KR20190029979A - Therapeutic method of hepatocyte growth factor overexpressing adipose tissue derived mesenchymal stem cells for Amyotrophic Lateral Sclerosis - Google Patents

Therapeutic method of hepatocyte growth factor overexpressing adipose tissue derived mesenchymal stem cells for Amyotrophic Lateral Sclerosis Download PDF

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KR20190029979A
KR20190029979A KR1020170117136A KR20170117136A KR20190029979A KR 20190029979 A KR20190029979 A KR 20190029979A KR 1020170117136 A KR1020170117136 A KR 1020170117136A KR 20170117136 A KR20170117136 A KR 20170117136A KR 20190029979 A KR20190029979 A KR 20190029979A
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mesenchymal stem
stem cells
growth factor
derived mesenchymal
adipose tissue
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한상훈
윤화영
이희우
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로얄에이알씨 주식회사
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Abstract

The present invention relates to a mammal, including human, adipose tissue derived mesenchymal stem cells having an effect of treating amyotrophic lateral sclerosis. In the present invention, a liver cell growth factor gene is introduced by a pMEX expression vector by specifically the following steps: (a) culturing mammal adipose tissue derived mesenchymal stem cells; (b) introducing a liver cell growth factor gene into the cultured mammal adipose tissue derived mesenchymal stem cells; (c) selecting the mammal adipose tissue derived mesenchymal stem cells in which the liver cell growth factor gene has been introduced; and (d) selectively culturing the selected mammal adipose tissue derived mesenchymal stem cells. As a result of reverse transcription polymerase chain reaction analysis, compared to a control group, a significant increased expression in mammal, including human, adipose tissue derived mesenchymal stem cells in which the liver cell growth factor is over-expressed has been identified. As a result of enzyme immunoassay, compared to a control group, over-expression, by 7.7 times, in mammal, including human, adipose tissue derived mesenchymal stem cells in which the liver cell growth factor has been over-expressed has been identified. Mammal, including human, adipose tissue derived mesenchymal stem cells in which the liver cell growth factor has been over-expressed enhances motoneurons of an SOD1 G93A gene mutant mouse and contributes to delaying the time point of symptom expression.

Description

간세포성장인자 유전자가 도입된 지방유래 중간엽줄기세포 및 이를 이용한 근위축성측삭경화증 치료방법{Therapeutic method of hepatocyte growth factor overexpressing adipose tissue derived mesenchymal stem cells for Amyotrophic Lateral Sclerosis}      [0001] The present invention relates to an adipose-derived mesenchymal stem cell into which a hepatocyte growth factor gene is introduced, and a method for treating amyotrophic lateral sclerosis using the same.

본 발명은 근위축성측삭경화증의 치료효능이 있는 사람을 포함하는 포유동물지방유래 중간엽줄기세포에 관한 것으로 상세하게는 간세포성장인자 유전자를 도입한 포유동물 지방유래 중간엽줄기세포를 이용한 근위축성측삭경화증 (Amyotrophic Lateral Sclerosis: ALS, 루게릭병) 환자를 치료하기 위한 세포치료방법에 관한 것이다.The present invention relates to a mammalian adipose derived mesenchymal stem cell including a human having a therapeutic effect for amyotrophic lateral sclerosis, and more particularly, to a mammalian adipose derived mesenchymal stem cell using mammalian adipose derived mesenchymal stem cells transfected with a hepatocyte growth factor gene The present invention relates to a cell therapy method for treating patients with amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease).

현대의학이 많은 발전을 이루었지만 장기 및 조직이 완전히 파괴된 경우에는 기존의 약물적인 요법이나 수술적인 요법으로는 그 근본적인 치료에 한계가 있는 것이 현실이다. 그 대안으로서 장기를 이식하는 방법이 있기는 하나 장기를 공여 받는 것도 현실적으로는 극히 어려우며, 장기 이식 차원에서도 해결할 수 없는 질병들이 존재한다. 그 대표적인 질병에 근위축성측삭경화증(ALS)이 있다. Modern medicine has developed a lot but if the organs and tissues are completely destroyed, it is a reality that there is a limit to the fundamental treatment by existing pharmacologic therapy or surgery. As an alternative, there are ways to transplant organs, but in reality it is extremely difficult to donate organs, and there are diseases that can not be solved even at the organ transplant level. The representative disease is amyotrophic lateral sclerosis (ALS).

근위축성측삭경화증(ALS)은 루게릭병으로 잘 알려진 질병으로서 매우 파괴적인 신경퇴행성 질환이다. 골격근의 위약과 위축, 호흡근 마비 등의 증상이 동반되며, 상위운동뉴런과 하위운동뉴런 모두에 심각한 영향을 미치는 것으로 알려져 있다. 여러 기전들이 질병의 발병에 기여할 수 있다고 제시되어 왔으나, 운동뉴런 사멸의 원인과 기전은 아직 명확하게 밝혀지지 않았다. 그 동안 여러 가지 약제와 줄기세포 치료 관련 임상실험들이 진행되었으나, 치료제로서 효과가 입증된 사례는 없었다. Amyotrophic lateral sclerosis (ALS) is a well-known disease of Lou Gehrig's disease and is a very destructive neurodegenerative disease. It is known that skeletal muscle is accompanied by symptoms such as placebo, atrophy and respiratory paralysis, and has a serious effect on both upper and lower motor neurons. Several mechanisms have been suggested to contribute to the onset of the disease, but the cause and mechanism of motor neuron death has not yet been elucidated. In the meantime, various drug and stem cell treatment clinical trials have been carried out, but there has been no proven effectiveness as a therapeutic agent.

ALS는 퇴행성 신경계 질환이며, 대뇌 및 척수와 같은 중추신경계의 운동 신경원이 선택적으로 파괴되기 때문에 운동 신경의 자극에 대해 근육들이 적절한 운동 기능을 나타내지 못해 "운동 신경원 질환"으로도 불리고, 1930년대 이 질병을 앓았던 운동선수 이름에 기원하여 "루게릭병(Lou Gehrig's disease)"으로도 불린다. 이 ALS 질환은 뇌의 신경 및 신체의 운동 근육 상태가 올바른 기능을 하고 있지 못한 질병이다.ALS is a degenerative nervous system disease, and since the motor neurons of the central nervous system such as the cerebrum and spinal cord are selectively destroyed, the muscles do not exhibit proper exercise function to the motor nerve stimulation and are called "motor neuron disease" "Lou Gehrig's disease", also known as "Lou Gehrig's disease". This ALS disease is a disease in which the muscular state of the brain and nerves of the body are not functioning properly.

ALS에 관한 정확한 발병 원인은 아직까지 밝혀지지 않았지만, 현재까지 ALS 발병 가능성은 다음과 같이 예측할 수 있다.Although the precise cause of ALS is not yet known, the possibility of ALS can be predicted as follows.

첫째, 대뇌, 뇌간 척수 등과 같은 중추신경계의 생물학적 기능을 유지하는 데 필요한 신경아교세포(neuroglia)와 신경세포 사이의 신경 전달물질 중에서 흥분성 신경 전달물질인 글루타민산은 뇌에서 형성되어 신경을 통해 근육에 신호를 전달하는 분자로서 한 번 분비되면 신속히 제거되어야 하는데, 활성 산소가 글루타민산의 파괴를 차단함으로써 글루타민산이 오히려 과잉 증가되고 그로 인하여 운동 신경이 파괴되면서 ALS가 발생될 수 있다. 둘째, 운동 신경 세포막에 위치한 "전위의존성 칼슘 통로"에 대한 항체가 형성되어 항원-항체 반응에 따른 일련의 생물학적 과정에 의해 운동신경세포가 파괴될 수 있다. 셋째, 운동선수들이 ALS 질환에 걸릴 위험도가 일반인보다 매우 높고, 그 발생 시기도 일반인보다 젊은 시기에 발생되고 있으며, 일부 지역에서 발생되고 있는 ALS 환자수가 다른 지역에 비해 훨씬 많을 만큼 환경 속의 특정 인자가 ALS질환을 유도시키고 있다. 넷째, 운동 신경 세포의 원활한 기능을 유지하기 위해서는 신경 영양인자의 적절한 공급이 계속 필요한데, 신경을 성장시키거나 상처가 난 세포를 회복시키는 데 필요한 영양성분이 결핍됨으로써 운동 신경 세포가 파괴될 수 있다. 다섯째, 극소수의 환자는 부모로부터 ALS 질환을 유전되고 있음이 확인되었고, 가족성 ALS 환자에서 SOD효소의 유전자 돌연변이 현상을 분석했을 때, 약 60개의 돌연변이가 보고되고 있으며, 그 일부는 동물실험을 통하여 ALS질환과 유사한 증상이 확인됨에 따라 ALS질환의 발병기전에 관한 연구가 보다 활발히 진행되고 있다. 여섯째, 바이러스 감염과 같은 환경오염으로 인해 ALS의 발병이 유도될 수 있다.First, among neurotransmitters between neuroglia and neurons necessary for maintaining the biological functions of the central nervous system such as the cerebrum and brain stem spinal cord, glutamate, an excitatory neurotransmitter, is formed in the brain and signals to the muscles through the nerves Once it is secreted as a molecule to be delivered, it must be removed promptly, and active oxygen may block the breakdown of glutamic acid, resulting in an excessive increase of glutamic acid, resulting in the destruction of motor nerves resulting in ALS. Second, an antibody against the "dislocation-dependent calcium channel" located in the motor neuron cell membrane may be formed and the motor neuron cell may be destroyed by a series of biological processes depending on the antigen-antibody reaction. Third, the athletes' risk for ALS disease is much higher than that of the general population, and it occurs at a younger age than the general population, and the number of ALS patients in some areas is much higher than in other regions. Leading to ALS disease. Fourth, in order to maintain the smooth function of motor neurons, adequate supply of neurotrophic factors is necessary, and motor nerve cells may be destroyed by lack of nutrients necessary for nerve growth or repair of wounded cells. Fifth, a small number of patients were found to have inherited ALS disease from their parents. About 60 mutations were reported when gene mutation of SOD enzyme was analyzed in familial ALS patients. As symptoms similar to ALS disease have been identified, studies on the pathogenesis of ALS disease have been actively conducted. Sixth, the occurrence of ALS can be induced by environmental pollution such as virus infection.

이와 같이 여러 가지 원인으로 인해 발생된 ALS는 시간이 흐름에 따라 그 증상이 점점 뚜렷해지고 초기에 뇌간에 위치한 운동신경세포의 손상이 인접 부위로 확산되어 상하지 근력 약화, 근 위축, 근섬유속성 연축 등이 초래된다. 일반적으로 ALS는 발병 초기에 경부 척수에 위치하는 운동 신경 세포가 주로 손상되는 사지형(limb type)과 뇌간에 위치하는 운동 신경 세포가 주로 손상되는 구형(bulbar type)으로 구분되는데, 대뇌의 운동 신경 세포도 손상 받아 상부 운동 신경원 장애에 따른 임상징후를 나타내기 때문에, ALS에서 특징적으로 관찰되는 임상 양상을 상하지 근력 약화, 구마비, 호흡 장애, 상지운동기능 장애, 하지 운동기능 장애 등으로 구분한다.In this way, ALS caused by various causes becomes more evident as time goes by, and the damage of motor neurons located in the brain stem is diffused to the adjacent region in the early stage, and weakness of muscular weakness, muscle atrophy, Lt; / RTI > In general, ALS is divided into limb type, in which motor nerve cells located in the cervical spinal cord are mainly damaged, and bulbar type, in which motor nerve cells located in the brainstem are mainly damaged. In addition, the clinical manifestations of ALS can be classified into weakness, weakness, paralysis, respiratory disturbance, upper limb movement dysfunction, and lower limb movement dysfunction because the cells are damaged and show clinical signs according to upper motor neuron dysfunction.

블랙홀과 빅뱅 이론 등으로 유명한 물리학자 스티븐 호킹 박사는 40년을 넘게 루게릭병을 앓다가 결국 전신마비가 된 상태이며, 한국에서도 많은 유명 인사들이 루게릭병을 앓고 있는 것이 현실임에도 불구하고 특별한 치료법이나 수술적인 요법이 전무한 실정이다. 또한, 이 질병은 젊은 연령대에서 발병한다는 점에서 육체적, 정신적으로 심한 고통을 주고 있다.Dr. Stephen Hawking, a physicist famous for his theory of black holes and big bangs, has been suffering from Lou Gehrig's disease for over 40 years and eventually has become paralyzed. Even though many famous people in Korea are suffering from Lou Gehrig's disease, There is no such therapy. In addition, the disease is caused by a young age, giving physical and mental pain.

더욱이, 현재로서는 ALS진단을 위한 특이적인 검사 방법도 전무한 상태이며, 환자가 운동 신경 세포의 손상을 보일 경우 ALS질환으로 의심되고 있다. 이처럼 ALS로 의심되는 환자가 있으면 안구 운동 장애, 감각 장애, 방광 및 항문 장애, 또는 지능장애 등과의 연관성 여부를 추가적으로 확인하고 있다.Furthermore, there is no specific test for ALS diagnosis at present, and ALS disease is suspected when the patient shows damage to motor neurons. The presence of suspected ALS patients further confirms the association with ocular movement disorders, sensory disturbances, bladder and anal disorders, or intellectual disabilities.

현재 ALS에 관한 연구가 조금씩 진전되어 운동 신경 세포의 글루타민산 과도 생산을 억제시킬 수 있는 약이 개발되어 병의 상태를 약간 지연시킬 수 있게 되었지만, 일단 파괴된 신경은 원래의 상태로 돌아오지 않기 때문에 ALS환자의 증상이 개선되기는 현실적으로 불가능하다. 즉, 환자의 상태가 더 이상 나빠지지 않도록 시간적인 약간의 여유만을 제공해 주는 것이 전부이다.At present, studies on ALS have progressed little by little, and drugs capable of inhibiting glutamate transient production of motor neurons have been developed to delay the disease state. However, since the destroyed neurons do not return to their original state, ALS It is practically impossible to improve the patient's symptoms. In other words, it is all about providing only a small amount of time so that the patient's condition does not deteriorate further.

한편, 줄기세포(stem cell)란 아직 운명이 결정되지 않은 미숙한 세포로, 체내의 장기나 조직이 외부로부터 공격이나 손상을 받게 되면 스스로 그 손상부위를 찾아가 분화시키는 능력을 가진 세포를 말한다. 줄기세포는 그 기원에 따라 배아 줄기세포(embryonic stem cell), 성체 줄기세포(adult stem cell), 제대혈 줄기세포(neonatal stem cell)로 나뉜다. 현재, 줄기세포를 골수에서 분리 및 배양하는 것은 쉬운 일이지만, 골수의 획득이 용이치 않고, 타인의 줄기세포를 이식할 경우 면역 거부 반응이 발생하는 문제를 해결하는 것이 현실적으로 어려운 것으로 알려져 있다. On the other hand, a stem cell is an immature cell whose fate has not yet been determined, and refers to a cell capable of locating and differentiating a damaged organs or organ itself if the organ or tissue is attacked or damaged from the outside. Stem cells are divided into embryonic stem cells, adult stem cells, and neonatal stem cells depending on their origins. Although it is easy to isolate and cultivate stem cells from the bone marrow, it is difficult to solve the problem of immune rejection when the bone marrow is acquired and the stem cells of other persons are transplanted.

중간엽 줄기세포는 일반적으로 골수(bone marrow)에서 조혈작용을 돕는 지지세포(stroma)로서 미분화상태를 유지하면서 뼈, 연골, 지방, 및 근육세포를 포함한 여러 가지 중배엽성 세포(mesodermal lineage cell)로 분화하는 특성을 가지기 때문에 인공조직을 개발하기 위한 재료로서 매우 유용하다. 중간엽 줄기세포가 뇌에서 신경교세포로 분화하는 잠재력을 지닌다고 보고되면서(Azizi., et al., Proc. Natl.Acad. Sci. USA, 94, 4080-4085(1998); 및 Kopen., et.al., Proc. Natl. Acad. Sci. USA, 96, 10711-10716(1999)) 중간엽 줄기세포를 이용하여 중추신경계의 질병을 치료할 수 있다는 가능성이 제기된 바 있다Mesenchymal stem cells are generally stromal cells that support hematopoiesis in the bone marrow. They are mesodermal lineage cells, including bone, cartilage, fat, and muscle cells, while maintaining their undifferentiated state. It is very useful as a material for developing an artificial tissue. As mesenchymal stem cells have been reported to have the potential to differentiate into glial cells in the brain (Azizi, et al., Proc. Natl. Acad Sci USA, 94, 4080-4085 (1998); USA, 96, 10711-10716 (1999)). It has been suggested that mesenchymal stem cells can be used to treat diseases of the central nervous system

(대한민국 특허공개) 제2006-0119064호(Korean Patent Publication) No. 2006-0119064 (대한민국 특허공개) 제2005-0036136호(Korean Patent Publication) No. 2005-0036136

본 발명의 목적은 근위축성측삭경화증의 치료효능이 있는 포유동물지방유래 중간엽줄기세포를 제공하기 위한 것이다. 상기의 목적을 위해서 사람을 포함하는 포유동물지방유래 중간엽줄기세포에 간세포성장인자 유전자를 도입한 포유동물지방유래 중간엽줄기세포를 이용하여 근위축성측삭경화증(Amyotrophic Lateral Sclerosis: ALS, 루게릭병)을 치료하기 위한 세포치료 방법을 제공하고자 하는 것이다.It is an object of the present invention to provide mammalian adipose-derived mesenchymal stem cells having therapeutic effect of amyotrophic lateral sclerosis. For the above-mentioned purpose, amyotrophic lateral sclerosis (ALS) is induced by using mammalian adipose derived mesenchymal stem cells into which a hepatocyte growth factor gene has been introduced into mammalian adipose-derived mesenchymal stem cells, The present invention provides a method for treating a cell.

본 발명은 간세포 성장인자(hepatocyte growth factor; HGF) 유전자가 도입된 포유동물지방유래 중간엽줄기세포를 이용하여 근위축성측삭경화증을 치료하기 위한 세포치료에 관한 것이다. 간세포성장인자 유전자는 pMEX발현벡터에 의해 도입되며, 구체적인 과정은 먼저 (a) 포유동물지방유래 중간엽줄기세포를 배양하는 단계, (b) 상기 배양된 포유동물지방유래 중간엽줄기세포에 간세포성장인자 유전자를 도입하는 단계, (c) 상기 간세포성장인자 유전자가 도입된 포유동물지방유래 중간엽줄기세포를 선별하는 단계, (d) 상기 선별된 포유동물지방유래 중간엽줄기세포를 선택적으로 배양하는 단계로 구성된다.The present invention relates to a cell therapy for treating amyotrophic lateral sclerosis using mammalian adipose derived mesenchymal stem cells into which a hepatocyte growth factor (HGF) gene has been introduced. The hepatocyte growth factor gene is introduced by a pMEX expression vector. Specifically, (a) the step of culturing the mesenchymal stem cells derived from the mammalian adipose, (b) the step of culturing the mesenchymal stem cells (C) selecting a mesenchymal stem cell derived from a mammalian adipose tissue into which the hepatocyte growth factor gene has been introduced, (d) selectively culturing the mesenchymal stem cells derived from the selected mammalian adipose .

상기와 같은 공정으로 사람을 포함하는 포유동물지방유래 중간엽줄기세포에 간세포성장인자 유전자를 도입하고, 상기 간세포성장인자 유전자가 도입된 포유동물지방유래 중간엽줄기세포주를 유효성분으로 하는 신경계질환 예방 및 치료용 조성물을 제조하며, 상기 신경계질환은 근위축성측삭경화증(Amyotrophic Lateral Sclerosis: ALS, 루게릭병)을 치료를 주 목적으로 하고, 그 외에 상기 신경계 질환은 파킨슨병(Parkinson's disease), 알츠하이머병(Alzheimer disease), 헌팅톤 무도병(Huntington's chorea), 간질(epilepsy), 정신분열증(schizophrenia), 급성 뇌졸중(acute stroke), 만성 뇌졸중(chronic stroke) 또는 척수손상(spinal cord injuries) 등을 포함하는 신경 질환 중 하나이거나 뇌졸중 후 수반되는 만성적인 신체 장애를 포함한다.The above process introduces a hepatocyte growth factor gene into a mammalian adipose-derived mesenchymal stem cell, which comprises a human, and administers the mammalian adipose-derived mesenchymal stem cell line into which the hepatocyte growth factor gene has been introduced as an active ingredient, Wherein the nervous system disease is amyotrophic lateral sclerosis (ALS), and the nervous system disease is Parkinson ' s disease, Alzheimer ' s disease, Alzheimer's disease, Huntington's chorea, epilepsy, schizophrenia, acute stroke, chronic stroke, or spinal cord injuries. Or a chronic physical disability that is accompanied by a stroke.

본원발명은 간세포성장인자가 과발현된 사람을 포함하는 포유동물지방유래중간엽줄기세포는 pMEX발현벡터에 간세포성장인자의 형질주입을 통해서 만들어진다.. In the present invention, mesenchymal stem cells derived from a mammalian adipose-derived mesenchymal stem cell comprising a human overexpressing a hepatocyte growth factor are produced by transfection of a hepatocyte growth factor into a pMEX expression vector.

본원발명에서 역전사중합효소연쇄반응분석으로 분석한 결과 간세포성장인자가 과발현된 사람을 포함하는 포유동물지방유래중간엽줄기세포가 대조군에 비해서 현저하게 발현이 증가한 것을 확인하였으며, 효소면역측정법 결과에서는 대조군에 비해 간세포성장인자가 과발현된 사람을 포함하는 포유동물지방유래중간엽줄기세포가 7.7배 이상 과발현되었음을 확인하되었다. 또한 간세포성장인자가 과발현된 사람을 포함하는 포유동물지방유래중간엽줄기세포가 SOD1 G93A 유전자 변이 마우스의 운동뉴런 기능을 증진시키고, 증상발현 시점을 늦춰주는데 기여하였다.As a result of analysis by reverse transcription polymerase chain reaction analysis in the present invention, it was confirmed that the expression of mesenchymal stem cells in mammalian adipose-derived mesenchymal stem cells including human with overexpression of hepatocyte growth factor was remarkably increased compared to the control group. In the enzyme immunoassay, And that overexpression of mesenchymal stem cells in mammalian adipose-derived mesenchymal stem cells, including those with overexpression of hepatocyte growth factor, was overexpressed. In addition, mesenchymal stem cells from mammalian adipose tissue, including those with overexpression of hepatocyte growth factor, promoted motor neuron function in SOD1 G93A mutant mice and contributed to delaying the onset of symptoms.

그리고 간세포성장인자가 과발현된 사람을 포함하는지방유래 중간엽줄기세포를 이용한 유전자 도입 세포치료가 운동뉴런의 증식 향상과 세포사멸 억제의 측면에서 강력한 신경 관련 잠재력을 가지고 있음을 보여주고 있으며, 난치성 질병인 근위축성측삭경화증에 대한 새로운 치료방법으로서 가능성을 제시하였다.In addition, it has been shown that transgenic cell therapy using adipose derived mesenchymal stem cells, including those with overexpression of hepatocyte growth factor, has a strong neuronal potential in terms of proliferation enhancement and cell death inhibition of motor neurons, Suggesting the possibility of a new treatment method for amyotrophic lateral sclerosis.

도 1은 본원발명에서 mRNA와 protein level에서 간세포 성장인자가 과발현되었는지를 확인하기 위해서 RT-PCR(역전사중합효소 연쇄반응)과 ELISA (효소면역측정법)를 실시한 결과를 나타낸 도면이다.
도 2는 본원발명에서 간세포 성장인자가 과발현된 지방조직유래중간엽줄기세포가 motor neuron proliferation(운동뉴런 증식)에 미치는 효과를 나타낸 도면이다.
도 3은 본원발명에서 cell cycle(세포주기) 분석을 실시한 결과를 나타낸 도면이다.
도 4는 본원발명에서 Apoptosis(세포자멸)가 진행되고 있는 motor neuron에 HGF-hATMSC의 세포자멸 억제효과를 나타낸 도면이다.
도 5는 본원발명에서 HGF-hATMSC의 치료적인 효과를 확인하기 위해서 saline, hATMSC, HFG-hATMSC 투여 그룹으로 나누어 각각의 세포를 streotaxic injector를 이용하여, 척수내에 주입하는 사진이다.
도 6은 본 발명에서 이식된 HGF-hATMSCs 가 관찰되는 사진이다.
도 7은 본 발명에서 HGF-hATMSC를 이식한 mouse가 hATMSC와 control에 비해서 증상 발현 시작 시점이 늦춰짐을 확인할 수 있다.
도 8은 본 발명에서 HGF-hATMSC가 SOD1 G93A transgenic mice의 motor neuron function을 증진시키고, 증상발생시점을 늦춰주는데 기여함을 보여주고 있다.FIG. 1 is a graph showing the results of RT-PCR (Reverse Transcription Polymerase Chain Reaction) and ELISA (enzyme immunoassay) in order to confirm whether hepatocyte growth factor was overexpressed at mRNA and protein level in the present invention.
FIG. 2 is a graph showing the effect of mesenchymal stem cells derived from adipose tissue overexpressing hepatocyte growth factor on motor neuron proliferation (motor neuron proliferation) in the present invention.
FIG. 3 is a view showing a result of a cell cycle (cell cycle) analysis in the present invention. FIG.
FIG. 4 is a graph showing the effect of HGF-hATMSC on apoptosis in motor neuron undergoing apoptosis (apoptosis) in the present invention.
FIG. 5 is a photograph showing injection of saline, hATMSC, and HFG-hATMSC into the spinal cord using a streotaxic injector in order to confirm the therapeutic effect of HGF-hATMSC in the present invention.
Fig. 6 is a photograph showing HGF-hATMSCs transplanted in the present invention. Fig.
FIG. 7 shows that the mice transplanted with HGF-hATMSC delayed the onset of symptom onset compared to hATMSC and control.
FIG. 8 shows that HGF-hATMSC improves the motor neuron function of SOD1 G93A transgenic mice and contributes to delaying the onset of symptoms in the present invention.

이하, 본 발명을 실시예에 의하여 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

본 발명은 간세포성장인자 유전자를 도입한 포유동물지방유래 중간엽줄기세포를 이용하여 한 근위축성측삭경화증(Amyotrophic Lateral Sclerosis: ALS, 루게릭병)을 치료하기 위한 세포치료 방법에 관한 것이다.The present invention relates to a cell therapy method for treating amyotrophic lateral sclerosis (ALS) using mammalian adipose derived mesenchymal stem cells into which a hepatocyte growth factor gene has been introduced.

발명에서 용어, "간세포 성장인자(hepatocyte growth factor; HGF)"는 SF(scatter factor; 산란인자)라고도하며, 중간엽 세포(mesenchymal cell)에 의해 생산되는 다기능성 이종이량체(heterodimer) 폴리펩티드를 의미한다. 상기 간세포 성장인자는 N-말단 핑거 도메인(finger domain) 및 4개의 크링글 도메인(Kringle domain)을 포함하는 69 kDa의 알파-체인, 및 키모트립신-유사 세린 프로테아제의 프로테아제 도메인과 유사성을 갖는 34kDa 베타-체인을 포함하는 구조로 이루어져 있다The term " hepatocyte growth factor (HGF) " as used herein refers to a multifunctional heterodimer polypeptide produced by mesenchymal cells, also referred to as SF (scatter factor) do. The hepatocyte growth factor has a 69 kDa alpha-chain comprising an N-terminal finger domain and four Kringle domains, and a 34 kDa beta-chain having similarity to the protease domain of chymotrypsin-like serine protease - It consists of a structure containing chains.

간세포성장인자의 여러 가지 기전이 신경세포의 기능을 도와주고, 신경 관련 질병의 진행 속도를 늦출 수 있음을 알수 있다. 중간엽줄기세포는 생체 내에서 적응능력과 분화능력을 가지고 있기 때문에 국소적으로나 전신적으로 적용하였을 때 해당 질병부위에 작용하여, 치료효과가 있다. 또한 중간엽줄기세포의 신경세포에 대한 치료적인 잠재효과도 있으며 간세포성장인자와 중간엽줄기세포 각각의 치료적인 잠재력을 바탕으로 유전자(간세포성장인자) 도입 줄기세포 치료가 운동뉴런의 기능과 퇴행에 보다 높은 시너지 효과가 있다.Several mechanisms of hepatocyte growth factor can help nerve cell function and slow down the progression of neurological diseases. Since mesenchymal stem cells have adaptive and differentiative capacity in vivo, they act on localized or systemically applied disease sites and have a therapeutic effect. In addition, there is a therapeutic potential effect on mesenchymal stem cell neurons and based on the therapeutic potential of each of the stem cell growth factor and mesenchymal stem cell, stem cell therapy with gene (hepatocyte growth factor) There is a higher synergy effect.

발명의 구체적인 하나의 양태로서, 간세포성장인자 유전자는 벡터에 클로닝되어 포유동물지방유래 중간엽줄기세포에 도입되는 것이 바람직하다.In a specific embodiment of the invention, the hepatocyte growth factor gene is preferably cloned into a vector and introduced into mammalian adipose-derived mesenchymal stem cells.

본 발명에서 용어, "벡터"는 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 발현 벡터로서, 벡터는 플라스미드 벡터, 코즈미드 벡터를 포함하며, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절요소를 포함하는 유전자 작제물을 의미한다.As used herein, the term " vector " refers to an expression vector capable of expressing a desired protein in a suitable host cell, the vector including a plasmid vector, a cosmid vector, and an essential regulatory element operably linked to the expression of the gene insert Gene constructs.

본 발명에서 용어, "작동가능하게 연결된(operably linked)"은 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질을 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 것을 의미한다. 재조합 벡터와의 작동적 연결은 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다.As used herein, the term " operably linked " means that a nucleic acid sequence encoding a desired protein and a nucleic acid expression control sequence are functionally linked to perform a general function. The operative linkage with the recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage are made using enzymes generally known in the art.

본 발명의 구체적인 실시예에서는 유전자의 도입을 위해 진뱅크 수탁번호 U63842의 유전자 서열 중 coding region (55~768bp)을 pMSCV-puro 플라스미드에 클로닝하고, 상기 pMSCV-puro 플라스미드를 벡토로 하여중간엽줄기세포주에 형질전환시켰다. 바람직하게 상기 벡터가 삽입된 중간엽줄기세포는 사람을 포함하는 포유동물지방유래 중간엽줄기세포주이다.In a specific embodiment of the present invention, the coding region (55 to 768 bp) of the gene sequence of Genbank Accession No. U63842 was cloned into the pMSCV-puro plasmid and the pMSCV-puro plasmid was used as a vector to introduce the mesenchymal stem cell line ≪ / RTI > Preferably, the mesenchymal stem cell into which the vector is inserted is a mammalian adipose derived mesenchymal stem cell line containing human.

본 발명의 구체적인 실시예에서는 간세포 성장인자 유전자의 도입을 위해 진뱅크 수탁번호 NM_000601.4의 유전자서열 중 coding region (166~2352 bp)을 pShuttle-CMV에 클로닝한 후 pAdEasy-1과 재조합을 통하여 pAd-HGF를 얻었다. 상기 플라스미드를 PacI 제한효소를 이용하여 선형화 한 후(linearize) 포유동물지방유래 중간엽줄기세포주를 형질전환시켰다. 바람직하게 상기 벡터가 삽입된 중간엽줄기세포주는 사람을 포함하는 포유동물지방유래 중간엽줄기세포주이다.For the introduction of the hepatocyte growth factor gene, the coding region (166-2352 bp) of the gene sequence of Jinbank Accession No. NM_000601.4 was cloned into pShuttle-CMV and recombined with pAdEasy-1 to introduce pAd -HGF. The plasmid was linearized using PacI restriction enzyme and then transformed into mammalian adipose-derived mesenchymal stem cell line. Preferably, the mesenchymal stem cell line into which the vector is inserted is a mammalian adipose derived mesenchymal stem cell line including a human.

본 발명의 "유전자가 도입된 포유동물지방유래 중간엽줄기세포"에서 유전자 도입은 형질전환 방법을 이용한다. 상기 간세포성장인자 유전자를 도입하기 위한 형질전환 방법은 당업계의 통상적인 방법에 따라 용이하게 수행할 수 있다.In the " gene-derived mammalian adipose derived mesenchymal stem cells " of the present invention, the transfection method is used for gene transfer. The transformation method for introducing the hepatocyte growth factor gene can be easily performed according to a conventional method in the art.

본 발명에서 용어, "형질전환"은 외부DNA 또는 외부DNA를 함유하는 벡터가 숙주에 도입되어 DNA가 염색체의 인자로서 또는 염색체 통합 완성에 의해, 인위적으로 유전적인 변화를 일으키는 현상을 의미한다. 일반적으로 형질전환방법에는 DNA의 CaCl2 침전법, CaCl2 방법에 환원물질인 DMSO(dimethyl sulfoxide)를 사용함으로써 효율을 높인 Hanahan 방법, 전기천공법(electroporation), 인산칼슘 침전법, 원형질 융합법, 실리콘 카바이드 섬유를 이용한 교반법, 아그로 박테리아 매개된 형질전환법, PEG를 이용한 형질전환법, 덱스트란 설페이트, 리포펙타민을 포함한 리포좀 및 건조/억제 매개된 형질전환 방법 등이 있다. 본 발명의 실시예에서는 플라스미드 벡터와 간세포성장인자 유전자가 함유된 벡터를 줄기세포에 감염시켜 형질전환시켰다.The term " transformed " in the present invention means a phenomenon in which an external DNA or a vector containing an external DNA is introduced into a host to cause an artificial genetic change as a factor of chromosome integration or completion of chromosome integration. Generally, transformation methods include DNA precipitation method of CaCl 2, DMSO (dimethyl sulfoxide) as a reducing agent in the CaCl 2 method, Hanahan method of increasing efficiency, electroporation, calcium phosphate precipitation method, Agrobacterium-mediated transformation, Transformation using PEG, Liposomes including dextran sulfate, Lipofectamine, and drying / inhibition-mediated transformation methods. In the example of the present invention, a vector containing a plasmid vector and a hepatocyte growth factor gene was transfected into a stem cell.

상기 서술한 바와 같이, 간세포성장인자 유전자가 도입된 포유동물지방유래 중간엽줄기세포주는 그 종류가As described above, the mammalian adipose-derived mesenchymal stem cell line into which the hepatocyte growth factor gene has been introduced is classified into

제한되지 않으며, 특정한 조직의 세포로 분화하게 되는 분화능을 가진 세포주이면 어떠한 세포주라도 본 발명의 세포주가 될 수 있다. And any cell line capable of differentiating into a cell of a specific tissue may be a cell line of the present invention.

본 발명은 간세포성장인자가 과발현된 포유동물지방유래 중간엽줄기세포의 운동뉴런에 대한 증식과 세포사멸 억제효과에 관한 생체 외 실험과 근위축성측삭경화증 마우스 모델에 대한 치료적인 효과에 관한 생체 내 실험으로 나누어 진행하였다. The present invention relates to in vitro experiments on the proliferation and cell death inhibitory effect on the motor neurons of mammalian adipose-derived mesenchymal stem cells overexpressing hepatocyte growth factor and in vivo experiments on the therapeutic effect on the mouse model of amyotrophic lateral sclerosis Respectively.

도 1은 본원발명에서 mRNA와 protein level에서 간세포 성장인자가 과발현되었는지를 확인하기 위해서 RT-PCR(역전사중합효소 연쇄반응)과 ELISA (효소면역측정법)를 실시한 결과를 나타낸 도면이고, 도 2는 본원발명에서 간세포 성장인자가 과발현된 지방조직유래중간엽줄기세포가 motor neuron proliferation(운동뉴런 증식)에 미치는 효과를 나타낸 도면이며, 도 3은 본원발명에서 cell cycle(세포주기) 분석을 실시한 결과를 나타낸 도면이다.1 is a graph showing the results of RT-PCR (Reverse Transcription Polymerase Chain Reaction) and ELISA (enzyme immunoassay) in order to confirm whether hepatocyte growth factor was overexpressed at mRNA and protein level in the present invention, FIG. 3 is a graph showing the effect of mesenchymal stem cells derived from adipose tissue overexpressing hepatocyte growth factor on motor neuron proliferation (motor neuron proliferation), and FIG. 3 is a graph showing the results of cell cycle analysis FIG.

도 4는 본원발명에서 Apoptosis(세포자멸)가 진행되고 있는 motor neuron에 HGF-hATMSC의 세포자멸 억제효과를 나타낸 도면이고, 도 5는 본원발명에서 HGF-hATMSC의 치료적인 효과를 확인하기 위해서 saline, hATMSC, HFG-hATMSC 투여 그룹으로 나누어 각각의 세포를 streotaxic injector를 이용하여, 척수내에 주입하는 사진이며, 도 6은 본 발명에서 이식된 HGF-hATMSCs 가 관찰되는 사진이다.FIG. 4 is a graph showing the effect of HGF-hATMSC on apoptosis in motor neuron undergoing apoptosis (apoptosis) in the present invention. FIG. 5 is a graph showing the effect of HGF- hATMSC and HFG-hATMSC, and each cell is injected into the spinal cord using a streotaxic injector. Fig. 6 is a photograph showing the HGF-hATMSCs transplanted in the present invention.

도 7은 본 발명에서 HGF-hATMSC를 이식한 mouse가 hATMSC와 control에 비해서 증상 발현 시작 시점이 늦춰짐을 확인할 수 있으며, 도 8은 본 발명에서 HGF-hATMSC가 SOD1 G93A transgenic mice의 motor neuron function을 증진시키고, 증상발생시점을 늦춰주는데 기여함을 보여주고 있다.FIG. 7 shows that the mice transplanted with HGF-hATMSC in the present invention slowed the onset of symptom onset compared to hATMSC and control. FIG. 8 shows that HGF-hATMSC enhances motor neuron function of SOD1 G93A transgenic mice And to delay the onset of symptoms.

상기 도면을 보다 더 상세하게 설명하면 도 1은 mRNA와 protein level에서 간세포 성장인자가 과발현 여부를 확인하기 위해서 RT-PCR(역전사중합효소 연쇄반응)과 ELISA (효소면역측정법)를 실시한 결과, 사람지방조직유래중간엽줄기세포에 비해서 현저하게 과발현된 것을 확인할 수 있다. 간세포 성장인자 항체를 이용한 면역형광분석에서도 간세포 성장인자의 과발현을 확인할 수 있다.More specifically, FIG. 1 shows RT-PCR (reverse transcription polymerase chain reaction) and ELISA (enzyme immunoassay) in order to confirm the overexpression of hepatocyte growth factor at mRNA and protein level. As a result, Induced stem cell proliferation compared with mesenchymal stem cells derived from tissue-derived mesenchymal stem cells. Immunofluorescence analysis using hepatocyte growth factor antibody can also confirm overexpression of hepatocyte growth factor.

도 2는 간세포 성장인자가 과발현된 지방조직유래중간엽줄기세포가 motor neuron proliferation(운동뉴런 증식)에 미치는 효과를 확인하기 위해서, NSC34 cells끼리, NSC34cell과 hATMSC(사람지방유래중간엽줄기세포), NSCcell34과 HGF-hATMSC(간세포 성장인자가 과발현된 지방조직유래중간엽줄기세포) 를 세그룹으로 나누어 indirect co-culture(간접공생배양)한 후, WST-1 proliferation assay와 trypan blue exclusion assay를 진행한 결과, day1, day 2, day3에 HGF-hATMSC와 indirect co-culture한 그룹의 NSC34 cell의 수가 현저하게 증가되었음을 확인할 수 있었다.FIG. 2 is a graph showing the effect of mesenchymal stem cells derived from adipose tissue overexpressing hepatocyte growth factor on motor neuron proliferation (motoneuron proliferation) among NSC34 cells, NSC34cell and hATMSC (mesenchymal stem cells from human adipose tissue) NSCcell34 and HGF-hATMSC (mesenchymal stem cells derived from adipose tissue overexpressing hepatocyte growth factor) were divided into three groups and indirectly co-cultured, followed by WST-1 proliferation assay and trypan blue exclusion assay As a result, it was confirmed that the number of NSC34 cells in the group co-cultured with HGF-hATMSC significantly increased in day 1, day 2 and day 3.

도 3은 cell cycle(세포주기) 분석을 실시한 결과, 휴지기인 G0/G1기는 control에 비해서 hATMSC coculture 그룹이, hATMSC 그룹에 비해서, HGF-hATMSC 그룹이 상대적으로 cell population이 감소하였으며, DNA 합성기인 S기, 유사분열 단계인 G2/M에는 각각 세포수가 증가한 것을 확인 할 수 있었다. 또한, Cell cycle에서 G1에서 S기로의 진행의 조절과 DNA 복제신호에 매우 중요한 역할을하는 Cyclin D1의 과발현은 그만큼 Cell cycle이 활발히 진행되고 있음을 의미한다. Cell cycle에서 proliferation(증식) 과정을 촉진시키는 HGF receptor인 phospho c-Met의 과발현은 NSC34cell의 증식이 활발히 진행되고 있음을 의미한다. 이러한 결과는 HGF-hATMSC가 motor neuron의 증식 능력을 향상시키고 있음을 의미한다.FIG. 3 shows that the cell population of HAT-HATMSC group was lower than that of hATMSC group and hATMSC coculture group as compared with the control group. In the cell cycle analysis, And G2 / M, which is the mitotic phase, respectively. In addition, regulation of progression from G1 to S in the cell cycle and overexpression of cyclin D1, which plays an important role in the DNA replication signal, indicates that the cell cycle is actively progressing. Overexpression of the HGF receptor phospho c-Met, which promotes proliferation in the cell cycle, indicates that the proliferation of NSC34 cells is actively undergoing. These results indicate that HGF-hATMSC improves the proliferation of motor neurons.

도 4는 Apoptosis(세포자멸)가 진행되고 있는 motor neuron에 HGF-hATMSC의 세포자멸 억제효과를 확인하기 위해서, NSC34 cells에 apoptosis를 유발한 후, control, hATMSC, HGF-hATMSC와 indirect co-culture 한 후, survival cell assay를 진행하였다. Apoptosis를 유발한 NSC34cell의 viability가 HGF-hATMSC 와 co-culture한그룹이 현저하게 높음을 확인할 수 있다. 또한 Annexin V와 PI staining 후, flow cytometry 분석을 시행한 결과 Annexin V positive cell이 control 에 비해서 hATMSC 그룹이, hATMSC 그룹에 비해서 HGF-hATMSC 그룹이 현저하게 감소되어 있는 것을 확인할 수 있었다. 또한 Western blot analysis 결과 활성화된 Caspase-3와 PARP의 발현량의 감소, 그리고 phospho-cMET의 발현량의 증가 또한 HGF-hATMSC가 NSC 34cell의 apoptosis를 억제할 수 있음을 뒷받침한다.FIG. 4 shows apoptosis induction in NSC34 cells and indirect co-culture with control, hATMSC, and HGF-hATMSC in order to confirm the apoptosis inhibitory effect of HGF-hATMSC on motor neurons undergoing apoptosis (apoptosis) The survival cell assay was performed. The viability of NSC34 cells that induced apoptosis was significantly higher in the group co-cultured with HGF-hATMSC. In addition, after the Annexin V and PI staining, flow cytometry analysis showed that the HATMSC group had significantly reduced HGF-hATMSC group compared to the hATMSC group. Western blot analysis also confirmed that HGF-hATMSC could inhibit the apoptosis of NSC 34 cells by decreasing expression of caspase-3 and PARP and increasing expression of phospho-cMET.

[표 1] Immunophenotyping of HGF-hATMSCs by flow cytometry [Table 1] Immunophenotyping of HGF-hATMSCs by flow cytometry

Figure pat00001
Figure pat00001

* Data show the average values from three independent experiments and are expressed as the Mean±SD* Data show the average values from three independent experiments and are expressed in the mean ± SD

상기 [표 1]은 immunophenotyping characteristics을 평가하기 위해서 flow cytometry를 이용하여 stem cell surface marker 의 발현을 확인한 결과로서, 양성표지마커와 음성표지 마커 모두 컨트롤과 차이가 없는 것으로 보아 HGF gene transfection이 stem cell의 특성을 변화시키지 않는 것을 알 수 있다. 이러한 결과는 transfection에 의해 HGF가 과발현된 HGF-hATMAC가 성공적으로 만들어졌음을 의미한다.Table 1 shows the expression of stem cell surface markers using flow cytometry in order to evaluate the immunophenotyping characteristics. As a result, there was no difference between control and positive marker markers and HGF gene transfection, It can be seen that the characteristics are not changed. These results indicate that HGF-overexpressed HGF-hATMAC was successfully produced by transfection.

도 5와 도 6은 ALS(근위축성측삭경화증) mouse 모델인 SOD1 G93A transgenic mouse에 HGF-hATMSC의 치료적인 효과를 확인하기 위해서, saline, hATMSC, HFG-hATMSC 투여 그룹으로 나누어 각각의 세포를 streotaxic injector를 이용하여 척수내에 주입하고, green fluorescent nanoparticles이 포함된 세포가 제대로 이식되었는지 확인하기 위해서 fluorescence imaging analysis분석을 통해 확인하였다.Fig. 5 and Fig. 6 show that the SOD1 G93A transgenic mouse model of ALS (mouse with amyotrophic lateral sclerosis) mice was divided into two groups: saline, hATMSC, and HFG-hATMSC, in order to examine the therapeutic effect of HGF-hATMSC. Were injected into the spinal cord and confirmed by fluorescence imaging analysis analysis to confirm that cells containing green fluorescent nanoparticles were properly transplanted.

도 7과 도 8은 세포를 이식한 후, 증상 발현과 신경 기능을 평가하기 위해서 disease onset point와 rotarod failure를 평가하였다. HGF-hATMSC를 이식한 mouse가 hATMSC와 control에 비해서 증상 발현 시작 시점이 늦춰짐을 확인 할 수 있었으며, rotarod failure의 평균 시간도 늦어짐을 확인하였습니다. 이러한 결과는 HGF-hATMSC가 SOD1 G93A transgenic mice의 motor neuron function을 증진시키고, 증상발생시점을 늦춰주는데 기여함을 보여준다. 게다가 평균 survival time도 control 그룹에 비해서 유의적으로 증가하는 것을 확인할 수 있었다. 7 and 8, after cell transplantation, evaluated disease onset point and rotarod failure to assess symptom development and neurological function. The mice transplanted with HGF-hATMSC were found to be delayed in the onset of symptom onset compared to hATMSC and control, and the average time of rotarod failure was found to be slow. These results demonstrate that HGF-hATMSC promotes motor neuron function in SOD1 G93A transgenic mice and contributes to slowing the onset of symptoms. In addition, the mean survival time was significantly increased compared to the control group.

본원발명에서 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포는 pMEX발현벡터에 간세포성장인자의 형질주입을 통해서 만들어졌다. 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포가 성공적으로 만들어졌는지 확인하기 위해서 역전사중합효소 연쇄반응(RT-PCR), 효소면역측정법(ELISA), 그리고 간세포 성장인자 항체를 이용한 면역형광분석을 시행하였다. 역전사중합효소연쇄반응분석 결과, 대조군에 비해서 현저하게 발현이 증가한 것을 확인하였으며, 효소면역측정법 결과, 대조군에 비해 7.7배 이상 과발현되었음을 확인하였다. In the present invention, mesenchymal stem cells derived from mammalian adipose tissue overexpressing hepatocyte growth factor were produced by transfection of hepatocyte growth factor into pMEX expression vector. (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence analysis using hepatocyte growth factor antibody to determine whether mesenchymal stem cells from hepatocyte growth factor-overexpressing mammalian fat were successfully produced . As a result of reverse transcription polymerase chain reaction, it was confirmed that the expression was remarkably increased as compared with the control group, and the result of enzyme immunoassay was over 7.7 times over that of the control group.

면역형광분석 결과 간세포성장인자의 발현을 직접 눈으로 확인할 수 있었다. 또한 간세포성장인자가 과발현된 포유동물지방유래 중간엽줄기세포의 유세포분석 결과, 줄기세포 양성표지 마커는 각각 99% 이상, 음성표지 마커는 2% 미만으로 확인되어 대조군과 큰 차이가 없는 것을 확인할 수 있었다. 이러한 결과는 유전자 도입을 통해 간세포성장인자가 과발현된 사람지방유래 중간엽줄기세포가 성공적으로 제작되었음을 의미한다. 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포가 운동뉴런 증식에 미치는 효과를 확인하기 위해서, 세 그룹(①NSC34세포(마우스운동뉴런 세포), ②NSC34세포와 포유동물지방유래중간엽줄기세포, ③NSC34 세포와 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포) 으로 나누어 간접공생배양(indirect coculture)한 후, WST-1 세포증식측정법과 트립판블루세포배제측정법(trypan blue exclusion assay)을 진행한 결과, 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포와 공생배양한 그룹의 NSC34세포수가 현저하게 증가되었음을 확인할 수 있었다. 마찬가지로 각각 공생배양한 그룹에서 얻은 NSC34세포를 유세포분석 방법을 통하여 세포주기분석을 시행하였다. 대조군의 경우, 휴지기인 GO/G1 기, 유전자 합성기인 S기, 유사분열 단계인 G2/M기에 각각, 73.27±4%, 10.68±2%, 15.23±1.5%, 포유동물지방유래중간엽줄기세포 그룹은 각각, 63.1±2.1%, 15.1±1.83%, 20.4±2%, 간세포 성장인자가 과발현된 포유동물지방유래중간엽줄기세포 그룹은 각각, 57.12±1.5%, 17.23±1.1%, 24.1±1.3% 의 결과를 나타내었다. 세포주기분석 결과, G0/G1기는 대조군에 비해 포유동물지방유래 중간엽줄기세포 그룹에서, 포유동물지방유래중간엽줄기세포 그룹에 비해 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포 그룹에서, 상대적으로 세포수가 감소하였으며, S기, G2/M기에는 각각 세포수가 유의적으로 증가한 것을 확인할 수 있었다. 세포주기에서 G1기에서 S기로의 진행의 조절과 유전자 복제신호에 매우 중요한 역할을 하는 Cyclin D1의 과발현과 활성화 c-Met의 과발현은 NSC34세포의 증식이 활발히 진행되고 있음을 의미한다. 이러한 결과는 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포가 운동뉴런의 증식능력을 향상시키고 있음을 의미한다. 세포자멸이 진행되고 있는 운동뉴런에 간세포 성장인자가 과발현된 포유동물지방유래중간엽줄기세포의 세포자멸 억제효과를 확인하기 위해서, NSC34세포에 세포자멸을 유발한 후, 각각의 그룹과 간접공생배양하여, WST-1세포증식측정법을 진행하였다. 세포자멸을 유발한 NSC34세포의 생존 세포수가 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포와 공생배양한 그룹이 현저하게 높음을 확인할 수 있었다(간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포 그룹은 각각 76±2%(1일), 68.4±3%(2일), 포유동물지방유래중간엽줄기세포 그룹은 각각 65.1±3%(1일), 48.3±2%(2일), 대조군은 각각 52.1±2% (1일), 25.2±1%(2일)). Annexin V 와 PI 염색 후 유세포분석을 시행한 결과, Annexin V 양성 세포수가 대조군(28.6±0.1%)에 비해 포유동물지방유래중간엽줄기세포 그룹(22.6±0.1%)에서, 포유동물 지방유래중간엽줄기세포 그룹에 비해 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포 그룹(8.6±0.2%)에서, 유의적으로 감소되어 있는 것을 확인할 수 있었다. Western-blot분석 결과, 대조군에 비해 포유동물지방유래중간엽줄기세포 그룹에서, 포유동물 지방유래중간엽줄기세포 그룹에 비해 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포 그룹에서, 활성화된 Caspase-3와 PARP의 발현이 상대적으로 감소되어 있었으며, 활성화된 c-Met의 발현은 증가되었음을 확인할 수 있었다. 이러한 결과들은 간세포 성장인자가 과발현된 포유동물지방유래중간엽줄기세포가 NSC34세포의 세포자멸을 억제할 수 있음을 뒷받침한다.Immunofluorescence analysis confirmed the expression of hepatocyte growth factor directly. In addition, flow cytometry analysis of mammalian adipose-derived mesenchymal stem cells overexpressing hepatocyte growth factor showed that the stem cell positive marker was 99% or more and the negative marker was less than 2% there was. These results indicate that human adipose-derived mesenchymal stem cells overexpressing hepatocyte growth factor have been successfully produced through gene transfer. In order to examine the effects of mesenchymal stem cells overexpressing the hepatocyte growth factor on mesenchymal stem cell proliferation, three groups (①NSC34 cells (mouse motor neuron cells), ②NSC34 cells and mesenchymal stem cells from mammalian fats, (3) NSCC cells and mesenchymal stem cells from mammalian fat cells overexpressing hepatocyte growth factor. After indirect coculture, WST-1 cell proliferation assay and trypan blue exclusion assay As a result, it was confirmed that the number of NSC34 cells in the group cultured with mesenchymal stem cells derived from mammalian adipose tissue overexpressing hepatocyte growth factor was remarkably increased. Likewise, NSC34 cells obtained from each of the co-cultured groups were subjected to cell cycle analysis by flow cytometry. In the control group, 73.27 ± 4%, 10.68 ± 2% and 15.23 ± 1.5% were observed in the GO / G1, G, and G2 / M groups, respectively, The mesenchymal stem cell group derived from the mammalian adipose tissue overexpressing the hepatocyte growth factor was 57.12 ± 1.5%, 17.23 ± 1.1%, 24.1 ± 1.3%, respectively, in the groups of 63.1 ± 2.1%, 15.1 ± 1.83% and 20.4 ± 2% %. As a result of the cell cycle analysis, the G0 / G1 group was significantly higher in the mesenchymal stem cell group of mammalian adipose compared to the control group, and in the mesenchymal stem cell group of the mammalian adipose-derived mesenchymal stem cell group , The number of cells was relatively decreased, and the number of cells in S and G2 / M groups was significantly increased. Overexpression of Cyclin D1 and overexpression of activated c-Met, which play an important role in regulating the progression from the G1 phase to the S phase in the cell cycle and the gene replication signal, indicate that the proliferation of NSC34 cells is actively progressing. These results indicate that mesenchymal stem cells derived from mammalian adipose tissue overexpressing hepatocyte growth factor improve the proliferation ability of motor neurons. In order to confirm the effect of inhibiting apoptosis of mesenchymal stem cells derived from mammalian adipose tissue, which is overexpressing hepatocyte growth factor in apoptotic motor neurons, apoptosis of NSC34 cells was induced, And the WST-1 cell proliferation assay was performed. It was confirmed that the survival cell number of NSC34 cells inducing apoptosis was significantly higher in the group in which the mesenchymal stem cells were cultured with mesenchymal stem cells derived from mammalian fat cells overexpressing the hepatocyte growth factor The mesenchymal stem cell group in the mammalian adipose tissue was 65.1 ± 3% (1 day), 48.3 ± 2% (2 days) in the mesenchymal stem cell group, 76 ± 2% Day) and the control group was 52.1 ± 2% (1 day) and 25.2 ± 1% (2 days), respectively. Analysis of flow cytometry after Annexin V and PI staining showed that the number of Annexin V positive cells was significantly higher in the mesenchymal stem cell group (22.6 ± 0.1%) from the mammalian adipose tissue compared to the control group (28.6 ± 0.1%), (8.6. + -. 0.2%) in the mammalian adipose-derived mesenchymal stem cell group overexpressed hepatocyte growth factor as compared to the stem cell group. Western-blot analysis showed that in the mesenchymal stem cell group of the mammalian adipose-derived mesenchymal stem cell group compared to the control group, in the mesenchymal stem cell group of the mammalian adipose-derived mesenchymal stem cell group overexpressing the hepatocyte growth factor compared to the mesenchymal stem cell group of the mammalian adipose- Expression of caspase-3 and PARP was relatively decreased, and the expression of activated c-Met was increased. These results support that mesenchymal stem cells from mammalian adipose tissue overexpressing hepatocyte growth factor can inhibit apoptosis of NSC34 cells.

근위축성측삭경화증 마우스 모델인 SOD1 G93A 유전자 변이 마우스에 대한 간세포성장인자가 과발현된 포유동물지방유래중간엽 줄기세포의 치료적인 효과를 확인하기 위해서, 세 그룹(①대조군 (생리식염수), ②포유동물지방유래중간엽줄기세포, 그리고 ③간세포 성장인자가 과발현된 포유동물지방유래 중간엽줄기세포)으로 나누어, 각각의 세포를 척수 내 주입하였다. 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포를 이식한 마우스 그룹이 포유동물 지방유래중간엽줄기세포를 이식한 마우스 그룹과 대조군에 비해서 증상발현 시작시점(각각, 111±2.7일, 108±1.6일, 101±2.4일)이 늦어짐을 확인할 수 있었으며, 신경기능 저하시점(각각, 131.7±3.2일, 128.1±3.1일, 120.8±2.9일)도 늦어짐을 확인하였다. 마우스의 평균수명(각각, 141.6±4.1일, 135.8±2.7일, 127±4.1일) 또한 유의적으로 증가한 것을 확인할 수 있었다. 이러한 결과는 간세포성장인자가 과발현된 포유동물지방유래중간엽줄기세포가 SOD1 G93A 유전자 변이 마우스의 운동뉴런 기능을 증진시키고, 증상발현 시점을 늦춰주는데 기여할 수 있음을 보여준다. In order to confirm the therapeutic effect of mesenchymal stem cells derived from mammalian adipose tissue overexpressing hepatocyte growth factor (SOD1 G93A) mutant mouse model of amyotrophic lateral sclerosis, three groups (① control group (saline), ② mammal And mesenchymal stem cells from mammalian adipose tissue overexpressing hepatocyte growth factor), and each cell was injected into the spinal cord. Mice transplanted with mesenchymal stem cells overexpressing hepatocyte growth factor had significantly higher incidences of symptom onset (111 ± 2.7 days, 108, and 108 days, respectively) than those of mice transplanted with mesenchymal stem cells ± 1.6 days and 101 ± 2.4 days, respectively), and it was confirmed that the time point of neurological depression (131.7 ± 3.2 days, 128.1 ± 3.1 days, 120.8 ± 2.9 days) was also delayed. The mean lifespan of the mice (141.6 ± 4.1 days, 135.8 ± 2.7 days, and 127 ± 4.1 days, respectively) were also significantly increased. These results show that mesenchymal stem cells derived from mammalian fat cells overexpressing hepatocyte growth factor can contribute to the enhancement of motor neuron function in SOD1 G93A mutant mice and to delay the onset of symptoms.

결론적으로, 간세포성장인자가 과발현된 포유동물지방유래 중간엽줄기세포를 이용한 유전자 도입 세포치료가 운동뉴런의 증식 향상과 세포자멸 억제의 측면에서 강력한 신경 관련 잠재력을 가지고 있음을 보여주고 있으며, 난치성 질병인 근위축성측삭경화증에 대한 새로운 치료방법으로서 가능성을 제시하였다.In conclusion, it has been shown that transgenic cell therapy using mammalian adipose-derived mesenchymal stem cells overexpressing hepatocyte growth factor has potent nerve-related potential in terms of proliferation enhancement of motor neurons and inhibition of apoptosis, Suggesting the possibility of a new treatment method for amyotrophic lateral sclerosis.

Claims (5)

지방유래 중간엽줄기세포를 이용한 세포치료방법에 있어서,
상기 세포치료는 pMEX발현벡터에 간세포성장인자의 형질주입을 통해 제조된 간세포성장인자 유전자가 도입되어 간세포성장인자가 과발현된 사람을 포함하는 포유동물지방유래중간엽줄기세포를 이용한 세포치료방법
A cell therapy method using adipose derived mesenchymal stem cells,
The cell treatment may be a cell therapy method using mesenchymal stem cells derived from a mammalian adipose-derived mesenchymal stem cell, which includes a human having a hepatocyte growth factor gene overexpressed by introducing a hepatocyte growth factor gene prepared by transfection of a hepatocyte growth factor into a pMEX expression vector
(a) 포유동물지방유래 중간엽줄기세포를 배양하는 단계;
(b) 상기 배양된 포유동물지방유래 중간엽줄기세포에 형질전환 방법으로 간세포성장인자 유전자를 도입하는 단계;
(c) 상기 간세포성장인자 유전자가 도입된 포유동물지방유래 중간엽줄기세포를 선별하는 단계;
(d) 상기 선별된 포유동물지방유래 중간엽줄기세포를 선택적으로 배양하는 단계를 포함하는 포유동물지방유래 중간엽줄기세포주의 제조 방법.
(a) culturing mammalian adipose derived mesenchymal stem cells;
(b) introducing a hepatocyte growth factor gene into the cultured mammalian adipose-derived mesenchymal stem cells by a transformation method;
(c) selecting mammalian adipose derived mesenchymal stem cells into which the hepatocyte growth factor gene has been introduced;
(d) selectively culturing the selected mesenchymal stem cells derived from the mammalian adipose tissue.
제1항의 포유동물지방유래 중간엽줄기세포주를 유효성분으로 포함하는 신경계 질환 예방 또는 치료용 조성물.
A composition for preventing or treating a neurological disease, comprising the mammalian adipose derived mesenchymal stem cell line of claim 1 as an active ingredient.
제3항의 포유동물지방유래 중간엽줄기세포주를 유효성분으로 포함하는 신경계 질환 예방 또는 치료용 조성물.
A composition for preventing or treating a neurological disease, comprising the mammalian adipose derived mesenchymal stem cell line of claim 3 as an active ingredient.
제3항 또는 제4항에 있어서,
상기 신경계질환은 근위축성측삭경화증(Amyotrophic Lateral Sclerosis: ALS, 루게릭병)을 치료를 포함하는 신경질환 중 하나인 것을 특징으로 하는 신경계질환 예방 또는 치료용 조성물
The method according to claim 3 or 4,
Wherein the nervous system disease is one of neurological diseases including amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) treatment
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KR20050036136A (en) 2003-10-15 2005-04-20 허서구 Online delivery service system of gifts of celebration or condolence and method of the same
KR20060119064A (en) 2005-05-18 2006-11-24 한훈 Composition for cell therapy of amyotrophic lateral sclerosis using mannitol and stem cell derived from umbilical cord blood

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021034162A1 (en) * 2019-08-22 2021-02-25 서울대학교 산학협력단 Pharmaceutical composition comprising late-passage human mesenchymal stem cells induced to differentiate to glia-like cells as active ingredient for treatment of stroke

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