In order to investigate the role of MAPK gene in adaptation of Dunaliella salina to hypersaline environment, the Ds MAPK gene of D. salina was amplified by PCR. After inverted insertion of the open reading flame ofDs ...In order to investigate the role of MAPK gene in adaptation of Dunaliella salina to hypersaline environment, the Ds MAPK gene of D. salina was amplified by PCR. After inverted insertion of the open reading flame ofDs MAPK gene into downstream sequence of 35S promoter of plant expression vector, an antisense expression vector of Ds MAPK gene was successfully constructed and introduced into D. salina cells by LiAc/PEG-mediated method. The expression of Ds MAPK gene in transgenic D. salina was analyzed by semi-quantitative RT-PCR method. The results showed that the expression of Ds MAPK gene in D. salina was significantly inhibited at the transcriptional level. The study laid the foundation for further identification of the function of Ds MAPK gene.展开更多
A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from geno...A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.展开更多
Objective：Hyperglycemia stimulates secretion of transforming growth factor-βl （TGF-βl） in cultured glomerular mesangial cells, thereby increases production of extracellular matrix （ECM）. We examined the effect ...Objective：Hyperglycemia stimulates secretion of transforming growth factor-βl （TGF-βl） in cultured glomerular mesangial cells, thereby increases production of extracellular matrix （ECM）. We examined the effect of antisense mRNA for Smad2 on high glucose-induced ECM production in rat mesangial cells. Methods..A mammalian expression vector, pES2a, which expresses antisense Smad2 mRNA and green fluorescent protein （EGFP）, was transfected into mesangial cells. Following incubation in high glucose medium, EGFP expression and Smad2 mRNA level were determined by fluorescence microscopy and PCR, respectively. Secreted fibronectin and type IV collagen were assessed by enzyme-linked immunosorbent assay. Results ：Within 48 h of incubation in high glucose medium, Smad2 mRNA level significantly increased by 1.6 fold in association with increases in prodtaction of both fibronectin （from [45.86±2.73] to [84.19±6.81] ng/ml） and type IV collagen （from [16. 28±0. 90] to [55.27±4.75] ng/ml） in nontransfected cells （P〈0.05）. In pES2a-transfected cells, the high glucose-induced increase in Smad2 mRNA was abrogated completely, in parallel with significant suppression of the high glucose-indtmed increase in fibronectinproduction （[54.44±4.99] ng/ml） and type Ⅳ collagen （[20.96±2.47] ng/ml）. An empty vector was without effects. Coneluslon：These findings demonstrate that Smad2 plays a critical role in mediating high glucose-stimulated ECM production in mesangial cells, indicating that inhibition of Smad2 activity by antisense Smad2 mRNA may be an effective means to attenuate glomerular matrix accumulation in diabetic nephropathy.展开更多
A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3...A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3'-5' and / or 5'-3' orientation. The vectors were ligated with EGF and EGFR fragments by T-4 Ligase. The recombinant retroviral vectors were then packaged with packaging cell line PA317 through calcium phosphate mediated transfection. The viral supernatant of transfected PA317 cell lines were used to infect the human pancreatic carcinoma cell line PC-7. The resultant transformant cell lines: PC-7 / AS-EGF, PC-7 / S-EGFR, PC-7 / AS-EGFR and PC-7 / pBabe were tested for their endogenous EGF and EGFR mRNA expressions, cell growth rate, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice. The results showed that there were noticeable inhibitions of cell growth, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice in PC-7 / AS-EGF and PC-7 / AS-EGFR transformant cell lines. The endogenous EGF mRNA expression was blocked in PC-7 / AS-EGF cell line and the endogenous EGFR mRNA was significantly down-regulated in PC-7 / AS-EGFR cell line.展开更多
Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of...Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy展开更多
基金Supported by National Natural Science Foundation of China(31472260,30972240)
文摘In order to investigate the role of MAPK gene in adaptation of Dunaliella salina to hypersaline environment, the Ds MAPK gene of D. salina was amplified by PCR. After inverted insertion of the open reading flame ofDs MAPK gene into downstream sequence of 35S promoter of plant expression vector, an antisense expression vector of Ds MAPK gene was successfully constructed and introduced into D. salina cells by LiAc/PEG-mediated method. The expression of Ds MAPK gene in transgenic D. salina was analyzed by semi-quantitative RT-PCR method. The results showed that the expression of Ds MAPK gene in D. salina was significantly inhibited at the transcriptional level. The study laid the foundation for further identification of the function of Ds MAPK gene.
文摘A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.
文摘Objective：Hyperglycemia stimulates secretion of transforming growth factor-βl （TGF-βl） in cultured glomerular mesangial cells, thereby increases production of extracellular matrix （ECM）. We examined the effect of antisense mRNA for Smad2 on high glucose-induced ECM production in rat mesangial cells. Methods..A mammalian expression vector, pES2a, which expresses antisense Smad2 mRNA and green fluorescent protein （EGFP）, was transfected into mesangial cells. Following incubation in high glucose medium, EGFP expression and Smad2 mRNA level were determined by fluorescence microscopy and PCR, respectively. Secreted fibronectin and type IV collagen were assessed by enzyme-linked immunosorbent assay. Results ：Within 48 h of incubation in high glucose medium, Smad2 mRNA level significantly increased by 1.6 fold in association with increases in prodtaction of both fibronectin （from [45.86±2.73] to [84.19±6.81] ng/ml） and type IV collagen （from [16. 28±0. 90] to [55.27±4.75] ng/ml） in nontransfected cells （P〈0.05）. In pES2a-transfected cells, the high glucose-induced increase in Smad2 mRNA was abrogated completely, in parallel with significant suppression of the high glucose-indtmed increase in fibronectinproduction （[54.44±4.99] ng/ml） and type Ⅳ collagen （[20.96±2.47] ng/ml）. An empty vector was without effects. Coneluslon：These findings demonstrate that Smad2 plays a critical role in mediating high glucose-stimulated ECM production in mesangial cells, indicating that inhibition of Smad2 activity by antisense Smad2 mRNA may be an effective means to attenuate glomerular matrix accumulation in diabetic nephropathy.
文摘A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3'-5' and / or 5'-3' orientation. The vectors were ligated with EGF and EGFR fragments by T-4 Ligase. The recombinant retroviral vectors were then packaged with packaging cell line PA317 through calcium phosphate mediated transfection. The viral supernatant of transfected PA317 cell lines were used to infect the human pancreatic carcinoma cell line PC-7. The resultant transformant cell lines: PC-7 / AS-EGF, PC-7 / S-EGFR, PC-7 / AS-EGFR and PC-7 / pBabe were tested for their endogenous EGF and EGFR mRNA expressions, cell growth rate, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice. The results showed that there were noticeable inhibitions of cell growth, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice in PC-7 / AS-EGF and PC-7 / AS-EGFR transformant cell lines. The endogenous EGF mRNA expression was blocked in PC-7 / AS-EGF cell line and the endogenous EGFR mRNA was significantly down-regulated in PC-7 / AS-EGFR cell line.
文摘Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy
