Non-thermal atmospheric-pressure plasma keloid treatment device, and use thereof

Abstract

The present invention relates to a non-thermal atmospheric-pressure plasma keloid treatment device and a use thereof. The non-thermal atmospheric-pressure plasma keloid treatment device according to the present invention has effects of inhibiting collagen deposition in keloid fibroblasts and mobility thereof, and thus, is expected to be greatly useful for preventing and treating keloids.

Claims

1. A method of preventing or treating keloids of a subject in need thereof, the method comprising: (a) a step of treating a skin of the subject with plasma for at least 30 seconds using a non-thermal atmospheric-pressure plasma keloid treatment device, wherein the non-thermal atmospheric-pressure plasma keloid treatment device comprises a power supply; a plasma generator; a gas supply path; and a housing, wherein the power supply comprises a high-voltage transformer, the plasma generator comprises an electrode, a metal electrode, a dielectric, and a reaction part, the plasma generator comprises a non-thermal atmospheric-pressure plasma source with multiple nozzles, and the dielectric is constituted of a quartz or ceramic tube; and (b) a step of administering an expression inhibitor of EGFR or STAT3 gene to the subject.

2. The method according to claim 1, wherein the expression inhibitor of EGFR or STAT3 gene of step (b) is selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and microRNA specific to EGFR or STAT3 gene.

3. The method according to claim 2, wherein the non-thermal atmospheric-pressure plasma keloid treatment device has a required power of 10 to 50 kHz and a power peak of 0.1 to 10 kV.

4. The method according to claim 1, wherein the non-thermal atmospheric-pressure plasma keloid treatment device uses argon and nitrogen gases.

5. The method according to claim 1, wherein the non-thermal atmospheric-pressure plasma keloid treatment device has a required power of 10 to 50 kHz and a power peak of 0.1 to 10 kV.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates results of cell mobility according to plasma treatment in NF and KF according to an embodiment of the present invention.

(2) FIGS. 2A-2C illustrate results of FITC Annexin V/PI cell death staining performed to investigate cell viability changes according to plasma treatment in NF and KF according to an embodiment of the present invention.

(3) FIG. 3 illustrates results of MTT staining performed to investigate cell viability changes according to plasma treatment in NF and KF according to an embodiment of the present invention.

(4) FIGS. 4A and 4B illustrate results of Western blotting performed to investigate protein expression changes according to plasma treatment in NF and KF according to an embodiment of the present invention.

(5) FIG. 5 illustrates results of immunofluorescence staining performed to investigate protein expression changes according to plasma treatment in NF and KF according to an embodiment of the present invention.

(6) FIG. 6 illustrates cell mobility changes in NF and KF after being treated with EGFR-specific siRNA according to an embodiment of the present invention.

(7) FIG. 7 illustrates cell mobility changes in NF and KF after being treated with STAT3-specific siRNA according to an embodiment of the present invention.

(8) FIG. 8 illustrates protein expression changes in NF and KF after being treated with EGFR or STAT3-specific siRNA according to an embodiment of the present invention.

(9) FIG. 9 illustrates results of RT-PCR performed to investigate expression changes in Collagen Type 1, Collagen Type 3 and TGF-β according to plasma treatment in NF and KF according to an embodiment of the present invention.

(10) FIG. 10 illustrates results of Sircol collagen assay performed to investigate changes in collagen deposition levels according to plasma treatment in NF and KF according to an embodiment of the present invention.

(11) FIG. 11 illustrates results of immunofluorescence staining performed to investigate changes in collagen deposition levels according to plasma treatment in NF and KF according to an embodiment of the present invention.

(12) FIG. 12 illustrates changes in collagen deposition levels after treating NF and KF with a combination of plasma and STAT3-specific siRNA according to an embodiment of the present invention.

BEST MODE

(13) Hereinafter, the present invention will be described in more detail with reference to the following Examples. It will be apparent to those skilled in the art that Examples are merely for concretely explaining the invention and therefore, there is no intent to limit the invention to Examples.

Example 1. Manufacture of Non-Thermal Atmospheric-Pressure Plasma Keloid Treatment Device

(14) A non-thermal atmospheric-pressure plasma keloid treatment device including a power supply, a plasma generator, a gas supply path, and a housing was manufactured. Here, the plasma generator includes an electrode, a metal electrode, a dielectric, and a reaction part, the power supply includes a high-voltage transformer, the plasma generator includes a non-thermal atmospheric-pressure plasma source with multiple nozzles, and the dielectric is constituted of a quartz or ceramic tube. In a non-limiting embodiment, the plasma generator of the present invention may include a high-voltage LF power module with a pulse width modulation controller IC to produce plasma under atmospheric pressure.

(15) When the plasma generator for treating keloids of the present invention is used to treat keloids, a required power is preferably 10 to 50 kHz, more preferably 20 to 30 kHz, most preferably 25 kHz. A power peak by the high-voltage transformer is preferably 0.1 to 10 kV, more preferably 1 to 5 kV, most preferably 3 kV. A gas used in the plasma generation apparatus is argon and nitrogen, but the present invention is not limited thereto.

Example 2. Confirmation of Effects of Non-Thermal Atmospheric-Pressure Plasma Keloid Treatment Device

Example 2-1. Culture of Normal Fibroblasts or Keloid Fibroblasts

(16) Fibroblasts isolated from skin tissue, diagnosed as keloid, spread beyond an original wound boundary for one or more years even after a wounded skin tissue was recovered were termed “keloid fibroblasts (KF).” Fibroblasts isolated from normal skin tissue were used as a control and termed “normal fibroblasts (NF).”

(17) KF and NF were cultured in RPMI-1640 medium containing % by volume of FBS and 1% by volume of an antibiotic/antimicrobial under 5% by volume of CO.sub.2 in a 37° C. wet environment. Passage culture was performed when KF proliferated to a density of 80 to 90%. The passage was performed using trypsin. In examples of the present invention, cells of F2 to F7 generations were only used.

Example 2-2. Confirmation of Cell Mobility According to Plasma Treatment in NF and KF

(18) To investigate plasma effect on cell migration, as an important cellular mechanism in wound healing of NF and KF-mediated keloid formation, wound healing analysis was performed. For this, NF or KF were cultured at a density of 5×10.sup.5/well in a 12-well culture plate. The center of cells forming a monolayer sheet was scraped with a tip, followed by washing to remove cell debris due to the scraping, treating with plasma for 10 or 30 seconds using nitrogen and argon gases, and additionally culturing for 24 hours, followed by measuring cell migration.

(19) As experimental results, treatment with plasma for 10 seconds did not cause significant differences in both NF and KF, compared to a control (a non-treated group or a group treated only with gas). When treated with plasma for 30 seconds, migration of NF increased by 28% compared to a control, but migration of KF rather decreased by 36%. These results are shown in FIG. 1.

Example 2-3. Confirmation of Cell Viability According to Plasma Treatment in NF and KF

(20) Whether molecular mechanisms related to cell viability according to plasma treatment in NF and KF were changed was confirmed.

(21) NF and KF were cultured and treated with plasma in the same manner as in Example 2-2 and were additionally cultured for 24 or 48 hours, followed by being subjected to FITC Annexin V/PI cell death staining (Annexin-V and propidium iodide staining, BD Biosciences) according to the manufacturer's protocol and being measured by means of a flow cytometer. Results are shown in FIGS. 2A-2C.

(22) In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT, Sigma-Aldrich) staining was carried out to quantitatively measure cell proliferation rates. After staining, optical measurements were carried out at 540 nm using a microplate reader (Bio-Tek, Winooski, Vt., USA), and conversion into a percentage was made based on a control non-treated with plasma. Results are shown in FIG. 3.

(23) From results of FIGS. 2A-2C and 3, it was confirmed that plasma treatment did not induce a quantitative increase in NF or KF cells and did not significantly affect cell viability.

Example 2-4. Confirmation of Protein Expression Changes According to Plasma Treatment in NF and KF

(24) NF and KF were cultured and treated with plasma (10 seconds, 30 seconds, or 60 seconds) in the same manner as in Example 2-2 to measure intercellular protein expression changes. As antibodies for Western blotting, EGFR, E-cadherin, p-STAT-3 (Y705), STAT-3, p-ERK, ERK, Collagen Type 1, and α-tubulin (Cell Signaling Technology, Danvers, Mass., 1:1000) were used, and immunofluorescence staining was carried out using an anti-p-STAT3 antibody to observe an intercellular position of p-STAT3.

(25) Western blotting results are shown in FIGS. 4A and 4B, and immunofluorescence staining results are shown in FIG. 5.

(26) As experimental results, EGFR expression in NF treated with plasma was relatively maintained constant, but EGFR expression in KF treated with plasma was significantly reduced. The expression of E-cadherin was decreased in NF, but increased in KF. The expression level of p-ERK was not changed in NF, but was decreased in KF. Specifically, after being treated with plasma, the expression levels of p-STAT3 and Collagen Type 1 were higher in NF, but, compared to respective controls, were decreased in KF. Such results indicate that plasma treatment interferes with migration of keloid cells by inhibiting the pathways of EGFR, STAT3 and p-ERK in KF.

(27) From immunofluorescence staining results, it was confirmed that plasma treatment caused a decrease in p-STAT3 expression in KF, but caused an increase in p-STAT3 expression in NF. However, translocation of STAT3 into a nucleus was not observed in any cell types.

Example 2-5. Confirmation of Migration Inhibition Mechanism in KF According to Plasma Treatment

Example 2-5-1. Confirmation of Cell Migration Mechanism Using EGFR siRNA and STAT3 siRNA

(28) To assess the roles of EGFR and STAT3 in cell migration, NF and KF were treated with EGFR or STAT3-specific siRNA, and then cell mobility and protein expression changes were investigated. For transfection with siRNA, Lipofectamine 2000 (Gibco/Invitrogen) reagent was used, a medium was removed after culturing cells for 24 hours, and the cells were washed with PBS and treated with siRNA for 24 or 48 hours. EGFR-specific siRNA, STAT3-specific siRNA, and a control siRNA were obtained from Genolution Pharmaceuticals (Seoul, Korea). Cell mobility analysis was carried out in the same manner as in Example 2-2, and protein expression change analysis was carried out in the same manner as in Example 2-4 using EGFR, p-EGFR, p-AKT (Ser473), AKT, p-ERK, ERK, p-STAT-3 (Y705), STAT-3, Collagen Type 1, Vimentin, E-cadherin, and a-tubulin (Cell Signaling Technology, Danvers, Mass., 1:1000). Results of the EGFR-specific siRNA treatment are shown in FIG. 6, results of the STAT3-specific siRNA treatment are shown in FIG. 7, and results of the protein expression change analysis are shown in FIG. 8.

(29) As experimental results, cell migration inhibition by EGFR-specific siRNA was effective in both NF and KF, and cell migration by STAT3-specific siRNA was also similarly inhibited in both NF and KF. From the protein expression change analysis, it was confirmed that the expressions of EGFR, p-AKT, p-STAT3, and Collagen Type 1 in NF and KF were reduced by treating with EGFR-specific siRNA. When STAT3-specific siRNA was simultaneously treated, the expression of Collagen Type 1 was reduced, but the expression levels of EGFR and p-AKT were not affected. Such results indicate that inhibition of EGFR and STAT3 may play a central epidemiological role in the inhibition of cell migration by plasma treatment.

Example 2-5-2. Confirmation of Cell Migration Mechanism in NF and KF Treated with Plasma

(30) Based on the results of Example 2-5-1, it was confirmed whether plasma treatment suppressed collagen generation in KF.

(31) First, the expression levels of Collagen Type 1, Collagen Type 3 and TGF-β in NF and KF treated with plasma for 10 seconds, 30 seconds, or 60 seconds in the same manner as in Example 2-2 were assessed by RT-PCR. Collagen deposition increased through activation of TGF-β signal transduction represents a major mechanism of keloid formation. Primer sequences for RT-PCR are summarized in Table 1 below. Particularly, a sample was denatured at 94° C. for 3 minutes, and then amplified for 30 seconds at 94° C., 62° C. and 72° C., and then elongated for 5 minutes at 72° C. A final product was separated by electrophoresis on a 1.5% agarose gel, and bands were detected using ultraviolet light (Bio-Rad, Hercules, Calif., USA). Results are shown in FIG. 9.

(32) TABLE-US-00001 TABLE 1 Target gene Direction Sequence Collagen F(forward 5′-GGG CAA GAC AGT GAT TGA Type 1 direction) ATA-3′ (SEQ ID NO: 1) R(reverse 5′-ACG TCC AAG CCG AAT TCC direction) T-3′ (SEQ ID NO: 2) Collagen F(forward 5′-AGG TCC TGC GGG TAA CAC Type 3 direction) T-3′ (SEQ ID NO: 3) R(reverse 5′-ACT TTC ACC CTT GAC ACC direction) CTG-3′ (SEQ ID NO: 4) TGF-β F(forward 5′-CCG ACT ACT ACG CCA direction) AGG-3′ (SEQ ID NO: 5) R(reverse 5′-AGT GAA CCC GTT GAT direction) CA-3′ (SEQ ID NO: 6) GAPDH F(forward 5′-ACC ACA GTC CAT GCC ATC direction) AC-3′ (SEQ ID NO: 7) R(reverse 5′-TCC ACC ACC CTG TTG CTG direction) TA-3′ (SEQ ID NO: 8)

(33) As experimental results, the expression levels of Collagen Type 1, Collagen Type 3 and TGF-β mRNA in NF after being treated with plasma were not significantly changed. However, all of the three mRNA types in KF were significantly reduced.

(34) Next, to investigate whether the deposition of soluble collagen at a cellular level by plasma treatment was reduced, total soluble collagen in a cell culture supernatant was quantified. For this, Sircol collagen assay (Biocolor, Belfast, UK) was carried out. Particularly, cells were cultured in a 60 mm.sup.2 culture plate for 24 hours, 400 μl of Sirius red stain, as an anionic dye specifically reacting with a basic collagen side chain group, was added to a supernatant, and then additional culture was performed while gently rotating for 30 minutes at room temperature, and then centrifugation was performed at 12,000 g for 10 minutes, and then a collagen binding dye was re-dissolved after addition of 1 ml of 0.5 M NaOH, and then an absorbance at 540 nm was measured by enzyme-linked immunosorbent assay (Bio-Tek, Winooski, Vt., USA). Absorbance is directly proportional to the amount of collagen newly formed in a cell culture supernatant.

(35) As experimental results, it was confirmed that, by the plasma treatment, a soluble collagen content was slightly reduced in KF, but rather slightly increased in NF. Results are shown in FIG. 10.

(36) In addition, it was confirmed that the expression level changes in Collagen Type 1 by the plasma treatment were the same as the immunofluorescence staining results. These results are shown in FIG. 11.

Example 2-6. Confirmation of Collagen Production Change in NF and KF Treated with Combination of Plasma and siRNA

(37) NF and KF were treated with a combination of plasma and STAT3-specific siRNA. As results, it was confirmed that, when treated with a combination of plasma and STAT3-specific siRNA, the generation of collagen was significantly reduced, compared to the cases of treating with only plasma or STAT3-specific siRNA. These results are shown in FIG. 12.

(38) From the results of Examples 1 and 2, it was confirmed that cell mobility in KF was significantly reduced by plasma treatment, which was caused by collagen synthesis reduction in cells. In addition, it was confirmed that the generation of keloid could be further significantly inhibited when treated with a combination of plasma and STAT3-specific siRNA.