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Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.

Zhu P, Yu H, Huang S, Xiang H, Li F, Zheng W - Mol Med Rep (2015)

Bottom Line: The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells.When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently.In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.

ABSTRACT
Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316‑hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription‑quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl‑thiazolyl‑tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently. In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

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Identification of plasmid pDC316-hTK1-hTIMP1. (A) PCR reaction detection assay. Lane 1, negative control; Lane 2, mMCV-hTIMP1 (1,338 bp) gene obtained from pDC316-hTIMP1-EGFP plasmid (positive control); Lane 3, mMCV-hTIMP1 (1,338 bp) obtained from recombinant plasmid pDC316-hTK1-hTIMP1. (B) Double digestion assay by Bg1II/SalI: Lane 1, corresponding to double digested plasmid pDC316-hTK1-hTIMP1 (1,338 bp and 4.7 kb). (C) Sequence analysis of the cloned PCR products confirmed hTK1 and hTIMP1 cDNA with primer pDC316-cexu-F. PCR, polymerase chain reaction; TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
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f1-mmr-12-04-5671: Identification of plasmid pDC316-hTK1-hTIMP1. (A) PCR reaction detection assay. Lane 1, negative control; Lane 2, mMCV-hTIMP1 (1,338 bp) gene obtained from pDC316-hTIMP1-EGFP plasmid (positive control); Lane 3, mMCV-hTIMP1 (1,338 bp) obtained from recombinant plasmid pDC316-hTK1-hTIMP1. (B) Double digestion assay by Bg1II/SalI: Lane 1, corresponding to double digested plasmid pDC316-hTK1-hTIMP1 (1,338 bp and 4.7 kb). (C) Sequence analysis of the cloned PCR products confirmed hTK1 and hTIMP1 cDNA with primer pDC316-cexu-F. PCR, polymerase chain reaction; TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.

Mentions: The mCMV-hTIMP1 PCR fragment from pDC316-hTIMP1-EGFP was cloned to predigested plasmid pDC316-hTK1 by double-digestion (Bg1II/SalI), and subjected to ligation. The positive recombinant plasmid was extracted and a PCR reaction was performed using the aforementioned primers. The 1,338 bp fragment was observed on 1% agarose using a UV transilluminator (Fig. 1A). Subsequently, the recombinant plasmid was analyzed by double-digestion (Bg1II/SalI, Fig. 1B) and sequencing (Fig. 1C) providing the expected results. The hTK1 and hTIMP1 cDNA sequences are shown in Fig. 1C. A comparison of the hTK1 and hTIMP1 gene sequence with available sequences in gene bank revealed that the obtained sequence had a 100% homology with hTK1 and hTIMP1, and indicated that mMCV-hTIMP1 was correctly cloned into the plasmid pDC316-hTK1.


Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.

Zhu P, Yu H, Huang S, Xiang H, Li F, Zheng W - Mol Med Rep (2015)

Identification of plasmid pDC316-hTK1-hTIMP1. (A) PCR reaction detection assay. Lane 1, negative control; Lane 2, mMCV-hTIMP1 (1,338 bp) gene obtained from pDC316-hTIMP1-EGFP plasmid (positive control); Lane 3, mMCV-hTIMP1 (1,338 bp) obtained from recombinant plasmid pDC316-hTK1-hTIMP1. (B) Double digestion assay by Bg1II/SalI: Lane 1, corresponding to double digested plasmid pDC316-hTK1-hTIMP1 (1,338 bp and 4.7 kb). (C) Sequence analysis of the cloned PCR products confirmed hTK1 and hTIMP1 cDNA with primer pDC316-cexu-F. PCR, polymerase chain reaction; TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581792&req=5

f1-mmr-12-04-5671: Identification of plasmid pDC316-hTK1-hTIMP1. (A) PCR reaction detection assay. Lane 1, negative control; Lane 2, mMCV-hTIMP1 (1,338 bp) gene obtained from pDC316-hTIMP1-EGFP plasmid (positive control); Lane 3, mMCV-hTIMP1 (1,338 bp) obtained from recombinant plasmid pDC316-hTK1-hTIMP1. (B) Double digestion assay by Bg1II/SalI: Lane 1, corresponding to double digested plasmid pDC316-hTK1-hTIMP1 (1,338 bp and 4.7 kb). (C) Sequence analysis of the cloned PCR products confirmed hTK1 and hTIMP1 cDNA with primer pDC316-cexu-F. PCR, polymerase chain reaction; TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
Mentions: The mCMV-hTIMP1 PCR fragment from pDC316-hTIMP1-EGFP was cloned to predigested plasmid pDC316-hTK1 by double-digestion (Bg1II/SalI), and subjected to ligation. The positive recombinant plasmid was extracted and a PCR reaction was performed using the aforementioned primers. The 1,338 bp fragment was observed on 1% agarose using a UV transilluminator (Fig. 1A). Subsequently, the recombinant plasmid was analyzed by double-digestion (Bg1II/SalI, Fig. 1B) and sequencing (Fig. 1C) providing the expected results. The hTK1 and hTIMP1 cDNA sequences are shown in Fig. 1C. A comparison of the hTK1 and hTIMP1 gene sequence with available sequences in gene bank revealed that the obtained sequence had a 100% homology with hTK1 and hTIMP1, and indicated that mMCV-hTIMP1 was correctly cloned into the plasmid pDC316-hTK1.

Bottom Line: The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells.When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently.In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.

ABSTRACT
Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316‑hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription‑quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl‑thiazolyl‑tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently. In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

Show MeSH
Related in: MedlinePlus