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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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Related in: MedlinePlus

Packaging and verification of recombinant adenovirus Ad-hTK1-hTIMP1. (A) Morphological changes in Ad-hTK1-hTIMP1 transfected HEK293A cells (magnification, ×100): The partial plaque formation of HEK293A cells after 10 days under an inverted microscope. (B) Identification of mMCV-hTIMP1 fragment using PCR. Lane 1, negative control; Lane 2: positive control, PCR product of pDC316-hTK1-hTIMP1; Lane 3: 1,338 bp PCR products detected from Ad-hTK1-hTIMP1. PCR, polymerase chain reaction; TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
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f2-mmr-12-04-5671: Packaging and verification of recombinant adenovirus Ad-hTK1-hTIMP1. (A) Morphological changes in Ad-hTK1-hTIMP1 transfected HEK293A cells (magnification, ×100): The partial plaque formation of HEK293A cells after 10 days under an inverted microscope. (B) Identification of mMCV-hTIMP1 fragment using PCR. Lane 1, negative control; Lane 2: positive control, PCR product of pDC316-hTK1-hTIMP1; Lane 3: 1,338 bp PCR products detected from Ad-hTK1-hTIMP1. PCR, polymerase chain reaction; TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.

Mentions: With the aid of the AdMax system, the recombinant adenovirus Ad-hTK1-hTIMP1 was prepared in HEK293A cells. A viral plaque became visible at 9–11 days after transfection, as is shown in Fig. 2A. The target gene was confirmed to be correctly cloned in the adenovirus vector using a PCR assay with corresponding primers of the hTK1 and hTIMP1 gene, as shown in Fig. 2B. Following plaque purification, the titration of the recombinant adenovirus Ad-hTK1-hTIMP1 was achieved up to 1.26×1010 IU/ml using a TCID assay.


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)

Packaging and verification of recombinant adenovirus Ad-hTK1-hTIMP1. (A) Morphological changes in Ad-hTK1-hTIMP1 transfected HEK293A cells (magnification, ×100): The partial plaque formation of HEK293A cells after 10 days under an inverted microscope. (B) Identification of mMCV-hTIMP1 fragment using PCR. Lane 1, negative control; Lane 2: positive control, PCR product of pDC316-hTK1-hTIMP1; Lane 3: 1,338 bp PCR products detected from Ad-hTK1-hTIMP1. 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

f2-mmr-12-04-5671: Packaging and verification of recombinant adenovirus Ad-hTK1-hTIMP1. (A) Morphological changes in Ad-hTK1-hTIMP1 transfected HEK293A cells (magnification, ×100): The partial plaque formation of HEK293A cells after 10 days under an inverted microscope. (B) Identification of mMCV-hTIMP1 fragment using PCR. Lane 1, negative control; Lane 2: positive control, PCR product of pDC316-hTK1-hTIMP1; Lane 3: 1,338 bp PCR products detected from Ad-hTK1-hTIMP1. PCR, polymerase chain reaction; TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
Mentions: With the aid of the AdMax system, the recombinant adenovirus Ad-hTK1-hTIMP1 was prepared in HEK293A cells. A viral plaque became visible at 9–11 days after transfection, as is shown in Fig. 2A. The target gene was confirmed to be correctly cloned in the adenovirus vector using a PCR assay with corresponding primers of the hTK1 and hTIMP1 gene, as shown in Fig. 2B. Following plaque purification, the titration of the recombinant adenovirus Ad-hTK1-hTIMP1 was achieved up to 1.26×1010 IU/ml using a TCID assay.

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