KR20230047305A - Pharmaceutical composition for preventing or treating of cognitive dysfunction comprising stem cells overexpressing neprlysin, conditioned medium thereof, and exosome purified from those - Google Patents
Pharmaceutical composition for preventing or treating of cognitive dysfunction comprising stem cells overexpressing neprlysin, conditioned medium thereof, and exosome purified from those Download PDFInfo
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Abstract
본 발명은 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀을 유효성분으로 포함하는 인지기능장애의 예방 또는 치료용 약학적 조성물에 관한 것이다. 구체적으로, 본 발명에 따른 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 또는 이들로부터 분리된 엑소좀은 신경세포를 보호하고, 동물모델에서 인지기능장애와 관련된 질환의 원인으로 인정받고 있는 아밀로이드베타의 축적을 억제하며, 아세틸콜린의 발현 수준을 증가시키고, 동물모델의 인지기능을 개선시킴으로써, 상기 줄기세포, 이의 배양액 또는 이들로부터 분리된 엑소좀은 인지기능장애의 치료에 유용하게 사용될 수 있다.The present invention relates to a pharmaceutical composition for preventing or treating cognitive dysfunction comprising stem cells overexpressing neprilysin, their culture medium, and exosomes isolated therefrom as active ingredients. Specifically, the stem cells overexpressing neprilysin according to the present invention, their culture medium, or exosomes isolated therefrom protect neurons and prevent amyloid beta, which is recognized as a cause of diseases related to cognitive dysfunction in animal models. By inhibiting accumulation, increasing the expression level of acetylcholine, and improving cognitive function in animal models, the stem cells, their cultures, or exosomes isolated from them can be usefully used for the treatment of cognitive dysfunction.
Description
본 발명은 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀을 유효성분으로 포함하는 인지기능장애의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating cognitive dysfunction comprising stem cells overexpressing neprilysin, their culture medium, and exosomes isolated therefrom as active ingredients.
인지기능장애는 사고 처리과정에 문제가 발생한 것으로, 추론능력의 상실, 망각, 학습장애, 집중력 문제 및 지능의 저하나, 이외의 다른 정신기능의 감소를 포함한다. 인지기능장애는 태아의 발육기간이나 출산, 출산 직후 또는 살아오면서 발생한 질환에 의해 생길 수 있고, 그 원인을 밝힐 수 없는 경우도 많다. 다만, 인지기능장애의 초기 원인에 해당하는 것으로 염색체 이상, 유전, 영양실조, 출산 전 약물노출, 중금속 노출, 저혈당증, 신생아 황달, 갑상선 기능 저하증, 외상, 태아 내 산소저하, 난산, 조산 등이 알려져 있다.Cognitive dysfunction is a problem in the process of thinking, and includes loss of reasoning ability, forgetting, learning difficulties, concentration problems, and decline in intelligence or other mental functions. Cognitive dysfunction can be caused by diseases that occur during the development of the fetus, childbirth, immediately after childbirth, or during life, and in many cases, the cause cannot be identified. However, chromosomal abnormalities, genetics, malnutrition, exposure to drugs before childbirth, exposure to heavy metals, hypoglycemia, neonatal jaundice, hypothyroidism, trauma, hypoxia in the fetus, dystocia, and premature birth are known to be early causes of cognitive dysfunction. there is.
인지기능장애의 하나인 치매는 정상적인 노화와 구분되는 병적 현상으로, 알츠하이머병, 혈관성 치매, 알콜 중독 등에 의한 치매, 외상에 의한 치매 등으로 구분된다. 특히, 치매 중 가장 흔한 질환으로 알려진 알츠하이머병은 대개 만성적이고 진행성으로 나타나며 점진적인 기억, 판단, 언어능력 등의 인지적 기능 감퇴와 일상생활능력, 인격, 행동양상의 장애를 보인다. 알츠하이머병의 발병 원인과 기전에 대해서는 아직까지 명확히 밝혀지지 않았으나, 신경전달물질인 아세틸콜린의 합성 감소, 아밀로이드베타의 축적, 타우(Tau) 단백질의 과인산화로 인한 신경세포의 손상이 주된 원인으로 알려져 있다.Dementia, one of the cognitive dysfunctions, is a pathological phenomenon distinct from normal aging, and is classified into Alzheimer's disease, vascular dementia, dementia caused by alcoholism, etc., and dementia caused by trauma. In particular, Alzheimer's disease, which is known as the most common disease among dementias, is usually chronic and progressive, showing gradual decline in cognitive function such as memory, judgment, and language ability, and impairment of daily living ability, personality, and behavioral patterns. Although the cause and mechanism of Alzheimer's disease have not been clarified yet, it is known that the main causes are damage to nerve cells due to decreased synthesis of acetylcholine, an accumulation of amyloid beta, and hyperphosphorylation of tau protein, which is a neurotransmitter. .
알츠하이머병 환자의 뇌조직을 관찰해보면 콜린성 신경 손상이 심각한 것이 확인되는데, 이에 기초한 알츠하이머 병리를 콜린가설(cholinergic hypothesis in Alzheimer's disease)이라 한다. 최근 콜린가설을 바탕으로 아세틸콜린의 기능저하를 유도하는 아세틸콜린에스터라제의 활성을 저해시키는 약물을 알츠하이머병 치료제로서 개발하고자 하는 시도가 많이 이루어지고 있다. 현재 알츠하이머병의 치료제로서 사용되고 있는 약물도 아세틸콜린에스터라제 저해제인 타크린(tacrine), 도네페질(donepezil), 리바스티그민(rivastigmine), 갈란타민(galantamine) 등이 있다.Observation of the brain tissues of patients with Alzheimer's disease confirms that the cholinergic nerve damage is severe, and the pathology of Alzheimer's based on this is called the cholinergic hypothesis in Alzheimer's disease. Recently, based on the choline hypothesis, many attempts have been made to develop a drug that inhibits the activity of acetylcholinesterase, which induces a decrease in the function of acetylcholine, as a therapeutic agent for Alzheimer's disease. Drugs currently used as a treatment for Alzheimer's disease include acetylcholinesterase inhibitors such as tacrine, donepezil, rivastigmine, and galantamine.
관련하여, 대한민국 공개특허 제10-2022-0061046호는 알츠하이머병 또는 인지기능장애 치료를 위한 도네페질 및 실데나필 병용 요법에 관한 것으로, 도네페질 및 실데나필 병용 투여가 도네페질 단독 투여 대비 알츠하이머병 또는 인지기능장애 개선 효과가 우수하므로, 이들의 치료에 활용될 수 있음을 개시하고 있다.In this regard, Korean Patent Publication No. 10-2022-0061046 relates to a combination therapy of donepezil and sildenafil for the treatment of Alzheimer's disease or cognitive dysfunction. Since the effect of improving disorders is excellent, it is disclosed that it can be used for their treatment.
본 발명의 목적은 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀의 용도를 제공하는 것이다.An object of the present invention is to provide uses of stem cells overexpressing neprilysin, their culture medium, and exosomes isolated therefrom.
상기 목적을 달성하기 위하여, 본 발명은 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀으로 구성된 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인지기능장애의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention is a method for preventing or treating cognitive dysfunction comprising at least one selected from the group consisting of stem cells overexpressing neprilysin, cultures thereof, and exosomes isolated therefrom as an active ingredient. It provides a pharmaceutical composition for use.
또한, 본 발명은 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀으로 구성된 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인지기능장애의 예방 또는 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing or improving cognitive dysfunction comprising at least one selected from the group consisting of stem cells overexpressing neprilysin, culture medium thereof, and exosomes isolated therefrom as an active ingredient. to provide.
본 발명에 따른 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 또는 이들로부터 분리된 엑소좀은 신경세포를 보호하고, 동물모델에서 인지기능장애와 관련된 질환의 원인으로 인정받고 있는 아밀로이드베타의 축적을 억제하며, 아세틸콜린의 발현 수준을 증가시키고, 동물모델의 인지기능을 개선시킴으로써, 상기 줄기세포, 이의 배양액 또는 이들로부터 분리된 엑소좀은 인지기능장애의 치료에 유용하게 사용될 수 있다.Stem cells overexpressing neprilysin according to the present invention, their culture medium, or exosomes isolated from them protect nerve cells and inhibit the accumulation of amyloid beta, which is recognized as a cause of diseases related to cognitive dysfunction in animal models In addition, by increasing the expression level of acetylcholine and improving the cognitive function of animal models, the stem cells, their cultures, or exosomes isolated from them can be usefully used for the treatment of cognitive dysfunction.
도 1은 본 발명의 일 실시예에서 양막 줄기세포(A) 및 상기 줄기세포 배양액(B)에서 네프릴라이신의 발현을 웨스턴블롯으로 확인한 결과 도면이다.
도 2는 본 발명의 일 실시예에서 양막 줄기세포 배양액 내 존재하는 엑소좀의 수 및 평균 크기를 확인한 결과 그래프이다.
도 3은 본 발명의 일 실시예에서 양막 줄기세포(A) 및 상기 줄기세포 배양액(B)에서 네프릴라이신의 발현을 확인한 웨스턴블롯 결과를 정량적으로 나타낸 그래프이다.
도 4는 본 발명의 일 실시예에서 양막 줄기세포 배양액(NCM), 낮은 산소농도에서 배양된 엑소좀 풍부 배양액(ERCM), 홍삼 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+RGE) 및 산삼 배양근 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+MGARE)의 아밀로이드베타 펩타이드에 의한 세포독성 억제 효과를 확인한 결과 그래프이다.
도 5는 본 발명의 일 실시예에서 양막 줄기세포 배양액(NCM), 낮은 산소농도에서 배양된 엑소좀 풍부 배양액(ERCM), 홍삼 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+RGE) 및 산삼 배양근 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+MGARE)의 인지기능 개선 효과를 수동회피실험으로 확인한 결과 그래프이다.
도 6은 본 발명의 일 실시예에서 양막 줄기세포 배양액(NCM), 낮은 산소농도에서 배양된 엑소좀 풍부 배양액(ERCM), 홍삼 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+RGE) 및 산삼 배양근 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+MGARE)의 인지기능 개선 효과를 수중미로시험으로 확인한 결과 그래프이다.
도 7은 본 발명의 일 실시예에서 양막 줄기세포 배양액(NCM), 낮은 산소농도에서 배양된 엑소좀 풍부 배양액(ERCM), 홍삼 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+RGE) 및 산삼 배양근 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+MGARE)에 의한 수중미로시험에서 수영패턴 분석 결과를, 동물모델의 뇌조직 내 아밀로이드베타 축적 수준과 비교한 결과 도면이다.
도 8은 본 발명의 일 실시예에서 양막 줄기세포 배양액(NCM), 낮은 산소농도에서 배양된 엑소좀 풍부 배양액(ERCM), 홍삼 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+RGE) 및 산삼 배양근 추출물을 첨가하여 배양된 엑소좀 풍부 배양액(ERCM+MGARE)의 동물모델의 뇌조직 내 아세틸콜린 생산 촉진효과를 확인한 결과 그래프이다.1 is a diagram showing the results of western blot analysis of the expression of neprilysin in amnion stem cells (A) and the stem cell culture medium (B) in one embodiment of the present invention.
Figure 2 is a graph showing the results of confirming the number and average size of exosomes present in the amnion stem cell culture medium in one embodiment of the present invention.
3 is a graph showing quantitatively Western blot results confirming the expression of neprilysin in amnion stem cells (A) and the stem cell culture medium (B) in one embodiment of the present invention.
Figure 4 is an embodiment of the present invention, in one embodiment of the present invention, amnion stem cell culture medium (NCM), exosome rich culture medium cultured at low oxygen concentration (ERCM), exosome rich culture medium cultured by adding red ginseng extract (ERCM + RGE) and wild ginseng It is a graph as a result of confirming the cytotoxicity inhibitory effect by the amyloid beta peptide of the exosome-rich culture medium (ERCM + MGARE) cultured by adding cultured root extract.
Figure 5 is an embodiment of the present invention, in one embodiment of the present invention, amnion stem cell culture medium (NCM), exosome rich culture medium cultured at low oxygen concentration (ERCM), exosome rich culture medium cultured by adding red ginseng extract (ERCM + RGE) and wild ginseng The graph is the result of confirming the cognitive function improvement effect of the exosome-rich culture medium (ERCM + MGARE) cultured with the addition of cultured root extract through a passive avoidance experiment.
Figure 6 is an embodiment of the present invention, in one embodiment of the present invention, amnion stem cell culture medium (NCM), exosome rich culture medium cultured at low oxygen concentration (ERCM), exosome rich culture medium cultured by adding red ginseng extract (ERCM + RGE) and wild ginseng The graph is the result of confirming the cognitive function improvement effect of the exosome-rich culture medium (ERCM + MGARE) cultured with the addition of the cultured root extract through the water maze test.
7 is an amnion stem cell culture medium (NCM), exosome-rich culture medium (ERCM) cultured at low oxygen concentration, exosome-rich culture medium (ERCM + RGE) cultured by adding red ginseng extract and wild ginseng in one embodiment of the present invention. It is a drawing comparing the swimming pattern analysis result in the water maze test by the exosome-rich culture medium (ERCM + MGARE) cultured with the addition of the cultured muscle extract and the amyloid beta accumulation level in the brain tissue of the animal model.
Figure 8 is an embodiment of the present invention, in one embodiment of the present invention, amnion stem cell culture medium (NCM), exosome rich culture medium cultured at low oxygen concentration (ERCM), exosome rich culture medium cultured by adding red ginseng extract (ERCM + RGE) and wild ginseng This is a graph as a result of confirming the effect of promoting acetylcholine production in the brain tissue of an animal model of an exosome-rich culture medium (ERCM + MGARE) cultured by adding cultured root extract.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀으로 구성된 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인지기능장애의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising at least one selected from the group consisting of stem cells overexpressing neprilysin, their culture, and exosomes isolated therefrom as an active ingredient. .
본 명세서에서 사용된 용어, "줄기세포(stem cell)"는 상대적으로 발생이 덜된 미분화된 세포로서 특정 조직의 세포로 분화할 수 있는 능력을 지닌 세포를 의미한다. 줄기세포는 분화 능력을 기준으로 만능줄기세포(pluripotent stem cell), 다분화능 줄기세포(multipotent stem cell) 또는 단분화능 줄기세포(unipotent stem cell)로 나눌 수 있다. 또한, 상기 줄기세포는 기원이 되는 세포에 따라 배아 줄기세포(embryonic stem cell), 성체 줄기세포(adult stem cell) 또는 인간 체세포로부터 제조된 유도만능 줄기세포(induced pluripotent stem cell, iPSC)로 구분될 수 있다. 구체적으로, 본 발명에 따른 줄기세포는 성체 줄기세포일 수 있다. 상기 용어, 성체 줄기세포는 성체의 조직 또는 기관에 존재하고, 원하는 세포로 분화가 가능하며 자기복제(self-renew)를 할 수 있는 미분화된 세포를 의미한다. 일례로, 본 발명에 따른 줄기세포는 양막 유래 성체줄기세포일 수 있다.As used herein, the term "stem cell" refers to a cell that has the ability to differentiate into a cell of a specific tissue as an undifferentiated cell that is relatively less developed. Stem cells can be divided into pluripotent stem cells, multipotent stem cells, or unipotent stem cells based on their differentiation ability. In addition, the stem cells can be classified into embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPSC) prepared from human somatic cells, depending on the cell of origin. can Specifically, the stem cells according to the present invention may be adult stem cells. As used herein, adult stem cells refer to undifferentiated cells that are present in adult tissues or organs, capable of differentiating into desired cells, and capable of self-renewal. For example, the stem cells according to the present invention may be amniotic membrane-derived adult stem cells.
본 발명에 따른 배양액은 상기 서술한 바와 같은 줄기세포를 낮은 산소 농도하에서 배양함으로써 수득될 수 있다. 상기 낮은 산소 농도는 통상적인 대기하에서의 평균 산소농도인 약 20%보다 낮은 농도일 수 있다. 구체적으로, 상기 낮은 산소 농도는 10% 이하, 0.1 내지 10%, 0.1 내지 8%, 0.1 내지 5%, 0.1 내지 4%, 1 내지 10%, 1 내지 8%, 1 내지 5%, 1 내지 4%, 2 내지 10%, 2 내지 8%, 2 내지 5% 또는 2 내지 4%일 수 있다. 또한, 상기 배양은 1 내지 80시간, 1 내지 70시간, 1 내지 60시간, 1 내지 50시간, 10 내지 80시간, 10 내지 70시간, 10 내지 60시간, 10 내지 50시간, 20 내지 80시간, 20 내지 70시간, 20 내지 60시간, 20 내지 50시간, 30 내지 80시간, 30 내지 70시간, 30 내지 60시간, 30 내지 50시간, 40 내지 80시간, 40 내지 70시간, 40 내지 60시간 또는 40 내지 50시간 동안 수행될 수 있다.The culture solution according to the present invention can be obtained by culturing the stem cells as described above under low oxygen concentration. The low oxygen concentration may be a concentration lower than about 20%, which is an average oxygen concentration under a normal atmosphere. Specifically, the low oxygen concentration is 10% or less, 0.1 to 10%, 0.1 to 8%, 0.1 to 5%, 0.1 to 4%, 1 to 10%, 1 to 8%, 1 to 5%, 1 to 4% %, 2 to 10%, 2 to 8%, 2 to 5% or 2 to 4%. In addition, the culture is 1 to 80 hours, 1 to 70 hours, 1 to 60 hours, 1 to 50 hours, 10 to 80 hours, 10 to 70 hours, 10 to 60 hours, 10 to 50 hours, 20 to 80 hours, 20 to 70 hours, 20 to 60 hours, 20 to 50 hours, 30 to 80 hours, 30 to 70 hours, 30 to 60 hours, 30 to 50 hours, 40 to 80 hours, 40 to 70 hours, 40 to 60 hours or It may be performed for 40 to 50 hours.
상기와 같이 배양된 배양액은 통상적인 조건에서 배양된 배양액과 비교하여, 더 높은 수준의 엑소좀을 포함할 수 있다. 즉, 상기 배양액은 엑소좀 풍부 배양액일 수 있다. 상기 용어, "엑소좀 풍부 배양액(exosome-rich conditioned medium, ERCM)"은 상기 서술한 바와 같은 특징을 갖는 줄기세포를 배양하여 수득된 배양액을 의미한다. 즉, 본 발명에 따른 약학적 조성물은 줄기세포 또는 이의 배양액뿐 아니라 이들에 포함되는 엑소좀에 의해서도 목적하는 효과를 달성할 수 있다. The culture medium cultured as described above may contain a higher level of exosomes compared to the culture medium cultured under conventional conditions. That is, the culture medium may be an exosome-rich culture medium. The term "exosome-rich conditioned medium (ERCM)" refers to a culture medium obtained by culturing stem cells having the above-described characteristics. That is, the pharmaceutical composition according to the present invention can achieve the desired effect not only by stem cells or their culture medium, but also by exosomes included in them.
상기 용어, "엑소좀(exosome)"은 여러 종류의 세포로부터 분비되는 막 구조의 세포외소포체(extracellular vesicle, EV)를 의미한다. 상기 엑소좀은 다른 세포나 조직에 결합하여 막 구성요소, 단백질, miRNA(micro RNA)를 전달하는 등 다양한 기능을 하며, 일반적으로 약 50 내지 200 ㎚의 평균 직경을 가질 수 있다. 상기 엑소좀은 이에 포함되는 마커 단백질을 이용하여 확인할 수 있다. 상기 마커 단백질은 통상의 기술분야에 알려진 모든 종류의 마커 단백질을 포함할 수 있다.The term "exosome" refers to membrane-structured extracellular vesicles (EVs) secreted from various types of cells. The exosomes bind to other cells or tissues and perform various functions such as delivering membrane components, proteins, and miRNA (micro RNA), and may generally have an average diameter of about 50 to 200 nm. The exosome can be identified using a marker protein included therein. The marker protein may include all types of marker proteins known in the art.
또한, 본 발명에 따른 배양액은 삼(參) 추출물을 포함하는 배지에서 줄기세포를 배양하여 수득된 것일 수 있다. 상기 삼은 통상의 기술분야에 알려진 모든 종류의 삼을 포함할 수 있고, 예를 들어, 상기 삼은 인삼, 산삼, 홍삼, 산양삼, 장뇌삼, 수삼 및 산삼 배양근으로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다. 상술한 바와 같은 삼을 이용하여 추출물을 제조하는 것은 통상의 기술분야에 잘 알려져 있다.In addition, the culture solution according to the present invention may be obtained by culturing stem cells in a medium containing hemp extract. The ginseng may include all types of ginseng known in the art, and for example, the ginseng may be at least one selected from the group consisting of ginseng, wild ginseng, red ginseng, wild ginseng, camphor ginseng, fresh ginseng, and wild ginseng cultured roots. It is well known in the art to prepare an extract using hemp as described above.
일례로, 상기 삼 추출물은 하기의 단계를 포함하는 제조방법에 의해 제조될 수 있다:As an example, the hemp extract may be prepared by a manufacturing method comprising the following steps:
1) 삼에 추출용매를 가하여 추출물을 제조하는 단계;1) preparing an extract by adding an extraction solvent to hemp;
2) 단계 1)의 추출물을 여과하는 단계; 및2) filtering the extract of step 1); and
3) 단계 2)의 여과된 여과물을 감압농축한 후 건조하는 단계.3) Concentrating the filtered filtrate of step 2) under reduced pressure and then drying.
또한, 상기 추출용매는 물, 알코올 또는 이의 혼합물일 수 있다. 상기 알코올은 C1 내지 C2의 저급 알코올일 수 있고, 구체적으로, 상기 알코올은 에탄올, 메탄올 또는 주정일 수 있다. 상기 추출용매는 통상의 기술자에 의해 적절한 양으로 첨가될 수 있다. 상기 추출방법은 진탕추출, 냉침추출, 환류추출 또는 초임계추출일 수 있고, 추출 조건은 방법에 따라 통상의 기술자에 의해 적절히 조절될 수 있다. 상기 추출은 1회 또는 그 이상 반복될 수 있다. 한편, 상기 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용할 수 있다. 또한, 상기 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조일 수 있다.In addition, the extraction solvent may be water, alcohol or a mixture thereof. The alcohol may be a C 1 to C 2 lower alcohol, and specifically, the alcohol may be ethanol, methanol or alcohol. The extraction solvent may be added in an appropriate amount by a person skilled in the art. The extraction method may be shaking extraction, cold brew extraction, reflux extraction or supercritical extraction, and extraction conditions may be appropriately adjusted by a person skilled in the art according to the method. The extraction may be repeated one or more times. Meanwhile, the vacuum concentration in step 3) may use a vacuum concentrator or a vacuum rotary evaporator. In addition, the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying.
본 발명에 따른 배양액은 필요에 따라 불순물을 제거하기 위해 여과되거나, 농축될 수 있다. 또한, 상기 배양액으로부터 엑소좀을 분리하는 방법 또한 통상의 기술분야에 자명하다. 상기 방법은 필요에 따라 통상의 기술자에 의해 적절히 변형되어 수행될 수 있다.The culture medium according to the present invention may be filtered or concentrated to remove impurities, if necessary. In addition, a method for isolating exosomes from the culture medium is also apparent in the art. The method may be appropriately modified and performed by a person skilled in the art if necessary.
본 명세서에서 사용된 용어, "네프릴라이신(neprilysin)"은 MME(membrane metallo-endopeptidase), NEP(neutral endopeptidase), CD10(cluster of differentiation 10), CALLA(common acute lymphoblastic leukemia antigen) 등으로도 알려진 효소 단백질로서, 소수성 잔기의 아미노기 쪽에서 펩타이드를 절단하는 아연-의존성 메탈로프로테아제(zinc-dependent metalloprotease)를 의미한다. 상기 네프릴라이신은 글루카곤(glucagon), 엔케팔린(enkephalins), 뉴로텐신(neurotensin), 옥시토신(oxytosin) 등과 같은 펩타이드 호르몬을 비활성화시키는 것으로 알려져 있다.As used herein, the term "neprilysin" is also known as MME (membrane metallo-endopeptidase), NEP (neutral endopeptidase), CD10 (cluster of differentiation 10), CALLA (common acute lymphoblastic leukemia), and the like. As an enzyme protein, it refers to a zinc-dependent metalloprotease that cleaves a peptide at the amino group of a hydrophobic residue. The neprilysin is known to inactivate peptide hormones such as glucagon, enkephalins, neurotensin, and oxytosin.
상기 네프릴라이신의 아미노산 서열 또는 상기 아미노산 서열을 암호화하는 핵산 서열은 통상의 기술분야에 잘 알려져 있다. 또한, 상기 네프릴라이신 단백질은 이와 동일하거나 이에 상응하는 생물학적 활성을 유지하는 한, 공지된 서열에서 하나 또는 그 이상의 아미노산 또는 핵산이 첨가, 결실 또는 치환된 것일 수 있다. 이때, 아미노산 또는 핵산의 치환은 전체 단백질의 전하, 즉, 극성 또는 소수성에 영향을 주지 않거나 약하게 주는 범위 내에서 수행되는 보존적 치환일 수 있다. 또한, 상기 네프릴라이신 단백질은 공지된 아미노산 또는 핵산 서열과 80% 이상, 90% 이상, 95% 이상, 97% 이상 또는 99% 이상의 상동성을 가질 수 있다. 또한, 상기 네프릴라이신 단백질은 이와 동일하거나 이에 상응하는 생물학적 활성을 유지하는 한, 네프릴라이신 단백질의 일부만을 포함할 수 있다. 일례로, 상기 네프릴라이신은 서열번호 1로 기재된 아미노산 서열로 구성될 수 있다.The amino acid sequence of neprilysin or a nucleic acid sequence encoding the amino acid sequence is well known in the art. In addition, the neprilysin protein may have one or more amino acids or nucleic acids added, deleted, or substituted in a known sequence as long as the same or equivalent biological activity is maintained. At this time, the amino acid or nucleic acid substitution may be a conservative substitution performed within a range that does not affect or weakly affects the charge of the entire protein, that is, polarity or hydrophobicity. In addition, the neprilysin protein may have a homology of at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% to a known amino acid or nucleic acid sequence. In addition, the neprilysin protein may contain only a part of the neprilysin protein as long as it maintains the same or corresponding biological activity. In one example, the neprilysin may be composed of the amino acid sequence described in SEQ ID NO: 1.
본 발명에 따른 네프릴라이신을 과발현하는 줄기세포 배양액은 상기 서술한 바와 같이 배양 조건을 조절함으로써 네프릴라이신의 발현 증가를 유도한 것일 수 있으나, 네프릴라이신을 암호화하는 유전자가 도입된 줄기세포를 배양한 배양액도 포함할 수 있다. 상기 네프릴라이신을 암호화하는 유전자를 줄기세포에 도입하는 방법은 통상의 기술분야에 잘 알려져 있으며, 필요에 따라 통상의 기술자에 의해 적절히 변형된 방법으로 수행될 수 있다. 일례로, 상기 네프릴라이신을 암호화하는 유전자를 줄기세포에 도입하는 방법은 미세주입법, 칼슘 포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염볍, DEAE-덱스트란 처리법 등을 포함할 수 있다.The stem cell culture solution overexpressing neprilysin according to the present invention may be one in which an increase in neprilysin expression is induced by controlling the culture conditions as described above, but stem cells into which a gene encoding neprilysin has been introduced may be used. A cultured broth may also be included. The method of introducing the gene encoding neprilysin into stem cells is well known in the art, and can be performed by a person skilled in the art by appropriately modified methods, if necessary. For example, a method of introducing the gene encoding neprilysin into stem cells may include a microinjection method, a calcium phosphate precipitation method, an electroporation method, a liposome-mediated transfection method, a DEAE-dextran treatment method, and the like.
상기 인지기능장애는 인지기능의 장애로 발생하거나, 질환의 발병에 의해 인기지능에 장애가 발생하는 것으로 알려진 모든 종류의 질환을 포함할 수 있다. 구체적으로, 상기 인지기능장애는 아밀로이드베타의 발현 또는 응집 수준이 정상보다 높거나 높을 위험이 있는 질환일 수 있다. 예를 들어, 상기 인지기능장애는 치매, 알츠하이머병, 노인성 치매, 초로기치매, 파킨슨병, 헌팅턴병, 경도인지장애, 대뇌 아밀로이드 맥관병증, 다운증후군, 아밀로이드성 뇌졸중, 혈관성 뇌졸중, 전신성 아밀로이드병, 더취(Dutch)형 아밀로이드증, 니만-픽병, 다발성 경화증(multiple sclerosis), 루이소체 치매, 크루츠펠트-야곱병 또는 전두측두엽 치매일 수 있다.The cognitive dysfunction may include all kinds of diseases known to occur due to cognitive function disorders or to cause cognitive impairment due to the onset of the disease. Specifically, the cognitive dysfunction may be a disease in which the expression or aggregation level of amyloid beta is higher than normal or at risk of being higher. For example, the cognitive dysfunction is dementia, Alzheimer's disease, senile dementia, early dementia, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloidogenic stroke, vascular stroke, systemic amyloidosis, Dutch ( Dutch) type amyloidosis, Niemann-Pick disease, multiple sclerosis, Lewy body dementia, Creutzfeldt-Jakob disease or frontotemporal dementia.
본 발명에 따른 약학적 조성물은 조성물 전체 중량에 대하여 유효성분인 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀을 10 내지 95 중량%로 포함할 수 있다. 또한, 본 발명의 약학적 조성물은 상기 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 추가로 포함할 수 있다.The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of stem cells overexpressing neprilysin as an active ingredient, their culture medium, and exosomes isolated therefrom, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include at least one active ingredient exhibiting the same or similar function in addition to the above active ingredient.
본 발명의 약학적 조성물은 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 이의 혼합물을 포함할 수 있다. 약학적으로 허용가능한 담체는 조성물을 생체 내에 전달하는데 적합한 것이면 모두 사용할 수 있다. 구체적으로, 상기 담체는 Merck Index, 13th ed., Merck & Co. Inc.에 기재된 화합물, 식염수, 멸균수, 링거액, 덱스트로스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 또는 이의 혼합물일 수 있다. 또한, 필요에 따라 항산화제, 완충액, 정균제 등과 같은 통상의 첨가제를 첨가할 수 있다.The pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof commonly used in biological preparations. Any pharmaceutically acceptable carrier may be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is described in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatic agents and the like may be added as needed.
상기 조성물을 제제화하는 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 첨가할 수 있다.When formulating the composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.
본 발명의 조성물은 경구용 제제 또는 비경구용 제제로 제형화될 수 있다. 경구용 제제로는 고형 제제 및 액상 제제가 포함될 수 있다. 상기 고형 제제는 정제, 환제, 산제, 과립제, 캡슐제 또는 트로키제일 수 있고, 이러한 고형 제제는 상기 조성물에 적어도 하나 이상의 부형제를 첨가하여 조제할 수 있다. 상기 부형제는 전분, 탄산칼슘, 수크로스, 락토오스, 젤라틴 또는 이의 혼합물일 수 있다. 또한, 상기 고형 제제는 윤활제를 포함할 수 있고, 그 예로는 마그네슘 스티레이트, 탈크 등이 있다. 한편, 상기 액상 제제는 현탁제, 내용액제, 유제 또는 시럽제일 수 있다. 이때, 상기 액상 제제에는 습윤제, 감미제, 방향제, 보존제 등과 같은 부형제가 포함될 수 있다.The composition of the present invention may be formulated as an oral preparation or a parenteral preparation. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, pill, powder, granule, capsule or troche, and such a solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or mixtures thereof. In addition, the solid preparation may include a lubricant, examples of which include magnesium stearate and talc. Meanwhile, the liquid formulation may be a suspension, internal solution, emulsion or syrup. In this case, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, and preservatives.
상기 비경구용 제제는 주사제, 좌제, 호흡기 흡입용 분말, 호흡기 점적 도포용 용액, 스프레이용 에어로졸제, 파우더 및 크림 등을 포함할 수 있다. 상기 주사제는 멸균된 수용액, 비수성용제, 현탁용제, 유제 등을 포함할 수 있다. 이때, 비수성용제 또는 현탁용제로서는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름이나, 에틸올레이트와 같이 주사가능한 에스테르 등이 사용될 수 있다.The preparation for parenteral use may include injections, suppositories, respiratory inhalation powders, respiratory instillation solutions, spray aerosols, powders and creams, and the like. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, and the like. At this time, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used as non-aqueous solvents or suspending solvents.
본 발명의 조성물은 목적하는 방법에 따라 경구 또는 비경구로 투여될 수 있다. 비경구 투여는 복강내, 직장내, 비강내, 피하, 정맥, 근육내 또는 흉부내 주사 방식을 포함할 수 있다.The composition of the present invention may be administered orally or parenterally, depending on the desired method. Parenteral administration may include intraperitoneal, intrarectal, intranasal, subcutaneous, intravenous, intramuscular or intrathoracic injection modes.
상기 조성물은 약학적으로 유효한 양으로 투여될 수 있다. 이는 질환의 종류, 중증도, 약물의 활성, 약물에 대한 환자의 민감도, 투여 시간, 투여 경로, 치료기간, 동시에 사용되는 약물 등에 따라 달라질 수 있다. 그러나, 바람직한 효과를 위해서, 본 발명에 따른 약학적 조성물에 포함되는 유효성분의 양은 0.0001 내지 1,000 ㎎/㎏, 구체적으로 0.001 내지 500 ㎎/㎏일 수 있다. 상기 투여는 하루에 1회 또는 수회일 수 있다.The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the administration time, the route of administration, the treatment period, and the drugs used simultaneously. However, for desirable effects, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 1,000 mg/kg, specifically 0.001 to 500 mg/kg. The administration may be once or several times a day.
본 발명의 조성물은 단독 또는 다른 치료제와 병용하여 투여될 수 있다. 병용 투여 시, 투여는 순차적 또는 동시일 수 있다.The compositions of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, administration can be sequential or simultaneous.
또한, 본 발명은 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀으로 구성된 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인지기능장애의 예방 또는 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing or improving cognitive dysfunction comprising at least one selected from the group consisting of stem cells overexpressing neprilysin, culture medium thereof, and exosomes isolated therefrom as an active ingredient. to provide.
본 발명에 따른 건강기능식품에 포함되는 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀은 상기 서술한 바와 같은 특징을 포함할 수 있다.Stem cells overexpressing neprilysin included in the health functional food according to the present invention, their culture medium, and exosomes isolated therefrom may have the above-described characteristics.
상기 건강기능식품은 이의 유효성분인 네프릴라이신을 과발현하는 줄기세포, 이의 배양액 및 이들로부터 분리된 엑소좀을 식품에 그대로 첨가하거나, 다른 식품 또는 식품 성분과 함께 사용될 수 있다. 이때, 첨가되는 유효성분의 함량은 목적에 따라 결정될 수 있고, 일반적으로는 전체 식품 중량의 0.01 내지 90 중량부일 수 있다.The health functional food can be added to food as it is or used together with other food or food ingredients, stem cells overexpressing neprilysin, their active ingredient, their culture medium, and exosomes isolated from them. At this time, the content of the active ingredient added may be determined according to the purpose, and may generally be 0.01 to 90 parts by weight of the total food weight.
상기 건강기능식품의 형태 및 종류는 특별히 제한되지 않는다. 구체적으로, 상기 건강기능식품은 정제, 캅셀, 분말, 과립, 액상 및 환의 형태일 수 있다. 상기 건강기능식품은 추가성분으로서 여러 가지 향미제, 감미제 또는 천연 탄수화물을 포함할 수 있다. 상기 감미제는 천연 또는 합성 감미제일 수 있고, 천연 감미제의 예로는 타우마틴, 스테비아 추출물 등이 있다. 한편, 합성 감미제의 예로는 사카린, 아스파르탐 등이 있다. 또한, 상기 천연 탄수화물은 모노사카라이드, 디사카라이드, 폴리사카라이드, 올리고당 및 당알코올 등일 수 있다.The shape and type of the health functional food is not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The health functional food may include various flavors, sweeteners, or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include thaumatin and stevia extract. On the other hand, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides and sugar alcohols.
본 발명의 건강기능식품은 상기 서술한 추가성분 외에, 영양제, 비타민, 전해질, 풍미제, 착색제, 펙스탄 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 등을 더 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합으로 사용될 수 있다. 상기 첨가제의 비율은 본 발명의 조성물의 100 중량부당 0.01 내지 0.1 중량부의 범위에서 선택될 수 있다.The health functional food of the present invention, in addition to the above-mentioned additional ingredients, nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectanes and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, and preservatives , glycerin, alcohol, and the like may be further included. These components may be used independently or in combination. The ratio of the additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 본 발명을 하기 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 이들에 의해 본 발명이 제한되는 것은 아니다. 본 발명의 청구범위에 기재된 기술적 사상과 실질적으로 동일한 구성을 갖고 동일한 작용 효과를 이루는 것은 어떠한 것이라도 본 발명의 기술적 범위에 포함된다.Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto. Anything that has substantially the same configuration and achieves the same effect as the technical concept described in the claims of the present invention is included in the technical scope of the present invention.
실시예 1. 엑소좀 풍부 배양액의 제조Example 1. Preparation of exosome-rich culture medium
인간 양막 줄기세포(AMSC)를 다음과 같이 배양하여 상기 세포 및 이를 배양한 배양배지에 엑소좀 함량을 증진시켰다.Human amnion stem cells (AMSC) were cultured as follows to increase the content of exosomes in the cells and the culture medium in which they were cultured.
먼저, 고려대학교 안암병원 기관윤리위원회(Institutional Review Board, IRB)의 심의를 거쳐 건강한 산모의 동의서를 받고, 제왕절개 출산 후 양막을 채취하였다. 채취된 양막으로부터 통상적인 방법으로 양막줄기세포를 수득하고, 이를 깁코(GIBCO) DMEM/F12 배양배지(Life Technologies)를 이용하여 5% 이산화탄소 및 20% 산소 조건하에서 24시간 동안 배양하였다. 배양된 세포를 1×106 개/㎖의 농도로 새로운 배지에 분주하고, 3%의 산소농도 하에서 48시간 동안 배양하였다. 배양 후, 배양액을 취하여 0.2 ㎛의 포어(pore) 크기를 갖는 필터로 여과하고, 비바 플로우(viva flow)를 사용하여 30배 농축하였다. 그 결과, 양막 줄기세포로부터 엑소좀 풍부 배양액(exosome-rich conditioned medium, ERCM)을 수득하였다.First, consent was obtained from healthy mothers after deliberation by the Institutional Review Board (IRB) of Korea University Anam Hospital, and amniotic membranes were collected after delivery by caesarean section. Amnion stem cells were obtained from the collected amnion by a conventional method, and cultured for 24 hours under 5% carbon dioxide and 20% oxygen conditions using GIBCO DMEM/F12 culture medium (Life Technologies). The cultured cells were dispensed into a new medium at a concentration of 1×10 6 cells/ml, and cultured for 48 hours under an oxygen concentration of 3%. After culturing, the culture medium was taken, filtered through a filter having a pore size of 0.2 μm, and concentrated 30 times using a viva flow. As a result, an exosome-rich conditioned medium (ERCM) was obtained from amnion stem cells.
실시예 2. 홍삼 추출물을 사용한 엑소좀 풍부 배양액의 제조Example 2. Preparation of exosome-rich culture medium using red ginseng extract
배지에 1 ㎎/㎖의 홍삼 추출물을 첨가한 것을 제외하고는, 상기 실시예 1과 동일한 조건 및 방법으로 배양하여 엑소좀 풍부 배양액을 제조하였다.Exosome-rich culture was prepared by culturing under the same conditions and methods as in Example 1, except that 1 mg/ml red ginseng extract was added to the medium.
실시예 3. 산삼 배양근 추출물을 사용한 엑소좀 풍부 배양액의 제조Example 3. Preparation of exosome-rich culture medium using wild ginseng cultured root extract
배지에 1 ㎎/㎖의 산삼 배양근 추출물을 첨가한 것을 제외하고는, 상기 실시예 1과 동일한 조건 및 방법으로 배양하여 엑소좀 풍부 배양액을 제조하였다.An exosome-rich culture medium was prepared by culturing under the same conditions and methods as in Example 1, except that 1 mg/ml wild ginseng cultured root extract was added to the medium.
실시예 4. 정상 배양액의 제조Example 4. Preparation of normal culture medium
1×106개/㎖의 농도로 분주한 세포를 20%의 산소 조건하에서 배양한 것을 제외하고는, 상기 실시예 1과 동일한 조건 및 방법으로 배양하여 정상 배양액을 제조하였다.A normal culture solution was prepared by culturing under the same conditions and method as in Example 1, except that the cells dispensed at a concentration of 1×10 6 cells/ml were cultured under 20% oxygen conditions.
실험예 1. 네프릴라이신 발현 수준 확인Experimental Example 1. Confirmation of neprilysin expression level
상기에서 수득된 양막줄기세포의 배양액과, 줄기세포 자체에 발현된 네프릴라이신(neprlysin, NEP)의 발현 수준을 웨스턴 블롯 방법으로 확인하였다.The expression levels of neprlysin (NEP) expressed in the culture medium of the amnion stem cells obtained above and in the stem cells themselves were confirmed by Western blotting.
먼저, 포유동물 단백질 추출 시약(mammalian protein extraction reagent, Pierce)을 사용하여 배양된 양막줄기세포로부터 단백질을 추출하고, BCA(bicinchoninic acid, Bradford) 방법으로 총 단백질량을 정량하였다. 정량한 단백질 및 상기 실시예에서 제조된 양말줄기세포 배양액을 SDS-폴리아크릴아마이드 겔에서 전기영동하여 분리하고 PVDF 막(polyvinyllidine difluoride membrane)으로 이동시켰다. 상기 PVDF 막에 항-NEP 항체(1:5,000, Abcam)를 처리하고 실온에서 1시간 동안 반응시키고, 이를 TBS-T 완충액으로 세척하였다. 여기에 2차 항체인 IgG-HRP-결합된 전체 항체(IgG-HRP-linked whole antibody)를 처리하고 실온에서 다시 1시간 동안 반응시켰다. 반응이 끝난 후, PVDF 막을 ECL 플러스 웨스턴 블롯팅 디텍션 시스템(ECL plus western blotting detection system, GE HealthCare)으로 확인하고, 루미노이미져(LuminoImager, Fuji Film)로 분석하였다. 그 결과, 네프릴라이신의 발현 정도를 촬영한 결과 사진을 도 1에, 상기 결과를 정량적으로 나타낸 그래프를 도 2에 나타내었다.First, proteins were extracted from cultured amnion stem cells using a mammalian protein extraction reagent (Pierce), and the total protein amount was quantified by the BCA (bicinchoninic acid, Bradford) method. The quantified proteins and the sock stem cell culture solution prepared in the above example were separated by electrophoresis on an SDS-polyacrylamide gel and transferred to a PVDF membrane (polyvinyllidine difluoride membrane). The PVDF membrane was treated with an anti-NEP antibody (1:5,000, Abcam), reacted at room temperature for 1 hour, and washed with TBS-T buffer. Here, the secondary antibody, IgG-HRP-linked whole antibody, was treated and reacted at room temperature for another 1 hour. After the reaction was over, the PVDF membrane was identified with an ECL plus western blotting detection system (GE HealthCare) and analyzed with a lumino imager (LuminoImager, Fuji Film). As a result, a photograph of the expression level of neprilysin is shown in FIG. 1 and a graph quantitatively showing the result is shown in FIG. 2 .
도 1 및 2에 나타난 바와 같이, 낮은 산소농도의 조건하에서 배양된 양막 줄기세포 및 상기 양막 줄기세포의 배양액에서 네프릴라이신의 발현 수준이 정상 조건에서 배양한 경우에 비해 유의적으로 증가하였다. 특히, 낮은 산소농도 하에서 홍삼 추출물 또는 산삼 배양근 추출물을 첨가하여 배양한 경우에는 네프릴라이신의 발현 수준이 더욱 현저히 증가하였다.As shown in FIGS. 1 and 2 , the expression level of neprilysin in amnion stem cells cultured under low oxygen concentration conditions and in the culture medium of the amnion stem cells was significantly increased compared to that when cultured under normal conditions. In particular, when the red ginseng extract or wild ginseng cultured root extract was added and cultured under low oxygen concentration, the expression level of neprilysin increased more remarkably.
실험예 2. 엑소좀 수 확인Experimental Example 2. Confirmation of the number of exosomes
상기 제조된 양막 줄기세포 배양액 내 존재하는 엑소좀의 수를 통상적인 방법으로 NTA(nanoparticle tracking analysis)를 수행함으로써 확인하고, 그 결과를 도 3 및 표 1에 나타내었다.The number of exosomes present in the prepared amnion stem cell culture medium was confirmed by performing NTA (nanoparticle tracking analysis) in a conventional manner, and the results are shown in FIG. 3 and Table 1.
도 3에 나타난 바와 같이, 배양액 내 존재하는 엑소좀의 평균 크기는 77 ㎜였다. 한편, 표 1에 나타난 바와 같이, 정상 배양액 내에서 배양액 1 ㎖ 당 1.35×109개의 많은 엑소좀이 존재하였으며, 양막 줄기세포를 낮은 산소조건에서 배양한 경우에는 정상 배양액을 기준으로 엑소좀의 수가 현저히 증가하였다. 특히, 홍삼 추출물 또는 산삼 배양근 추출물을 첨가하여 배양한 배양액의 경우 정상 배양액과 비교하여 각각 13.11배 및 30.74배까지 엑소좀의 수가 더욱 현저히 증가하하였다.As shown in Figure 3, the average size of exosomes present in the culture medium was 77 mm. On the other hand, as shown in Table 1, there were 1.35×10 9 exosomes per 1 ml of culture medium in normal culture medium, and when amnion stem cells were cultured under low oxygen conditions, the number of exosomes based on normal culture medium was markedly increased. In particular, in the case of the culture medium with the addition of the red ginseng extract or wild ginseng root extract, the number of exosomes increased significantly by 13.11 times and 30.74 times, respectively, compared to the normal culture medium.
실험예 3. 신경세포 보호 확인Experimental Example 3. Confirmation of neuronal protection
상기 수득된 양막 줄기세포 배양액의 신경세포 보호 효과를, 인지기능 형성에 관여하는 아세틸콜린(acetylcholine, ACh) 합성효소인 콜린 아세틸트랜스페라제(choline acetyltransferase, ChAT)를 발현하는 신경줄기세포주(F3.ChAT)를 사용하여 MTT 분석을 수행함으로써 확인하였다.The neural cell protection effect of the obtained amnion stem cell culture was evaluated in a neural stem cell line (F3. It was confirmed by performing MTT assay using ChAT).
먼저, F3.ChAT 세포주(KCTC 12885BP, 한국)를 1×106 개/㎖가 되도록 96웰 플레이트에 분주하였다. 이를 하룻밤 동안 배양한 후, 10 ㎕/㎖의 상기 실시예에서 제조된 배양액 및 5 μM의 아밀로이드베타(Aβ)를 처리하고, 24시간을 추가로 배양하였다. 배양이 끝난 후, 세포에 50 ㎕/㎖의 MTT를 첨가하고, 2시간을 동일한 조건으로 배양하였다. 이후, 배양배지를 제거하고 100 ㎕/㎖의 DMSO(dimethylsulfoxide)를 첨가하여 30분 동안 방치함으로써 포마잔(formazan)을 생성시켰다. 생성된 포마잔을 용출시켜 570 ㎚ 파장에서 흡광도를 측정하고, 측정된 흡광도 값으로부터 세포 생존율을 계산하여 도 4에 나타내었다.First, the F3.ChAT cell line (KCTC 12885BP, Korea) was seeded in a 96-well plate at a concentration of 1×10 6 cells/ml. After culturing it overnight, it was treated with 10 μl/ml of the culture medium prepared in the above example and 5 μM of amyloid beta (Aβ), and further cultured for 24 hours. After the incubation, 50 μl/ml of MTT was added to the cells and cultured for 2 hours under the same conditions. Thereafter, the culture medium was removed, and 100 µl/ml of dimethylsulfoxide (DMSO) was added and left for 30 minutes to generate formazan. Produced formazan was eluted, absorbance was measured at a wavelength of 570 nm, and cell viability was calculated from the measured absorbance value, which is shown in FIG. 4 .
도 4에 나타난 바와 같이, 아밀로이드베타 처리에 의해 감소한 세포 생존율이 낮은 산소 조건에서 배양된 양막 줄기세포 배양액에 의해 78.8%로 유의하게 증가하였다. 특히, 홍삼 추출물 또는 산삼 배양근 추출물을 함께 처리하여 배양된 양막 줄기세포 배양액의 경우에는 세포 생존율이 각각 82.4% 및 105.6%까지 현저히 증가하였다. As shown in Figure 4, the cell viability decreased by amyloid beta treatment was significantly increased to 78.8% by the amnion stem cell culture medium cultured under low oxygen conditions. In particular, in the case of amnion stem cell culture medium treated with red ginseng extract or wild ginseng cultured root extract, the cell viability was significantly increased to 82.4% and 105.6%, respectively.
따라서, 상기 결과로부터 본 발명에 따른 배양액이 아밀로이드베타에 의한 독성으로부터 신경세포 보호 효과를 나타냄을 알 수 있었다.Therefore, from the above results, it can be seen that the culture solution according to the present invention exhibits an effect of protecting neurons from toxicity caused by amyloid beta.
실험예 4. 인지기능 개선 확인Experimental Example 4. Confirmation of cognitive function improvement
치매 동물모델을 사용하여 상기에서 제조된 양막 줄기세포 배양액의 인지기능 개선 효과를 다음과 같은 방법으로 확인하였다.The cognitive function improvement effect of the amnion stem cell culture medium prepared above was confirmed by the following method using an animal model of dementia.
4-1. 약물의 투여4-1. administration of drugs
실험은 충북대학교 실험동물연구지원센터의 동물실험윤리위원회(Institutional Animal Care and Use Committee, IACUC)의 승인하에 동 기관의 표준작업지침서(Standard Operation Procedures, SOP)에 따라 수행되었다.Experiments were conducted in accordance with the Standard Operation Procedures (SOP) of the institution under the approval of the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Research Support Center of Chungbuk National University.
먼저, 6주령의 수컷 ICR 마우스(25 내지 30 g)는 에테르(ether)를 사용하여 흡입마취시킨 후, 뇌심부 고정장치(stereotaxic frame)에 고정하고, 각 마리당 1 ㎍/㎕의 아밀로이드베타를 5 ㎕씩 주사하여 치매를 유발하였다. 이때, 아밀로이드베타는 브레그마(bregma)로부터 뒤로 2.0 ㎜, 측면으로 2.9 ㎜ 및 배측으로 3.0 ㎜ 위치인 뇌실에 주사하였다. 24시간 후, 0.1 ㎖의 상기에서 제조된 배양액을 혈관내로 주사하였다.First, 6-week-old male ICR mice (25 to 30 g) were anesthetized with inhalation using ether, fixed in a stereotaxic frame, and 1 μg/μl amyloid beta was administered to each mouse for 5 Dementia was induced by injecting each μl. At this time, amyloid beta was injected into the ventricle located 2.0 mm posteriorly, 2.9 mm laterally, and 3.0 mm dorsally from bregma. After 24 hours, 0.1 ml of the culture solution prepared above was intravascularly injected.
4-2. 수동회피시험(passive avoidance test)4-2. passive avoidance test
수동회피시험은 아밀로이드베타를 투여하고 48시간 후부터 당일에 30분 간격으로 7회 수행되었다. 구체적으로, 어두운 방과 밝은 방으로 나뉜 수동회피상자(Med Associated)의 밝은 방에 동물을 위치시키고, 동물이 어두운 방으로 이동하면 1 ㎃로 2초 동안 전기충격을 가했다. 이후, 같은 시험에서 어두운 방에서의 전기충격으로 인한 기억을 확인하기 위해 밝은 방에서 동물이 체류하는 시간을 측정하여 기억력을 평가하였다. 그 결과, 밝은 방에서의 체류 시간을 도 5에 나타내었다.The passive avoidance test was performed 7 times at 30-minute intervals on the same day from 48 hours after administration of amyloid beta. Specifically, the animal was placed in a bright room of a passive avoidance box (Med Associated) divided into a dark room and a bright room, and when the animal moved to the dark room, an electric shock was applied at 1 mA for 2 seconds. Thereafter, in the same test, memory was evaluated by measuring the amount of time the animal stayed in a bright room to confirm the memory caused by the electric shock in the dark room. As a result, the residence time in the bright room is shown in FIG. 5 .
도 5에 나타난 바와 같이, 아밀로이드베타 처리에 의해 감소된 밝은 방에서의 체류 시간이 낮은 산소 조건에서 배양된 양막 줄기세포 배양액에 의해서 유의적으로 증가하였다. 이는 홍삼 추출물 또는 산삼 배양근 추출물을 함께 처리하여 배양된 양막 줄기세포 배양액에 의해 더욱 개선되었으며, 특히 산삼 배양근 추출물을 처리하여 배양된 양막 줄기세포 배양액은 정상 개체 수준으로 밝은 방에서의 체류시간이 회복되었다.As shown in FIG. 5 , the residence time in a bright room, which was reduced by amyloid beta treatment, was significantly increased by the amnion stem cell culture medium cultured under low oxygen conditions. This was further improved by the amnion stem cell culture medium treated with red ginseng extract or wild ginseng culture root extract together and cultured. In particular, the amnion stem cell culture medium treated with wild ginseng culture root extract recovered the residence time in a bright room to the level of normal individuals. .
4-3. 수중미로시험(water-maze test)4-3. Water-maze test
수중미로시험은 아밀로이드베타를 투여하고 매일 1회씩 7일 동안 수행되었다. 구체적으로, 원형의 수조에 물을 채우고 이를 22±2℃의 온도로 유지하면서 수면을 스티로폼으로 채워 수면 아래의 피신용 플랫폼(platform)을 가리는 히든 플랫폼 장치(hidden platform device)를 설치하였다. 동물을 수조에 넣고, 수조 주변의 표지물을 기억하여 수조 내 일정한 장소에 위치한 플랫폼을 찾아갈때까지의 소요시간을 측정하였다. 이때, 180초가 경과하여도 플랫폼을 찾지 못할 경우에는 동물을 30초 동안 플랫폼에 머물게 하여 기억을 유도하였고, CCTV 추적 시스템(CCTV tracking system)을 사용하여 수영거리 및 패턴을 확인하였다. 그 결과, 플랫폼을 찾아가는데 소요되는 시간을 도 6에 나타내었다.The water maze test was performed once a day for 7 days after administration of amyloid beta. Specifically, a hidden platform device was installed to cover a platform for hiding under the water surface by filling a circular water tank with water and maintaining it at a temperature of 22±2° C. while filling the water surface with Styrofoam. The animals were placed in a tank, and the time required to find a platform located at a certain place in the tank by remembering the markers around the tank was measured. At this time, if the platform was not found even after 180 seconds, the animal was allowed to stay on the platform for 30 seconds to induce memory, and the swimming distance and pattern were confirmed using a CCTV tracking system. As a result, the time required to find the platform is shown in FIG. 6 .
도 6에 나타난 바와 같이, 아밀로이드베타 처리에 의해 증가된 플랫폼까지의 소요시간이 낮은 산소 조건에서 배양된 양막 줄기세포 배양액에 의해 유의적으로 감소하였다. 이는 홍삼 추출물 또는 산삼 배양근 추출물을 함께 처리하여 배양된 양막 줄기세포 배양액에 의해 더욱 개선되었으며, 특히 산삼 배양근 추출물을 처리하여 배양된 양막 줄기세포 배양액은 정상 개체와 유사한 수준으로 플랫폼을 찾아가는데 소요되는 시간을 단축시켰다.As shown in FIG. 6 , the time required to reach the platform, which was increased by amyloid beta treatment, was significantly reduced by the amnion stem cell culture medium cultured under low oxygen conditions. This was further improved by the amnion stem cell culture medium treated with red ginseng extract or wild ginseng culture root extract together and cultured. shortened
실험예 5. 뇌조직 내 아밀로이드베타 함량 확인Experimental Example 5. Confirmation of amyloid beta content in brain tissue
상기에서 양막 줄기세포 배양액의 인지기능 개선 효과를 확인한 동물모델의 뇌조직을 이용하여 조직 내 아밀로이드베타의 함량을 웨스턴블롯으로 확인하였다.The content of amyloid beta in the tissue was confirmed by Western blot using the brain tissue of the animal model in which the cognitive function improvement effect of the amnion stem cell culture medium was confirmed.
먼저, 상기 실시예에서 인지기능 개선 효과를 확인한 마우스를 실험 종료 24시간 후에 희생시키고, 차가운 식염수로 뇌를 충분히 관류하여 뇌조직을 적출하였다. 적출된 뇌조직의 중량을 측정하고, 액체질소에 동결시켜 실험전까지 -80℃에 보관하였다. 측정된 뇌 무게를 기준으로 10배 부피의 차가운 PBS를 첨가하고 균질기(homogenizer)를 사용하여 균질화하였다. 1차 항체로서 항-아밀로이드베타 항체(1:1,000, Abcam)를 사용한 것을 제외하고, 상기 균질액을 이용하여 실험예 1에 기재된 것과 동일한 방법 및 조건으로 웨스턴블롯을 수행하였다. 그 결과, 뇌조직 내 아밀로이드베타 발현 수준을 확인한 결과를, 상기 수중미로시험에서 마우스의 수영패턴을 분석한 결과와 함께 도 7에 나타내었다.First, the mice whose cognitive function improvement effect was confirmed in the above example were sacrificed 24 hours after the end of the experiment, and the brain tissue was extracted by sufficiently perfusing the brain with cold saline. The weight of the extracted brain tissue was measured, frozen in liquid nitrogen, and stored at -80 ° C until the experiment. Based on the measured brain weight, 10 times the volume of cold PBS was added and homogenized using a homogenizer. Western blotting was performed in the same manner and conditions as described in Experimental Example 1 using the homogenate, except that an anti-amyloid beta antibody (1:1,000, Abcam) was used as the primary antibody. As a result, the result of confirming the expression level of amyloid beta in the brain tissue is shown in FIG. 7 together with the result of analyzing the swimming pattern of the mouse in the water maze test.
도 7에 나타난 바와 같이, 정상동물과 비교하여 아밀로이드베타를 투여한 마우스는 플랫폼을 찾기까지 시간이 오래걸렸으며, 아밀로이드베타 발현 수준 또한 현저히 높았다. 한편, 양막 줄기세포 배양액을 투여한 경우, 플랫폼을 찾는 시간이 유의적으로 감소하였으며, 아밀로이드베타의 발현 수준 또한 감소하였다. 이는 정상 조건에서 배양된 배양액과 비교하여, 낮은 산소조건에서 배양한 배양액에서 현저하였고, 특히 홍삼 추출물 또는 산삼 배양근 추출물을 첨가하여 배양한 배양액은 더욱 현저한 효과를 나타내었다.As shown in Figure 7, compared to normal animals, mice administered with amyloid beta took a long time to find the platform, and the amyloid beta expression level was also significantly higher. On the other hand, when the amnion stem cell culture medium was administered, the time to find the platform was significantly decreased, and the expression level of amyloid beta was also decreased. This was remarkable in the culture medium cultured under low oxygen conditions compared to the culture medium cultured under normal conditions, and in particular, the culture medium cultured with the addition of red ginseng extract or wild ginseng cultured root extract showed a more remarkable effect.
따라서, 상기 결과로부터 양막 줄기세포 배양액이 치매의 원인으로 알려진 아밀로이드베타의 축적으로 인한 인지기능 장애를 회복시키는 것을 알 수 있었다.Therefore, from the above results, it was found that the amnion stem cell culture medium restores cognitive dysfunction due to the accumulation of amyloid beta known as the cause of dementia.
실험예 6. 뇌조직 내 아세틸콜린 확인Experimental Example 6. Confirmation of acetylcholine in brain tissue
상기에서 양막 줄기세포 배양액의 인지기능 개선 효과를 확인한 동물모델의 뇌조직을 이용하여 조직 내 아세틸콜린의 함량을 앰플렉스 레드 아세틸콜린/아세틸콜린에스테라제 어세이 키트(Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit, Invitrogen)를 사용하여 확인하였다. 실험은 상기 실시예에서 뇌조직을 균질화시킨 균질액을 사용하여 키트 제조사의 프로토콜에 따라 수행하였다. 그 결과, 아세틸콜린의 발현 수준을 확인한 결과를 도 8에 나타내었다.Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit) , Invitrogen). The experiment was performed according to the protocol of the kit manufacturer using the homogenate obtained by homogenizing the brain tissue in the above example. As a result, the results of confirming the expression level of acetylcholine are shown in FIG. 8 .
도 8에 나타난 바와 같이, 아밀로이드베타를 투여한 마우스는 아세틸콜린의 발현 수준이 정상 마우스와 비교하여 현저히 낮아졌다. 그러나, 이는 양막 줄기세포 배양액 처리에 의해 회복되었으며, 특히 산삼 배양근 추출물을 처리하여 배양한 배양액의 경우 정상 마우스와 비슷한 정도로 아세틸콜린의 수준이 회복되었다.As shown in FIG. 8, the expression level of acetylcholine in mice administered with amyloid beta was significantly lower than that of normal mice. However, this was recovered by treatment with the amnion stem cell culture medium, and in particular, in the case of the culture medium treated with wild ginseng culture root extract, the level of acetylcholine was restored to a similar extent to that of normal mice.
Claims (9)
A pharmaceutical composition for preventing or treating cognitive dysfunction comprising, as an active ingredient, at least one selected from the group consisting of stem cells overexpressing neprilysin, culture medium thereof, and exosomes isolated therefrom.
According to claim 1, wherein the stem cells are amniotic membrane-derived stem cells, a pharmaceutical composition for the prevention or treatment of cognitive dysfunction.
According to claim 1, wherein the culture medium obtained by culturing the stem cells under an oxygen concentration of 10% or less, a pharmaceutical composition for the prevention or treatment of cognitive dysfunction.
According to claim 1, wherein the culture medium is obtained by culturing stem cells in a medium containing hemp (參) extract, a pharmaceutical composition for the prevention or treatment of cognitive dysfunction.
The pharmaceutical composition for preventing or treating cognitive dysfunction according to claim 4, wherein the ginseng is at least one selected from the group consisting of ginseng, wild ginseng, red ginseng, wild ginseng, camphor ginseng, fresh ginseng and cultured wild ginseng roots.
The pharmaceutical composition for preventing or treating cognitive dysfunction according to claim 1, wherein the disease associated with cognitive dysfunction is a disease in which the expression or aggregation level of amyloid beta is higher than normal or at high risk.
The method of claim 1, wherein the disease associated with cognitive dysfunction is dementia, Alzheimer's disease, senile dementia, early dementia, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down's syndrome, amyloidogenic stroke, vascular stroke, systemic A pharmaceutical composition for the prevention or treatment of amyloid disease, Dutch-type amyloidosis, Niemann-Pick disease, multiple sclerosis, Lewy body dementia, Creutzfeldt-Jacob disease or frontotemporal dementia, cognitive dysfunction.
The pharmaceutical composition for preventing or treating cognitive dysfunction according to claim 1, wherein the neprilysin is composed of the amino acid sequence represented by SEQ ID NO: 1.
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| CN202211190388.7A CN115887497A (en) | 2021-09-30 | 2022-09-28 | Use of stem cells overexpressing neprilysin, their culture medium and exosomes isolated from them |
| EP22198922.1A EP4159228A1 (en) | 2021-09-30 | 2022-09-30 | Pharmaceutical composition containing neprilysin-overexpressing stem cell, conditioned medium thereof, and exosome isolated therefrom as active ingredient for prevention or treatment of cognitive impairment |
| US17/937,096 US20230096790A1 (en) | 2021-09-30 | 2022-09-30 | Pharmaceutical composition containing neprilysin-overexpressing stem cell, conditioned medium thereof, and exosome isolated therefrom as active ingredient for prevention or treatment of cognitive impairment |
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