KR102494223B1 - Therapeutic method and system of Bio-Plasma in malignant brain tumor glioblastoma - Google Patents
Therapeutic method and system of Bio-Plasma in malignant brain tumor glioblastoma Download PDFInfo
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- A—HUMAN NECESSITIES
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- H—ELECTRICITY
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Abstract
본 발명은 뇌정위적 방법으로 교모세포종 세포주인 U87-effluc를 이식하고 일주일 뒤 plasma를 direct하게 마우스 뇌에 처리하고 in vivo live imaging을 통해 종양이 증식하는 정도를 비교 분석하고, 추가적으로 마우스의 생존율을 확인하여 치료 효과 및 안전성을 검증한다. In the present invention, U87-effluc, a glioblastoma cell line, is implanted in a brain stereotaxic method, and a week later, plasma is directly treated in the mouse brain, and the degree of tumor proliferation is compared and analyzed through in vivo live imaging, and additionally, the survival rate of the mouse is confirmed. to verify the therapeutic effect and safety.
Description
본 발명은 악성 뇌종양 교모세포종의 치료에 관한 것으로, 좀 더 상세하게는 바이오 플라즈마를 이용한 치료 방법 및 장치에 관한 것이다. The present invention relates to the treatment of malignant brain tumor glioblastoma, and more particularly, to a treatment method and apparatus using bio-plasma.
교모세포종은 성인에게 나타나는 원발성 뇌종양 중 가장 흔한 것으로 악성도가 가장 높다. 일반 종양의 경우 5년 생존율이 평균 53.4%, 일반 뇌종양은 60%인데 반하여 교모세포종은 약 5%로 진단 후 평균 수명이 12~15개월로서 예후가 불량하고 질병의 진행이 매우 빠르며 (국가암정보센터, 2012) 현재 표준 치료인 수술, 방사선과 테모달(Temozolomide)의 병용 치료에도 불구하고 평균 생존기간이 12개월 미만으로 가장 치료가 어려운 뇌종양으로 알려져 있다. 뇌종양의 경우 발생율이 다른 종양에 비해 적지만 발생 부위의 기능학적 특성에 의해 언어, 인지 및 신체 능력의 저하로 환자의 삶의 질이 떨어지고 이러한 문제는 환자 개인의 넘어 범국가적인 부담으로 이어지고 있다. 따라서 교모세포종에 대한 새로운 치료법의 제시 또는 항암 효과를 높이는 새로운 치료 방향성의 제시가 시급한 실정이다.Glioblastoma is the most common primary brain tumor in adults and has the highest malignancy. In the case of general tumors, the average 5-year survival rate is 53.4%, and general brain tumors are 60%, whereas glioblastoma is about 5%. Center, 2012) Despite the current standard treatment of surgery, radiation, and combination treatment with Temodal (Temozolomide), it is known as the most difficult brain tumor with an average survival time of less than 12 months. In the case of brain tumor, the incidence rate is lower than other tumors, but due to the functional characteristics of the site of occurrence, the quality of life of patients is reduced due to the deterioration of language, cognition, and physical abilities. Therefore, it is urgent to present a new treatment method for glioblastoma or a new treatment direction that enhances the anticancer effect.
수술과 약물에 의한 치료 외에 항체에 의한 치료가 나와있고, 공개특허 10-2016-0097578호는 교류 자기장에 의한 뇌종양 세포의 선별적 억제에 대해 기재한다. 그러나 효과면에서 비교적 소극적인 전이억제 수준으로, 적극적인 치료 효과에는 미치지 못하는 것으로 보인다. In addition to treatment by surgery and drugs, treatment by antibodies is shown, and Publication Patent Publication No. 10-2016-0097578 describes selective inhibition of brain tumor cells by alternating magnetic fields. However, in terms of effectiveness, it seems to be a relatively passive level of metastasis inhibition, which does not reach the active therapeutic effect.
한편, 플라즈마(Plasma)란 고체, 액체, 기체 단계에서 에너지를 더 받게 되면 초고온에서 음전하를 가진 전자와 양전하를 띤 이온으로 분리된 상태를 말한다. 원시대기 실험에서 보는 바와 같이 플라즈마 방전이 일어나면 액체 안에서 아미노산이 생성되고 이것으로부터 단백질이 만들어지는 생명현상의 일부가 진행되므로 이를 생명의 원천이라고 말한다. 이러한 플라즈마는 진공 챔버를 사용하여 고온/고압 상태에서 방전되지만 상온/상압상태의 플라즈마를 발생하는 기술이 개발되면서 이를 이용한 생물학과의 융합학문이 탄생하였고 이를 바이오 플라즈마라 명명한다. 플라즈마에서 발생된 유리기 (free radicals)등의 물질에 의해 암세포와 미생물의 사멸을 촉진하며 특히 정상세포에는 사멸효과가 거의 없기 때문에 부작용없는 치료법으로서 각광받기 시작했고 이를 제4의 치료법이라 부르기 시작했다.On the other hand, plasma refers to a state in which negatively charged electrons and positively charged ions are separated at ultra-high temperatures when more energy is received in the solid, liquid, and gas phases. As seen in the primitive atmosphere experiment, when plasma discharge occurs, amino acids are created in the liquid, and a part of the life phenomenon in which proteins are made from this proceeds, so this is called the source of life. Such plasma is discharged at high temperature/high pressure using a vacuum chamber, but as a technology for generating plasma at room temperature/normal pressure was developed, a convergence study with biology was born using this technology, which is called bio-plasma. It promotes the death of cancer cells and microorganisms by substances such as free radicals generated from plasma, and since there is almost no killing effect on normal cells, it began to be in the limelight as a treatment without side effects, and it began to be called the fourth treatment.
본 발명은 교모세포종 orthotopic xenograft 마우스 동물 모델을 이용하여 바이오-플라즈마 처리 효과를 확인하고, 그에 따른 치료방안을 제공하고자 한다. The present invention is to confirm the bio-plasma treatment effect using a glioblastoma orthotopic xenograft mouse animal model, and to provide a treatment plan accordingly.
상기 목적에 따라 본 발명은 뇌정위적 방법으로 교모세포종 세포주인 U87-effluc를 이식하고 일주일 뒤 plasma를 direct하게 마우스 뇌에 처리하고 in vivo live imaging을 통해 종양이 증식하는 정도를 비교 분석하고, 추가적으로 마우스의 생존율을 확인하여 치료 효과 및 안전성을 검증한다. According to the above object, the present invention transplants U87-effluc, a glioblastoma cell line, by a brain stereotactic method, and one week later, plasma is directly treated in the mouse brain, and the degree of tumor proliferation is compared and analyzed through in vivo live imaging, and additionally mouse The survival rate is checked to verify the treatment effect and safety.
본 발명에 따르면, 질소 가스를 방전가스로 포함하는 플라즈마를 뇌종양 부위에 직접 처리함으로써, 정상 세포에 대한 영향없이 뇌종양 세포만을 선별적으로사멸시켜 뇌종양을 치료할 수 있다.According to the present invention, it is possible to treat a brain tumor by selectively killing only brain tumor cells without affecting normal cells by directly treating the brain tumor with plasma containing nitrogen gas as a discharge gas.
플라즈마 처리는 외과적인 뇌종양 제거 수술과 병행될 수도 있고, 뇌종양 발생 부위의 스킨에 필요한 정도의 절개 구멍을 형성한 후, 플라즈마를 주입하여 실시될 수 있어, 비교적 간단한 시술의 치료법이 만들어질 수 있다. Plasma treatment may be combined with surgical brain tumor removal, or it may be performed by injecting plasma after forming a necessary degree of incision in the skin of the area where the brain tumor occurs, so a relatively simple treatment can be made.
도 1은 본 발명의 뇌종양 치료에 적용될 수 있는 플라즈마 발생장치의 일 실시예로 플라즈마 제트 장치를 보여준다.
도 2는 플라즈마 제트 장치에 인가되는 방전 전압과 전류를 보여주는 그래프이다.
도 3은 플라즈마 제트 장치에서 발생된 플라즈마 활성종을 분석한 분광 스펙트럼이다.
도 4는 플라즈마 제트로 세포를 직접 처리하는 실험예를 도시한다.
도 5는 교모세포종 세포에 대한 바이오-플라즈마 처리로 인한 세포사멸 증가 결과를 보여준다.
도 6은 교모세포종 세포에 대한 바이오-플라즈마 처리에 따른 신호전달 경로를 보여준다.
도 7 및 도 8은 바이오-플라즈마의 처리된 세포의 생체 내 종양 성장 억제 능력을 보여준다.
도 9는 바이오-플라즈마 직접 처리에 의한 뇌종양 치료 효과를 영상으로 보여준다. 1 shows a plasma jet device as an embodiment of a plasma generating device that can be applied to brain tumor treatment according to the present invention.
2 is a graph showing discharge voltage and current applied to the plasma jet device.
3 is a spectral spectrum of plasma active species generated in the plasma jet device.
4 shows an experimental example of directly treating cells with a plasma jet.
Figure 5 shows the result of increasing apoptosis due to bio-plasma treatment for glioblastoma cells.
Figure 6 shows the signal transduction pathway according to the bio-plasma treatment for glioblastoma cells.
7 and 8 show the ability of bio-plasma to inhibit tumor growth in vivo of treated cells.
9 shows the brain tumor treatment effect by bio-plasma direct treatment as an image.
이하, 첨부도면을 참조하여 본 발명의 바람직한 실시예에 대해 상세히 설명한다.Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
실험 방법 및 재료Experimental methods and materials
세포 배양cell culture
인간 뇌종양 교모세포종 세포주 U87은 ATCC사를 통해 구입하였고 동물실험에서 영상촬영을 위한 U87-effluc 세포는 이전 논문에서 사용한 세포를 사용하였다. 정상 astrocyte 세포주는 Lonza를 통해 구입해서 사용하였다. 모든 세포주는 Dulbecco's Modified Eagle Medium (DMEM; Life Technologies, Carlsbad, CA, USA)에 10% FBS (Gibco, Grand Island, NY, USA) and 1% antibiotic (penicillin/streptomycin; Gibco) 를 첨가한 배지와 5% CO2, 37℃조건에서 배양되었다. Human brain tumor glioblastoma cell line U87 was purchased from ATCC, and U87-effluc cells for imaging in animal experiments were used in previous papers. Normal astrocyte cell lines were purchased and used through Lonza. All cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% antibiotic (penicillin/streptomycin; Gibco) and 5 % CO 2 , cultured at 37°C.
바이오-플라즈마 처리 Bio-plasma treatment
바이오 플라즈마 소스로는 도 1과 같은 soft jet를 이용하였다. A soft jet as shown in FIG. 1 was used as a bioplasma source.
도 1의 고전압 전극은 속이 빈 금속 바늘로 이루어져 있으며, 이는 질소가스를 운반하는 동시에 고전압 전극의 역할을 한다. 석영튜브는 접지된 금속 경통 내부에 설치되며, 가스 운반과 동시에 플라즈마 방전을 위한 유전체 역할을 한다. The high voltage electrode of FIG. 1 is composed of a hollow metal needle, which serves as a high voltage electrode while delivering nitrogen gas. The quartz tube is installed inside the grounded metal barrel and serves as a dielectric for gas transport and plasma discharge.
방전가스로는 2 lpm의 질소가스 (99.999%)를 이용하며, 도 1의 노즐부(테이퍼링 된 단부를 뜻한다)로부터 약 5 mm의 길이를 갖는 플라즈마 제트가 형성된다(도 4 참조). Nitrogen gas (99.999%) of 2 lpm is used as the discharge gas, and a plasma jet having a length of about 5 mm is formed from the nozzle part (meaning the tapered end) of FIG. 1 (see FIG. 4).
플라즈마 방전은 2 kV의 전압과 30 kHz의 주파수를 가진 인버터에 의해서 동작시켰으며, 연속 방전에 따른 온도상승을 피하기 위해 도 2(a)와 같은 동작비율을 가진다. 오프 듀티 시간이 온 듀티 시간에 비해 5 내지 7배 더 길게 할 수 있다. Plasma discharge was operated by an inverter with a voltage of 2 kV and a frequency of 30 kHz, and had an operating ratio as shown in FIG. 2 (a) to avoid temperature rise due to continuous discharge. The off-duty time can be 5 to 7 times longer than the on-duty time.
플라즈마 방전시의 전류, 전압 그래프는 도 2(b)와 같으며, 1 kV의 전압에서 플라즈마가 일어나며. 이 때 약 200 mA 의 방전 전류를 갖는다.The graph of current and voltage during plasma discharge is shown in FIG. 2 (b), and plasma occurs at a voltage of 1 kV. At this time, it has a discharge current of about 200 mA.
분광분석을 통해, soft jet의 플라즈마내에 일산화질소(NO-r band; 200 - 300 nm)와 질소종(N2 second positive system; 300 - 400 nm, N2 first negative system; 400 - 420 nm, N2 first positive system; 500 - 800 nm)이 존재하는 것을 알 수 있다(도 3 참조).Through spectroscopic analysis, nitrogen monoxide (NO-r band; 200 - 300 nm) and nitrogen species (N 2 second positive system; 300 - 400 nm, N 2 first negative system; 400 - 420 nm, N 2 first positive system; 500 - 800 nm) can be seen (see FIG. 3).
바이오-플라즈마는 soft jet을 이용하여 FBS가 첨가되지 않은 DMEM 배지에 10, 30, 60 및 180초 처리 후 세포에 처리하였다. 대조군으로 바이오-플라즈마가 처리되지 않은 배지를 사용하였다.Bio-plasma was applied to cells after 10, 30, 60, and 180 seconds of treatment in DMEM medium without FBS using a soft jet. As a control, a medium not treated with bio-plasma was used.
세포생존율 분석Cell viability analysis
세포생존율은 Alamar Blue (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA)를 이용하여 제조사 프로토콜에 따라 측정하였다. Alamar blue 시약 처리 3시간 후 플레이트 리더기 (Biotek, Winooski, VT, USA)를 이용하여 570-560 nm (excitation 파장) 과 590 nm (emission 파장) 에서 측정하였고 모든 실험은 독립적으로 3번 이상 반복하고 분석하였다.Cell viability was measured using Alamar Blue (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. After 3 hours of Alamar blue reagent treatment, it was measured at 570-560 nm (excitation wavelength) and 590 nm (emission wavelength) using a plate reader (Biotek, Winooski, VT, USA), and all experiments were independently repeated at least three times and analyzed did
활성산소 분석Active oxygen analysis
활성산소는 ROS 및 RNS 검출 키드 (Invitrogen)을 사용하였고 제조사의 프로토콜에 따라 실험을 수행하였다. 간략하게, 2′7′-diacetate (H2DCF DA; Invitrogen)가 세포를 투과하여 세포내 ROS에 의해 산화되어 밝은 녹색 형광을 방출하는 것을 공초점 현미경을 통해 이미지를 확보하였다. ROS and RNS detection kits (Invitrogen) were used, and the experiment was performed according to the manufacturer's protocol. Briefly, 2′7′-diacetate (H2DCF DA; Invitrogen) penetrated cells and was oxidized by intracellular ROS to emit bright green fluorescence. Images were obtained using a confocal microscope.
세포사멸 분석Apoptosis assay
Annexin V-FITC and PI staining (BD, San Jose, CA, USA) 검출 키트를 이용하였고 제조사의 프로토콜에 따라 실험을 수행하였다. 세포를 수집하고 DPBS로 세척한 후 V-FITC and PI로 염색하고 유세포 분석법으로 분석하였다. Annexin V-FITC and PI staining (BD, San Jose, CA, USA) detection kit was used and the experiment was performed according to the manufacturer's protocol. Cells were collected, washed with DPBS, stained with V-FITC and PI and analyzed by flow cytometry.
신호전달 경로 분석Signal transduction pathway analysis
단백질은 RIPA buffer를 이용하여 분리하였고 이전 논문과 동일한 방법으로 웨스턴 블롯을 수행하였다. 일차 항체는 phosphor-p38 (p-p38), p-38, cleaved caspase3, survivin, cleave parp, b-actin을 사용하였고 2차 항체는 HRP (horseradish peroxidase)-conjugated를 사용하였다. 화학발광 (ChemiDocTM System; BIO-RAD) 방법을 통해 band 강도를 검출하고 image J를 통해 정량 분석하였다. Proteins were separated using RIPA buffer and Western blotting was performed in the same manner as in the previous paper. Phosphor-p38 (p-p38), p-38, cleaved caspase3, survivin, cleaved parp, and b-actin were used as primary antibodies, and horseradish peroxidase (HRP)-conjugated secondary antibodies were used. The band intensity was detected through the chemiluminescence (ChemiDocTM System; BIO-RAD) method and quantitatively analyzed through image J.
두개내 동소위 교모세포종 세포 이식 Intracranial orthotopic glioblastoma cell transplantation
모든 동물실험은 국제실험동물사용관리 가이드(NIH publication No. 80-23, revised in 1996)에 따라 서울대학교 동물실험윤리위원회에 승인 [IACUC number:]을 받았다. 7주령 암컷 BALB/c-nude 쥐 (JungAng Lab Animal, Seoul, South Korea)를 30mg/kg Zoletil (Virbac, Carros, France) and 10mg/kg xylazine (Bayer, Leverkusen, Germany)를 복강 주사하여 마취하였다. 세포의 이식 전 U87-effluc 세포는 60초 처리하였고 처리하지 않은 세포를 대조군으로 사용하였다. 생물발광영상에 이전에 사용했던 방법과 동일하게 측정하였다. 바이오-플라즈마 처리 또는 처리하지 않은 (1.0 × 105) 세포를 3μl의 PBS 에 희석하여 stereotaxic flame 장비를 사용하여 뇌정위적 방법으로 26 두께의 해밀턴 주사기를 이용하여 1μl/min 속도로 이식하였다. 뇌정위적 좌표는 브레그마에서 뒤쪽으로 1mm 오른쪽으로 2mm이고 깊이는 듀라에서 3mm 로 하였다. All animal experiments were approved [IACUC number:] by the Animal Experimentation Ethics Committee of Seoul National University in accordance with the International Laboratory Animal Use Guidelines (NIH publication No. 80-23, revised in 1996). 7-week-old female BALB/c-nude rats (JungAng Lab Animal, Seoul, South Korea) were anesthetized with intraperitoneal injection of 30 mg/kg Zoletil (Virbac, Carros, France) and 10 mg/kg xylazine (Bayer, Leverkusen, Germany). U87-effluc cells were treated for 60 seconds before cell transplantation, and untreated cells were used as a control. It was measured in the same way as previously used for bioluminescence imaging. Bio-plasma treated or untreated (1.0 × 10 5 ) The cells were diluted in 3 μl of PBS and transplanted at a rate of 1 μl / min using a 26-thick Hamilton syringe in a brain stereotactic method using a stereotaxic flame equipment. Stereotactic coordinates were 1 mm posterior to bregma and 2 mm to the right, and the depth was 3 mm in dura.
마우스 두개내 바이오-플라즈마 직접 처리법 Mouse intracranial bio-plasma direct treatment method
두개내 동소위 교모세포종 U87-effluc 세포 이식 7일 후, 바이오-플라즈마를 마우스 뇌에 직접 처리한다. 마우스를 마취시키고 stereotaxic flame 장비를 사용하여 뇌를 고정 후 스킨을 절개하여 뇌를 노출시킨다. '두개내 동소위 교모세포종 세포 이식' 방법에서 언급된 뇌종양 세포를 이식 방법과 동일한 좌표에 (브레그마에서 뒤쪽으로 1mm 오른쪽으로 2mm이고 깊이는 듀라에서 3mm) 바이오-플라즈마를 도 4에서와 같이 처리한다. 7 days after intracranial orthotopic glioblastoma U87-effluc cell transplantation, bio-plasma was applied directly to the mouse brain. After anesthetizing the mouse and fixing the brain using a stereotaxic flame device, the skin is incised to expose the brain. The brain tumor cells mentioned in the 'intracranial orthotopic glioblastoma cell transplantation' method are treated with bio-plasma at the same coordinates as the transplantation method (1 mm posterior from bregma, 2 mm to the right, and 3 mm deep from dura) as shown in FIG. do.
생체 영상 기술과 생존율 분석 Vital Imaging Technology and Survival Analysis
뇌종양 크기의 변화를 측정하기 위해서 생물발광영상을 이용한 비침습적인 생체내 모니터링을 실시하였다 (각 그룹당 5마리). 동물실험은 종양 볼륨 크기 측정을 위한 실험과 동일하게 계획하였다. 쥐의 뇌는 CCD camera (Caliper Life Sciences)가 장착된 IVIS-100 imaging system (Xenogen Corporation, Alameda, CA)을 이용하여 세포 이식 2, 7, 14, 21, 28, 33일째 촬영하였다. 쥐들은 150mg/kg D-Luciferin (Caliper Life Sciences, Hopkinton, MA)을 복강 주사하였고 2% isoflurane (Piramal Healthcare, Bethlehem, PA,) 와 100% O2로 마취하였다. 영상은 3분에서 5분 생물발광신호를 측정하여 Living Image software (Xenogen Corporation)로 분석하였다. 생물발광영상은 관심영역에서의 발광강도를 산출하여 정량하였다. 모든 동물들은 생존율 35일의 종료시점을 두고 안락사 하였다.Non-invasive in vivo monitoring using bioluminescence imaging was performed to measure changes in brain tumor size (5 animals in each group). The animal experiment was planned in the same way as the experiment for measuring the tumor volume size. The brains of the rats were photographed on the 2nd, 7th, 14th, 21st, 28th, and 33rd days of cell transplantation using an IVIS-100 imaging system (Xenogen Corporation, Alameda, CA) equipped with a CCD camera (Caliper Life Sciences). Mice were intraperitoneally injected with 150 mg/kg D-Luciferin (Caliper Life Sciences, Hopkinton, MA) and anesthetized with 2% isoflurane (Piramal Healthcare, Bethlehem, PA,) and 100% O2. The image was analyzed by Living Image software (Xenogen Corporation) by measuring the bioluminescence signal from 3 to 5 minutes. Bioluminescence images were quantified by calculating the luminescence intensity in the region of interest. All animals were euthanized at the end of survival of 35 days.
종양 볼륨과 면역형광 분석Tumor volume and immunofluorescence analysis
쥐들은 조직학 분석을 위해 종양세포 이식 35일째 희생시켰다. 관류 후, 냉동 조직 절단을 이전에 보고한 바와 같이 수행하였다. 조직들은 종양크기 분석을 위해 헤마톡실린-에오신 염색하였다. 면역형광 분석시 사용한 1차 항체는 다음과 같다. Phosphor-p38 (p-p38), p-38, cleaved caspase3, survivin, cleave PARP 양성 세포들은 적어도 무작위적인 3군데를 형광현미경으로 관찰하고 counting하여 정량하였다. Mice were sacrificed on day 35 of tumor cell implantation for histological analysis. After perfusion, frozen tissue sectioning was performed as previously reported. Tissues were hematoxylin-eosin stained for tumor size analysis. Primary antibodies used in immunofluorescence analysis are as follows. Phosphor-p38 (p-p38), p-38, cleaved caspase3, survivin, and cleaved PARP-positive cells were observed at least three random locations under a fluorescence microscope and counted for quantification.
통계분석statistical analysis
모든 실험 결과들은 적어도 3번 이상의 각각의 독립된 실험으로부터 표준편차로 산출하거나 대조군의 백분율로 표현하였다. 통계분석은 two-tailed Student's t-test 또는 ANOVA 분석 방법을 이용하였다. 생존율 결과는 그래프패드 프리즘 5 소프트웨어 (GraphPad Software, San Diego, CA) 를 이용하여 Kaplan-Meier 생존그래프로 나타냈고 log-rank test로 분석하였다. 0.05 이하의 유의 확률(p value)을 통계학적으로 유의미한 것으로 고려하였다.All experimental results were calculated as standard deviations from at least three independent experiments or expressed as a percentage of control. Statistical analysis was performed using two-tailed Student's t-test or ANOVA analysis method. Survival results were expressed as Kaplan-Meier survival graphs using GraphPad Prism 5 software (GraphPad Software, San Diego, CA) and analyzed by log-rank test. A significance probability (p value) of 0.05 or less was considered statistically significant.
결과result
교모세포종 세포주에 바이오-플라즈마 처리에 의한 세포생존율 억제 및 활성산소 증가Inhibition of cell viability and increase of active oxygen by bio-plasma treatment of glioblastoma cell lines
Astrocyte에 바이오-플라즈마 처리시 세포 형태와 크기에 변화가 없었다. 반면에, U87 세포에 바이오-플라즈마 처리시 세포 모양 및 형태가 수축 및 둥근 모양이 관찰되었고 이는 처리 시간 의존적으로 확인되었다. 세포생존율 비교시 정상세포에는 180초 처리에도 전혀 세포 생존율에 변화가 없었던 반면 U87세포의 경우에는 180 초 처리시 세포 생존율이 급격히 감소하는 것을 확인할 수 있었다. 또한, U87 세포에 바이오-플라즈마 처리에 따른 ROS와 RNA 분석시 시간 의존적으로 증가하는 것을 관찰 할 수 있었다. When astrocytes were treated with bio-plasma, there was no change in cell shape or size. On the other hand, when U87 cells were treated with bio-plasma, shrinkage and roundness of cell shape and shape were observed, which was confirmed in a treatment time-dependent manner. When comparing cell viability, normal cells showed no change in cell viability even after 180 seconds of treatment, whereas in the case of U87 cells, it was confirmed that cell viability rapidly decreased after 180 seconds of treatment. In addition, it was observed that U87 cells were increased in a time-dependent manner when ROS and RNA were analyzed according to bio-plasma treatment.
교모세포종 세포에 바이오-플라즈마 처리에 의한 세포사멸 증가Increased apoptosis in glioblastoma cells by bio-plasma treatment
Astrocyte에 바이오-플라즈마 처리시 세포사멸이 거의 관찰되지 않았다. 반면에, U87 세포의 경우 60초와 180초 처리시 초기 apoptosis가 현저히 증가하는 것이 관찰되었고 후기 apoptosis도 증가하였다. 또한 세포주기 관찰시 180초 처리시 G2 arrest가 유도되는 것을 확인할 수 있었다.When astrocytes were treated with bio-plasma, almost no apoptosis was observed. On the other hand, in the case of U87 cells, a significant increase in early apoptosis was observed when treated with 60 seconds and 180 seconds, and late apoptosis also increased. In addition, when observing the cell cycle, it was confirmed that G2 arrest was induced when treated for 180 seconds.
교모세포종 세포에 바이오-플라즈마 처리에 의한 신호전달 경로Signal transduction pathway by bio-plasma treatment in glioblastoma cells
U87 세포에 바이오-플라즈마 처리시 p38의 인산화가 증가되며 이를 통해 cleaved caspase-3가 증가, survivin은 감소, cleaved PARP는 감소되는 것이 명확히 관찰되었다. P38 인산화의 증가는 apoptosis 관련 단백질들의 발현 조절을 통해 세포 사멸을 유도하는 것으로 추측된다.When U87 cells were treated with bio-plasma, phosphorylation of p38 was increased, and it was clearly observed that cleaved caspase-3 increased, survivin decreased, and cleaved PARP decreased. Increased phosphorylation of P38 is presumed to induce apoptosis by regulating the expression of apoptosis-related proteins.
바이오-플라즈마의 처리된 세포의 생체 내 종양 성장 억제 능력Ability of bio-plasma to inhibit tumor growth in vivo of treated cells
바이오-플라즈마 처리된 U87-effluc 세포의 생체 내 종양 크기의 변화를 분석하였다. 바이오-플라즈마 처리하지 않은 control 그룹의 경우에는 U87-effluc 시그널이 점점 커졌다. 반면에, 바이오-플라즈마를 처리한 경우에서 U87-effluc 시그널이 14일 이후 증가율이 유의미하게 낮았으며 7-14일째 증가하는 비율도 control 그룹과 비교하여 낮았다. 또한, 35일 조직을 확보해서 종양 크기를 분석시 바이오-플라즈마 처리한 마우스 모델에서 종양 크기가 현저히 줄어들어 있는 것이 관찰되었다. 바이오-플라즈마 처리된 U87-effluc의 생체 내 변화를 면역형광 실험을 통해 확인하였다. 뇌종양조직에서 p38의 인산화가 증가되며 cleaved caspase-3가 증가, survivin은 감소, cleaved PARP는 감소되는 것이 관찰되었으며 이러한 결과는 이전의 in vitro 웨스턴 블롯 데이터와 일치 하였다.Changes in tumor size in vivo of bio-plasma-treated U87-effluc cells were analyzed. In the case of the control group not treated with bio-plasma, the U87-effluc signal gradually increased. On the other hand, in the case of bio-plasma treatment, the increase rate of U87-effluc signal after 14 days was significantly low, and the increase rate on days 7-14 was also low compared to the control group. In addition, it was observed that the tumor size was significantly reduced in the bio-plasma-treated mouse model when the tissue was obtained on day 35 and the tumor size was analyzed. In vivo changes of U87-effluc treated with bio-plasma were confirmed through immunofluorescence experiments. In brain tumor tissue, p38 phosphorylation was increased, cleaved caspase-3 was increased, survivin was decreased, and cleaved PARP was decreased. These results were consistent with previous in vitro Western blot data.
바이오-플라즈마 직접 처리에 의한 뇌종양 치료 효과Brain tumor treatment effect by bio-plasma direct treatment
교모세포종 마우스 동물 모델에 바이오-플라즈마를 처리하고 뇌종양이 커지는 정도를 영상을 통해 확인하였다. 바이오-플라즈마를 처리하지 않은 그룹 마우스의 뇌종양의 크기는 점점 커지는 반면에 바이오-플라즈마 처리 그룹 마우스의 뇌종양 크기는 커지지 않거나 점점 줄어드는 것을 관찰할 수 있었다. A glioblastoma mouse animal model was treated with bio-plasma, and the extent of brain tumor enlargement was confirmed through images. It was observed that the size of brain tumors of the mice of the bio-plasma treatment group did not increase or gradually decreased, while the size of the brain tumors of the mice of the group not treated with bio-plasma gradually increased.
상기에서, 플라즈마 발생장치는 반드시 플라즈마 제트에 한정되지 않으며, 유전장벽방전 플라즈마 발생장치로서 방전 가스를 공급할 수 있는 형태이면 적용가능하다. In the above, the plasma generating device is not necessarily limited to a plasma jet, and as a dielectric barrier discharge plasma generating device, any form capable of supplying a discharge gas is applicable.
만일, 뇌종양이 발생한 부위에 대해 외과적인 수술로 종양 제거를 실시한 후 후처리를 실시할 경우, 플라즈마 제트 외에 넓은 면적에 대해 플라즈마 처리할 수 있는 유전장벽방전 플라즈마 발생장치를 사용할 수 있다. 예를 들면, 본 출원인에 의한 공개번호 1020200017903호의 플라즈마 샤워기를 적용할 수도 있다. 플라즈마 샤워기는 일정 두께를 갖는 유전체에 하나 이상의 관통공을 타공하고 타공부 둘레 전부 또는 일부를 따라 유전체 상면과 이면에 서로 투영되는 부분에 전극을 설치하여 관통공 안으로 방전 가스를 주입하고 관통공 둘레 상하에 배치된 전극에 전압을 인가하여 방전 가스로부터 플라즈마 방전을 일으킨다. 대면적화하기 쉽고 플라즈마 제트처럼 방전 가스를 공급하기도 유리하여 뇌종양의 제거를 위한 외과적 수술 부위에 대한 전후 처리 또는 외과적 제거 없이 절개 후 플라즈마 처리에 적용될 수 있다. If post-processing is performed after surgically removing the tumor from the area where the brain tumor has occurred, a dielectric barrier discharge plasma generator capable of plasma-treating a large area can be used in addition to the plasma jet. For example, a plasma shower of Publication No. 1020200017903 by the present applicant may be applied. The plasma shower machine drills one or more through-holes in a dielectric having a certain thickness, installs electrodes along all or part of the circumference of the perforated part where the upper and lower surfaces of the dielectric are projected to each other, injects discharge gas into the through-hole, and Plasma discharge is generated from the discharge gas by applying a voltage to the electrodes disposed on the . It is easy to make a large area and it is advantageous to supply discharge gas like a plasma jet, so it can be applied to a surgical site for brain tumor removal before and after treatment or plasma treatment after incision without surgical removal.
한편, 뇌종양의 지름이 작고, 외과적 수술 제거를 하지 않고, 뇌종양 부위의 스킨을 제거하여 뇌종양 부위를 노출한 상태에서 플라즈마 처리할 경우, 좁은 직경 부위에 대한 처리이므로 플라즈마 제트가 적절하게 사용될 수 있다. 즉, 두피에 일종의 작은 구멍을 내어 구멍을 통해 뇌종양 부위에 플라즈마가 접근할 수 있게 하는 경우는 플라즈마 제트가 유리하며, 플라즈마 제트의 노즐의 직경을 스킨 절개 구멍의 직경과 비슷한 사이즈로 맞출 수 있다. On the other hand, when the diameter of the brain tumor is small and the plasma treatment is performed in a state where the brain tumor site is exposed by removing the skin of the brain tumor site without surgical removal, the plasma jet can be appropriately used because it is a treatment for a narrow diameter site. . That is, a plasma jet is advantageous when making a small hole in the scalp to allow plasma to access the brain tumor site through the hole, and the diameter of the nozzle of the plasma jet can be adjusted to a size similar to the diameter of the skin incision hole.
플라즈마 발생장치의 인가전압과 전류는 예시적이나, 10 내지 50kHz 주파수의 1 내지 2kV 전압, 100 내지 500mA의 전류가 흐를 수 있다. The applied voltage and current of the plasma generator are exemplary, but a voltage of 1 to 2 kV with a frequency of 10 to 50 kHz and a current of 100 to 500 mA may flow.
방전 가스는 질소를 포함하며, 필요에 따라 비활성 가스를 혼합할 수도 있다. The discharge gas contains nitrogen, and may be mixed with an inert gas if necessary.
플라즈마 처리시간 역시 예시적인 것으로, 180초 이상의 처리시간을 설정할 수 있다. 여전히 비교적 짧은 시간의 시술로도 명백한 효과를 볼 수 있다. The plasma processing time is also exemplary, and a processing time of 180 seconds or more may be set. You can still see obvious results with a relatively short procedure.
상기 실험에서 보인 바와 같이, 질소를 방전 가스로 한 바이오 플라즈마는 교모세포종 사멸 효과가 있고 정상 세포에는 영향이 없어, 종양에 대해 직접적인 플라즈마 처리가 종양 치료가 될 수 있다.As shown in the above experiment, bioplasma using nitrogen as a discharge gas has an effect of killing glioblastoma and has no effect on normal cells, so direct plasma treatment for tumors can be a tumor treatment.
한편, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예는 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 등가개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.Meanwhile, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical spirit or essential features. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention is indicated by the following claims rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention. do.
Claims (7)
질소 가스를 포함한 방전 가스에 플라즈마를 발생시켜 동물의 뇌종양 부위에 플라즈마를 처리하여 뇌종양 세포를 사멸시키는 것을 특징으로 하는 사람을 제외한 동물 뇌종양 치료방법.The skin of the brain of an animal with a brain tumor is incised to expose the brain tumor,
A method of treating brain tumors in animals other than humans, characterized in that the brain tumor cells are killed by generating plasma in a discharge gas including nitrogen gas and treating the brain tumor area with plasma.
고전압이 인가되는 속이 빈 바늘형 전극;
상기 바늘형 전극을 에워싸는 유전체 관; 및
상기 유전체 관을 에워싸는 관형 접지 전극;을 포함하고,
상기 바늘형 전극의 내부를 통해 질소 가스를 포함한 방전 가스를 공급하고, 상기 바늘형 전극에 전압을 인가하여, 발생되는 플라즈마를 뇌종양이 있는 부위에 조사하여 치료하되, 바늘형 전극에 인가되는 전압은 듀티 온 타임보다 긴 듀티 오프 타임을 갖고 동작하여 과열을 방지하는 것을 특징으로 하는 플라즈마 뇌종양 치료시스템.A plasma treatment system that treats a brain tumor by directly plasma treating a brain tumor area,
a hollow needle-type electrode to which a high voltage is applied;
a dielectric tube surrounding the needle-shaped electrode; and
A tubular ground electrode surrounding the dielectric tube; includes,
A discharge gas containing nitrogen gas is supplied through the inside of the needle-shaped electrode, and a voltage is applied to the needle-shaped electrode to irradiate and treat the generated plasma to the brain tumor. The voltage applied to the needle-shaped electrode is A plasma brain tumor treatment system characterized in that it operates with a duty-off time longer than the duty-on time to prevent overheating.
[6] The plasma brain tumor treatment system according to claim 5, wherein the jet plasma emitted from the nozzle unit is aimed to approach the brain tumor through a hole formed by incising the scalp.
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