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[Transfection of recombinant bone morphogenetic protein-7 expressing plasmid into cultured human renal tubular epithelial cells attenuates the extracellular matrix accumulation induced by transforming growth factor-beta].
Zhonghua Yi Xue Za Zhi. 2006 Feb 28; 86(8):544-8.ZY

Abstract

OBJECTIVE

To investigate the effects of bone morphogenetic protein (BMP)-7 on the extracellular matrix (ECM) accumulation induced by transforming growth factor (TGF)-beta.

METHODS

Mouse full length BMP-7 cDNA was ligated into a eukaryotic expression vector pcDNA3.1. Restriction enzymatic analyses and DNA sequencing were used to confirm the accuracy of the BMP-7 expressing plasmid thus constructed. The recombinant expression plasmid pcDNA3.1-BMP-7 was transfected into cultured human renal tubular epithelial cells of the line HK-2 mediated by liposome. Positive clones were selected so as to obtain the human renal epithelial cells with stable transfection. These HK-2 cells were cultured and divided into 5 groups to be treated with 5 ng/ml TGF-beta, blank plasmid pcDNA3.1, blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta, pcDNA3.1-BMP-7, pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta, and an additional grin the cells and the supernatant of the cell culture fluid were collected. The expression level of BMP-7 protein was determined by Western blotting. RT-PCR and ELISA were used to determine the mRNA and protein expression of collagen (Col) I and III, and fibronectin (FN) in the human renal tubular epithelial cells and supernatant of different groups.

RESULTS

The recombinant plasmid pcDNA3.1-BMP-7 was successfully constructed. The cell mRNA expression levels of Col I and III and FN of the 5 ng/ml TGF-beta group and blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta group were all significantly higher than those of the blank plasmid pcDNA3.1 group, pcDNA3.1-BMP-7 group, and control group (all P < 0.05). The cell mRNA expression levels of Col I and FN of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta were all significantly lower than those of the TGF-beta group (all P < 0.05). The cell mRNA expression level of Col III of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta was lower, however, not significantly, than that of the TGF-beta group. The supernatant FN levels of the 5 ng/ml TGF-beta group and pcDNA3.1 + 5 ng/ml TGF-beta group were both significantly higher than that of the control group (both P < 0.05), and the supernatant FN level of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta group was significantly lower that that of the 5 ng/ml TGF-beta group (P < 0.05).

CONCLUSION

Over-expression of BMP-7 significantly inhibits the increased syntheses of collagen I and III, and fibronectin induced by TGF-beta. BMP-7 exerts its antifibrotic effect partially through blocking the TGF-beta-induced accumulation of extracellular matrix in human renal tubular epithelial cells.

Authors+Show Affiliations

Li Y
Department of Nephrology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Chen N
No affiliation info available
Yu HJ
No affiliation info available
Dong XP
No affiliation info available
Huang QH
No affiliation info available

MeSH

AnimalsBlotting, WesternBone Morphogenetic Protein 7Bone Morphogenetic ProteinsCell LineCollagen Type ICollagen Type IIIEnzyme-Linked Immunosorbent AssayEpithelial CellsExtracellular MatrixFibronectinsGene ExpressionHumansKidney GlomerulusMicePlasmidsRNA, MessengerReverse Transcriptase Polymerase Chain ReactionTransfectionTransforming Growth Factor beta

Pub Type(s)

English Abstract
Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

16681885

Citation

Li, Ya, et al. "[Transfection of Recombinant Bone Morphogenetic Protein-7 Expressing Plasmid Into Cultured Human Renal Tubular Epithelial Cells Attenuates the Extracellular Matrix Accumulation Induced By Transforming Growth Factor-beta]." Zhonghua Yi Xue Za Zhi, vol. 86, no. 8, 2006, pp. 544-8.
Li Y, Chen N, Yu HJ, et al. [Transfection of recombinant bone morphogenetic protein-7 expressing plasmid into cultured human renal tubular epithelial cells attenuates the extracellular matrix accumulation induced by transforming growth factor-beta]. Zhonghua Yi Xue Za Zhi. 2006;86(8):544-8.
Li, Y., Chen, N., Yu, H. J., Dong, X. P., & Huang, Q. H. (2006). [Transfection of recombinant bone morphogenetic protein-7 expressing plasmid into cultured human renal tubular epithelial cells attenuates the extracellular matrix accumulation induced by transforming growth factor-beta]. Zhonghua Yi Xue Za Zhi, 86(8), 544-8.
Li Y, et al. [Transfection of Recombinant Bone Morphogenetic Protein-7 Expressing Plasmid Into Cultured Human Renal Tubular Epithelial Cells Attenuates the Extracellular Matrix Accumulation Induced By Transforming Growth Factor-beta]. Zhonghua Yi Xue Za Zhi. 2006 Feb 28;86(8):544-8. PubMed PMID: 16681885.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Transfection of recombinant bone morphogenetic protein-7 expressing plasmid into cultured human renal tubular epithelial cells attenuates the extracellular matrix accumulation induced by transforming growth factor-beta]. AU - Li,Ya, AU - Chen,Nan, AU - Yu,Hai-jin, AU - Dong,Xiao-pei, AU - Huang,Qiu-hua, PY - 2006/5/10/pubmed PY - 2008/2/27/medline PY - 2006/5/10/entrez SP - 544 EP - 8 JF - Zhonghua yi xue za zhi JO - Zhonghua Yi Xue Za Zhi VL - 86 IS - 8 N2 - OBJECTIVE: To investigate the effects of bone morphogenetic protein (BMP)-7 on the extracellular matrix (ECM) accumulation induced by transforming growth factor (TGF)-beta. METHODS: Mouse full length BMP-7 cDNA was ligated into a eukaryotic expression vector pcDNA3.1. Restriction enzymatic analyses and DNA sequencing were used to confirm the accuracy of the BMP-7 expressing plasmid thus constructed. The recombinant expression plasmid pcDNA3.1-BMP-7 was transfected into cultured human renal tubular epithelial cells of the line HK-2 mediated by liposome. Positive clones were selected so as to obtain the human renal epithelial cells with stable transfection. These HK-2 cells were cultured and divided into 5 groups to be treated with 5 ng/ml TGF-beta, blank plasmid pcDNA3.1, blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta, pcDNA3.1-BMP-7, pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta, and an additional grin the cells and the supernatant of the cell culture fluid were collected. The expression level of BMP-7 protein was determined by Western blotting. RT-PCR and ELISA were used to determine the mRNA and protein expression of collagen (Col) I and III, and fibronectin (FN) in the human renal tubular epithelial cells and supernatant of different groups. RESULTS: The recombinant plasmid pcDNA3.1-BMP-7 was successfully constructed. The cell mRNA expression levels of Col I and III and FN of the 5 ng/ml TGF-beta group and blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta group were all significantly higher than those of the blank plasmid pcDNA3.1 group, pcDNA3.1-BMP-7 group, and control group (all P < 0.05). The cell mRNA expression levels of Col I and FN of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta were all significantly lower than those of the TGF-beta group (all P < 0.05). The cell mRNA expression level of Col III of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta was lower, however, not significantly, than that of the TGF-beta group. The supernatant FN levels of the 5 ng/ml TGF-beta group and pcDNA3.1 + 5 ng/ml TGF-beta group were both significantly higher than that of the control group (both P < 0.05), and the supernatant FN level of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta group was significantly lower that that of the 5 ng/ml TGF-beta group (P < 0.05). CONCLUSION: Over-expression of BMP-7 significantly inhibits the increased syntheses of collagen I and III, and fibronectin induced by TGF-beta. BMP-7 exerts its antifibrotic effect partially through blocking the TGF-beta-induced accumulation of extracellular matrix in human renal tubular epithelial cells. SN - 0376-2491 UR - https://www.unboundmedicine.com/medline/citation/16681885/[Transfection_of_recombinant_bone_morphogenetic_protein_7_expressing_plasmid_into_cultured_human_renal_tubular_epithelial_cells_attenuates_the_extracellular_matrix_accumulation_induced_by_transforming_growth_factor_beta]_ DB - PRIME DP - Unbound Medicine ER -