KR101101835B1 - Compositions for Enhancing Viability and Proliferation of Stem Cell - Google Patents
Compositions for Enhancing Viability and Proliferation of Stem Cell Download PDFInfo
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- KR101101835B1 KR101101835B1 KR1020090046247A KR20090046247A KR101101835B1 KR 101101835 B1 KR101101835 B1 KR 101101835B1 KR 1020090046247 A KR1020090046247 A KR 1020090046247A KR 20090046247 A KR20090046247 A KR 20090046247A KR 101101835 B1 KR101101835 B1 KR 101101835B1
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- stem cells
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- pluripotent
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Abstract
본 발명은 서열목록 제1서열에 기재된 ADC(arginine decarboxylase) 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 포함하는 줄기세포 생존능 또는 증식능 개선용 조성물 및 상기 서열목록에 기재된 ADC코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 이용하여 줄기세포 생존능 또는 증식능을 개선하는 방법에 관한 것이다. 본 발명은 줄기세포에 ADC 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 형질도입 함으로써, 상기 줄기세포의 세포사멸(apoptosis) 및 산화적 스트레스(oxidative stress)로 의한 손상을 억제하고, 줄기세포의 생존능 또는 증식능을 매우 효과적으로 개선시키는 효과를 나타낸다. 또한, 본 발명은 유전자 치료, 즉 줄기세포를 이용하는 신경질환의 예방 및 치료법에 응용시킬 수 있으며, 상기 신경질환 예방 및 치료 효능을 가지는 의약으로서의 기초적인 자료를 제공한다.The present invention provides a composition for improving stem cell viability or proliferation, including a gene carrier comprising an ADC (arginine decarboxylase) coding nucleotide sequence described in SEQ ID NO: 1, and a gene carrier comprising an ADC coding nucleotide sequence described in the sequence list. It relates to a method for improving stem cell viability or proliferation using. The present invention, by transducing a gene transporter containing an ADC coding nucleotide sequence to stem cells, thereby inhibiting damage caused by apoptosis and oxidative stress of the stem cells, stem cell viability or proliferation ability It has the effect of improving very effectively. In addition, the present invention can be applied to gene therapy, that is, the prevention and treatment of neurological diseases using stem cells, and provides basic data as a medicament having the effects of preventing and treating the neurological diseases.
ADC, 알기닌 탈탄산효소, 줄기세포, 생존, 증식 ADC, Arginine Decarboxylase, Stem Cells, Survival, Proliferation
Description
본 발명은 줄기세포 생존능 및 증식능 개선용 조성물과 줄기세포 생존능 및 증식능을 개선하는 방법에 관한 것이다.The present invention relates to a composition for improving stem cell viability and proliferation and a method for improving stem cell viability and proliferation.
아르기닌 탈탄산효소(arginine decarboxylase: ADC)는 L-아르기닌을 효소적 탈탄산화시켜 아그마틴을 생성하는 효소이다3,4.Arginine decarboxylase (ADC) is an enzyme that produces agmatine by enzymatic decarbonization of L-arginine 3,4 .
효소학적인 면에서, 아르기닌 탈탄산효소(arginine decarboxylase: ADC)는 화학적 반응을 촉매하는 효소이며, 또 다른 이름은 SpeA 및 L-아르기닌 카르복실-리아제라고 한다. 아르기닌 탈탄산효소(arginine decarboxylase: ADC)는 L-아르기닌을 효소적 탈탄산화시켜 아그마틴과 이산화탄소(CO2)를 생성한다3,4. 이 효소는 리아제군(family of lyases) 특히 카르복실-리아제에 속하며 탄소-탄소 결합을 절단한다. 또한, 요소 회로, 아미노 그룹의 대사 및 글루타메이트 대사에 관여하 며, 조효소로서 피리독살 포스페이트(pyridoxal phosphate)를 이용한다. ADC는 랫트와 인간 뇌 부위에서 폭넓게 발현된다.In enzymatic terms, arginine decarboxylase (ADC) is an enzyme that catalyzes chemical reactions and another name is SpeA and L-arginine carboxyl-lyase. Arginine decarboxylase (ADC) produces enzymatic decarbonation of L-arginine to produce agmatine and carbon dioxide (CO 2 ) 3,4 . This enzyme belongs to the family of lyases, especially carboxyl-lyases, and cleaves carbon-carbon bonds. It is also involved in urea cycle, amino group metabolism and glutamate metabolism, and uses pyridoxal phosphate as coenzyme. ADCs are widely expressed in rat and human brain regions.
아그마틴은 내재성 클로니딘-대체 물질, α2-아드레날린(adrenergic)과 이미다졸린(imidazoline)의 리셉터에 대한 효능제, 및 NMDA(N-methyl-D-aspartate) 리셉터의 길항제이다7.Agmatine is an endogenous clonidine-replacement substance, an agonist for α2-adrenergic and imidazoline receptors, and an antagonist of NMDA (N-methyl-D-aspartate) receptors 7 .
현재 아르기닌 탈탄산화 효소를 발현하는 재조합체(recombinant)를 이용하여 아그마틴을 생산하는 방법(미국특허공개공보 제20040086985호), 외인성(exogenous) 아르기닌 탈탄산화 효소 유전자를 식물에 형질 도입하여 식물의 스트레스를 억제시키는 방법(일본특허공개 제2006-325554호) 및 아르기닌 탈탄산화 효소를 이용하여 암을 치료하는 방법이 보고되었다(미국특허등록 제06261557호). A method for producing agmatine using a recombinant expressing an arginine decarboxylase (US Patent Publication No. 20040086985), stress of a plant by introducing an exogenous arginine decarboxylase gene into a plant Has been reported (Japanese Patent Laid-Open Publication No. 2006-325554) and a method for treating cancer using arginine decarbonase (US Patent No. 06261557).
그러나, 현재까지 아르기닌 탈탄산효소(ADC) 유전자를 이용하여 줄기세포 특히 다능성 신경줄기세포주의 생존능 또는 증식능을 향상시킬 수 있다는 보고는 발표된 바 없다.However, no reports have been made so far that arginine decarboxylase (ADC) genes may be used to improve the viability or proliferation of stem cells, particularly pluripotent neural stem cell lines.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 인간 줄기세포의 생존율 및 증식율을 개선하고자 노력하였고, 그 결과 인간 ADC (arginine decarboxylase) 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 줄기세포에 형질 도입시켜 궁극적으로 줄기세포의 생존율 또는 증식율이 매우 효과적으로 향상되는 것을 발견함으로써, 본 발명을 완성하게 되었다.The present inventors have tried to improve the survival rate and proliferation rate of human stem cells, and as a result, the gene carrier containing human ADC (arginine decarboxylase) coding nucleotide sequence is transduced into the stem cells and ultimately the survival or proliferation rate of stem cells is very effective. By discovering the improvement, the present invention has been completed.
따라서 본 발명의 목적은 ADC 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 포함하는 줄기세포 생존능 및 증식능 개선용 조성물을 제공하는 데 있다.Accordingly, an object of the present invention is to provide a composition for improving stem cell viability and proliferation, including a gene carrier including an ADC coding nucleotide sequence.
본 발명의 다른 목적은 ADC코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 이용하여 줄기세포 생존능 및 증식능을 개선하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for improving stem cell viability and proliferation by using a gene carrier including an ADC-coding nucleotide sequence.
본 발명의 또 다른 목적은 뇌질환, 특히 뇌신경질환의 예방 또는 치료방법을 제공하는 데 있다.Still another object of the present invention is to provide a method for preventing or treating brain diseases, particularly cerebral nerve diseases.
본 발명의 또 다른 목적은 뇌질환, 특히 뇌신경질환 예방 또는 치료용 약물(medicament)을 제조하기 위한 신규한 용도(use)를 제공하는 데 있다.Another object of the present invention is to provide a novel use for the manufacture of a drug (medicament) for the prevention or treatment of brain diseases, in particular cerebral neurological diseases.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 서열목록 제1서열에 기재된 ADC(arginine decarboxylase) 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 포함하는 줄기세포 생존능 및 증식능 개선용 조성물을 제공한다.According to an aspect of the present invention, the present invention provides a composition for improving stem cell viability and proliferation, including a gene carrier comprising an ADC (arginine decarboxylase) coding nucleotide sequence described in SEQ ID NO: 1.
본 발명의 다른 양태에 따르면, 본 발명은 상기 조성물을 투여대상(subject)에게 투여하는 단계를 포함하는 뇌질환, 특히 뇌신경질환 예방 및 치료방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for preventing and treating a brain disease, in particular a brain neurological disease, comprising administering the composition to a subject.
본 발명의 또 다른 양태에 따르면, 본 발명은 뇌질환, 특히 뇌신경질환 예방 또는 치료용 약물(medicament)을 제조하기 위한 ADC 유전자를 포함하는 유전자 전달체가 포함된 조성물의 용도(use)를 제공한다.According to another aspect of the invention, the invention provides the use of a composition comprising a gene carrier comprising an ADC gene for the manufacture of a medicament for the prevention or treatment of brain diseases, in particular cerebral neurological diseases.
본 발명자들은 인간 줄기세포의 생존율 또는 증식율을 개선하고자 노력하였고, 그 결과 인간 ADC (arginine decarboxylase) 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 줄기세포에 형질 도입시켜 궁극적으로 줄기세포의 생존율 또는 증식율이 매우 효과적으로 향상되는 것을 발견함으로써, 본 발명을 완성하게 되었다.The present inventors have tried to improve the survival rate or proliferation rate of human stem cells, and as a result, the gene carrier containing human ADC (arginine decarboxylase) coding nucleotide sequence is transduced into the stem cells and ultimately the survival or proliferation rate of the stem cells is very effective. By discovering the improvement, the present invention has been completed.
본 발명의 줄기세포 생존율 또는 증식률 개선에 있어서, ADC 유전자가 사용되며 이는 다양한 재조합 바이러스 벡터에 삽입되어 이용된다. 이 경우, ADC 유전자는 적합한 발현 컨스트럭트에 포함된다. 상기 발현 컨스트럭트에서 ADC 유전자는 프로머터에 작동적으로 연결된다. 본 명세서에서, 용어 “작동적으로 결합된”은 핵산 발현 조절 서열 (예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상 기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다. 본 발명에 있어서, ADC 유전자에 결합된 프로모터는, 바람직하게는 동물세포, 보다 바람직하게는 포유동물 세포에서 작동하여 ADC 유전자의 전사를 조절할 수 있는 것으로서, 포유동물 바이러스로부터 유래된 프로모터 및 포유동물 세포의 지놈으로부터 유래된 프로모터를 포함하며, 예컨대, CMV (cytomegalo virus) 프로모터, 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk 프로모터, RSV 프로모터, EF1 알파 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터, 인간 GM-CSF 유전자의 프로모터, 암세포 특이적 프로모터 (예컨대, TERT 프로모터, PSA 프로모터, PSMA 프로모터, CEA 프로모터, E2F 프로모터 및 AFP 프로모터) 및 조직 특이적 프로모터 (예컨대, 알부민 프로모터)를 포함하나, 이에 한정되는 것은 아니다. 가장 바람직하게는, SV40 프로모터이다.In improving the stem cell survival rate or proliferation rate of the present invention, the ADC gene is used, which is inserted into various recombinant viral vectors. In this case, the ADC gene is included in a suitable expression construct. In the expression construct the ADC gene is operably linked to the promoter. As used herein, the term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, whereby The regulatory sequences will control the transcription and / or translation of said other nucleic acid sequences. In the present invention, the promoter coupled to the ADC gene is preferably capable of controlling transcription of the ADC gene by operating in animal cells, more preferably mammalian cells, promoters and mammalian cells derived from mammalian viruses. Promoters derived from the genome of, for example, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HS tk promoter, RSV promoter, EF1 alpha promoter, metallothionein Promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, cancer cell specific promoter (e.g. , TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter and AFP promoter) and tissue specific promoters (eg, albumin promoters). Most preferably, it is the SV40 promoter.
본 발명의 바람직한 구현예에 따르면, ADC 유전자가 포함되는 재조합 바이러스는 레트로바이러스, 배큘로바이러스, 아데노바이러스, 아데노-관련 바이러스(Adeno-associated viruses: AAV), 렌티바이러스, 헤르페스 심플렉스 바이러스 또는 배시니아 바이러스이고, 가장 바람직하게는 레트로바이러스이다.According to a preferred embodiment of the invention, the recombinant virus comprising the ADC gene is a retrovirus, baculovirus, adenovirus, Adeno-associated viruses (AAV), lentivirus, herpes simplex virus or basinia Viruses, most preferably retroviruses.
(a) 레트로바이러스 (a) retrovirus
레트로바이러스는 자신의 유전자를 숙주의 게놈으로 삽입시키고, 대량의 외 래 유전 물질을 운반할 수 있으며, 감염시킬 수 있는 세포의 스펙트럼이 넓기 때문에 유전자 전달 벡터로서 많이 이용되고 있다.Retroviruses are widely used as gene transfer vectors because they insert their genes into the host's genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells that can infect them.
레트로바이러스 벡터를 구축하기 위하여, ADC 유전자는 레트로바이러스의 서열 대신에 레트로바이러스 게놈에 삽입되어 복제 불능의 바이러스를 생산한다. 바이리온을 생산하기 위하여, gag, pol 및 env 유전자를 포함하지만 LTR (long terminal repeat)와 Ψ서열이 없는 패키징 세포주를 구축한다 (Mann et al., Cell, 33:153-159(1983)). ADC 유전자를 포함하는 재조합 플라스미드를 상기 세포주에 이입하면, Ψ서열은 재조합 플라스미드의 RNA 전사체의 생산을 가능하게 하며, 이 전사체는 바이러스로 패키징되고, 바이러스는 배지로 배출된다 (Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513(1988)). 재조합 레트로바이러스를 함유하는 배지를 수집하고 농축하여 유전자 전달 시스템으로 이용한다.To construct the retroviral vector, the ADC gene is inserted into the retroviral genome instead of the sequence of the retrovirus to produce a nonreplicating virus. To produce the virion, a packaging cell line containing the gag, pol and env genes but no LTR (long terminal repeat) and Ψ sequences is constructed (Mann et al., Cell , 33: 153-159 (1983)). When the recombinant plasmid containing the ADC gene is introduced into the cell line, the Ψ sequence enables the production of the RNA transcript of the recombinant plasmid, which is packaged into a virus and the virus is discharged into the medium (Nicolas and Rubinstein " Retroviral vectors, "In: Vectors: A survey of molecular cloning vectors and their uses , Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988)). The medium containing the recombinant retrovirus is collected, concentrated and used as a gene delivery system.
2세대 레트로바이러스 벡터를 이용한 유전자 전달이 발표되었다. Kasahara et al. Science, 266:1373-1376(1994))는 몰로니 뮤라인 류케미아 바이러스의 변이체를 제조하였고, 여기에서 EPO (erythropoietin) 서열을 엔벨로프 부위에 삽입하여 새로운 결합 특성을 갖는 키메릭 단백질을 생산하였다. 본 발명의 유전자 치료도 이와 같은 2세대 레트로바이러스 벡터의 구축 전략에 따라 제조할 수 있다.Gene delivery using second generation retroviral vectors has been announced. Kasahara et al. Science , 266: 1373-1376 (1994)) prepared a variant of the Moloney murine leuchemia virus, in which an erythropoietin (EPO) sequence was inserted into the envelope site to produce a chimeric protein with new binding properties. The gene therapy of the present invention can also be prepared according to the construction strategy of this second generation retroviral vector.
(b) 아데노바이러스 (b) adenovirus
아데노바이러스는 중간 정도의 게놈 크기, 조작의 편의성, 높은 타이터, 광범위한 타깃세포 및 우수한 감염성 때문에 유전자 전달 벡터로서 많이 이용되고 있다. 게놈의 양 말단은 100-200 bp의 ITR (inverted terminal repeat)를 포함하며, 이는 DNA 복제 및 패키징에 필수적인 시스 엘리먼트이다. 게놈의 E1 영역 (E1A 및 E1B)은 전사 및 숙주 세포 유전자의 전사를 조절하는 단백질을 코딩한다. E2 영역 (E2A 및 E2B)은 바이러스 DNA 복제에 관여하는 단백질을 코딩한다.Adenoviruses are widely used as gene transfer vectors because of their moderate genome size, ease of manipulation, high titers, broad target cells and excellent infectivity. Both ends of the genome contain 100-200 bp of Inverted Terminal Repeat (ITR), which is a cis element essential for DNA replication and packaging. The El regions of the genome (E1A and E1B) encode proteins that regulate transcription and transcription of host cell genes. The E2 regions (E2A and E2B) encode proteins that are involved in viral DNA replication.
현재 개발된 아데노바이러스 벡터 중에서, E1 영역이 결여된 복제 불능 아데노바이러스가 많이 이용되고 있다. 한편, E3 영역은 통상적인 아데노바이러스 벡터에서 제거되어 외래 유전자가 삽입되는 자리를 제공한다(Thimmappaya, B. et al., Cell, 31:543-551(1982); 및 Riordan, J. R. et al., Science, 245:1066-1073(1989)). 따라서, ADC 유전자는 결실된 E1 영역 또는 E3 영역에 삽입되는 것이 바람직하고, 보다 바람직하게는 결실된 E3 영역에 삽입된다. 또한, 상기 삽입 서열들은 결실된 E4 영역에도 삽입될 수 있다. 본 명세서에서 바이러스 게놈 서열과 관련하여 사용되는 용어, “결실”은 해당 서열이 완전히 결실된 것뿐만 아니라, 부분적으로 결실된 것도 포함하는 의미를 가진다.Among the adenovirus vectors currently developed, non-replicating adenoviruses lacking the E1 region are widely used. On the other hand, the E3 region is removed from conventional adenovirus vectors to provide a site for insertion of foreign genes (Thimmappaya, B. et al., Cell , 31: 543-551 (1982); and Riordan, JR et al., Science , 245: 1066-1073 (1989). Thus, the ADC gene is preferably inserted into the deleted E1 region or E3 region, more preferably the deleted E3 region. The insertion sequences can also be inserted into the deleted E4 region. As used herein, the term "deletion" as used in connection with a viral genomic sequence has the meaning including not only a complete deletion of the sequence, but also a partial deletion.
아데노바이러스는 42개의 상이한 혈청형 및 A-F의 서브그룹을 갖는다. 이 중에서, 서브그룹 C에 속하는 아데노바이러스 타입 5가 아데노바이러스 벡터를 얻기 위한 가장 바람직한 출발물질이다. 아데노바이러스 타입 5에 대한 생화학적 및 유전적 정보는 잘 알려져 있다.Adenoviruses have 42 different serotypes and subgroups of A-F. Of these,
아데노바이러스에 의해 운반되는 외래 유전자는 에피좀과 동일한 방식으로 복제되며, 이에 숙주세포에 대해 유전적독성이 매우 낮다. Foreign genes carried by adenoviruses replicate in the same way as episomes, with very low genetic toxicity to host cells.
(c) AAV 벡터 (c) AAV vector
아데노-관련 바이러스 (AAV)는 비분열 세포을 감염시킬 수 있고, 다양한 종류의 세포에 감염할 수 있는 능력을 갖고 있기 때문에 본 발명의 유전자 전달 시스템으로 적합하다. AAV 벡터의 제조 및 용도에 대한 상세한 설명은 미국 특허 제 5,139,941 호 및 제 4,797,368 호에 상세하게 개시되어 있다.Adeno-associated virus (AAV) is suitable as the gene delivery system of the present invention because it can infect non-dividing cells and has the ability to infect various kinds of cells. Details of the preparation and use of AAV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368.
유전자 전달 시스템으로서의 AAV에 대한 연구는 LaFace et al, Viology, 162:483486(1988), Zhou et al., Exp. Hematol. (NY), 21:928-933(1993), Walsh et al, J. Clin. Invest., 94:1440-1448(1994) 및 Flotte et al., Gene Therapy, 2:29-37(1995)에 개시되어 있다. 최근에, AAV 벡터는 낭포성 섬유증의 치료제로서 임상 I을 실시하고 있다.Studies on AAV as a gene delivery system are described in LaFace et al, Viology , 162: 483486 (1988), Zhou et al., Exp. Hematol. (NY), 21: 928-933 (1993), Walsh et al, J. Clin. Invest. 94: 1440-1448 (1994) and Flotte et al., Gene Therapy , 2: 29-37 (1995). Recently, AAV vectors have undergone clinical I as a therapeutic agent for cystic fibrosis.
전형적으로, AAV 바이러스는 두 개의 AAV 말단 리피트가 옆에 위치되어 있는 목적의 유전자 서열 (Env 유전자 및 HPV L1 유전자)을 포함하는 플라스미드 (McLaughlin et al., J. Virol., 62:1963-1973(1988); 및 Samulski et al., J. Virol., 63:3822-3828(1989)) 및 말단 리피트가 없는 야생형 AAV 코딩 서열을 포함하는 발현 플라스미드 (McCarty et al., J. Virol., 65:2936-2945( 1991))를 동시 형질 전환시켜 제조된다. Typically, AAV viruses are characterized by plasmids (McLaughlin et al., J. Virol. , 62: 1963-1973, comprising a gene sequence of interest ( Env gene and HPV L1 gene) with two AAV terminal repeats located next to them) . 1988); and Samulski et al., J. Virol. , 63: 3822-3828 (1989) and expression plasmids containing wild type AAV coding sequences without terminal repeats (McCarty et al., J. Virol. , 65: 2936-2945 (1991)).
(d) 다른 바이러스 벡터 (d) other viral vectors
다른 바이러스 벡터들도 본 발명의 DNA 벡터에 이용할 수 있다. 배시니아 바이러스 (Puhlmann M. et al., Human Gene Therapy 10:649-657(1999); Ridgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses. Rodriguez and Denhardt, eds. Stoneham: Butterworth, 467-492(1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press, 117-148( 1986) 및 Coupar et al., Gene, 68:1-10(1988)), 렌티바이러스 (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)) 또는 헤르페스 심플렉스 바이러스 (Chamber R., et al., Proc. Natl. Acad. Sci USA 92:1411-1415(1995))로부터 유래된 벡터들도, ADC 유전자를 세포내로 운반할 수 있는 운반 시스템으로 이용할 수 있다.Other viral vectors can also be used in the DNA vector of the present invention. Basinian virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999); Ridgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses.Rodrigue and Denhardt, eds Stoneham: Butterworth, 467-492 (1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer.New York: Plenum Press, 117-148 (1986) and Coupar et al., Gene , 68: 1-10 (1988)), lentiviruses (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)) or vectors derived from the herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92: 1411-1415 (1995)) can also carry ADC genes intracellularly. Available as a transport system.
줄기세포는 크게 두 종류로 구별된다: 배아줄기세포 (ES) 및 배아생식세포 (EG)를 포함하는 전능성 줄기세포 (pluripotent stem cell)와 다능성 줄기세포 (multipotent stem cell).Stem cells are broadly divided into two types: pluripotent stem cells and multipotent stem cells, including embryonic stem cells (ES) and embryonic germ cells (EG).
본 발명의 바람직한 구현예에 따르면, 본 발명에서 이용하는 줄기세포는 전능성 줄기세포 또는 다능성 줄기세포이다. 보다 바람직하게는, 본 발명에서 이용하는 줄기세포는 다능성 중간엽 줄기세포, 다능성 조혈모 줄기세포, 다능성 신경 줄기세포, 다능성 간 줄기세포, 다능성 췌장 줄기세포 또는 다능성 표피줄기세포이며, 가장 바람직하게는 다능성 신경줄기세포를 이용한다.According to a preferred embodiment of the present invention, the stem cells used in the present invention are pluripotent stem cells or pluripotent stem cells. More preferably, the stem cells used in the present invention are pluripotent mesenchymal stem cells, pluripotent hematopoietic stem cells, pluripotent neural stem cells, pluripotent stem cells, pluripotent pancreatic stem cells or pluripotent epidermal stem cells Most preferably, pluripotent neural stem cells are used.
본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 줄기세포의 세포사멸(apoptosis)을 억제하는데 매우 효과적이다. 보다 구체적으로는, ADC 코딩하는 유전자 전달체를 신경줄기세포에 형질 도입시킴으로써, Erk1/2(extracellular signal-regulated kinases 1 and 2) 활성과 Bcl2(B-cell CLL/lymphoma 2)의 발현을 증가시키고 AIF (apoptosis inducible factor)의 발현을 억제하여 궁극적으로 다능성 신경줄기세포의 사멸을 매우 효과적으로 억제시킨다.According to a preferred embodiment of the present invention, the composition of the present invention is very effective in inhibiting apoptosis of stem cells. More specifically, transduction of ADC-encoding gene transporters into neural stem cells results in increased Erk1 / 2 (extracellular signal-regulated
본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 줄기세포의 세포주기를 G2-M기에 유지시킴으로써 줄기세포 특히 다능성 신경줄기세포의 증식능을 개선시킬 수 있다.According to a preferred embodiment of the present invention, the composition of the present invention can improve the proliferative capacity of stem cells, particularly pluripotent neural stem cells by maintaining the cell cycle of the stem cells in the G2-M phase.
본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 산화적 스트레스(oxidative stress)로 의한 줄기세포 손상을 억제하는 효능을 가진다. 보다 바람직하게는 다능성 성체줄기세포의 손상을 억제하고, 가장 바람직하게는 산화적 스트레스(oxidative stress)로 의한 신경줄기세포 손상을 억제하는 효능을 가진다.According to a preferred embodiment of the present invention, the composition of the present invention has the effect of inhibiting stem cell damage caused by oxidative stress (oxidative stress). More preferably, it has the effect of inhibiting damage to pluripotent adult stem cells, and most preferably to inhibit nerve stem cell damage caused by oxidative stress.
본 발명의 조성물은 뇌질환에 대한 유전자 치료에 응용이 될 수 있다. 바람직하게는, 신경 퇴행성 질환, 허혈 또는 재관류에 의한 질환, 정신 질환 및 산화적 스트레스이며, 가장 바람직하게는 산화적 스트레스(oxidative stress)에 의한 뇌신경질환이다.The composition of the present invention can be applied to gene therapy for brain diseases. Preferably, they are neurodegenerative diseases, diseases caused by ischemia or reperfusion, psychiatric diseases and oxidative stress, most preferably cerebral neurological diseases caused by oxidative stress.
상기 신경 퇴행성 질환은 알쯔하이머병, 헌팅톤 질병, 파킨슨씨 질병 및 근위축성 측삭 경화증이며, 상기 허혈 또는 재관류에 의한 질환은 허혈성 뇌졸중이고, 상기 정신질환은 우울증, 정신분열증 및 심적 외상후 스트레스 장애를 의미하 며, 상기 산화적 스트레스에 의한 뇌신경질환은 뇌신경세포의 산소부족에 따른 장애를 의미한다.The neurodegenerative diseases are Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis, the disease caused by ischemia or reperfusion is an ischemic stroke, and the mental disease means depression, schizophrenia and post-traumatic stress disorder. In addition, the cranial nerve disease caused by oxidative stress means a disorder caused by lack of oxygen in the cranial nerve cells.
본 발명의 조성물은 산화적 스트레스에 따른 뇌신경 세포의 손상에 의한 질환의 예방 또는 치료에 특히 유용하다. 본 발명의 조성물의 이와 같은 효능은 대체적으로 본 발명의 ADC 유전자의 신경 줄기세포 보호 작용을 통해 나타난다. 본 발명의 ADC 유전자에 의한 신경줄기세포의 보호 작용은 다양한 기전을 통해 발휘될 수 있으며, 예를 들어 신경 세포의 사멸을 억제함으로써 발휘되며, 이러한 신경 줄기세포의 사멸은 신경 줄기세포의 괴사 (necrosis) 및 아폽토시스 (apotosis)를 포함한다.The composition of the present invention is particularly useful for the prevention or treatment of diseases caused by damage of cerebral nerve cells due to oxidative stress. Such efficacy of the composition of the present invention is generally shown through the neuronal stem cell protective action of the ADC gene of the present invention. The protective action of neural stem cells by the ADC gene of the present invention can be exerted through various mechanisms, for example, by suppressing the death of nerve cells, the death of these neural stem cells necrosis of the neural stem cells (necrosis) And apoposis.
본 발명의 조성물이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 비경구 투여 방식으로 적용된다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and is preferably applied by parenteral administration.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention are prepared in unit dose form by being formulated with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 일반적인 투여량은 성인 기준으로 0.001-100 ㎎/kg 범위 내이다.Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.
본 발명의 또 다른 양태에 따르면, 본 발명은 서열목록 제1서열에 기재된 ADC(arginine decarboxylase) 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 줄기세포에 형질도입(transfection)시키는 단계를 포함하는 줄기세포의 생존능 및 증식능을 개선하는 방법을 제공한다.According to another aspect of the present invention, the present invention provides a viability of stem cells comprising the step of transfecting the stem cells gene transfer gene containing the ADC (arginine decarboxylase) coding nucleotide sequence described in SEQ ID NO: 1 And a method for improving proliferative capacity.
상기 줄기세포의 생존능 또는 증식능 개선하는 방법은 본 발명의 일 양태인 서열목록 제1서열에 기재된 ADC(arginine decarboxylase) 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 포함하는 조성물의 제조 단계를 포함하는 방법이기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the method for improving the viability or proliferation of the stem cells is a method comprising the step of preparing a composition comprising a gene carrier comprising an ADC (arginine decarboxylase) coding nucleotide sequence described in SEQ ID NO: 1 sequence of one embodiment of the present invention In order to avoid excessive complexity of this specification, the content common to both is abbreviate | omitted description.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(ⅰ) 본 발명은 서열목록 제1서열에 기재된 ADC(arginine decarboxylase) 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 포함하는 줄기세포 생존능 및 증식능 개선용 조성물, 및 상기 서열목록 제1서열에 기재된 ADC코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 이용하여 줄기세포 생존능 및 증식능을 개선하는 방법을 제공한다.(Iii) the present invention is a composition for improving stem cell viability and proliferation, including a gene carrier comprising an ADC (arginine decarboxylase) coding nucleotide sequence described in SEQ ID NO: 1, and the ADC coding nucleotide described in SEQ ID NO: 1 Provided is a method for improving stem cell viability and proliferation by using a gene carrier including a sequence.
(ⅱ) 본 발명은 줄기세포에 ADC 코딩 뉴클레오타이드 서열이 포함된 유전자 전달체를 형질도입 함으로써, 상기 줄기세포의 세포사멸(apoptosis) 및 산화적 스트레스(oxidative stress)로 의한 손상을 억제하고, 줄기세포의 생존능 및 증식능을 매우 효과적으로 개선시키는 효과를 나타낸다.(Ii) the present invention, by transducing a gene carrier containing an ADC coding nucleotide sequence to stem cells, thereby inhibiting damage caused by apoptosis and oxidative stress of the stem cells, It shows the effect of improving the viability and proliferation very effectively.
(ⅲ) 본 발명은 유전자 치료, 즉 줄기세포를 이용하는 신경질환의 예방 및 치료법에 응용시킬 수 있으며, 상기 신경질환 예방 및 치료 효능을 가지는 의약으로서의 기초적인 자료를 제공한다.(Iii) The present invention can be applied to gene therapy, that is, the prevention and treatment of neurological diseases using stem cells, and provides basic data as a medicament having the effects of preventing and treating the neurological diseases.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예Example
실시예 1: 신경줄기세포 적출Example 1 Neural Stem Cell Extraction
임신 14.5일차인 마우스(코아텍, 대한민국)로부터 태아를 얻어 뇌를 적출하였다. 적출한 뇌를 대뇌피질, 선조 및 해마 부위로 나누고, 기계적인 파이펫팅(pipetting)을 통해 단일세포로 분리하였다. 신경줄기세포 전용 배양배지인 NeuroCultNSC Basal Medium (Mouse)(STEM CELL Technologies, 미국)에 신경줄기세포 2 x 106 세포수/10 ㎖의 농도로 초대배양 하였다. 배양시작 후 6일차에 형성된 신경구(neurosphere)만을 선택하여 신경줄기세포로 이용하였다. Brains were harvested from fetuses from the 14.5th day of gestation (COREAC, Korea). The extracted brain was divided into cerebral cortex, progenitor and hippocampal regions, and separated into single cells by mechanical pipetting. Neuron stem cells were cultured in NeuroCultNSC Basal Medium (Mouse) (STEM CELL Technologies, USA) at a concentration of 2 x 10 6 cells / 10 ml. Only the neurospheres formed on day 6 after the start of culture were selected and used as neural stem cells.
실시예 2: Example 2: 인간 ADC(human arginine decarboxylase)Human arginine decarboxylase (ADC) 유전자를 포함하는 레트로바이러스 벡터 제조 Retroviral Vector Production Including Genes
본 연구를 위하여 인간 ADC(hADC) 유전자를 포함하는 레트로바이러스 벡터를 제조하였다. Retroviral vectors containing human ADC (hADC) genes were prepared for this study.
인간 ADC 유전자를 포함하는 재조합 레트로바이러스의 구축과정은 도 1 및 도 2에 기재되어 있다. hADC를 포함하는 pCMV-SPORT6 벡터(Invitrogen, 미국), 즉 hADC-pCMV-SPORT6으로부터 인간 ADC 유전자를 얻었다. 재조합 레트로바이러스 벡터 pLXSN(Clontech, 미국)를 EcoRI 및 XhoI으로 절단하고, 이 절단 부위에 인간 ADC 유전자를 삽입하였다. 신경줄기세포에 도입된 인간 ADC 유전자는 역전사중합효소연쇄반응(reverse transcription-polymerase chain reaction: RT-PCR)을 사용하여 검증하였다. 사용된 인간 ADC 유전자의 정방향 프라이머 서열은 ATG GCT GGC TAC CTG AGT G이며, 역방향 프라이머 서열은 TGA TGC TCG CTG GGG T이다. 반응 조건은 95℃ 5분을 시작으로 95℃ 1분, 50℃ 1분, 72℃ 1분을 30회 반복하였으며, 마지막으로 72℃ 10분을 반응 시켜 1378 bp의 인간 ADC 유전자 산물을 획득하였다.Construction of recombinant retroviruses comprising the human ADC gene is described in FIGS. 1 and 2. pCMV-SPORT6 vector containing hADC (Invitrogen, USA), that was obtained from the human gene ADC hADC-pCMV-SPORT6. Recombinant retroviral vector pLXSN (Clontech, USA) was digested with EcoR I and Xho I, and the human ADC gene was inserted at this cleavage site. Human ADC genes introduced into neural stem cells were verified using reverse transcription-polymerase chain reaction (RT-PCR). The forward primer sequence of the human ADC gene used is ATG GCT GGC TAC CTG AGT G and the reverse primer sequence is TGA TGC TCG CTG GGG T. Reaction conditions were repeated 95
실시예 3: Example 3: ADCADC 유전자의 신경줄기세포로의 도입 Introduction of genes into neural stem cells
초기 배양 후 6일차에 신경구에 BD 레트로-X 유니버셜 패키징 시스템(Rertro-X universal packaging system; BD Biosciences, 미국)을 이용하여, 신경구를 이루는 신경줄기세포에 형질도입 시켰다. 즉, 레트로바이러스 패키징 세포주 PT67(클론테크, 미국)을 이용하여 인간 ADC 유전자를 초기 배양한 신경줄기세포에 형질도입 시켰다. ADC 바이러스를 감염시킨 후 NeuroCult Chemical Dissociation Kit (Mouse)(STEM CELL Technologies, 미국)을 이용하여 신경구를 단일 신경줄기세포로 분리하였다. 신경줄기세포 전용 배양배지인 NeuroCultNSC Basal Medium (Mouse)에 5 x 105 세포수/ 10 ㎖의 농도로 신경줄기세포를 재배양 하 였다. On day 6 after the initial culture, the neurospheres were transduced into the neuronal stem cells forming the neurospheres using a BD Retro-X universal packaging system (BD Biosciences, USA). In other words, the human ADC gene was transduced into neural stem cells initially cultured using a retrovirus packaging cell line PT67 (Clontech, USA). After infection with ADC virus, neurospheres were isolated into single neural stem cells using NeuroCult Chemical Dissociation Kit (Mouse) (STEM CELL Technologies, USA). Neural stem cells were cultured in NeuroCultNSC Basal Medium (Mouse), a dedicated neuronal stem cell culture medium, at a concentration of 5 x 10 5 cells / 10 ml.
실시예 4: Example 4: ADCADC 과발현 신경줄기세포주(Neural stem cell: NSC)의 증식능 분석 Analysis of Proliferative Capacity of Overexpressed Neural Stem Cells (NSCs)
ADC 유전자를 포함하는 레트로바이스로 신경줄기세포에 감염시킨 후 14일차에 단일 세포로부터 형성된 신경구의 개수와 각 신경구의 크기를 측정하여 ADC 과발현이 신경줄기세포의 증식에 미치는 영향을 조사하였다. 대뇌피질에서 유래한 신경줄기세포로 형성된 신경구를 ADC 레트로바이러스로 감염시킨 후 신경구 평균 면적을 측정한 결과, 대조군과 비교했을 때 신경구의 평균 면적이 감소한 것으로 나타났다. 반면, 단일 세포에서 신경구를 형성한 수는 신경구를 ADC 레트로바이러스로 감염시킨 군에서 유의하게 증가하였다. 전체적인 신경구의 면적을 비교한 결과 ADC 레트로바이러스로 감염시킨 군에서 대조군에 비해 유의하게 증가됨을 확인하였다. 이는 ADC 유전자의 도입이 신경줄기세포의 증식을 촉진함을 알 수 있는 결과이다(도 3a-3c). (**: P 값 < 0.01 vs 대조군 , *: P 값 < 0.05 vs 대조군).After infecting neural stem cells with a retrovirus containing ADC gene, the number of neurospheres formed from single cells and the size of each neurosphere were measured on day 14 to investigate the effect of ADC overexpression on the proliferation of neural stem cells. After the infection of the neurospheres formed by the neural stem cells derived from the cerebral cortex with ADC retrovirus, the average area of the neurospheres was measured. In contrast, the number of neurosphere formation in single cells was significantly increased in the group infected with the ADC retrovirus. As a result of comparing the area of the whole neurosphere, it was confirmed that the ADC retrovirus-infected group was significantly increased compared to the control group. This result shows that the introduction of the ADC gene promotes the proliferation of neural stem cells (FIGS. 3A-3C). (**: P value <0.01 vs control, *: P value <0.05 vs control).
선조체(또는 선상체)에서 유래한 신경줄기세포로 형성된 신경구를 ADC 레트로바이러스로 감염시킨 결과, 신경구의 평균 면적이 대조군에 비해 유의하게 감소됨을 확인하였다(**: P 값 < 0.01 vs 대조군). 이와는 반대로 , 단일 세포로부터 신경구를 형성한 개수는 ADC 레트로바이러스로 감염시킨 군이 유의하게 늘어나는 양상을 보였다(**: P 값 < 0.01 vs 대조군). 이 두 개의 자료를 이용하여 전체 신경구의 면적을 비교한 결과 ADC 레트로바이러스로 감염시킨 군에서 유의하게 전 체 신경구의 면적이 늘어남을 확인하였다(*: P 값 < 0.05 vs 대조군). 이는 ADC 유전자의 도입이 신경줄기세포의 증식을 촉진함을 알 수 있는 결과이다.(도 4a-4c).As a result of infecting neurospheres formed with neural stem cells derived from striatum (or striatum) with ADC retrovirus, the average area of neurospheres was significantly reduced compared to the control group (**: P value <0.01 vs control). In contrast, the number of neurosphere formation from a single cell was significantly increased in the group infected with ADC retrovirus (**: P value <0.01 vs control). Comparing the area of all neurospheres using these two data, it was confirmed that the area of whole neurospheres increased significantly in the group infected with ADC retrovirus (*: P value <0.05 vs control). This result shows that the introduction of ADC gene promotes the proliferation of neural stem cells (FIGS. 4A-4C).
실시예 5: ADC 과발현 줄기세포주의 세포주기 분석 Example 5: Cell Cycle Analysis of ADC Overexpressing Stem Cell Lines
FACS(fluorescence activated cell sorter)를 이용하여 ADC 레트로바이스 감염 후 14일차에 신경구를 구성하는 신경줄기세포의 세포주기를 분석하였다.The cell cycle of the neural stem cells constituting the neurospheres was analyzed 14 days after the ADC retroviral infection using FACS (fluorescence activated cell sorter).
대뇌피질에서 기원한 신경줄기세포(NSC-NC)에 비해 ADC 과발현 신경줄기세포(NSC-ADC)에서 세포사멸(apoptosis) 정도가 감소하였고 G2-M기도 증가함을 보였다. 또한 선조체(또는 선상체) 유래 신경줄기세포(NSS-NC)에서도 ADC 과발현이 세포사멸을 감소시키고, G2-M기에 해당하는 세포의 양도 약 3배 정도 증가시킴을 알 수 있었다(도 5a-5d).Compared to neural stem cells derived from cerebral cortex (NSC-NC), ADC overexpressed neural stem cells (NSC-ADC) showed decreased apoptosis and increased G2-M airway. In the neural stem cells (NSS-NC) derived from the striatum, ADC overexpression reduced cell death and increased the amount of cells corresponding to the G2-M phase by about three times (FIGS. 5A-5D). .
따라서, ADC 과발현은 신경구의 크기는 감소시키고, 그 수는 증가시켰다. 또한 신경구의 중심 부위에 존재하는 신경줄기세포의 세포사멸을 감소시켰으며, 세포주기 중 G2-M기에 있는 세포의 수가 증가하였다. 이는 ADC 과발현으로 인해 각 신경구 당 살아 있는 세포의 수가 증가되었고, 이로 인해 단일 세포 부유물로 만들었을 때도 많은 신경줄기세포가 살아 있음을 의미한다. 여기에서 신경구의 개수가 증가한 것은 살아있는 많은 신경줄기세포들이 여전히 증식능을 가지고 있음을 말해주는 결과이다. 상대적으로 ADC 과발현 신경줄기세포들이 G2-M 기에 존재하는 세포들이 많은 것으로 나왔는데 이는 유전자를 도입하지 않은 줄기세포의 경우 에는 신경구가 일정 크기가 되고 나면 아래로 가라앉아 배지 조성에 따라 분화되는 성질을 보이나, ADC 유전자가 도입된 신경줄기세포에서는 아마도 신경구의 크기가 충분히 크지 않아서 세포들이 아직은 계속 증식하는 단계가 있음을 보여주는 결과라 판단되었다. 이에 비해 유전자가 도입되지 않은 줄기세포들은 이미 신경구가 충분히 증식한 단계에 있으므로 증식능이 ADC 과발현 신기능 줄기세포에 비해 상대적으로 적게 나왔을 수도 있다.Thus, ADC overexpression reduced the size of neurospheres and increased the number. In addition, cell death of neural stem cells in the central region of neurospheres was reduced, and the number of cells in the G2-M phase of the cell cycle was increased. This means that ADC overexpression increases the number of viable cells per neuron, resulting in many neural stem cells alive even when made into a single cell suspension. An increase in the number of neurospheres is a consequence of the fact that many living neural stem cells still have proliferative capacity. Relatively, ADC overexpressing neural stem cells were found to be present in G2-M phase cells. In the case of stem cells without genes, the neurospheres settled down after a certain size and differentiated according to medium composition. In neural stem cells into which the ADC gene was introduced, it was probably the result that the size of the neurosphere was not large enough to show that the cells are still growing. In contrast, stem cells without genes are already at the stage of fully proliferating neurospheres, so their proliferative capacity may be relatively low compared to ADC overexpressing renal function stem cells.
실시예 6: ADC 과발현 줄기세포주의 분화능 분석Example 6: Analysis of Differentiation Capacity of ADC Overexpressing Stem Cell Lines
ADC 과발현이 신경줄기세포의 성체 신경세포로의 분화에 미치는 영향을 확인하였다. 신경아교세포 표지인자인 GFAP와 신경세포 표지인자인 MAP2 모두 잘 발현 되는 것으로 확인되었다(도 6a). 또한 극히 일부에서 희소돌기아교세포의 표지인자인 Olig2에 대해 양성 반응이 일어났지만 매우 소수라서 발현이 되었다고 말하기는 어려울 것 같다. 이에 비해 정상 신경줄기세포의 경우 GFAP 표지인자에 양성 반응인 경우는 잘 나타난 반면, MAP2 양성반응은 관찰이 어려웠다(도 6a). Olig2에 대한 결과는 비교가 불가할 정도로 소수였다. 또한 많은 신경줄기세포들이 아직은 미분화 상태임을 나타내는 줄기세포 표지 인자인 Sox2와 Nestin 양성반응 세포들을 통해 알 수 있었다(도 6b).The effect of ADC overexpression on the differentiation of neural stem cells into adult neurons was confirmed. Neuroglial marker GFAP and neuronal marker MAP2 were confirmed to be well expressed (Fig. 6a). It is also difficult to say that only a few responded positively to Olig2 , a marker of oligodendrocytes , but it was very few. In contrast, the normal neural stem cells showed a positive response to the GFAP marker, whereas MAP2 positive reaction was difficult to observe (Fig. 6a). The results for Olig2 were minor in comparison. In addition, many neural stem cells were identified through Sox2 and Nestin positive cells, stem cell markers indicating that they are still undifferentiated (Fig. 6b).
이상의 결과를 종합해 보면 ADC 과발현으로 인해 신경줄기세포가 성체 신경계세포로 분화를 하는 분화능력 자체에는 영향이 없는 것으로 판단되며, 오히려 분화되는 경향, 즉 ADC 과발현이 일어난 신경줄기세포들이 정상 신경줄기세포들보다 상대적으로 신경세포로의 분화가 많아졌음을 확인할 수 있었다. 한 가지 새로운 사실은 분화 속도가 매우 빠르다는 것이다(도 6c). ADC 과발현이 분화의 시기를 앞당기는 것으로 판단되었다. ADC 레트로바이러스 감염 후 정상 줄기세포 배양배지에서 3일간 배양 후의 모습을 보면 ADC 과발현이 작은 크기의 신경구도 분화를 유도함을 알 수 있었다. 이는 실제 세포 치료시에 열악한 미세 환경(micro environment)에 줄기세포 이식시 세포 정착에 매우 유리하게 작용할 것이라 예상되었다. In conclusion, ADC overexpression does not affect neural stem cells differentiation into adult neuronal cells itself due to ADC overexpression, rather, the tendency to differentiate, that is, the neural stem cells with ADC overexpression are more relative than normal neural stem cells. As a result, it was confirmed that differentiation into neurons increased. One new fact is that the differentiation rate is very fast (FIG. 6C). ADC overexpression was thought to accelerate the differentiation period. After three days of culture in normal stem cell culture medium after ADC retrovirus infection, ADC overexpression induced differentiation of small-sized neurospheres. This is expected to be very advantageous for cell settlement when transplanting stem cells into poor micro environment in actual cell treatment.
실시예 7: 신경줄기세포 손상 및 손상방어 기전 관련 단백질 발현 분석Example 7 Analysis of Protein Expression Related to Neural Stem Cell Injury and Defense Mechanism
신경줄기세포 손상시 ADC 과발현이 신경줄기세포의 손상방어에 미치는 영향과 그 기전을 확인하였다. 신경줄기세포 손상은 H2O2 (200 μM)를 이용하여 4시간 동안 실시하였다. ADC 과발현이 신경줄기세포사멸을 감소시킨다는 것을 DNA 분석을 통해 알 수 있었다. 신경줄기세포에서 손상에 의해 아폽토시스가 일어날 경우, 전체 유전자들이 다양한 크기의 절편들로 파괴된다. 따라서 신경세포내의 전체 유전자의 확인을 통해 ADC 과발현이 신경줄기세포사멸을 감소시킴을 알 수 있었다. 이러한 효과는 Erk1/2(extracellular signal-regulated kinases 1 and 2) 활성과 Bcl2(B-cell CLL/lymphoma 2)의 발현을 증가시킴으로써 나타난 결과이며, 단백질 분석(Immunoblotting)을 통해 확인할 수 있었다(도 7b). 또한, AIF (apoptosis inducible factor)의 발현이 감소되었음을 확인하였다. 단백질 분석에서 사용된 1차 항체는 Erk1/2: 토끼 항-Erk1/2 (1: 1000, Cell Signaling, 미국), ph-Erk1/2: 토끼 항-포스포-Erk1/2 (1:1000, Cell Signaling, 미국), Bcl-2: 토끼 항-Bcl-2 (1:1000, Santa Cruz, 미국), AIF: 토끼 항-AIF (1:1000, Cell Signaling, 미국), 액틴(Actin): 마우스 모노 항-액틴(1:2000, Santa Cruz, 미국)이며, 퍼옥시다아제-컨쥬게이티드 항-토끼 또는 항-마우스 2차 항체(GE, 미국)를 사용한 후 단백질의 발현 정도를 ECL 단백질 검출 키트(GE, 미국)를 사용하여 확인하였다.The effect of ADC overexpression on neuroprotective stem cell damage and its mechanism were evaluated. Neural stem cell damage was performed for 4 hours using H 2 O 2 (200 μM). DNA analysis showed that ADC overexpression reduced neural stem cell death. When apoptosis occurs due to damage in neural stem cells, whole genes are broken into fragments of various sizes. Therefore, it was found that ADC overexpression reduced neural stem cell death through identification of all genes in neurons. This effect Erk1 / 2 (extracellular signal-regulated
장시간의 손상모델로서 신경줄기세포에 H2O2 (200 μM) 손상을 24시간 동안 지속한 후, 배양 배지 1 ㎖ 당 2-5 ㎍ Hoechst 염료를 추가하고 세포를 30분 동안 37℃에 유지시켰다. 그 다음 PI액을 Hoechst 염료와 동일한 양으로 첨가하고 형광 및 UV 필터막이 장착된 현미경(Olympus, 일본)으로 관찰하였다. Hoechst 33258(살아있는 세포의 핵을 파란색으로 염색함)과 PI(propidium iodide, 죽은 세포의 핵을 붉은 색으로 염색함) 이중 염색을 통해 거의 대부분의 신경줄기세포가 사멸한 것을 확인 할 수 있었으며, 다만 ADC 과발현 신경줄기세포에서만 일부가 살아있음을 확인하였다(도 7c).After 2 hours of H 2 O 2 (200 μM) damage to neural stem cells as a long-term damage model, 2-5 μg Hoechst dye was added per ml of culture medium and the cells were kept at 37 ° C. for 30 minutes. PI solution was then added in the same amount as Hoechst dye and observed under a microscope (Olympus, Japan) equipped with a fluorescent and UV filter membrane. Hoechst 33258 (staining the nuclei of living cells in blue) and PI (propidium iodide (staining the nuclei of dead cells) in red) double staining confirmed that most neural stem cells were killed, but ADC Only some of the overexpressed neural stem cells were confirmed to be alive (FIG. 7C).
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3. del Zoppo G, Ginis I, Hallenbeck JM, Iadecola C, Wang X, Feuerstein GZ:and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol. 2000 ;10:95-112. Del Zoppo G, Ginis I, Hallenbeck JM, Iadecola C, Wang X, Feuerstein GZ: and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol. 2000; 10: 95-112.
4. Tabor, C.W. & Tabor, H.(1984) : Agmatine is an endogenous polyamine derived from enzymatic decarboxylation of L-arginine decarboxylase (ADC). 4. Tabor, C.W. & Tabor, H. (1984): Agmatine is an endogenous polyamine derived from enzymatic decarboxylation of L-arginine decarboxylase (ADC).
5. Chao CC, Hu S, Ehrlich L, Peterson PK: Interleukin-1 and tumor necrosis factor-alpha synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-D-aspartate receptors. Brain Behav Immun. 1995 ;9:355-65.5. Chao CC, Hu S, Ehrlich L, Peterson PK: Interleukin-1 and tumor necrosis factor-alpha synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-D-aspartate receptors. Brain Behav Immun. 1995; 9: 355-65.
6. PILETZ, J.E., et al. 1995. Comparison of the properties of agmatine and endogenous clonidine-displacing substance at imidazoline and alpha-2 adrenergic receptors J. Pharmacol. Exp. Ther. 272: 581-587.6. PILETZ, J.E., et al. 1995. Comparison of the properties of agmatine and endogenous clonidine-displacing substance at imidazoline and alpha-2 adrenergic receptors J. Pharmacol. Exp. Ther. 272: 581-587.
7. PINTHONG, D., et al. 1995. Agmatine recognizes alpha 2-adrenoceptor binding sites but neither activates nor inhibits alpha 2-adrenoceptors. Naunyn Schmiedeberg's Arch. Pharmacol. 351: 10-16. 7. PINTHONG, D., et al. 1995. Agmatine recognizes alpha 2-adrenoceptor binding sites but neither activates nor inhibits alpha 2-adrenoceptors. Naunyn Schmiedeberg's Arch. Pharmacol. 351: 10-16.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
도 1은 ADC 유전자를 포함하는 재조합 레트로바이러스 벡터의 제조에 관한 것이다. 이것은 hADC-pCMV-SPORT6으로부터 얻은 인간 ADC 클론이다. 재조합 레트로바이러스 벡터 pLXSN를 절단하기 위하여 EcoRI 와 XhoI 부위를 이용하였다. 도 1에서, 5’LTR은 5’MoMuSV(Moloney murine sarcoma virus) LTR(long terminal repeat) 서열이고, Ψ+는 연장된 패키징 시그널이며, PSV40은 SV40 프로모터이고, 3’LTR은 3’MoMuSV LTR 서열이고, ColE1 ori는 Col E1 복제원점이다(pLXSN의 1575 bp에 TAG 종결 코돈이 존재하며 여기서 목표 단백질의 발현이 종결된다).1 relates to the production of recombinant retroviral vectors comprising the ADC gene. This is a human ADC clone obtained from hADC-pCMV-SPORT6. EcoR I and Xho I sites were used to cut the recombinant retroviral vector pLXSN. In FIG. 1, 5'LTR is a 5'MoMuSV (Moloney murine sarcoma virus) long terminal repeat (LTR) sequence, Ψ + is an extended packaging signal, P SV40 is an SV40 promoter, and 3'LTR is a 3'MoMuSV LTR Sequence, ColE1 ori is the Col E1 origin of replication (the TAG stop codon is present at 1575 bp of pLXSN where expression of the target protein is terminated).
도 2는 ADC 유전자를 포함하는 레트로바이러스의 제조 및 생산에 관한 것이다. 초기 신경줄기세포 유전자를 도입하기 위하여 ADC 유전자를 발현시키는 BD 레트로-X 유니버셜 패키징 시스템(BD Retro-X universal packaging system)을 이용하였다. 바이러스 유전자를 발현하는 신경줄기세포를 G418을 이용하여 선별하였다. ADC 유전자에 대한 특이적인 프라이머를 사용하여 RT-PCR을 실시하였다. ADC 유전자 245 bp 단편이 레트로바이러스 패키지 세포인 ADC가 도입된 PT67와 ADC가 도입된 신경줄기세포들에서 확인되었다.2 relates to the production and production of retroviruses comprising the ADC gene. In order to introduce the early neural stem cell gene, a BD Retro-X universal packaging system expressing an ADC gene was used. Neural stem cells expressing viral genes were selected using G418. RT-PCR was performed using primers specific for the ADC gene. The 245 bp fragment of the ADC gene was identified in PT67 with ADC, a retroviral package cell, and with neural stem cells with ADC.
도 3a-3c는 ADC 유전자가 포함되지 않은 대뇌피질 유래 신경줄기세포로부터 형성된 신경구(이하, ‘NSC-NC’라고 표기)와 대뇌피질 유래 ADC 과발현 신경줄기세포(이하, ‘NSC-ADC’라고 표기)로부터 형성된 신경구의 면적 및 개수를 나타낸 결과이다. 도 3a는 NSC-NC과 NSC-ADC로부터 형성된 신경구의 평균 크기를 나타낸 결과이며, 도 3b는 NSC-NC과 NSC-ADC에서 단일세포로부터 형성된 신경구의 총 개수를 나타낸 것이고, 도 3c는 NSC-NC과 NSC-ADC로부터 형성된 신경구의 총 면적에 대한 결과이다. Average area of Neurosphere(㎛2): 신경구의 2D(2 Dimention)평균 크기; Numbers of Spheres: 신경줄기세포에서 형성된 신경구의 수; Total area of Neurospheres(㎛2): 신경구의 총 면적(2D). -NC는 ADC 유전자가 포함되지 않은 것을 의미하고, -ADC는 ADC 유전자가 과발현 되는 것을 의미한다.Figures 3a-3c are neurospheres formed from cortical-derived neural stem cells that do not contain the ADC gene (hereinafter referred to as 'NSC-NC') and cortical-derived ADC overexpressing neural stem cells (hereinafter referred to as 'NSC-ADC') This is the result showing the area and number of the formed neurospheres. Figure 3a is a result showing the average size of neurons formed from NSC-NC and NSC-ADC, Figure 3b shows the total number of neurospheres formed from single cells in NSC-NC and NSC-ADC, Figure 3c is NSC-NC And the total area of neurospheres formed from NSC-ADC. Average area of Neurosphere (μm 2 ): 2D (average dimention) average size of neurospheres; Numbers of Spheres: Number of neurospheres formed in neural stem cells; Total area of Neurospheres (μm 2 ): Total area of neurospheres (2D). -NC means that the ADC gene is not included, -ADC means that the ADC gene is overexpressed.
도 4a-4c는 ADC 유전자가 포함되지 않은 선조체(striatum) 유래 신경줄기세포로부터 형성된 신경구(NSS-NC)와 선조체 유래 ADC 과발현 신경줄기세포(NSS-ADC)로부터 형성된 신경구의 면적 및 개수를 나타낸 결과이다. 도 4a는 대조군과 ADC 유전자가 포함된 신경줄기세포로부터 형성된 신경구의 평균 크기를 나타낸 결과이며, 도 4b는 대조군과 ADC 유전자가 포함된 단일 신경줄기세포로부터 형성된 신경구의 총 개수를 나타낸 것이고, 도 4c는 대조군과 ADC 유전자가 포함된 신경줄기세포로부터 형성된 신경구의 총 면적에 대한 결과이다. Average area of Neurosphere(um2): 신경구의 2D(2 Dimention)평균 크기; Numbers of Spheres: 신경줄기세포에서 형성된 신경구의 수; Total area of Neurospheres(um2): 신경구의 총 면적(2D). -NC는 ADC 유전자가 포함되지 않은 것을 의미하고, -ADC는 ADC 유전자가 과발현되는 것을 의미한다.Figures 4a-4c is a result showing the area and number of neurospheres formed from striatum-derived neural stem cells (NSS-NC) and ADC overexpressed neural stem cells (NSS-ADC) not containing ADC gene. Figure 4a is a result showing the average size of the neurospheres formed from neural stem cells containing the control and ADC gene, Figure 4b shows the total number of neurospheres formed from a single neural stem cells containing the control and ADC gene, Figure 4c And the total area of neurospheres formed from neural stem cells containing ADC gene. Average area of Neurosphere (um 2 ): 2D (average dimention) average size of neurospheres; Numbers of Spheres: Number of neurospheres formed in neural stem cells; Total area of Neurospheres (um 2 ): Total area of neurospheres (2D). -NC means that the ADC gene is not included, -ADC means that the ADC gene is overexpressed.
도 5a-5d는 NSC-NC와 NSC-ADC, NSS-NC 및 NSS-ADC의 세포사멸된 세포수와 세 포주기를 FACS(fluorescence activated cell sorter)를 이용하여 각각 나타낸 결과이다. NSC-ADC는 NSC-NC보다 세포사멸된 세포수가 억제되었고, G2-M기의 세포주기를 가지는 세포수가 증가하였다(도 5a-5b). 또한, NSS-ADC 역시 NSC-ADC와 동일한 양상을 나타내었다(도 5c-5d).5A-5D show the apoptotic cell counts and cell cycles of NSC-NC and NSC-ADC, NSS-NC and NSS-ADC, respectively, using FACS (fluorescence activated cell sorter). NSC-ADC suppressed apoptosis compared to NSC-NC and increased the number of cells having a cell cycle of the G2-M phase (FIGS. 5A-5B). In addition, NSS-ADC also showed the same aspect as NSC-ADC (Figs. 5c-5d).
도 6a-6c는 ADC 과발현이 신경줄기세포의 성체 신경세포로의 분화에 미치는 영향을 나타낸 이미지이다. 도 6a는 ADC 과발현 신경줄기세포가 신경교세포(glia cell) 및 신경세포(neuron)로 분화되는 것을 나타낸 것이며. 도 6b는 대부분의 ADC 과발현 신경줄기세포들이 미분화 상태임을 나타낸 것이고, 도 6c는 ADC 과발현 신경줄기세포들이 정상 신경줄기세포보다 세포부착성의 증가와 분화속도가 더 빠르다는 것을 타낸 이미지이다. GFAP: Glial fibrillary acidic protein (신경교세포 표지 마커); MAP2: microtubule-associated protein 2 (신경세포 표지 마커); SOX2: SRY (sex determining region Y)-box 2 (전능성 줄기세포 표지 마커); Nestin: type VI intermediate filament protein (신경 줄기 세포 표지 마커).6A-6C are images showing the effect of ADC overexpression on the differentiation of neural stem cells into adult neurons. 6a shows that ADC overexpressing neural stem cells are differentiated into glial cells and neurons. 6B shows that most ADC overexpressing neural stem cells are in an undifferentiated state, and FIG. 6C is an image showing that ADC overexpressing neural stem cells have an increased cell adhesion and differentiation rate faster than normal neural stem cells. GFAP: Glial fibrillary acidic protein (glial cell marker); MAP2: microtubule-associated protein 2 (nerve cell marker marker); SOX2: sex determining region Y (SRY) -box 2 (pluripotent stem cell marker); Nestin: type VI intermediate filament protein (nerve stem cell marker).
도 7a-7c는 신경줄기세포 손상 및 손상방어 기전 관련 단백질 발현 분석 결과이다. 도 7a는 ADC 과발현이 신경줄기세포의 세포사멸을 억제시키는 효과에 대하여 확인(confirmation)하기 위한 DNA 래더 분석이며 ADC가 과발현 신경줄기세포는 세포사멸이 억제되어 DNA 절편(fragment)이 거의 나타나지 않았다. 도 7b는 H2O2 손상 4시간 후 ADC 과발현 신경줄기세포 및 정상 신경줄기세포의 웨스턴 블롯팅(western blotting)을 한 결과이고, 도 7c는 H2O2 손상 24시간 후 ADC 과발현 신 경줄기세포 및 정상 신경줄기세포를 Hoechst-PI로 염색한 결과이다. 세포사멸된 세포는 PI에 의하여 레드로 염색되었고, 살아있는 세포는 Hoechst에 의하여 블루로 염색되었다.Figures 7a-7c is a result of analysis of protein expression related to neuronal stem cell damage and defense mechanisms. Figure 7a is a DNA ladder analysis to confirm the effect of ADC overexpression to inhibit the apoptosis of neural stem cells and ADC overexpressed neural stem cells suppressed apoptosis showed little DNA fragment (fragment). Figure 7b is the result of Western blotting of ADC overexpressing neural stem cells and normal
<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> Compositions for Enhancing Viability and Proliferation of Stem Cell <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1378 <212> DNA <213> Homo sapiens <220> <221> gene <222> (1)..(1378) <223> human ADC gene CDS <400> 1 atggctggct acctgagtga atcggacttt gtgatggtgg aggagggctt cagtacccga 60 gacctgctga aggaactcac tctgggggcc tcacaggcca ccacggacga ggtagctgcc 120 ttcttcgtgg ctgacctggg tgccatagtg aggaagcact tttgctttct gaagtgcctg 180 ccacgagtcc ggccctttta tgctgtcaag tgcaacagca gcccaggtgt gctgaaggtt 240 ctggcccagc tggggctggg ctttagctgt gccaacaagg cagagatgga gttggtccag 300 catattggaa tccctgccag taagatcatc tgcgccaacc cctgtaagca aattgcacag 360 atcaaatatg ctgccaagca tgggatccag ctgctgagct ttgacaatga gatggagctg 420 gcaaaggtgg taaagagcca ccccagtgcc aagatggttc tgtgcattgc taccgatgac 480 tcccactccc tgagctgcct gagcctaaag tttggagtgt cactgaaatc ctgcagacac 540 ctgcttgaaa atgcgaagaa gcaccatgtg gaggtggtgg gtgtgagttt tcacattggc 600 agtggctgtc ctgaccctca ggcctatgct cagtccatcg cagacgcccg gctcgtgttt 660 gaaatgggca ccgagctggg tcacaagatg cacgttctgg accttggtgg tgscttccct 720 ggcacagaag gggccaaagt gagatttgaa gagattgctt ccgtgatcaa ctcagccttg 780 gacctgtact tcccagaggg ctgtggcgtg gacatctttg ctgagctggg gcgctactac 840 gtgacctcgg ccttcactgt ggcagtcagc atcattgcca agaaggaggt tctgctagac 900 cagcctggca gggaggagga aaatggttcc acctccaaga ccatcgtgta ccaccttgat 960 gagggcgtgt atgggatctt caactcagtc ctgtttgaca acatctgccc tacccccatc 1020 ctgcagaaga aaccatccac ggagcagccc ctgtacagca gcagcctgtg gggcccggcg 1080 gttgatggct gtgattgcgt ggctgagggc ctgtggctgc cgcaactaca cgtaggggac 1140 tggctggtct ttgacaacat gggcgcctac actgtgggca tgggttcccc cttttggggg 1200 acccaggcct gccacatcac ctatgccatg tcccgggtgg cctgggaagc gctgcgaagg 1260 cagctgatgg ctgcagaaca ggaggatgac gtggagggtg tgtgcaagcc tctgtcctgc 1320 ggctgggaga tcacagacac cctgtgcgtg ggccctgtct tcaccccagc gagcatca 1378 <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> Compositions for Enhancing Viability and Proliferation of Stem Cell <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1378 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (1378) <223> human ADC gene CDS <400> 1 atggctggct acctgagtga atcggacttt gtgatggtgg aggagggctt cagtacccga 60 gacctgctga aggaactcac tctgggggcc tcacaggcca ccacggacga ggtagctgcc 120 ttcttcgtgg ctgacctggg tgccatagtg aggaagcact tttgctttct gaagtgcctg 180 ccacgagtcc ggccctttta tgctgtcaag tgcaacagca gcccaggtgt gctgaaggtt 240 ctggcccagc tggggctggg ctttagctgt gccaacaagg cagagatgga gttggtccag 300 catattggaa tccctgccag taagatcatc tgcgccaacc cctgtaagca aattgcacag 360 atcaaatatg ctgccaagca tgggatccag ctgctgagct ttgacaatga gatggagctg 420 gcaaaggtgg taaagagcca ccccagtgcc aagatggttc tgtgcattgc taccgatgac 480 tcccactccc tgagctgcct gagcctaaag tttggagtgt cactgaaatc ctgcagacac 540 ctgcttgaaa atgcgaagaa gcaccatgtg gaggtggtgg gtgtgagttt tcacattggc 600 agtggctgtc ctgaccctca ggcctatgct cagtccatcg cagacgcccg gctcgtgttt 660 gaaatgggca ccgagctggg tcacaagatg cacgttctgg accttggtgg tgscttccct 720 ggcacagaag gggccaaagt gagatttgaa gagattgctt ccgtgatcaa ctcagccttg 780 gacctgtact tcccagaggg ctgtggcgtg gacatctttg ctgagctggg gcgctactac 840 gtgacctcgg ccttcactgt ggcagtcagc atcattgcca agaaggaggt tctgctagac 900 cagcctggca gggaggagga aaatggttcc acctccaaga ccatcgtgta ccaccttgat 960 gagggcgtgt atgggatctt caactcagtc ctgtttgaca acatctgccc tacccccatc 1020 ctgcagaaga aaccatccac ggagcagccc ctgtacagca gcagcctgtg gggcccggcg 1080 gttgatggct gtgattgcgt ggctgagggc ctgtggctgc cgcaactaca cgtaggggac 1140 tggctggtct ttgacaacat gggcgcctac actgtgggca tgggttcccc cttttggggg 1200 acccaggcct gccacatcac ctatgccatg tcccgggtgg cctgggaagc gctgcgaagg 1260 cagctgatgg ctgcagaaca ggaggatgac gtggagggtg tgtgcaagcc tctgtcctgc 1320 ggctgggaga tcacagacac cctgtgcgtg ggccctgtct tcaccccagc gagcatca 1378
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| JP2006325554A (en) | 2005-05-30 | 2006-12-07 | Toyobo Co Ltd | Method for imparting stress-preventing effect by inducing gene expression |
| KR20090056707A (en) * | 2007-11-30 | 2009-06-03 | (주)미래생명공학연구소 | Anti-apoptotic gene introduced embryonic stem cell and method of manufacturing the same |
| KR20100124090A (en) * | 2009-05-18 | 2010-11-26 | 연세대학교 산학협력단 | Pharmaceutic compositions for prevention or treatment of brain diseases |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2010137890A3 (en) | 2011-04-21 |
| KR20100127912A (en) | 2010-12-07 |
| WO2010137890A2 (en) | 2010-12-02 |
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