated with clinical stage, histopathological grade and lymph node metastasis. It was concluded that the high expression of MTA2 was associated with more aggressive behaviors of epithelial ovarian cancer. MTA2 provides a novel indicator of ovarian cancer.
基金theMajorStateBasicResearchDevelopmentProgramofChina (No G19990 5 42 0 3)
文摘OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2. CONCLUSIONS: Our data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.
