WO2021241846A1 - Prevention and treatment method against viruses using blue light - Google Patents
Prevention and treatment method against viruses using blue light Download PDFInfo
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- WO2021241846A1 WO2021241846A1 PCT/KR2021/001529 KR2021001529W WO2021241846A1 WO 2021241846 A1 WO2021241846 A1 WO 2021241846A1 KR 2021001529 W KR2021001529 W KR 2021001529W WO 2021241846 A1 WO2021241846 A1 WO 2021241846A1
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- virus
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- A61N5/0613—Apparatus adapted for a specific treatment
- A61N5/0624—Apparatus adapted for a specific treatment for eliminating microbes, germs, bacteria on or in the body
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K13/00—Devices for grooming or caring of animals, e.g. curry-combs; Fetlock rings; Tail-holders; Devices for preventing crib-biting; Washing devices; Protection against weather conditions or insects
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Definitions
- the present invention relates to a method for preventing and treating viruses using blue light.
- Viruses have DNA or RNA as their genome and are surrounded by proteins. Viruses cannot reproduce independently, replicate in host cells, and multiply through intercellular infection. Virtually all living things, such as animals, plants, and bacteria, each have a virus that infects them, which causes various diseases such as AIDS and the flu. Diseases caused by viruses typically include acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus, hepatitis or liver cancer caused by hepatitis virus, skin diseases caused by herpesvirus, tumors, and MERS virus (MERS virus). ) caused by respiratory diseases. Methods of treating viruses include delaying or blocking the infection of the virus by damaging or killing the virus-infected host cells.
- AIDS acquired immunodeficiency syndrome
- MERS virus MERS virus
- Corona virus is one of the three major viruses that cause colds in humans along with adenovirus and rhinovirus, and is an RNA virus with a gene size of 27 to 32 kb that can infect humans through various routes.
- the surface of the virus particle protrudes like a protrusion, and this shape resembles a crown, so it was named after the Latin word “corona” meaning crown. It accounts for 10 to 30% of adult colds that occur mainly in the cold winter, and the main symptom is a nasal cold accompanied by a headache, sore throat, or cough. Since the coronavirus was first discovered in chickens in the 1930s, it has been found in animals such as dogs, pigs, and birds, and in humans in the 1960s.
- Corona virus has been found in both animals and humans, and as the area of human activity expands, the virus that was prevalent only among animals causes genetic mutations in order to survive and is passed on to humans. Examples include SARS (bats and civets), MERS (bats and camels), and COVID-19 (probably bats).
- SARS baths and civets
- MERS baths and camels
- COVID-19 probably bats.
- the coronaviruses discovered so far are classified into four genera: Alpha, Beta, Gamma, and Delta.
- alpha is further divided into types 1a and 1b
- beta is divided into types 2a, 2b, 2c, and 2d. Of these, alpha and beta infect humans and animals, and gamma and delta infect animals.
- coronaviruses There are a total of 7 types of coronaviruses that have been identified so far, including HCoV 229E ⁇ HCoV NL63 ⁇ HCoV OC43 ⁇ HCoV HKU1 ⁇ SARS-CoV ⁇ MERS-CoV ⁇ SARS-CoV-2. Of these, four (229E, OC43, NL63, and HKU1) caused only mild symptoms similar to colds. However, SARS (severe acute respiratory syndrome), MERS (MERS-CoV), and coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) It can cause disease and cause many deaths.
- SARS severe acute respiratory syndrome
- MERS MERS
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- Coronavirus Disease 2019 is a new type of coronavirus (SARS-CoV-2) that first emerged in Wuhan, China in December 2019 and has spread throughout China and around the world.
- SARS-CoV-2 coronavirus
- the COVID-19 virus has a very high transmission rate and is particularly highly contagious. After being infected with the COVID-19 virus, after an incubation period of about 2 to 14 days (estimated), respiratory symptoms such as fever (37.5 degrees), cough or shortness of breath, and pneumonia appear as the main symptoms, but asymptomatic infections are not uncommon.
- viruses that invade the respiratory system are known to invade the respiratory system in a similar way, and depending on the characteristics of the virus, it is highly contagious because of its fast dissemination power, or it is high in invasiveness to the host by quickly killing host cells. looks like A common invasive route, etc., enters the mucosal epithelial cells in the respiratory tract via droplets or airflow, and redness, ulceration, and secretion of the pharyngeal mucosa are found.
- Complications include bronchitis, pneumonia, and sepsis in the elderly or patients with underlying diseases with weakened immunity. Therefore, there is a need for a treatment that reduces the inflammatory response caused by viral infection and inhibits viral activity.
- the present inventors did not kill the host cell, reduce the inflammatory response, and as a result of studying a method for inhibiting growth or killing an infected virus, when irradiated with blue light, the death rate and inflammatory response of the host cell are reduced while reducing the infection By confirming that it is possible to inhibit the proliferation of viruses, the present invention is submitted with this content.
- an object of the present invention is to provide a light therapy device for treating or preventing viruses, including an LED or a laser light source in a blue region.
- the present invention may provide an apparatus for light therapy for treating or preventing a virus, including an LED or a laser light source in a blue region.
- the blue region may be 400 nm to 500 nm.
- the LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
- the virus may be a virus causing respiratory diseases.
- the present invention may provide a method of treating a virus comprising; irradiating the above-described device for phototherapy to animals other than humans.
- the device for phototherapy may be irradiated to any one site selected from the group consisting of the whole body, lungs, airways, nasal mucosa, and oral mucosa of an animal other than a human infected with a virus.
- the blue region may be 400 nm to 500 nm.
- the LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
- the virus may be a virus causing respiratory diseases.
- the blue light of the present invention does not kill the virus-infected host cell, suppresses its inflammatory response, and has the effect of inhibiting the growth and killing of the infected virus.
- 3 is a result of cell viability 24 hours and 48 hours after irradiating the blue light of the present invention to Vero E6 cells.
- 5 is a result of measuring the cell viability and intracellular virus expression level 24 hours after irradiating the blue light of the present invention to Vero E6 cells infected with SARS-CoV2 virus.
- Figure 6 is the result of irradiating the blue light of the present invention to Vero E6 cells infected with SARS-CoV2 virus, and then extracting vRNA from the supernatant after 24 hours (left) and 72 hours (right), and measuring its quantification cycle (Cq) value. .
- Figure 8 shows the blue light of the present invention at 10 J/m 2 SARS-CoV2 virus-infected Vero E6 cells were irradiated once with overlay media containing 0.6% agarose, and the plaques and uninfected cells generated 72 hours later. This is the result of measuring the number of unstained plaques by staining.
- Figure 9 shows the results of measuring the titer of the virus by irradiating the blue light of the present invention at 10 J/m 2 to MDCK cells infected with H1N1 virus twice, then extracting vRNA from the supernatant after 72 hours, calculating its (Ct) value. am.
- Viruses that invade the respiratory tract are highly contagious because they spread quickly.
- the mortality rate increases due to complications such as bronchitis, pneumonia, and sepsis. Therefore, there is a need for a treatment that reduces the inflammatory response caused by viral infection and inhibits viral activity.
- the present inventors did not die in the host cell, reduce the inflammatory response, and as a result of studying a method for inhibiting or killing the infected virus, when irradiated with blue light, the death rate of the host cell does not decrease, and the inflammatory response is It was confirmed that it reduces, inhibits the growth of, and kills the infected virus, and completed the present invention.
- the present invention may provide an apparatus for light therapy for treating or preventing a virus, including an LED or a laser light source in a blue region.
- plaque staining with uninfected cells showed that the number of plaques was statistically higher than that of the control group. was significantly reduced by about 80% or more (FIG. 7). As described above, it was confirmed that the blue light energy level can affect virus inactivation without affecting apoptosis in cells infected with SARS-CoV2 virus.
- the virus After exposing blue light ⁇ J to MDCK cells infected with H1N1 virus for 30 minutes each at 0 hpi (post-infection hour) and 24 hpi time zones, the virus was allowed to grow for 72 hours, and the supernatant was recovered to determine the Ct value of vRNA. was calculated to determine the virus titer. As a result of the measurement, the virus history was statistically decreased in the control group after one or two blue light irradiation. Through this, it was confirmed that the blue light energy level can affect the virus inactivation in H1N1 virus-infected cells.
- the blue region may be 400 nm to 500 nm. Preferably, it may be 425 to 475 nm, and more preferably, 450 nm.
- the LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
- the virus may be a virus causing respiratory diseases, preferably adenovirus, influenza virus, parainfluenza virus, PIV, respiratory syncytial virus (RSV), bocavirus ( Human bocavirus, hBoV), metapneumovirus (human metapneumovirus, hMPV), may be any one selected from the group consisting of rhinovirus (rhinovirus) and coronavirus (coronavirus), more preferably corona virus or influenza virus. .
- the coronavirus is a group consisting of SARS (SARS-CoV, severe acute respiratory syndrome), MERS (MERS-CoV, Middle East respiratory syndrome) and coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) It may be any one selected from, and more preferably, coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2).
- influenza virus may be influenza virus type A, influenza virus type B, influenza type C, influenza virus type D, togotovirus, quaranza virus and salmon anemia virus, preferably influenza virus type A, more preferably may be N1H1.
- the present invention may provide a method of treating a virus comprising the step of irradiating the above-described device for phototherapy to animals other than humans.
- the device for phototherapy may be irradiated to any one site selected from the group consisting of the whole body, lungs, airways, nasal mucosa, and oral mucosa of an animal other than a human infected with a virus.
- light with a wavelength of 400 nm has a penetration depth of 1 mm or less
- light with a wavelength of 514 nm has a penetration depth of 0.5 to 2 mm
- light with a wavelength of 630 nm has a penetration depth of 1 to 6 mm
- 700 to 900 nm Wavelengths of light can penetrate deeper.
- the light source of the present invention can penetrate the skin of 1 mm to 2 mm, and when irradiated to the whole body, there is an effect of treating viruses floating in microvessels existing in the skin.
- the blue region may be 400 nm to 500 nm. Preferably, it may be 425 to 475 nm, and more preferably, 450 nm.
- the LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
- the virus may be a virus causing respiratory diseases, preferably adenovirus, influenza virus, parainfluenza virus (PIV), RS virus (respiratory syncytial virus, RSV), rhinovirus ( rhinovirus) and coronavirus (coronavirus) may be any one selected from the group consisting of, and more preferably may be a corona virus.
- the coronavirus is a group consisting of SARS (SARS-CoV, severe acute respiratory syndrome), MERS (MERS-CoV, Middle East respiratory syndrome) and coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) It may be any one selected from, and more preferably, coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2).
- CCD-1131Sk and CCD-18Co cells were cultured in a 96-well plate at a concentration of 5 x 10 3 , replaced with a medium without fetal bovine serum, and irradiated with 5, 10, 15 J/cm 2 of 450 nm blue light, After 48 hours, 10 ⁇ l MTT (5 mg/ml) was treated and the viability was measured by the MTT method.
- CCD-1131Sk and CCD-18Co cells were irradiated with blue light at 5, 10, 15 J/cm 2 for 48 hours and then incubated for 48 hours followed by MTT at a concentration of 5 mg/ml. Each ⁇ l was put into 96 wells and incubated for 4 hours. After the culture was completed, the culture medium was removed, and 100 ⁇ l of 2-propanol was added per well to dissolve formazan MTT, and absorbance was measured at 570 nm with an ELISA reader to compare with the control group. No morphological changes were observed in human skin cell lines and colon fibroblasts after blue light energy irradiation (FIG. 1).
- CT-26 cells were recovered and 7M urea, 2M Thiourea, 4%(w/v) 3-[(3-cholamidopropy) ) dimethyammonio]-1-propanesulfonate (CHAPS), 1% (w/v) dithiothreitol (DTT), 2% (v/v) pharmalyte, and 1 mM benzamidine were mixed with a 2DE lysis solution. Then, for protein extraction, vortexing was performed for 1 hour, centrifugation was performed at 15° C.
- IPG strips are reswelling composed of 7M urea, 2M thiourea, 2% 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% pharmalyte. The solution was reswelled at room temperature for 12 to 16 hours.
- IPG Strips were incubated with equilibration buffer (50mM Tris-Cl, pH6.8, 6M urea, 2% SDS, 30% glycerol) containing 1% DTT for 10 minutes, and immediately after 2.5% Incubation was continued for 10 minutes with equilibration buffer containing iodoacetamide.
- equilibration buffer 50mM Tris-Cl, pH6.8, 6M urea, 2% SDS, 30% glycerol
- Quantitative analysis for confirming the expression change of protein spots from the scanned image was performed using PDQuest software (version 7.0, BioRad). The quantity of each spot was normalized to the intensity of total valid sopts, and protein spots showing a significant change in expression more than double that of the control group were selected. After identifying spots by performing two-dimensional electrophoresis, TCTP, LASP1, Enol 1, and PLS3 expression in actual CT-26 cells were reconfirmed by Western blot based on the analyzed results.
- the primary antibody was reacted with anti-TCTP (cell signaling), anti-LASP1 (cell signaling), anti-Enol 1 (cell signaling), anti-PLS3 (Santacruz), and anti-GAPDH (cell signaling), It was reacted with a secondary antibody bound to horseradish peroxidase. Thereafter, the ECL kit (Millipore, USA) was treated according to the manufacturer's method to induce a developmental reaction. As a result, it was confirmed that blue light did not affect apoptosis and suppressed the expression of proteins (TCTP, LASP1, Enol 1, PLS3) related to inhibition of cell mobility and invasiveness ( FIG. 4 ).
- the expression of the DNA or mRNA level was also affected in the expression of the protein. Therefore, it can be considered that an energy level of less than 20 J/cm 2 of blue light does not affect apoptosis in virus-infected cells and can affect cell proliferation, inflammatory response, and protein expression such as kinases in a direction that is suppressed. .
- the decrease in protein expression after blue light irradiation is considered to be one of the important mechanisms in explaining the mechanism by which virus proliferation is inhibited. It is judged that it can cause the result of inhibition of proliferation and inactivation of function by being inhibited.
- SARS-CoV2 was confirmed by the Korea Centers for Disease Control and Prevention, and all experimental procedures were conducted in a domestic biosafety level 3 research facility (BL3). After culturing the monkey kidney cell line Vero E6 cells in DMEM medium supplemented with 10% fetal bovine serum, when 70-80% confluence is achieved, SARS-CoV2 is infected and periodically cytopathic effect (CPE) was observed. When cell mutations were clearly observed, the culture medium was collected, and the supernatant was filtered with a 0.45 ⁇ m filter using a centrifuge, then divided and stored at -80 °C. For quantification of SARS-CoV2 virus, the infectious virus titer was expressed as 50% tissue culture infectious dose (TCID50).
- TCID50 tissue culture infectious dose
- Virus RNA was extracted according to the provided method and confirmed by RT-PCR. After quantifying and diluting the extracted RNA, qRT-PCR was performed using a target specific primer.
- RNA 1 ⁇ g of total RNA was mixed with 2 ⁇ l 5X RT buffer, 0.5 ⁇ l RT Enzyme Mix, 0.5 ⁇ l primer Mix, and Nuclease-free water according to the manual of the ReverTra Ace TM qPCR RT kit in a total volume of 10 ⁇ l at 37 °C for 15 minutes. , after 5 min reaction at 98 °C, react at 4 °C to synthesize cDNA.
- RNA from intracellular or cell culture medium was extracted using the AccuPrep Viral RNA Extraction Kit (K-3033, Bioneer, Korea) provided by the manufacturer. Separated according to the method.
- Cq quantification cycle
- the cell supernatant was recovered 24 hours after irradiating 10J of blue light once or twice to measure the number of vRNA copies. After irradiation with blue light once or twice, the number of vRNA copies was statistically significantly reduced by about 50% or more compared to the control group (FIG. 7).
- Plaque reduction assay is a general method for quantifying and viewing viral infection, and was performed to check whether the intracellular virus suppression effect was observed as the number of vRNA copies decreased after BLED irradiation. Plaque shows host cells damaged by virus infection distinct from surrounding normal cells. When stained with vital dye, normal cells around the plaque are stained, but lesion cells infected with SARS-CoV-2 virus are colorless because the pigment is released. have white spots According to the theory that one infectious virus particle forms a single plaque, plaque was measured using overlay media after SARS-CoV-2 virus infection. Overlay media supplies nutrients and makes a gel-like medium to prevent the virus propagated in the first infected cells from spreading through the medium and infecting surrounding cells.
- the plaques generated 72 hours after irradiation with BLED 10J in cells infected with SARS-CoV-2 virus were reduced by more than 80% compared to the control group (FIG. 8).
- the blue light energy level can affect virus inactivation without affecting apoptosis in cells infected with SARS-CoV2 virus.
- the Mardin-Dardy canine kidney (MDCK) cell line is DMEM (Dulbecco's Modified Eagle's Medium) supplemented with penicillin (5 units/mL) / streptomysin (5 ⁇ g/mL) and 10% fetal bovine serum (Gibco, USA): Gibco, USA) was used as a basal medium, maintaining a 5% CO2 concentration, and cultured at 37°C in a CO2 incubator (Thermo Forma, USA).
- the antiviral activity test of H1N1 against LED-blue light exposure was performed twice in a 12-well plate in MDCK cells, which are host cells.
- virus growth media DMEM 0 + 1 ⁇ g/mL TPCK-treated trypsin + 1% P/S
- DMEM 0 + 1 ⁇ g/mL TPCK-treated trypsin + 1% P/S was added, and at 35 ° C, 5% CO 2 condition cultured.
- the Blue-LED generator was put together in the CO 2 incubator, and the Blue-LED test group was exposed for 30 minutes at 0 hpi (post-infection hour) and 24 hpi time zones, respectively.
- the negative control was performed under the same conditions as the test group after blocking blue light using aluminum foil.
- H1N1_PA_For 5 ⁇ -CGG TCC AAA TTC CTG CTG-3 ⁇ and H1N1_PA_Rev: 5 ⁇ -CAT TGG GTT CCT TCC ATC CA- 3 ⁇
- a reaction solution containing TB green ® Premix Ex Taq TM (Takara, Cat# RR420). Reaction conditions were 95°C 30 seconds (1 time), 95°C 5 seconds, 55°C 10 seconds, 72°C 20 seconds (45 times), and the virus titer was measured using Thermal Cycler Dice ® Real Time System III (Takara, TP950). measured.
- a standard curve was prepared by converting the Ct values obtained by performing plaque assay and RT-qPCR analysis by serially diluting H1N1 into PFU/mL units.
- the slope of the standard curve prepared by plotting the Ct values obtained by step-dilution was -4.065
- the y-intercept was 45.65
- R 2 was 0.991.
- the detection limit of H1N1 was set to a Ct value of 35.
- the H1N1 virus concentration was calculated and compared in non-LED and blue-LED. As a result, the H1N1 concentration in blue-LED was shown to have the effect of reducing 3.38 times at the level of the 12-well plate. was confirmed (FIG. 9).
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Abstract
The present invention relates to a light therapy apparatus which uses a LED or laser light source to irradiate the respiratory system and the like of animals with light in the blue region to thereby inhibit the growth of or kill viruses infiltrating the respiratory system and the like.
Description
본 출원은 2020년 05월 29일 출원된 대한민국 특허출원 제 10-2020-0065064호 및 2020년 12월 10일 출원된 대한민국 특허출원 제 10-2020-0171948호를 우선권으로 주장하고, 상기 명세서 전체는 본 출원의 참고문헌이다.This application claims priority to Korean Patent Application No. 10-2020-0065064 filed on May 29, 2020 and Korean Patent Application No. 10-2020-0171948 filed on December 10, 2020, and the entire specification is This application is incorporated herein by reference.
본 발명은 청색광을 이용한 바이러스를 예방 및 치료하는 방법에 관한 것이다. The present invention relates to a method for preventing and treating viruses using blue light.
바이러스는 DNA나 RNA를 유전체로 가지고 있고 단백질로 둘러싸인 구조이다. 바이러스는 독립적으로는 증식 불가능하며, 숙주세포 (host cell) 내에서 복제를 하고 세포간 감염을 통해 증식한다. 동물, 식물, 박테리아 등 거의 모든 생명체에는 각각 감염되는 바이러스가 존재하며, AIDS나 독감과 같은 다양한 질환의 원인이 된다. 바이러스에 의한 질환들은 대표적으로 인체면역결핍바이러스에 의한 후천성면역결핍증(AIDS), 간염바이러스 (Hepatitis virus)에 의한 간염 또는 간암, 헤르페스 바이러스 (Herpesvirus)에 의한 피부질환부터 종양, 메르스 바이러스 (MERS virus)에 의한 호흡기 질환이 있다. 바이러스를 치료하는 방법에는 바이러스에 감염된 숙주세포를 손상시키거나 죽여서 바이러스의 감염을 지연시키거나 차단할 수 있다. Viruses have DNA or RNA as their genome and are surrounded by proteins. Viruses cannot reproduce independently, replicate in host cells, and multiply through intercellular infection. Virtually all living things, such as animals, plants, and bacteria, each have a virus that infects them, which causes various diseases such as AIDS and the flu. Diseases caused by viruses typically include acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus, hepatitis or liver cancer caused by hepatitis virus, skin diseases caused by herpesvirus, tumors, and MERS virus (MERS virus). ) caused by respiratory diseases. Methods of treating viruses include delaying or blocking the infection of the virus by damaging or killing the virus-infected host cells.
코로나 바이러스는 아데노바이러스, 리노바이러스와 함께 사람에게 감기를 일으키는 3대 바이러스 중 하나로, 사람에게 다양한 경로를 통해 감염될 수 있는 유전자 크기 27~32kb의 RNA 바이러스이다. 전자 현미경으로 봤을 때, 바이러스 입자 표면이 돌기처럼 튀어나와 있는데 이 모양이 마치 왕관처럼 생겼다고 해서 라틴어로 왕관을 뜻하는 “corona”에서 파생돼 명명됐다. 주로 추운 겨울철에 발생하는 성인 감기의 10 ~ 30%를 차지하며, 두통이나 인후통, 기침을 동반한 코감기를 주 증상으로 한다. 코로나 바이러스는 1930년대 닭에서 처음으로 발견된 이후 개, 돼지, 조류 등의 동물에서 발견되었고, 1960년대에는 사람에서도 발견되었다. 코로나 바이러스는 동물과 사람 모두에게 발견되었고, 인간 활동 영역이 광범위해지면서 동물 사이에서만 유행하던 바이러스가 생존을 위해 유전자 변이를 일으켜 사람에게로 넘어오기도 한다. 예컨대 사스(박쥐와 사향 고양이), 메르스 (박쥐와 낙타), 코로나바이러스감염증-19(박쥐로 추정)가 이에 해당된다. 지금까지 발견된 코로나바이러스는 알파(Alpha)·베타(Beta)·감마(Gamma)·델타(Delta) 등 4속(屬)으로 분류된다. 여기서 알파는 다시 1a형과 1b형으로 나뉘고 베타는 2a, 2b, 2c, 2d형으로 나뉜다. 이 중 알파와 베타는 사람과 동물에게 감염되며, 감마와 델타는 동물에게 감염된다. 현재까지 확인된 인체 전염 코로나바이러스는 총 7종으로, HCoV 229E·HCoV NL63·HCoV OC43·HCoV HKU1·SARS-CoV·MERS-CoV·SARS-CoV-2가 이에 해당한다. 이 가운데 4종(229E, OC43, NL63, HKU1)은 감기와 비슷한 가벼운 증상만 일으킨다. 하지만 사스(SARS-CoV·중증급성호흡기증후군)와 메르스(MERS-CoV·중동호흡기증후군), 코로나바이러스감염증-19(SARS-CoV-2,severe acute respiratory syndrome coronavirus 2)는 중증 폐렴 등 심각한 호흡기 질환을 일으킬 수 있으며, 많은 사망자를 발생시킨다. Corona virus is one of the three major viruses that cause colds in humans along with adenovirus and rhinovirus, and is an RNA virus with a gene size of 27 to 32 kb that can infect humans through various routes. When viewed under an electron microscope, the surface of the virus particle protrudes like a protrusion, and this shape resembles a crown, so it was named after the Latin word “corona” meaning crown. It accounts for 10 to 30% of adult colds that occur mainly in the cold winter, and the main symptom is a nasal cold accompanied by a headache, sore throat, or cough. Since the coronavirus was first discovered in chickens in the 1930s, it has been found in animals such as dogs, pigs, and birds, and in humans in the 1960s. Corona virus has been found in both animals and humans, and as the area of human activity expands, the virus that was prevalent only among animals causes genetic mutations in order to survive and is passed on to humans. Examples include SARS (bats and civets), MERS (bats and camels), and COVID-19 (probably bats). The coronaviruses discovered so far are classified into four genera: Alpha, Beta, Gamma, and Delta. Here, alpha is further divided into types 1a and 1b, and beta is divided into types 2a, 2b, 2c, and 2d. Of these, alpha and beta infect humans and animals, and gamma and delta infect animals. There are a total of 7 types of coronaviruses that have been identified so far, including HCoV 229E · HCoV NL63 · HCoV OC43 · HCoV HKU1 · SARS-CoV · MERS-CoV · SARS-CoV-2. Of these, four (229E, OC43, NL63, and HKU1) caused only mild symptoms similar to colds. However, SARS (severe acute respiratory syndrome), MERS (MERS-CoV), and coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) It can cause disease and cause many deaths.
코로나-19 바이러스(COVID-19; Coronavirus Disease 2019)은 2019년 12월 중국 우한에서 처음 발생한 이후 중국 전역과 전 세계로 확산된, 새로운 유형의 코로나바이러스(SARS-CoV-2)이다. 코로나-19 바이러스는 대단히 높은 전파율을 보이며 전염성이 특히 높다. 코로나-19 바이러스에 감염되면 약 2~14일(추정)의 잠복기를 거친 뒤 발열(37.5도) 및 기침이나 호흡곤란 등 호흡기 증상과 폐렴이 주 증상으로 나타나지만 무증상 감염 사례도 드물지 않게 나오고 있다.Coronavirus Disease 2019 (COVID-19) is a new type of coronavirus (SARS-CoV-2) that first emerged in Wuhan, China in December 2019 and has spread throughout China and around the world. The COVID-19 virus has a very high transmission rate and is particularly highly contagious. After being infected with the COVID-19 virus, after an incubation period of about 2 to 14 days (estimated), respiratory symptoms such as fever (37.5 degrees), cough or shortness of breath, and pneumonia appear as the main symptoms, but asymptomatic infections are not uncommon.
일반적으로 호흡기를 침범하는 바이러스는 비슷한 경로로 호흡기계를 침범하게 되는 것으로 알려져 있으며, 바이러스의 특징에 따라 전파력이 빨라 전염력이 높거나, 숙주세포를 빨리 죽임으로써 숙주에게 미치는 침습성이 높거나 하는 것에 차이를 보인다. 공통된 침습경로 등은 비말이나 공기의 흐름을 타고 호흡기내 점막 상피세포로 유입되어, 인후두 점막의 발적, 궤양, 분비물 등이 발견되고 인후두가 염증에 의해 좁아지면서 부종이 심해지면 호흡이 힘들어지는 기도 폐쇄 증상을 보이고, 면역 능력이 떨어진 노인이나 기저질환을 지닌 환자의 경우 기관지염, 폐렴, 패혈증 등이 합병증으로 나타난다. 따라서 바이러스 감염으로 인한 염증반응 감소하고, 바이러스 활성을 저해하는 치료가 필요하다. In general, viruses that invade the respiratory system are known to invade the respiratory system in a similar way, and depending on the characteristics of the virus, it is highly contagious because of its fast dissemination power, or it is high in invasiveness to the host by quickly killing host cells. looks like A common invasive route, etc., enters the mucosal epithelial cells in the respiratory tract via droplets or airflow, and redness, ulceration, and secretion of the pharyngeal mucosa are found. Complications include bronchitis, pneumonia, and sepsis in the elderly or patients with underlying diseases with weakened immunity. Therefore, there is a need for a treatment that reduces the inflammatory response caused by viral infection and inhibits viral activity.
이에 본 발명자들은 숙주세포는 사멸하지 않고, 염증반응은 감소시키며, 감염된 바이러스에서는 성장을 억제하거나 사멸하는 방법을 연구한 결과, 청색광을 조사한 경우, 숙주세포의 사멸율과 염증반응은 감소시키면서, 감염된 바이러스의 증식을 억제시킬 수 있다는 것을 확인함으로써 이 내용으로 본원 발명을 제출하는 바이다. Accordingly, the present inventors did not kill the host cell, reduce the inflammatory response, and as a result of studying a method for inhibiting growth or killing an infected virus, when irradiated with blue light, the death rate and inflammatory response of the host cell are reduced while reducing the infection By confirming that it is possible to inhibit the proliferation of viruses, the present invention is submitted with this content.
따라서 본 발명은 상기 문제점을 해결하기 위하여 안출된 것으로, 그 목적은 청색 영역 내에 있는 LED 또는 레이저 광원을 포함하는, 바이러스를 치료 또는 예방하는 광선 치료용 장치를 제공하는 것이다. Accordingly, the present invention has been devised to solve the above problems, and an object of the present invention is to provide a light therapy device for treating or preventing viruses, including an LED or a laser light source in a blue region.
본 발명에서 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급하지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The technical problems to be achieved in the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. will be able
상기 목적을 달성하기 위한 구체적인 수단으로서 본 발명은 청색 영역 내에 있는 LED 또는 레이저 광원을 포함하는, 바이러스를 치료 또는 예방하는 광선 치료용 장치를 제공할 수 있다. As a specific means for achieving the above object, the present invention may provide an apparatus for light therapy for treating or preventing a virus, including an LED or a laser light source in a blue region.
상기 청색 영역은 400nm 내지 500nm일 수 있다. The blue region may be 400 nm to 500 nm.
상기 LED 또는 레이저 광원은 1.0 내지 20 J/cm2 의 조사량으로 조사될 수 있다. The LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
상기 바이러스는 호흡기 질환 원인 바이러스일 수 있다. The virus may be a virus causing respiratory diseases.
본 발명은 상기 서술한 광선 치료용 장치를 인간을 제외한 동물에게 조사하는 단계;를 포함하는 바이러스를 치료하는 방법을 제공할 수 있다. The present invention may provide a method of treating a virus comprising; irradiating the above-described device for phototherapy to animals other than humans.
상기 광선 치료용 장치를 바이러스에 감염된 인간을 제외한 동물의 전신, 폐, 기도, 코 점막 및 입 점막으로 이루어진 군으로부터 선택되는 어느 하나의 부위에 조사할 수 있다. The device for phototherapy may be irradiated to any one site selected from the group consisting of the whole body, lungs, airways, nasal mucosa, and oral mucosa of an animal other than a human infected with a virus.
상기 청색 영역은 400nm 내지 500nm일 수 있다. The blue region may be 400 nm to 500 nm.
상기 LED 또는 레이저 광원은 1.0 내지 20 J/cm2 의 조사량으로 조사될 수 있다. The LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
상기 바이러스는 호흡기 질환 원인 바이러스일 수 있다. The virus may be a virus causing respiratory diseases.
본 발명의 청색광은 바이러스에 감염된 숙주세포는 사멸시키지 않고, 이의 염증반응을 억제하며, 감염된 바이러스의 성장을 억제하고 사멸시키는 효과가 있다. The blue light of the present invention does not kill the virus-infected host cell, suppresses its inflammatory response, and has the effect of inhibiting the growth and killing of the infected virus.
도 1은 본 발명의 청색광을 CCD-18Co와 CCD-1131Sk 세포에 조사한 후, 48시간 뒤 현미경으로 관찰한 결과이다. 1 is a result of observation under a microscope after 48 hours after irradiating the blue light of the present invention to CCD-18Co and CCD-1131Sk cells.
도 2는 본 발명의 청색광을 CCD-18Co와 CCD-1131Sk 세포에 조사한 후, 48시간 뒤 세포 생존율의 결과이다. 2 is a result of cell viability 48 hours after irradiating the blue light of the present invention to CCD-18Co and CCD-1131Sk cells.
도 3은 본 발명의 청색광을 Vero E6세포에 조사한 후, 24 시간, 48시간 뒤 세포 생존율의 결과이다. 3 is a result of cell viability 24 hours and 48 hours after irradiating the blue light of the present invention to Vero E6 cells.
도 4은 본 발명의 청색광 마우스 대장암 세포주 CT-26 세포에 조사한 후, 단백질 발현 변화를 확인한 결과이다. 4 is a result of confirming the protein expression change after irradiating the blue light mouse colorectal cancer cell line CT-26 cells of the present invention.
도 5는 본 발명의 청색광을 SARS-CoV2 바이러스 감염된 Vero E6 세포에 조사한 후, 24시간 후에 세포 생존율 및 세포내 바이러스 발현 양을 측정한 결과이다. 5 is a result of measuring the cell viability and intracellular virus expression level 24 hours after irradiating the blue light of the present invention to Vero E6 cells infected with SARS-CoV2 virus.
도 6는 본 발명의 청색광을 SARS-CoV2 바이러스 감염된 Vero E6 세포에 조사한 후, 24시간 (좌), 72시간 (우) 후에 상등액내 vRNA 추출하여, 이의 quantification cycle (Cq) 값을 측정한 결과이다. Figure 6 is the result of irradiating the blue light of the present invention to Vero E6 cells infected with SARS-CoV2 virus, and then extracting vRNA from the supernatant after 24 hours (left) and 72 hours (right), and measuring its quantification cycle (Cq) value. .
도 7은 본 발명의 청색광을 10 J/m2로 SARS-CoV2 바이러스 감염된 Vero E6 세포에 1회, 2회 조사한 후, 24시간 뒤 세포 상등액의 vRNA 추출하여, 이의 copy 수를 측정한 결과이다. 7 is a result of measuring the number of copies by irradiating the blue light of the present invention at 10 J/m 2 to Vero E6 cells infected with SARS-CoV2 virus once or twice, then extracting vRNA from the cell supernatant 24 hours later.
도 8은 본 발명의 청색광을 10 J/m2로 SARS-CoV2 바이러스 감염된 Vero E6 세포에 0.6% agarose가 포함된 overlay media를 넣은 후 1회 조사한 후, 72시간 뒤 생성된 plaque와 감염되지 않은 세포를 염색하여 염색되지 않은 plaque 수를 측정한 결과이다.Figure 8 shows the blue light of the present invention at 10 J/m 2 SARS-CoV2 virus-infected Vero E6 cells were irradiated once with overlay media containing 0.6% agarose, and the plaques and uninfected cells generated 72 hours later. This is the result of measuring the number of unstained plaques by staining.
도 9는 본 발명의 청색광을 10 J/m2로 H1N1 바이러스에 감염된 MDCK 세포에 2회 조사한 후, 72시간 뒤 상등액내 vRNA 추출하여, 이의 (Ct) 값을 계산하여 바이러스의 역가를 측정한 결과이다. Figure 9 shows the results of measuring the titer of the virus by irradiating the blue light of the present invention at 10 J/m 2 to MDCK cells infected with H1N1 virus twice, then extracting vRNA from the supernatant after 72 hours, calculating its (Ct) value. am.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
호흡기를 침범하는 바이러스는 전파력이 빨라 전염력이 높다. 면역 능력이 떨어진 노인이나 기저질환을 지닌 환자가 호흡기를 침범하는 바이러스에 감염된 경우 기관지염, 폐렴, 패혈증과 같은 합병증으로 사망률이 증가된다. 따라서 바이러스 감염으로 인한 염증반응 감소하고, 바이러스 활성을 저해하는 치료가 필요하다. 이에 본 발명자들은 숙주세포에서는 사멸하지 않고, 염증반응은 감소시키며, 감염된 바이러스에서는 성장을 억제하거나 사멸하는 방법을 연구한 결과, 청색광을 조사한 경우, 숙주세포의 사멸율은 감소하지 않고, 염증반응은 감소시키며, 감염된 바이러스의 성장을 억제하고, 사멸시킨다는 것을 확인하고 본원 발명을 완성하였다. Viruses that invade the respiratory tract are highly contagious because they spread quickly. When the elderly with weakened immunity or patients with underlying diseases are infected with a virus that invades the respiratory tract, the mortality rate increases due to complications such as bronchitis, pneumonia, and sepsis. Therefore, there is a need for a treatment that reduces the inflammatory response caused by viral infection and inhibits viral activity. Accordingly, the present inventors did not die in the host cell, reduce the inflammatory response, and as a result of studying a method for inhibiting or killing the infected virus, when irradiated with blue light, the death rate of the host cell does not decrease, and the inflammatory response is It was confirmed that it reduces, inhibits the growth of, and kills the infected virus, and completed the present invention.
상기 목적을 달성하기 위한 구체적인 수단으로서 본 발명은 청색 영역 내에 있는 LED 또는 레이저 광원을 포함하는, 바이러스를 치료 또는 예방하는 광선 치료용 장치를 제공할 수 있다. As a specific means for achieving the above object, the present invention may provide an apparatus for light therapy for treating or preventing a virus, including an LED or a laser light source in a blue region.
450nm 청색광을 인간 피부 세포주 (CCD-1131Sk), 인간 결장 섬유아 세포주 (CCD-18Co) 및 원숭이 정상 신장세포주 (Vero E6)에 5, 10, 15 J/cm2 조사한 다음, 48시간 뒤 세포 생존율을 측정한 결과, 세포의 형태학적 변화는 관찰되지 않았고, 이의 세포 생존율을 측정한 결과, 두 세포 모두에서 대조군에 비해 통계적으로 차이가 나지 않았다 (도 1 및 도 2). 마우스 대장암 세포주 CT-26세포에 450nm 청색광을 30분간 11.3 J/cm2 조사 한 후, 상기 세포주 CT-26세포의 단백질 발현을 측정한 결과, 청색광은 세포 사멸에는 영향을 주지 않았고 세포 이동성 및 침윤성 억제 등과 관련된 단백질들 (TCTP, LASP1, Enol 1, PLS3) 발현이 억제되는 것을 확인하였다 (도 3). SARS-CoV2를 원숭이 신장세포주 Vero E6 세포에 감염시킨 뒤, 1.6, 5, 10J/cm2 조사 후, 24시간 후 세포 생존율 및 SARS-CoV2 바이러스 발현을 측정한 결과, 세포 생존율에는 대조군에 비해 변화되지 않으나, 세포내 SARS-CoV2 바이러스 발현은 상대적으로 감소되는 것을 확인하였다 (도 4). 청색광을 1.7, 5, 10 J/cm2 에너지량으로 조사하고 24시간, 72시간 후 세포내 혹은 세포 배양액에서 RNA를 추출하여 quantification cycle (Cq)을 측정한 결과, 대조군에 비해 청색광을 5J 과 10J 조사한 세포는 통계적으로 유의하게 감소하였다 (도 5). 청색광 10 J을 1회 혹은 2회 조사 후 24시간 뒤 세포 상등액을 회수하여 vRNA copy 수를 측정한 결과, 청색광 1회 혹은 2회 조사 후 vRNA copy 수는 대조군에 비해 통계적으로 유의하게 약 50% 이상 감소하였다 (도 6). SARS-CoV-2 바이러스에 감염된 세포에 0.6% agarose가 포함된 overlay media를 넣은 후 청색광 10 J을 1회 조사 후 72시간 뒤 감염되지 않은 세포와 plaque염색하여 측정한 결과 plaque 수는 대조군에 비해 통계적으로 유의하게 약 80% 이상 감소하였다 (도 7). 상기와 같이 청색광 에너지 준위는 SARS-CoV2 바이러스에 감염된 세포에 있어서 세포 사멸에는 영향을 주지 않고 바이러스 불활성화에 영향을 줄 수 있다는 것을 확인하였다. H1N1 바이러스에 감염된 MDCK 세포에 청색광 ~~J을 0 hpi (post-infection hour)와 24 hpi 시간대에서 각각 30분간 노출시킨 뒤, 72시간 동안 바이러스를 증식시킨 뒤 상층액을 회수하여 vRNA의 Ct 값을 계산하여 바이러스 역가를 측정하였다. 측정한 결과, 청색광 1회 또는 2회 조사 후 바이러스의 역사는 대조군에 통계적으로 감소하였다. 이를 통해 청색광 에너지 준위는 H1N1 바이러스에 감염된 세포에 있어서 바이러스 불활성화에 영향을 줄 수 있다는 것을 확인하였다. After irradiating 5, 10, and 15 J/cm 2 of 450 nm blue light to human skin cell line (CCD-1131Sk), human colon fibroblast cell line (CCD-18Co) and monkey normal kidney cell line (Vero E6), cell viability was measured after 48 hours. As a result of the measurement, the morphological change of the cells was not observed, and as a result of measuring the cell viability thereof, there was no statistical difference in both cells compared to the control group ( FIGS. 1 and 2 ). After irradiating 11.3 J/cm2 of 450 nm blue light to mouse colorectal cancer cell line CT-26 cells for 30 minutes, protein expression of the cell line CT-26 cells was measured. As a result, blue light did not affect apoptosis and inhibited cell mobility and invasion. It was confirmed that the expression of proteins (TCTP, LASP1, Enol 1, PLS3) related to the back is suppressed ( FIG. 3 ). After SARS-CoV2 infection with the monkey kidney cell line Vero E6 cells, 1.6, 5, 10J/cm2 irradiation, and 24 hours later, the cell viability and SARS-CoV2 virus expression were measured. As a result, the cell viability did not change compared to the control group. , it was confirmed that the intracellular SARS-CoV2 virus expression was relatively decreased ( FIG. 4 ). Blue light was irradiated with energy of 1.7, 5, and 10 J/cm2, and RNA was extracted from cells or cell culture after 24 hours or 72 hours to measure the quantification cycle (Cq). Cells were reduced statistically significantly ( FIG. 5 ). As a result of measuring the number of vRNA copies by collecting the cell supernatant 24 hours after irradiating 10 J of blue light once or twice, the number of vRNA copies after irradiating one or two times with blue light was statistically significantly higher than that of the control group by about 50% or more. decreased (Fig. 6). After putting the overlay media containing 0.6% agarose into SARS-CoV-2 virus-infected cells, and after irradiating 10 J with blue light once, 72 hours later, plaque staining with uninfected cells showed that the number of plaques was statistically higher than that of the control group. was significantly reduced by about 80% or more (FIG. 7). As described above, it was confirmed that the blue light energy level can affect virus inactivation without affecting apoptosis in cells infected with SARS-CoV2 virus. After exposing blue light ~~J to MDCK cells infected with H1N1 virus for 30 minutes each at 0 hpi (post-infection hour) and 24 hpi time zones, the virus was allowed to grow for 72 hours, and the supernatant was recovered to determine the Ct value of vRNA. was calculated to determine the virus titer. As a result of the measurement, the virus history was statistically decreased in the control group after one or two blue light irradiation. Through this, it was confirmed that the blue light energy level can affect the virus inactivation in H1N1 virus-infected cells.
상기 청색 영역은 400nm 내지 500nm일 수 있고. 바람직하게는 425 내지 475 nm일 수 있으며, 더 바람직하게는 450nm일 수 있다. The blue region may be 400 nm to 500 nm. Preferably, it may be 425 to 475 nm, and more preferably, 450 nm.
상기 LED 또는 레이저 광원은 1.0 내지 20 J/cm2 의 조사량으로 조사될 수 있다. The LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
상기 바이러스는 호흡기 질환 원인 바이러스일 수 있고, 바람직하게는 아데노바이러스 (adenovirus), 인플루엔자 바이러스 (influenza virus), 파라인플루엔자 바이러스 (parainfluenza virus, PIV), RS 바이러스 (respiratory syncytial virus, RSV), 보카바이러스(Human bocavirus, hBoV), 메타뉴모바이러스 (human metapneumovirus, hMPV), 라이노바이러스 (rhinovirus) 및 코로나바이러스 (coronavirus)로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며 더 바람직하게는 코로나 바이러스 또는 인플루엔자 바이러스 일 수 있다. The virus may be a virus causing respiratory diseases, preferably adenovirus, influenza virus, parainfluenza virus, PIV, respiratory syncytial virus (RSV), bocavirus ( Human bocavirus, hBoV), metapneumovirus (human metapneumovirus, hMPV), may be any one selected from the group consisting of rhinovirus (rhinovirus) and coronavirus (coronavirus), more preferably corona virus or influenza virus. .
상기 코로나 바이러스는 사스(SARS-CoV·중증급성호흡기증후군)와 메르스(MERS-CoV·중동호흡기증후군) 및 코로나바이러스감염증-19(SARS-CoV-2,severe acute respiratory syndrome coronavirus 2)로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며 더 바람직하게는 코로나바이러스감염증-19(SARS-CoV-2,severe acute respiratory syndrome coronavirus 2)일 수 있다. The coronavirus is a group consisting of SARS (SARS-CoV, severe acute respiratory syndrome), MERS (MERS-CoV, Middle East respiratory syndrome) and coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) It may be any one selected from, and more preferably, coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2).
상기 인플루엔자바이러스는 인플루엔자 바이러스 A형, 인플루엔자바이러스 B형, 인플루엔자 C형, 인플루엔자바이러스 D형, 토고토바이러스, 콰란자 바이러스 및 연어빈혈증 바이러스 일 수 있으며, 바람직하게는 인플루엔자 바이러스 A형일 있고, 더 바람직하게는 N1H1일 수 있다. The influenza virus may be influenza virus type A, influenza virus type B, influenza type C, influenza virus type D, togotovirus, quaranza virus and salmon anemia virus, preferably influenza virus type A, more preferably may be N1H1.
본 발명은 상기 서술한 광선 치료용 장치를 인간을 제외한 동물에게 조사하는 단계를 포함하는 바이러스를 치료하는 방법을 제공할 수 있다. The present invention may provide a method of treating a virus comprising the step of irradiating the above-described device for phototherapy to animals other than humans.
상기 광선 치료용 장치를 바이러스에 감염된 인간을 제외한 동물의 전신, 폐, 기도, 코 점막 및 입 점막으로 이루어진 군으로부터 선택되는 어느 하나의 부위에 조사할 수 있다. The device for phototherapy may be irradiated to any one site selected from the group consisting of the whole body, lungs, airways, nasal mucosa, and oral mucosa of an animal other than a human infected with a virus.
세포조직의 종류에 따라 다르지만, 400nm 파장의 광은 1mm 이하의 투과 깊이를, 514nm 파장의 광은 0.5~2mm의 투과 깊이를, 630nm 파장의 광은 1~6mm의 투과 깊이를 갖고, 700~900nm 파장의 광은 더 깊이 침투할수 있습니다. 본원 발명의 광원은 1mm 내지 2mm의 피부를 투과할 수 있으며, 전신에 조사할 경우, 피부에 존재하는 미세혈관 내에 부유하는 바이러스를 치료할 수 있는 효과가 있다. Although it depends on the type of cell tissue, light with a wavelength of 400 nm has a penetration depth of 1 mm or less, light with a wavelength of 514 nm has a penetration depth of 0.5 to 2 mm, and light with a wavelength of 630 nm has a penetration depth of 1 to 6 mm, and 700 to 900 nm Wavelengths of light can penetrate deeper. The light source of the present invention can penetrate the skin of 1 mm to 2 mm, and when irradiated to the whole body, there is an effect of treating viruses floating in microvessels existing in the skin.
상기 청색 영역은 400nm 내지 500nm일 수 있고. 바람직하게는 425 내지 475 nm일 수 있으며, 더 바람직하게는 450nm일 수 있다. The blue region may be 400 nm to 500 nm. Preferably, it may be 425 to 475 nm, and more preferably, 450 nm.
상기 LED 또는 레이저 광원은 1.0 내지 20 J/cm2 의 조사량으로 조사될 수 있다. The LED or laser light source may be irradiated with an irradiation amount of 1.0 to 20 J/cm 2 .
상기 바이러스는 호흡기 질환 원인 바이러스일 수 있고, 바람직하게는 아데노바이러스 (adenovirus), 인플루엔자 바이러스 (influenza virus), 파라인플루엔자 바이러스 (parainfluenza virus, PIV), RS 바이러스 (respiratory syncytial virus, RSV), 라이노바이러스 (rhinovirus) 및 코로나바이러스 (coronavirus)로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며 더 바람직하게는 코로나 바이러스 일 수 있다. The virus may be a virus causing respiratory diseases, preferably adenovirus, influenza virus, parainfluenza virus (PIV), RS virus (respiratory syncytial virus, RSV), rhinovirus ( rhinovirus) and coronavirus (coronavirus) may be any one selected from the group consisting of, and more preferably may be a corona virus.
상기 코로나 바이러스는 사스(SARS-CoV·중증급성호흡기증후군)와 메르스(MERS-CoV·중동호흡기증후군) 및 코로나바이러스감염증-19(SARS-CoV-2,severe acute respiratory syndrome coronavirus 2)로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며 더 바람직하게는 코로나바이러스감염증-19(SARS-CoV-2,severe acute respiratory syndrome coronavirus 2)일 수 있다. The coronavirus is a group consisting of SARS (SARS-CoV, severe acute respiratory syndrome), MERS (MERS-CoV, Middle East respiratory syndrome) and coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) It may be any one selected from, and more preferably, coronavirus infection-19 (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2).
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.below, The present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 청색광 조사 후 세포 독성 평가Example 1. Evaluation of cytotoxicity after blue light irradiation
450nm 청색광을 인간 피부 세포주 (CCD-1131Sk)와 인간 결장 섬유아 세포주 (CCD-18Co)에 5, 10, 15 J/cm2 조사 후 48시간 뒤 세포 생존율을 평가하였다. CCD-1131Sk 와 CCD-18Co 세포 5 x 103 농도로 96 well plate에 배양한 뒤 우 혈청(fetal bovine serum)이 없는 배지로 교체 후 450nm 청색광을 5, 10, 15 J/cm2 조사 한 후, 48시간 뒤 10 μl MTT (5mg/ml) 를 처리하여 생존율을 MTT 방법으로 측정하였다. MTT 분석은 Mosmann (1983)의 분석방법에 따라, CCD-1131Sk와 CCD-18Co 세포에 청색광을 5, 10, 15 J/cm2 조사 후 48시간동안 배양한 뒤 5 mg/ml 농도의 MTT를 10 μl 씩 96 well에 넣어 4시간동안 배양하였다. 배양이 완료된 후 배양액을 제거한 뒤 2-propanol을 well 당 100 μl 씩 넣어 formazan MTT를 녹인 다음 ELISA reader로 570 nm에서 흡광도를 측정하여 대조군과 비교 조사하였다. 청색광 에너지 조사 후 인간 피부세포주와 결장 섬유아세포의 형태학적 변화는 관찰되지 않았다(도 1). CCD-18Co와 CCD-1131Sk 세포에 청색광 에너지 5, 10, 15 J/cm2 조사 후 48시간 뒤 세포 생존율은 대조군과 비교하였을 때 통계적으로 유의한 차이가 관찰되지 않았다. 상기 농도의 청색광 에너지는 세포에 독성을 나타나지 않은 것을 확인하였다. CCD-18Co 세포에 청색광 에너지 5, 10, 15 J/cm2를 조사한 처리군의 세포 생존율은 대조군에 비해 각각 91.4, 92.2, 99.5%를 나타내었다. CCD-1131Sk 세포에서는 청색광 에너지 5, 10, 15 J/cm2를 조사한 처리군의 세포 생존율은 대조군에 비해 각각 93.4, 92.2, 101.5%를 나타내었다 (도 2). 상기 농도의 청색광 에너지의 세포 독성 평가는 바이러스 실험 시 숙주 세포로 사용되는 원숭이 신장 정상 세포주 Vero E6에서도 확인되었다. Vero E6 세포에 청색광 에너지 10, 15 J/cm2를 조사 후 24 시간 48시간 뒤 세포 생존율은 대조군과 비교하였을 때 각각 통계적으로 유의한 차이가 관찰되지 않았다. Vero E6 세포에 청색광 에너지 10 J/cm2를 조사 후 24 시간, 48시간 뒤 세포 생존율은 대조군과 비교하였을 때 102.04, 100.84%를 나타내었고, 15 J/cm2를 조사 후 24 시간, 48시간 뒤 세포 생존율은 102.06, 100.91%를 나타내었다 (도3).Cell viability was evaluated 48 hours after irradiating 5, 10, 15 J/cm 2 to human skin cell line (CCD-1131Sk) and human colon fibroblast cell line (CCD-18Co) with 450 nm blue light. CCD-1131Sk and CCD-18Co cells were cultured in a 96-well plate at a concentration of 5 x 10 3 , replaced with a medium without fetal bovine serum, and irradiated with 5, 10, 15 J/cm 2 of 450 nm blue light, After 48 hours, 10 μl MTT (5 mg/ml) was treated and the viability was measured by the MTT method. According to the analysis method of Mosmann (1983), CCD-1131Sk and CCD-18Co cells were irradiated with blue light at 5, 10, 15 J/cm 2 for 48 hours and then incubated for 48 hours followed by MTT at a concentration of 5 mg/ml. Each μl was put into 96 wells and incubated for 4 hours. After the culture was completed, the culture medium was removed, and 100 μl of 2-propanol was added per well to dissolve formazan MTT, and absorbance was measured at 570 nm with an ELISA reader to compare with the control group. No morphological changes were observed in human skin cell lines and colon fibroblasts after blue light energy irradiation (FIG. 1). After irradiating CCD-18Co and CCD-1131Sk cells with blue light energy of 5, 10, and 15 J/cm 2 , no statistically significant difference was observed in cell viability after 48 hours compared to the control group. It was confirmed that the blue light energy of the above concentration did not show toxicity to cells. Treatment group irradiated with blue light energy 5, 10, 15 J/cm 2 to CCD-18Co cells Cell viability was 91.4, 92.2, and 99.5%, respectively, compared to the control. In CCD-1131Sk cells, the treatment group irradiated with blue light energy of 5, 10, 15 J/cm 2 Cell viability was 93.4, 92.2, and 101.5%, respectively, compared to the control group (Fig. 2). The evaluation of the cytotoxicity of blue light energy at the above concentration was also confirmed in the normal monkey kidney Vero E6 cell line used as a host cell for virus experiments. After irradiating Vero E6 cells with blue light energy of 10, 15 J/cm 2 , no statistically significant difference was observed in cell viability after 24 hours and 48 hours compared to the control group. After irradiating Vero E6 cells with blue light energy of 10 J/cm 2 , the cell viability was 102.04, 100.84% after 24 and 48 hours compared to the control group, and 15 J/ cm 2 24 and 48 hours after irradiation. Cell viability was 102.06 and 100.91% (Fig. 3).
실시예 2. 청색광 조사 후 단백질의 발현 변화Example 2. Changes in protein expression after blue light irradiation
마우스 대장암 세포주 CT-26세포에 450nm 청색광을 30분간 11.3 J/cm2 조사 한 후, 상기 세포주 CT-26세포를 이차원 전기영동과 웨스턴 블랏팅을 이용하여 단백질 발현을 확인 하였다. 마우스 대장암 세포주 CT-26세포에 450nm 청색광을 30분간 11.3 J/cm2 조사 한 후, CT-26세포를 회수하여 7M urea , 2M Thiourea, 4%(w/v) 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate(CHAPS), 1%(w/v) dithiothreitol(DTT), 2%(v/v) pharmalyte, 1mM benzamidine로 구성된 2DE lysis solution과 혼합하였다. 그리고 단백질 추출을 위해서 1시간 동안 vortexing 하였으며, 15℃에서 15,000rpm으로 1시간 동안 원심 분리하여 상층액을 이차원 전기영동의 시료로 사용하였다. 단백질의 농도 측정은 Bradford 법으로 수행하였다(Bradford et al., Anal. Biochem., 1976, 72, 248). 일차 Isoelectric focusing(IEF)를 위하여 IPG strips은 7M urea, 2M thiourea, 2% 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate(CHAPS), 1% dithiothreitol(DTT), 1% pharmalyte로 구성된 reswelling 용액으로 상온에서 12 내지 16시간 정도 reswelling 되었다. Strip 당 시료는 각각 200ug씩을 사용하였으며, Amersham Biosciences 사의 Multiphore II system을 이용하여 제조회사의 사용 메뉴얼을 준수하여 20℃에서 IEF를 수행하였다. IEF 조건은 150V에서 3,500V까지의 도달시간을 3시간 되게 하였으며, 3,500V에서 26시간 지속되도록 하여 최종적으로 96kVh 가 되도록 설정하였다. 이차적으로 SDS-PAGE를 수행하기 전에 IPG Strips을 1% DTT를 함유한 equilibration buffer(50mM Tris-Cl, pH6.8, 6M urea, 2% SDS, 30% glycerol)로 10분간 incubation 하였으며, 곧바로 2.5% iodoacetamide를 함유한 equilibration buffer로 10분간 더 incubation 하였다. Equilibration이 완료된 strips을 SDS-PAGE gels(20x24cm, 10-16%) 위에 배열시키고, Hoefer DALT 2D system(Amersham Biosciences)을 이용하여 20℃에서 최종적으로 1.7kVh가 되게 전개하였다. 이차원전기영동이 완료된 이차원 젤의 단백질은 Oakley(Anal. Biochem. 1980, 105:361-363) 등의 방법에 따라 은염색으로 시각화되었으며, 질량분석기에 의한 단백질 동정을 위하여 glutaraldehyde 처리 단계는 생략되었다. 은염색된 이차원 젤은 AGFA 사의 Duoscan T1200 스캐너로 스캐닝되어 확장자가 TIFF 인 파일의 형태로 컴퓨터에 저장되었다. 스캐닝된 이미지로부터 단백질 spots의 발현변화 확인을 위한 정량적인 분석은 PDQuest software(version 7.0, BioRad)를 이용하여 수행하였다. 각 spot의 quantity는 total valid sopts의 intensity로 평준화(normalization)되었으며, 대조군에 비해 두 배 이상의 유의한 발현변화를 보여주는 단백질 spots을 선정하였다. 이차원 전기영동을 수행하여 spot를 동정한 뒤 분석된 결과를 바탕으로 실제 CT-26 세포에서 TCTP, LASP1, Enol 1, PLS3 발현을 웨스턴 블랏으로 재확인 하였다. 마우스 대장암 세포주 CT-26세포에 450nm 청색광을 30분간 11.3 J/cm2 조사 한 후, CT-26세포를 회수하여 RIPA (10 mM Tris, pH7.2, 150 mM NaCl , 1 % deoxycholate, 1 % Triton X-100, 0.1 % SDS, protease inhibitor cocktail solution)에서 용해하였고, 12000rpm, 4℃에서 20분 동안 원심분리하고 상층액을 수거한 뒤, Bradford분석 (Bio-Rad Laboratories, Hercules, CA)을 이용하여 단백질 정량하였다. 이후， 얻어진 세포 총 용해물 30 ug을 SDS 시료 버퍼와 혼합하여 가열한 다음， 8 %-15% SDS-PAGE 겔에 100V에서 2시간동안 전기영동 하였다. 전기영동을 통해 SDS-PAGE 상에서 분리된 단백질은 100% 메탄올에 10초간 미리 적신 후 증류수로 충분히 수화시킨 PVDF 멤브레인(polyvinyledene floride membrane)으로 80V에서 1시간동안 상기 전기영동 장치를 이용하여 분리한 단백질을 이동시키고, 5 % 스킴 밀크 (skim milk)로 블락킹 (blocking)하였다. 이 후 1차 항체 항-TCTP (cell signaling), 항-LASP1 (cell signaling), 항-Enol 1 (cell signaling), 항-PLS3 (Santacruz), 그리고 항-GAPDH (cell signaling)와 함께 반응 시켰고，홀스레디쉬 퍼올시데이즈 (horseradish peroxidase)가 결합된 2차 항체와 반응 시켰다. 이 후 ECL 키트 (Millipore, USA)를 제조사의 방법대로 처리하여 발생 반응을 유도하였다. 그 결과 청색광은 세포 사멸에는 영향을 주지 않았고 세포 이동성 및 침윤성 억제 등과 관련된 단백질들 (TCTP, LASP1, Enol 1, PLS3) 발현이 억제되는 것을 확인하였다 (도 4). 상기 단백질 발현에 있어 DNA 또는 mRNA 수준의 발현에도 영향을 주었을 것으로 추측할 수 있었다. 따라서 청색광 20 J/cm2이하의 에너지 준위는 바이러스에 감염된 세포에 있어서 세포 사멸에는 영향을 주지 않고 세포증식, 염증 반응, 키나제 등의 단백질 발현이 억제되는 방향으로 영향을 줄 수 있는 것으로 생각할 수 있다. 청색광 조사 후 단백질 발현의 감소는 바이러스 증식이 억제되는 기전을 설명하는데 있어 중요한 기전 중 하나로 판단되며, 숙주세포 측면에서는 염증의 완화, 세포 증식 억제를 설명할 수 있고 바이러스 측면에서는 숙주세포의 효소를 활용하는 것이 억제됨으로써 증식억제되고 기능의 불활성화를 가져오는 결과를 일으킬 수 있을 것으로 판단된다. After irradiating 11.3 J/cm2 of 450 nm blue light to the mouse colorectal cancer cell line CT-26 cells for 30 minutes, the protein expression of the CT-26 cell line cell line was confirmed using two-dimensional electrophoresis and western blotting. After irradiating 11.3 J/cm 2 of 450 nm blue light to mouse colorectal cancer cell line CT-26 cells for 30 minutes, CT-26 cells were recovered and 7M urea, 2M Thiourea, 4%(w/v) 3-[(3-cholamidopropy) ) dimethyammonio]-1-propanesulfonate (CHAPS), 1% (w/v) dithiothreitol (DTT), 2% (v/v) pharmalyte, and 1 mM benzamidine were mixed with a 2DE lysis solution. Then, for protein extraction, vortexing was performed for 1 hour, centrifugation was performed at 15° C. at 15,000 rpm for 1 hour, and the supernatant was used as a sample for two-dimensional electrophoresis. Protein concentration was measured by the Bradford method (Bradford et al., Anal. Biochem., 1976, 72, 248). For primary isoelectric focusing (IEF), IPG strips are reswelling composed of 7M urea, 2M thiourea, 2% 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% pharmalyte. The solution was reswelled at room temperature for 12 to 16 hours. 200 ug of each sample was used for each strip, and IEF was performed at 20° C. using the Multiphore II system of Amersham Biosciences in compliance with the manufacturer's instruction manual. The IEF condition was set so that the reaching time from 150V to 3,500V was 3 hours, and it was set to last for 26 hours at 3,500V to finally reach 96kVh. Secondarily, before performing SDS-PAGE, IPG Strips were incubated with equilibration buffer (50mM Tris-Cl, pH6.8, 6M urea, 2% SDS, 30% glycerol) containing 1% DTT for 10 minutes, and immediately after 2.5% Incubation was continued for 10 minutes with equilibration buffer containing iodoacetamide. Equilibration was completed strips were arranged on SDS-PAGE gels (20x24cm, 10-16%), and finally developed to 1.7kVh at 20°C using Hoefer DALT 2D system (Amersham Biosciences). The protein of the two-dimensional gel after the two-dimensional electrophoresis was completed was visualized with silver staining according to the method of Oakley (Anal. Biochem. 1980, 105:361-363) et al., and the glutaraldehyde treatment step was omitted for protein identification by mass spectrometry. The silver-stained two-dimensional gel was scanned with AGFA's Duoscan T1200 scanner and stored in a computer in the form of a file with the extension TIFF. Quantitative analysis for confirming the expression change of protein spots from the scanned image was performed using PDQuest software (version 7.0, BioRad). The quantity of each spot was normalized to the intensity of total valid sopts, and protein spots showing a significant change in expression more than double that of the control group were selected. After identifying spots by performing two-dimensional electrophoresis, TCTP, LASP1, Enol 1, and PLS3 expression in actual CT-26 cells were reconfirmed by Western blot based on the analyzed results. After irradiating 11.3 J/cm2 of 450 nm blue light to the mouse colon cancer cell line CT-26 cells for 30 minutes, the CT-26 cells were recovered and RIPA (10 mM Tris, pH7.2, 150 mM NaCl , 1% deoxycholate, 1% Triton). It was dissolved in X-100, 0.1 % SDS, protease inhibitor cocktail solution), centrifuged at 12000 rpm, 4° C. for 20 minutes, and the supernatant was collected, using Bradford analysis (Bio-Rad Laboratories, Hercules, CA). Protein was quantified. Then, 30 ug of the obtained total cell lysate was mixed with SDS sample buffer, heated, and then electrophoresed on an 8%-15% SDS-PAGE gel at 100V for 2 hours. Proteins separated on SDS-PAGE through electrophoresis were pre-wetted in 100% methanol for 10 seconds and then fully hydrated with distilled water. Proteins separated using the electrophoresis device at 80 V for 1 hour were used. It was moved and blocked with 5% skim milk. Thereafter, the primary antibody was reacted with anti-TCTP (cell signaling), anti-LASP1 (cell signaling), anti-Enol 1 (cell signaling), anti-PLS3 (Santacruz), and anti-GAPDH (cell signaling), It was reacted with a secondary antibody bound to horseradish peroxidase. Thereafter, the ECL kit (Millipore, USA) was treated according to the manufacturer's method to induce a developmental reaction. As a result, it was confirmed that blue light did not affect apoptosis and suppressed the expression of proteins (TCTP, LASP1, Enol 1, PLS3) related to inhibition of cell mobility and invasiveness ( FIG. 4 ). It could be inferred that the expression of the DNA or mRNA level was also affected in the expression of the protein. Therefore, it can be considered that an energy level of less than 20 J/cm 2 of blue light does not affect apoptosis in virus-infected cells and can affect cell proliferation, inflammatory response, and protein expression such as kinases in a direction that is suppressed. . The decrease in protein expression after blue light irradiation is considered to be one of the important mechanisms in explaining the mechanism by which virus proliferation is inhibited. It is judged that it can cause the result of inhibition of proliferation and inactivation of function by being inhibited.
실시예 3. 청색광의 조사에 따른 후 바이러스의 RNA 양의 변화Example 3. Changes in the amount of RNA in the virus after irradiation with blue light
SARS-CoV2는 질병관리본부로부터 확인되었고, 모든 실험 절차는 국내 생물안전 3등급 연구시설 (BL3) 에서 시행되었다. 원숭이 신장세포주 Vero E6 세포를 10% 우 혈청 (fetal bovine serum)을 첨가한 DMEM 배지에 배양한 후 70-80% confluence가 이루어지면 SARS-CoV2 를 감염시켜 주기적으로 세포병변 효과 (cytopathic effect, CPE)를 관찰하였다. 세포 변병이 확연히 관찰되면 배양액을 수거한 뒤, 원심분리기를 이용하여 상층액을 0.45 μm filter로 여과한 다음 소분하여 -80 ℃에 보관하였다. SARS-CoV2 바이러스의 정량을 위해 감염성 있는 바이러스 titer를 50% tissue culture infectious dose (TCID50)으로 나타내었다. SARS-CoV2 바이러스를 우혈청을 첨가한 DMEM 배지로 희석한 뒤 6 well plate (1 X 106 cells/well)에 배양된 세포에 접종하고, 음성 대조군으로 DMEM을 넣어 CO2 배양기에서 배양하면서 계속적으로 현미경으로 세포변병을 관찰하였다. Vero E6 세포를 6 well plate (1 X 106 cells/well)에 배양한 후 다음날 배양액을 제거한 뒤 PBS로 1회 세척하였다. 500 μl의 SARS-CoV2 바이러스를 0.01 MOI로 10분간 감염시킨 뒤 상층액을 제거하고 3% 우혈청이 첨가된 DMEM 로 교체하였다. 450nm 청색광을 SARS-CoV2 가 감염된 Vero E6 세포에 1.6, 5, 10J/cm2 조사 후, 24시간째 세포와 세포 배양액을 회수하여 AccuPrep Viral RNA Extraction Kit (K-3033, Bioneer, Korea)을 사용하여 제조사의 제공된 방법에 따라 바이러스 RNA를 추출한 뒤 RT-PCR로 확인하였다. 추출한 RNA를 정량하여 희석한 뒤 Target specific primer를 사용하여 qRT-PCR을 수행하였다. 1 μg의 total RNA를 ReverTra AceTM qPCR RT kit의 매뉴얼에 따라 2 μl 5X RT buffer, 0.5 μl RT Enzyme Mix, 0.5 μl primer Mix, Nuclease-free water와 혼합하여 총 10 μl 부피로 37 ℃의 15 분, 98 ℃의 5 분 반응 후 4 ℃에 반응하여 cDNA 합성한다. 이 후 합성된 10 ng cDNA, 2 μl forward primer ( 5’CAATGGTTTAACAGGCACAGG 3’), 2 μl reverse primer (5’CTCAAGTGTCTGTGGATCACG 3’), 10 μl 2X GreenStar Master Mix 를 넣어 총 20 μl 부피로 95 ℃의 3분 후 95 ℃의 10초, 56 ℃의 20초, 72 ℃의 30초로 40 cycle PCR을 수행하였다. BLED 조사 후 24시간 뒤 세포 생존율은 대조군에 비해 변화되지 않았으나, 세포 내 SARS-CoV2 바이러스 발현은 상대적으로 감소됨을 확인하였다 (도 5). 또한 청색광을 1.7, 5, 10 J/cm2 에너지량으로 조사하고 24시간, 72시간 후 세포내 혹은 세포 배양액에서 RNA를 AccuPrep Viral RNA Extraction Kit (K-3033, Bioneer, Korea)을 사용하여 제조사의 제공된 방법에 따라 분리하였다. 세포 상등액에서 vRNA 추출 후 RT-PCR 결과, 대조군에 비해 quantification cycle (Cq) 값이 증가함에 따라 바이러스 RNA 양은 청색광 에너지 5J 과 10J 조사 후 통계적으로 유의하게 감소하였다 (도 6). 상기의 결과에서 청색광을 10J로 조사한 후 세포내 SARS-CoV2 바이러스 발현이 감소됨에 따라, 청색광 10 J을 1회 혹은 2회 조사 후 24시간 뒤 세포 상등액을 회수하여 vRNA copy 수를 측정하였다. 청색광 1회 혹은 2회 조사 후 vRNA copy 수는 대조군에 비해 통계적으로 유의하게 약 50% 이상 감소하였다 (도 7). SARS-CoV2 was confirmed by the Korea Centers for Disease Control and Prevention, and all experimental procedures were conducted in a domestic biosafety level 3 research facility (BL3). After culturing the monkey kidney cell line Vero E6 cells in DMEM medium supplemented with 10% fetal bovine serum, when 70-80% confluence is achieved, SARS-CoV2 is infected and periodically cytopathic effect (CPE) was observed. When cell mutations were clearly observed, the culture medium was collected, and the supernatant was filtered with a 0.45 μm filter using a centrifuge, then divided and stored at -80 °C. For quantification of SARS-CoV2 virus, the infectious virus titer was expressed as 50% tissue culture infectious dose (TCID50). SARS-CoV2 virus and an inoculation to cultured at a rear 6 well plate (1 X 10 6 cells / well) diluted in DMEM media supplemented with bovine serum cells, put DMEM as a negative control continuously and cultured in a CO 2 incubator Cell mutations were observed under a microscope. After culturing Vero E6 cells in a 6 well plate (1 X 10 6 cells/well), the culture medium was removed the next day and washed once with PBS. After infecting 500 μl of SARS-CoV2 virus at 0.01 MOI for 10 minutes, the supernatant was removed and replaced with DMEM supplemented with 3% bovine serum. After irradiating 450nm blue light to the SARS-CoV2-infected Vero E6 cells at 1.6, 5, and 10J/cm2, the cells and cell culture were recovered 24 hours later, using the AccuPrep Viral RNA Extraction Kit (K-3033, Bioneer, Korea). Virus RNA was extracted according to the provided method and confirmed by RT-PCR. After quantifying and diluting the extracted RNA, qRT-PCR was performed using a target specific primer. 1 μg of total RNA was mixed with 2 μl 5X RT buffer, 0.5 μl RT Enzyme Mix, 0.5 μl primer Mix, and Nuclease-free water according to the manual of the ReverTra Ace TM qPCR RT kit in a total volume of 10 μl at 37 °C for 15 minutes. , after 5 min reaction at 98 °C, react at 4 °C to synthesize cDNA. Then, add the synthesized 10 ng cDNA, 2 μl forward primer ( 5'CAATGGTTTAACAGGCACAGG 3'), 2 μl reverse primer (5'CTCAAGTGTCTGTGGATCACG 3'), and 10 μl 2X GreenStar Master Mix to a total volume of 20 μl at 95 °C for 3 minutes. Afterwards, 40 cycle PCR was performed at 95 °C for 10 seconds, 56 °C for 20 seconds, and 72 °C for 30 seconds. 24 hours after BLED irradiation, the cell viability did not change compared to the control, but it was confirmed that the intracellular SARS-CoV2 virus expression was relatively reduced ( FIG. 5 ). In addition, after irradiating blue light with energy of 1.7, 5, 10 J/cm2, and after 24 hours or 72 hours, RNA from intracellular or cell culture medium was extracted using the AccuPrep Viral RNA Extraction Kit (K-3033, Bioneer, Korea) provided by the manufacturer. Separated according to the method. As a result of RT-PCR after vRNA extraction from the cell supernatant, as the quantification cycle (Cq) value increased compared to the control, the amount of viral RNA was statistically significantly decreased after irradiation with blue light energy of 5J and 10J (Fig. 6). According to the above results, as the intracellular SARS-CoV2 virus expression decreased after irradiating blue light with 10J, the cell supernatant was recovered 24 hours after irradiating 10J of blue light once or twice to measure the number of vRNA copies. After irradiation with blue light once or twice, the number of vRNA copies was statistically significantly reduced by about 50% or more compared to the control group (FIG. 7).
실시예 4. 청색광의 조사에 따른 후 세포 내 바이러스 억제 효과Example 4. Intracellular Virus Inhibitory Effect Following Irradiation of Blue Light
Plaque reduction assay는 바이러스 감염을 정량화하여 볼 수 있는 일반적인 방법으로, BLED 조사 후 vRNA copy수가 감소함에 따라 세포 내 바이러스 억제 효과가 있는지 확인하기 위해 실행하였다. Plaque는 바이러스 감염에 의해 손상된 숙주 세포가 주위의 정상세포와 구별되게 나타나는데, vital dye로 염색 하였을 때 plaque 주변의 정상세포는 염색되나 SARS-CoV-2 바이러스에 감염된 병변 세포들은 색소가 유리되어 무색으로 흰 반점을 띤다. 한 개의 감염성 있는 바이러스 입자는 하나의 plaque를 형성한다는 이론으로 SARS-CoV-2 바이러스 감염 후, overlay media를 이용하여 plaque를 측정하였다. Overlay media는 영양을 공급함과 동시에 최초의 감염 세포에서 증식된 바이러스가 배지를 통하여 퍼져나가 주위 세포를 2차 감염시키는 것을 방지하는 역할을 하도록 gel 상태의 배지를 만든다. Vero E6 세포를 24 well plate (3 X 105 cells/well)에 배양한 후 다음날 배양액을 제거한 뒤 PBS로 2회 세척하였다. 500 μl의 SARS-CoV2 바이러스를 50 pfu/well로 10분간 감염시킨 뒤 상층액을 제거하고 우혈청 없는 DMEM 로 교체하였다. 450nm 청색광을 SARS-CoV2 가 감염된 Vero E6 세포에 0.6% agarose가 포함된 DMEM을 1.5 ml 첨가한 후 10J/cm2 조사하여, 72시간째 crystal violet으로 염색하여 무색의 plaque 수를 계산하는 plaque reduction assay를 실행하였다. 그 결과, SARS-CoV-2 바이러스에 감염된 세포에서 BLED 10J 조사 후 72시간 뒤 생성된 plaque는 대조군에 비해 80% 이상 감소하였다 (도 8). 상기와 같이 청색광 에너지 준위는 SARS-CoV2 바이러스에 감염된 세포에 있어서 세포 사멸에는 영향을 주지 않고 바이러스 불활성화에 영향을 줄 수 있다는 것을 확인하였다. Plaque reduction assay is a general method for quantifying and viewing viral infection, and was performed to check whether the intracellular virus suppression effect was observed as the number of vRNA copies decreased after BLED irradiation. Plaque shows host cells damaged by virus infection distinct from surrounding normal cells. When stained with vital dye, normal cells around the plaque are stained, but lesion cells infected with SARS-CoV-2 virus are colorless because the pigment is released. have white spots According to the theory that one infectious virus particle forms a single plaque, plaque was measured using overlay media after SARS-CoV-2 virus infection. Overlay media supplies nutrients and makes a gel-like medium to prevent the virus propagated in the first infected cells from spreading through the medium and infecting surrounding cells. After culturing Vero E6 cells in a 24 well plate (3 X 10 5 cells/well), the culture medium was removed the next day and washed twice with PBS. After infecting 500 μl of SARS-CoV2 virus at 50 pfu/well for 10 minutes, the supernatant was removed and replaced with DMEM without bovine serum. After adding 1.5 ml of DMEM containing 0.6% agarose to SARS-CoV2-infected Vero E6 cells with 450 nm blue light, and irradiating 10 J/cm2, a plaque reduction assay was performed to count the number of colorless plaques by staining with crystal violet for 72 hours. was executed. As a result, the plaques generated 72 hours after irradiation with BLED 10J in cells infected with SARS-CoV-2 virus were reduced by more than 80% compared to the control group (FIG. 8). As described above, it was confirmed that the blue light energy level can affect virus inactivation without affecting apoptosis in cells infected with SARS-CoV2 virus.
실시예 5. 청색광 조사 후 H1N1 항바이러스 효과Example 5. H1N1 antiviral effect after blue light irradiation
5.1 세포의 배양 및 청색광의 조사 방법5.1 Cell culture and blue light irradiation method
Mardin-Dardy canine kidney (MDCK) 세포주는 penicillin (5 units/mL) / streptomysin (5 μg/mL)과 10% 우태아 혈청 (fetal bovine serum: Gibco, USA)이 첨가된 DMEM (Dulbecco's Modified Eagle's Medium: Gibco, USA)을 기본배지로 하여 5% CO2 농도를 유지하며 CO2 incubator (Thermo Forma, USA)에서 37℃ 조건으로 배양하였다. 숙주세포인 MDCK 세포를 12-well plate에서 LED-blue light노출에 대한 H1N1의 항바이러스 활성 시험을 2회 수행하였다. 12-well plate의 경우, 각 well에 2.0×105 cells/mL 농도의 세포현탁액 1 mL씩 주입하여 24시간 동안 5% CO2 농도를 유지하며 CO2 incubator (Thermo Forma, USA)에서 37℃ 조건으로 배양하였다. 배양된 세포는 PBS로 1회 세척하고, 인플루엔자 A의 아형인 H1N1 (ATCC, VR-1496)을 moi=0.1에 해당하는 농도로 희석하여 세포 표면에 접종하여 30분 간격으로 간헐적으로 흔들어주면서 37℃, 5% CO2 배양기에서 2시간 배양하여 바이러스를 감염시켰다. 2시간 후 바이러스액을 모두 제거하고 serum free medium으로 2회 세척한 후 virus growth media (DMEM0 + 1μg/mL TPCK-treated trypsin + 1% P/S) 1mL 넣고 35℃, 5% CO2 조건에서 배양하였다. 이 때, Blue-LED 발생장치를 CO2 배양기에 함께 넣어주어 Blue-LED 시험구는 0 hpi (post-infection hour)와 24 hpi 시간대에서 각각 30분간 노출시켰다. 음성대조구는 알루미늄 호일을 이용하여 blue light를 차단한 후 시험구와 동일한 조건하에서 수행하였다.The Mardin-Dardy canine kidney (MDCK) cell line is DMEM (Dulbecco's Modified Eagle's Medium) supplemented with penicillin (5 units/mL) / streptomysin (5 μg/mL) and 10% fetal bovine serum (Gibco, USA): Gibco, USA) was used as a basal medium, maintaining a 5% CO2 concentration, and cultured at 37°C in a CO2 incubator (Thermo Forma, USA). The antiviral activity test of H1N1 against LED-blue light exposure was performed twice in a 12-well plate in MDCK cells, which are host cells. In the case of a 12-well plate, 1 mL of a cell suspension with a concentration of 2.0×10 5 cells/mL is injected into each well to maintain a 5% CO 2 concentration for 24 hours and in a CO 2 incubator (Thermo Forma, USA) at 37°C. incubated with The cultured cells are washed once with PBS, diluted with influenza A subtype H1N1 (ATCC, VR-1496) to a concentration corresponding to moi=0.1, and inoculated on the cell surface at 37°C with intermittent shaking at 30-minute intervals , 5% CO 2 Incubated for 2 hours in an incubator to infect the virus. After 2 hours, all the virus solution was removed, washed twice with serum free medium, 1 mL of virus growth media (DMEM 0 + 1 μg/mL TPCK-treated trypsin + 1% P/S) was added, and at 35 ° C, 5% CO 2 condition cultured. At this time, the Blue-LED generator was put together in the CO 2 incubator, and the Blue-LED test group was exposed for 30 minutes at 0 hpi (post-infection hour) and 24 hpi time zones, respectively. The negative control was performed under the same conditions as the test group after blocking blue light using aluminum foil.
5.2 RT-qPCR 분석을 통한 바이러스 역가 측정 5.2 Determination of Virus Titer by RT-qPCR Analysis
72시간 동안 바이러스를 증식시킨 후 상층액을 수거하고, 300 μL의 상층액으로부터 RNiso Plus (Takara, Cat# 9109)를 이용하여 제조사의 프로토콜 방법대로 total RNA를 추출하였다. 추출된 RNA는 MMLV reverse transcriptase cDNA Synthesis Kit (Beamsbio, Cat.# 3201)를 이용하여 random 6-mer (Takara) 프라이머, dNTPs, RNase inhibitor를 포함한 20 μL 반응액으로 37℃에서 60분 동안 제조회사의 방법에 따라 cDNA를 합성하였다. 합성된 H1N1의 cDNA를 주형 2μL와 인플루엔자 A형 바이러스의 PA 유전자의 특이적인 primer set (H1N1_PA_For: 5`-CGG TCC AAA TTC CTG CTG-3` 및 H1N1_PA_Rev: 5`-CAT TGG GTT CCT TCC ATC CA-3`)가 포함된 20 μL 반응액을 TB green® Premix Ex TaqTM (Takara, Cat# RR420)를 이용하여 제조하였다. 반응조건은 95℃ 30초 (1회), 95℃ 5초, 55℃ 10초, 72℃ 20초 (45회), 이며 Thermal Cycler Dice® Real Time System III (Takara, TP950)이용하여 바이러스 역가를 측정하였다. 바이러스 정량을 위하여 H1N1을 단계 희석하여 plaque assay와 RT-qPCR 분석을 각각 수행하여 얻은 Ct 값을 PFU/mL 단위로 환산하여 표준곡선을 작성하였다. 단계희석하여 얻은 Ct 값을 플로팅하여 작성한 표준 곡선의 기울기는 -4.065, y 절편은 45.65 및 R2는 0.991이었다. H1N1의 검출한계는 Ct값 35으로 설정하였다. qPCR에서 얻어진 Ct값을 표준곡선에 대입하여 non-LED와 blue-LED에서 H1N1 바이러스 농도를 산출하여 비교한 결과, blue-LED에서 H1N1 농도는 12-well plate 수준에서 3.38배 감소하는 효과가 있음을 확인하였다 (도 9).After propagating the virus for 72 hours, the supernatant was collected, and total RNA was extracted from 300 μL of the supernatant according to the manufacturer's protocol using RNiso Plus (Takara, Cat# 9109). Extracted RNA was prepared by using the MMLV reverse transcriptase cDNA Synthesis Kit (Beamsbio, Cat. # 3201) in 20 μL reaction solution containing random 6-mer (Takara) primer, dNTPs, and RNase inhibitor at 37℃ for 60 minutes. cDNA was synthesized according to the method. 2μL of synthesized H1N1 cDNA template and primer set specific for influenza A virus PA gene (H1N1_PA_For: 5`-CGG TCC AAA TTC CTG CTG-3` and H1N1_PA_Rev: 5`-CAT TGG GTT CCT TCC ATC CA- 3`) was prepared using 20 μL of a reaction solution containing TB green ® Premix Ex Taq TM (Takara, Cat# RR420). Reaction conditions were 95°C 30 seconds (1 time), 95°C 5 seconds, 55°C 10 seconds, 72°C 20 seconds (45 times), and the virus titer was measured using Thermal Cycler Dice ® Real Time System III (Takara, TP950). measured. For virus quantification, a standard curve was prepared by converting the Ct values obtained by performing plaque assay and RT-qPCR analysis by serially diluting H1N1 into PFU/mL units. The slope of the standard curve prepared by plotting the Ct values obtained by step-dilution was -4.065, the y-intercept was 45.65, and R 2 was 0.991. The detection limit of H1N1 was set to a Ct value of 35. By substituting the Ct value obtained from qPCR into the standard curve, the H1N1 virus concentration was calculated and compared in non-LED and blue-LED. As a result, the H1N1 concentration in blue-LED was shown to have the effect of reducing 3.38 times at the level of the 12-well plate. was confirmed (FIG. 9).
Claims (9)
- 청색 영역 내에 있는 LED 또는 레이저 광원을 포함하는, 바이러스를 치료 또는 예방하는 광선 치료용 장치.A device for light therapy to treat or prevent a virus, comprising an LED or laser light source within the blue region.
- 제 1항에 있어서, 상기 청색 영역은 400nm 내지 500nm인, 바이러스를 치료 또는 예방하는 광선 치료용 장치.According to claim 1, wherein the blue region is 400nm to 500nm, a light therapy device for treating or preventing a virus.
- 제 1항에 있어서, 상기 LED 또는 레이저 광원은 1.0 내지 20 J/cm2 의 조사량으로 조사되는, 바이러스를 치료 또는 예방하는 광선 치료용 장치.According to claim 1, wherein the LED or laser light source is irradiated with an irradiation dose of 1.0 to 20 J/cm 2 , A device for treating or preventing viruses.
- 제 1항에 있어서, 상기 바이러스는 호흡기 질환 원인 바이러스인, 바이러스를 치료 또는 예방하는 광선 치료용 장치.According to claim 1, wherein the virus is a respiratory disease-causing virus, a device for phototherapy for treating or preventing a virus.
- 제 1항의 광선 치료용 장치를 인간을 제외한 동물에게 조사하는 단계;를 포함하는 바이러스를 치료하는 방법. A method of treating a virus comprising a; irradiating the device for phototherapy of claim 1 to animals other than humans.
- 제 5항에 있어서, 상기 광선 치료용 장치를 바이러스에 감염된 인간을 제외한 동물의 전신, 폐, 기도, 코 점막 및 입 점막으로 이루어진 군으로부터 선택되는 어느 하나의 부위에 조사하는, 바이러스를 치료하는 방법. The method of claim 5, wherein the light therapy device is irradiated to any one site selected from the group consisting of the whole body, lungs, airways, nasal mucosa, and oral mucosa of an animal other than a human infected with a virus. .
- 제 5항에 있어서, 상기 광선 치료용 장치의 청색 영역은 400nm 내지 500nm인, 바이러스를 치료하는 방법. The method of claim 5 , wherein the blue region of the device for phototherapy is 400 nm to 500 nm.
- 제 5항에 있어서, 상기 광선 치료용 장치의 LED 또는 레이저 광원은 1.0 내지 20 J/cm2 의 조사량으로 조사되는, 바이러스를 치료하는 방법.The method of claim 5, wherein the LED or laser light source of the device for phototherapy is irradiated with an irradiation dose of 1.0 to 20 J/cm 2 .
- 제 5항에 있어서, 상기 바이러스는 호흡기 질환 원인 바이러스인, 바이러스를 치료하는 방법. The method according to claim 5, wherein the virus is a virus causing respiratory diseases.
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| KR20200065064 | 2020-05-29 | ||
| KR10-2020-0065064 | 2020-05-29 | ||
| KR1020200171948A KR20210147847A (en) | 2020-05-29 | 2020-12-10 | Method for prevention and treatment for Virus using blue light |
| KR10-2020-0171948 | 2020-12-10 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1457893A (en) * | 2003-04-25 | 2003-11-26 | 周明非 | Atomizing type upper respiratory track virus inactivation instrument |
| KR101668561B1 (en) * | 2015-07-13 | 2016-10-21 | 원광대학교산학협력단 | Composition for photodynamic therapy of cancer diseases using gene expressing green fluorescent protein and rose bengal and method for photodynamic therapy using thereof |
| WO2017031367A1 (en) * | 2015-08-18 | 2017-02-23 | Aspyrian Therapeutics, Inc. | Compositions, combinations and related methods for photoimmunotherapy |
| US20170281966A1 (en) * | 2016-04-01 | 2017-10-05 | Mohamed A Basiony | Device to Kill Micro-Organisms Inside the Respiratory Tract |
| KR20180067499A (en) * | 2015-07-14 | 2018-06-20 | 비타빔 엘티디. | Methods and apparatus for sanitary, disinfection and sterilization |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1457893A (en) * | 2003-04-25 | 2003-11-26 | 周明非 | Atomizing type upper respiratory track virus inactivation instrument |
| KR101668561B1 (en) * | 2015-07-13 | 2016-10-21 | 원광대학교산학협력단 | Composition for photodynamic therapy of cancer diseases using gene expressing green fluorescent protein and rose bengal and method for photodynamic therapy using thereof |
| KR20180067499A (en) * | 2015-07-14 | 2018-06-20 | 비타빔 엘티디. | Methods and apparatus for sanitary, disinfection and sterilization |
| WO2017031367A1 (en) * | 2015-08-18 | 2017-02-23 | Aspyrian Therapeutics, Inc. | Compositions, combinations and related methods for photoimmunotherapy |
| US20170281966A1 (en) * | 2016-04-01 | 2017-10-05 | Mohamed A Basiony | Device to Kill Micro-Organisms Inside the Respiratory Tract |
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