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Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

Tobinaga S, Matsumoto K, Nagayasu T, Furukawa K, Abo T, Yamasaki N, Tsuchiya T, Miyazaki T, Koji T - Acta Histochem Cytochem (2015)

Bottom Line: In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells.Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection.Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Surgical Oncology, Department of Translational Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences , Nagasaki, Japan.

ABSTRACT
Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

No MeSH data available.


Related in: MedlinePlus

Protocol for mouse emphysema induction, gene transfection and mouse sacrifice. Emphysema was induced in mice by instillation of porcine pancreas elastase (PPE). A plasmid encoding the FLAG-tagged keratinocyte growth factor (pKGF-FLAG), or control FLAG was electroporated into quadriceps muscle either immediately post-PPE treatment (a), or pre-PPE treatment (b). Mice were sacrificed on the indicated days for analysis.
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Figure 1: Protocol for mouse emphysema induction, gene transfection and mouse sacrifice. Emphysema was induced in mice by instillation of porcine pancreas elastase (PPE). A plasmid encoding the FLAG-tagged keratinocyte growth factor (pKGF-FLAG), or control FLAG was electroporated into quadriceps muscle either immediately post-PPE treatment (a), or pre-PPE treatment (b). Mice were sacrificed on the indicated days for analysis.

Mentions: For gene transfer, mice were electroporated at the plasmid injection site using an Electro Square Porator (T820; BTX Inc., San Diego, CA, USA). Immediately after instillation of PPE, the bilateral quadriceps muscles were denuded, and 40 μg of the pKGF-FLAG or the pFLAG vector were injected once into each bilateral muscle. The muscles were held by a pair of electrode disks (10 mm in diameter and 5 mm apart) attached to tweezers (449-​10PRG; Meiwa Shoji, Tokyo, Japan), and electric pulses were delivered. Six square wave pulses of 75 V were administered for a duration of 20 ms at a rate of one pulse per second. The mice were sacrificed at various time-points after PPE administration with/without plasmid electroporation and the lungs were dissected out. As an additional test, plasmid was administered 4 days before PPE instillation to establish the prophylactic effect of KGF. Each experimental group consisted of 3 mice. The time scale of mouse treatments and sacrifice is shown in Figure 1. Following sacrifice, the lungs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) overnight and embedded in paraffin. Serial sections 4-μm thick were cut and stained with hematoxylin and eosin.


Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

Tobinaga S, Matsumoto K, Nagayasu T, Furukawa K, Abo T, Yamasaki N, Tsuchiya T, Miyazaki T, Koji T - Acta Histochem Cytochem (2015)

Protocol for mouse emphysema induction, gene transfection and mouse sacrifice. Emphysema was induced in mice by instillation of porcine pancreas elastase (PPE). A plasmid encoding the FLAG-tagged keratinocyte growth factor (pKGF-FLAG), or control FLAG was electroporated into quadriceps muscle either immediately post-PPE treatment (a), or pre-PPE treatment (b). Mice were sacrificed on the indicated days for analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4491498&req=5

Figure 1: Protocol for mouse emphysema induction, gene transfection and mouse sacrifice. Emphysema was induced in mice by instillation of porcine pancreas elastase (PPE). A plasmid encoding the FLAG-tagged keratinocyte growth factor (pKGF-FLAG), or control FLAG was electroporated into quadriceps muscle either immediately post-PPE treatment (a), or pre-PPE treatment (b). Mice were sacrificed on the indicated days for analysis.
Mentions: For gene transfer, mice were electroporated at the plasmid injection site using an Electro Square Porator (T820; BTX Inc., San Diego, CA, USA). Immediately after instillation of PPE, the bilateral quadriceps muscles were denuded, and 40 μg of the pKGF-FLAG or the pFLAG vector were injected once into each bilateral muscle. The muscles were held by a pair of electrode disks (10 mm in diameter and 5 mm apart) attached to tweezers (449-​10PRG; Meiwa Shoji, Tokyo, Japan), and electric pulses were delivered. Six square wave pulses of 75 V were administered for a duration of 20 ms at a rate of one pulse per second. The mice were sacrificed at various time-points after PPE administration with/without plasmid electroporation and the lungs were dissected out. As an additional test, plasmid was administered 4 days before PPE instillation to establish the prophylactic effect of KGF. Each experimental group consisted of 3 mice. The time scale of mouse treatments and sacrifice is shown in Figure 1. Following sacrifice, the lungs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) overnight and embedded in paraffin. Serial sections 4-μm thick were cut and stained with hematoxylin and eosin.

Bottom Line: In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells.Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection.Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Surgical Oncology, Department of Translational Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences , Nagasaki, Japan.

ABSTRACT
Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

No MeSH data available.


Related in: MedlinePlus