KR102253790B1 - An animal model having preeclampsia and method for producing the same - Google Patents
An animal model having preeclampsia and method for producing the same Download PDFInfo
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- KR102253790B1 KR102253790B1 KR1020190148651A KR20190148651A KR102253790B1 KR 102253790 B1 KR102253790 B1 KR 102253790B1 KR 1020190148651 A KR1020190148651 A KR 1020190148651A KR 20190148651 A KR20190148651 A KR 20190148651A KR 102253790 B1 KR102253790 B1 KR 102253790B1
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Abstract
Description
본 발명은 임신성 고혈압 동물모델 제조용 벡터 및 중간엽줄기세포와 이를 이용하여 제조한 임신성 고혈압 동물모델에 관한 것이다.The present invention relates to a vector and mesenchymal stem cells for preparing a gestational hypertension animal model, and a gestational hypertension animal model prepared using the same.
임신 중 고혈압성 장애(hypertensive disorders of pregnancy)는 임신과 합병된 고혈압, 자간전증, 자간증, 임신성 고혈압 등을 포함한다. 모태 및 태아의 주산기 사망의 주요한 원인이 되며, 전체 임산부의 5~8%를 차지할 정도로 큰 비중을 차지하고 있으며, 대표적인 주요 증상으로는 고혈압, 부종 및 임신중독성 요 단백질량의 증가, 심한 두통 및 갑작스런 체중 증가 등이 있다. 이러한 임신 중 고혈압성 장애는 대체로 20주 이후에 발생하고 조산의 주요한 원인이 되기도 한다. 그러나 현재까지 이의 정확한 발병원인과 치료방법은 명확하게 규명되어 있지 않으며, 분만 외에는 뚜렷한 치료 방법이 없다. Hypertensive disorders of pregnancy include hypertension combined with pregnancy, preeclampsia, eclampsia, and gestational hypertension. It is a major cause of maternal and fetal perinatal mortality, accounting for 5-8% of all pregnant women, and major symptoms include high blood pressure, edema and increased urine protein toxic to pregnancy, severe headaches and sudden symptoms. Weight gain and more. Such hypertensive disorders during pregnancy usually develop after 20 weeks and are also a major cause of premature birth. However, until now, its exact cause and treatment method have not been clearly identified, and there is no clear treatment method other than delivery.
임신 중 고혈압 (임신성 고혈압)은 임산부에게서 자주 발생하는 합병증으로 약 7-10%는 고혈압성 질환의 한 형태로 나타나게 되며, 이러한 고혈압 임산부 환자는 미숙아, 자궁내 태아 위협, 성장제한, 태반조기박리 등의 위협으로 고통을 받고 있다. 또한 그들은 정상임부들 보다 혈전성 소혈관질환, 응고장애, 뇌내출혈 및 다양한 장기의 손상(특히 신장과 간)의 위험성이 더 크다. 임신중 고혈압의 정의는 임부의 이완기압이 140mmHg 혹은 그 이상과 수축기압 90mmHg 혹은 그 이상일 때를 말한다. 이전의 NIH의 Working Group 보고에서는 임신중 고혈성 질환을 만성고혈압(Chronic hypertension), 임신성 고혈압(Gestational hypertension), 자간전증/자간증(Preeclampsia/Eclampsia) 및 만성 고혈압이 중첩된 자간전긍(Preeclampsia superimposed on chronic hypertension)을 포함하는 4가지 유형으로 분류한다 (Adapted from the national High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Am J Obstet Gynecol 2000;183:S1-S22.). 특히 자간전증은 임신 20주 이후에 일어나는 고혈압과 단백뇨가 발생하는 질환으로 산모와 태아에게 위험한 문제를 일으킬 수 있다.Hypertension during pregnancy (pregnant hypertension) is a frequent complication in pregnant women, and about 7-10% of them appear as a form of hypertensive disease, and these hypertensive pregnant women are premature infants, fetal threats in the uterus, growth restriction, early placental detachment, etc. Is suffering from the threat of They are also at greater risk for thrombotic small blood vessel disease, coagulation disorders, bleeding in the brain and damage to various organs (especially kidney and liver) than normal pregnant women. The definition of hypertension during pregnancy is when the diastolic pressure of a pregnant woman is 140mmHg or higher and the systolic pressure is 90mmHg or higher. In a previous NIH Working Group report, chronic hypertension, gestational hypertension, preeclampsia/eclampsia, and preeclampsia superimposed on chronic hypertension during pregnancy have been reported. ) (Adapted from the national High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy.Am
임신성 고혈압은 인간에서만 발생하는 고유 질환임으로 이를 체계적으로 이해하기 위한 동물 모델이 필요하나 아직 확립된 모델이 부족한 현실이다. 임신성 고혈압의 치료제로서 다양한 약물들이 연구되었으나 아직 확립된 약물은 없는 상황에서 분만만이 유일한 치료제로 인정받고 있다. 따라서, 이러한 문제를 극복하기 위해서는 임신성 고혈압 동물모델 확립이 절실히 필요하다. Gestational hypertension is a unique disease that occurs only in humans, so an animal model is needed to systematically understand it, but the established model is still lacking. Various drugs have been studied as a treatment for gestational hypertension, but delivery is the only treatment in the absence of established drugs. Therefore, in order to overcome this problem, it is urgently needed to establish an animal model of gestational hypertension.
본 발명의 목적은 임신성 고혈압 모델 제조용 바이러스 발현 벡터를 제공하는 것이다.It is an object of the present invention to provide a viral expression vector for preparing a gestational hypertension model.
또한, 본 발명의 목적은 상기 벡터가 형질도입된 바이러스를 제공하는 것이다.In addition, an object of the present invention is to provide a virus transduced with the vector.
또한, 본 발명의 목적은 상기 바이러스로 형질도입된 중간엽줄기세포를 제공하는 것이다.In addition, an object of the present invention is to provide a mesenchymal stem cell transduced with the virus.
또한, 본 발명의 목적은 상기 중간엽줄기세포를 포함하는 임신성 고혈압 모델 제조용 조성물을 제공하는 것이다.In addition, an object of the present invention is to provide a composition for preparing a gestational hypertension model comprising the mesenchymal stem cells.
또한, 본 발명의 목적은 임신성 고혈압 동물모델을 제공하는 것이다.In addition, it is an object of the present invention to provide an animal model of gestational hypertension.
또한, 본 발명의 목적은 임신성 고혈압 동물모델의 제조방법을 제공하는 것이다.In addition, an object of the present invention is to provide a method of manufacturing an animal model of gestational hypertension.
아울러, 본 발명의 목적은 임신성 고혈압 치료제의 스크리닝 방법을 제공하는 것이다.In addition, an object of the present invention is to provide a method for screening a therapeutic agent for gestational hypertension.
상기 과제를 해결하기 위하여, 본 발명은 sFlt-1 유전자를 포함하는 임신성 고혈압 모델 제조용 바이러스 발현 벡터를 제공한다.In order to solve the above problems, the present invention provides a viral expression vector for preparing a gestational hypertension model comprising the sFlt-1 gene.
또한, 본 발명은 상기 벡터가 형질도입된 바이러스를 제공한다.In addition, the present invention provides a virus transduced with the vector.
또한, 본 발명은 상기 바이러스로 형질도입된 중간엽줄기세포를 제공한다.In addition, the present invention provides a mesenchymal stem cell transduced with the virus.
또한, 본 발명은 상기 중간엽줄기세포를 포함하는 임신성 고혈압 모델 제조용 조성물을 제공한다.In addition, the present invention provides a composition for preparing a gestational hypertension model comprising the mesenchymal stem cells.
또한, 본 발명은 상기 조성물이 투여된 임신성 고혈압 동물모델을 제공한다.In addition, the present invention provides an animal model of gestational hypertension to which the composition is administered.
또한, 본 발명은 상기 임신성 고혈압 동물모델의 제조방법을 제공한다.In addition, the present invention provides a method of manufacturing the gestational hypertension animal model.
아울러, 본 발명은 상기 임신성 고혈압 동물모델을 이용한 임신성 고혈압 치료제의 스크리닝 방법을 제공한다.In addition, the present invention provides a screening method for a therapeutic agent for gestational hypertension using the gestational hypertension animal model.
본 발명에 따른 sFlt-1 유전자를 포함하는 렌티바이러스 발현 벡터를 포함하는 렌티바이러스로 감염된 중간엽줄기세포를 이용하여 제작한 임신성 고혈압 모델은 임신한 동물의 태반 조직에 생착한 중간엽줄기세포가 sFlt-1 유전자를 과발현함으로써 임신성 고혈압을 유발하므로, sFlt-1 유전자를 과발현하는 중간엽줄기세포를 임신성 고혈압 동물모델 제조용 세포 조성물로 이용할 수 있으며, 이로 제조된 동물모델은 임신성 고혈압의 병태생리 기전, 새로운 임신성 고혈압 치료 후보물질 개발, 임신성 고혈압과 향후 고혈압 발생과의 연관성 규명에 관한 연구 등에 유용하게 활용할 수 있다. The gestational hypertension model produced using a lentivirus-infected mesenchymal stem cell containing a lentiviral expression vector containing the sFlt-1 gene according to the present invention is characterized in that the mesenchymal stem cells engrafted in the placental tissue of a pregnant animal are sFlt. Since -1 gene overexpression causes gestational hypertension, mesenchymal stem cells overexpressing the sFlt-1 gene can be used as a cell composition for manufacturing an animal model of gestational hypertension. It can be usefully used for the development of candidates for the treatment of gestational hypertension and studies on the relationship between gestational hypertension and the occurrence of hypertension in the future.
도 1은 마우스 sFlt-1 유전자가 삽입된 렌티바이러스 발현 벡터의 제작 과정, 삽입된 sFlt-1 유전자의 염기서열 분석 및 제조된 렌티바이러스 입자의 역가를 측정한 결과를 나타낸 것이다.
도 2는 sFlt-1 유전자가 형질도입된 인간 지방조직 유래 중간엽줄기세포(human adipose tissue-derived mesenchymal stem cells, hATMSCs)의 제작 확인 및 줄기세포성을 검증한 도이다:
A: sFlt-1 유전자가 형질도입된 hATMSCs의 선별;
B: RT-PCR 방법으로 확인한 형질도입된 sFlt-1 hATMSCs에서의 sFlt-1 유전자 과발현; 및
C: 유세포분석기를 통한 면역 표현형 분석 결과.
도 3은 임신한 마우스에 투여한 GFP 표지된 hATMSCs의 태반 조직에서의 생착능 및 분포양상을 면역조직화학요법(immunohisotchemistry)을 통해 확인한 도이다.
도 4는 임신한 마우스에 sFlt-1 hATMSCs를 투여한 뒤 임신기간 동안 혈압의 변화를 측정한 도이다.
도 5는 sFlt-1 hATMSCs를 투여한 임신한 마우스 모체의 혈액 내 다양한 혈관생성 관련 인자들의 농도를 측정한 도이다.
도 6은 sFlt-1 hATMSCs를 투여한 임신한 마우스 모체 및 태아 뇌에서의 S100B 발현을 분석한 도이다.
도 7은 sFlt-1 hATMSCs를 투여한 임신한 마우스 모체의 간, 신장, 뇌, 태반 및 태아 뇌에서의 글루타치온의 발현을 분석한 도이다.
도 8은 sFlt-1 hATMSCs를 투여한 임신한 마우스 모체의 간, 신장, 뇌, 태반 및 태아 뇌에서의 락테이트/피루베이트의 발현을 분석한 도이다.
도 9는 본 발명의 sFlt-1 유전자가 삽입된 렌티바이러스 발현 벡터의 맵을 나타낸 도이다.FIG. 1 shows the results of the construction of a lentiviral expression vector into which a mouse sFlt-1 gene is inserted, a sequence analysis of the inserted sFlt-1 gene, and a titer of the prepared lentiviral particles.
FIG. 2 is a diagram illustrating the production confirmation and stem cellity of human adipose tissue-derived mesenchymal stem cells (hATMSCs) transduced with sFlt-1 gene:
A: selection of hATMSCs transduced with sFlt-1 gene;
B: sFlt-1 gene overexpression in transduced sFlt-1 hATMSCs confirmed by RT-PCR; And
C: Immuno phenotypic analysis result through flow cytometry.
3 is a diagram confirming the engraftment and distribution patterns of GFP-labeled hATMSCs administered to pregnant mice in placental tissues through immunohistochemistry.
4 is a diagram showing changes in blood pressure during pregnancy after administration of sFlt-1 hATMSCs to a pregnant mouse.
5 is a diagram measuring the concentration of various angiogenesis-related factors in the blood of a pregnant mouse mother administered sFlt-1 hATMSCs.
6 is a diagram illustrating the analysis of S100B expression in maternal and fetal brains of pregnant mice administered sFlt-1 hATMSCs.
7 is a diagram illustrating the analysis of glutathione expression in liver, kidney, brain, placenta and fetal brain of pregnant mouse mothers administered sFlt-1 hATMSCs.
8 is a diagram illustrating the analysis of lactate/pyruvate expression in liver, kidney, brain, placenta and fetal brain of pregnant mouse mothers administered sFlt-1 hATMSCs.
9 is a diagram showing a map of a lentiviral expression vector into which the sFlt-1 gene of the present invention is inserted.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technique or configuration well known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. However, the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of equality interpreted from the description of the claims to be described later and therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, terms used in the present specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of users or operators, or customs in the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the contents throughout the present specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 도입된다.All technical terms used in the present invention, unless otherwise defined, are used in the same meaning as those of ordinary skill in the art generally understand in the related field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention. The contents of all publications referred to herein by reference are incorporated into the present invention.
일 측면에서, 본 발명은 sFlt-1(soluble fms-like tyrosine kinase-1) 유전자를 포함하는 임신성 고혈압 모델 제조용 바이러스 발현 벡터에 관한 것이다.In one aspect, the present invention relates to a viral expression vector for preparing a gestational hypertension model comprising a sFlt-1 (soluble fms-like tyrosine kinase-1) gene.
일 구현예에서, sFlt-1 유전자는 서열번호 1의 염기서열을 포함할 수 있으며, sFlt-1 유전자를 포함하는 임신성 고혈압 모델 제조용 바이러스 발현 벡터는 도 9의 벡터맵으로 나타낼 수 있다.In one embodiment, the sFlt-1 gene may include the nucleotide sequence of SEQ ID NO: 1, and the viral expression vector for preparing a gestational hypertension model including the sFlt-1 gene may be represented by the vector map of FIG. 9.
일 구현예에서, 상기 바이러스는 아데노바이러스, 폭스 바이러스, 헤르페스 바이러스, 알파바이러스, 레트로바이러스, 피코르나바이러스, 바큘로바이러스, 및 이리도바이러스로 이루어진 군에서 선택될 수 있으며, 레트로바이러스에 속하는 렌티바이러스인 것이 더욱 바람직하다. 본 발명의 일 실시예에서 사용한 렌티바이러스는 세포분열이 활발히 일어나는 세포와 세포분열이 잘 일어나지 않는 세포 모두에 세포내로 감염율이 뛰어나며, 세포내에 형질도입되었을때 지놈(genome) 속에 삽입될 수 있기 때문에 지속적으로(long-term) 발현될 수 있는 장점이 있다.In one embodiment, the virus may be selected from the group consisting of adenovirus, pox virus, herpes virus, alpha virus, retrovirus, picornavirus, baculovirus, and iridovirus, and lentivirus belonging to retrovirus It is more preferable to be. The lentivirus used in an embodiment of the present invention has an excellent intracellular infection rate for both cells in which cell division is actively occurring and cells in which cell division does not occur, and can be inserted into the genome when transduced into cells. There is an advantage that it can be expressed long-term.
일 구현예에서, sFlt-1 유전자는 발현 조절 서열에 작동가능하게 연결되어 상기 벡터 내에 삽입될 수 있다. 본 발명에 있어, "작동 가능하게 연결된다(operably linked)"는 것은 핵산 발현 조절 서열 (예컨대, 프로모터, 시그널 서열, 또는 전사 조절인자 결합 위치의 어레이)과 다른 핵산 서열 사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절할 수 있다. 상기 조절 서열은 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열 및 전사 및 해독의 종결을 조절하는 서열을 포함한다.In one embodiment, the sFlt-1 gene can be operably linked to an expression control sequence and inserted into the vector. In the present invention, "operably linked" means a functional linkage between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence. And, whereby the control sequence can control the transcription and / or translation of the other nucleic acid sequence. Such regulatory sequences include any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
본 발명에서 사용되는 프로모터는 당업계에서 통상적으로 사용되는 벡터를 제조하기 위한 프로모터 서열을 제한없이 사용할 수 있다. 사용될 수 있는 프로모터는 예컨대, CMV (cytomegalovirus) 프로모터, NSE (neuronspecific enolase) 프로모터, SP6 프로모터, T3 프로모터, 또는 T7 프로모터 등일 수 있다.As for the promoter used in the present invention, a promoter sequence for producing a vector commonly used in the art may be used without limitation. A promoter that can be used may be, for example, a cytomegalovirus (CMV) promoter, a neurospecific enolase (NSE) promoter, an SP6 promoter, a T3 promoter, or a T7 promoter.
본 발명에 따른 sFlt-1 단백질은 혈관생성을 억제하는 특성을 갖는 티로신 키나아제 단백질로, VEGF 수용체 1 (Flt-1)의 또 다른 변이체인 sFlt-1은 혈관생성인자 혈관내피성장인자 및 태반성장인자와 결합하여 그들의 수용체와 신호전달을 차폐함으로써 혈관생성 및 성장을 감소시킨다. sFlt-1의 비정상적으로 높은 수치는 자간전증(preeclampsia)의 병인과 관련이 있음이 암시되어 왔다 [Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA (March 2003). "Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia". The Journal of Clinical Investigation. 111 (5): 649-58.]. sFlt-1의 발현은 저산소 상태에 의해 자극되며, 건강한 임신 상태에서 태반은 저산소 환경에서 발생하여 sFlt-1 발현이 20 배 증가한다. 조기에 발생하는 자간전증 환자의 경우는 sFlt-1 발현 최대 43 배까지 증가하는 것으로 추산되며, 자궁이 부족한 상태로 인해 더 심한 지방 저산소 상태로 이어질 수 있다 [Shibata E, Rajakumar A, Powers RW, Larkin RW, Gilmour C, Bodnar LM, Crombleholme WR, Ness RB, Roberts JM, Hubel CA (August 2005). "Soluble fms-like tyrosine kinase 1 is increased in preeclampsia but not in normotensive pregnancies with small-for-gestational-age neonates: relationship to circulating placental growth factor". The Journal of Clinical Endocrinology and Metabolism. 90 (8): 4895-903]. 모체 혈액에서 면역 측정법을 통해 sFlt-1 농도 측정은 자간전증의 예후 가능성을 추정할 수 있으며, 특히 sFlt-1의 증가와 VEGF의 감소는 자간전증 증상이 나타나기 적어도 5 주 전에 감지될 수 있어 조기 진단 및 치료가 용이해질 수 있다.The sFlt-1 protein according to the present invention is a tyrosine kinase protein having properties that inhibit angiogenesis, and sFlt-1, another variant of VEGF receptor 1 (Flt-1), is an angiogenesis factor, vascular endothelial growth factor, and placental growth factor. Binds to and shields their receptors and signaling, thereby reducing angiogenesis and growth. Abnormally high levels of sFlt-1 have been suggested to be associated with the etiology of preeclampsia [Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW. , Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA (March 2003). "Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia". The Journal of Clinical Investigation. 111 (5): 649-58.]. The expression of sFlt-1 is stimulated by hypoxia, and in a healthy pregnancy, the placenta occurs in a hypoxic environment, resulting in a 20-fold increase in sFlt-1 expression. In patients with preeclampsia that develop early, it is estimated that sFlt-1 expression increases up to 43 times, and a condition in which the uterus is deficient can lead to more severe fatty hypoxia [Shibata E, Rajakumar A, Powers RW, Larkin RW. , Gilmour C, Bodnar LM, Crombleholme WR, Ness RB, Roberts JM, Hubel CA (August 2005). "Soluble fms-
본 발명의 sFlt-1 단백질 유전자는 상기 공지된 서열로부터 이를 특이적으로 인식할 수 있는 프라이머를 제작하고, 이를 이용하여 중합효소연쇄증폭반응을 통해 상기 유전자를 증폭하고 이를 상술한 바와 같은 렌티바이러스 발현벡터에 도입하여 렌티바이러스를 조립한 후 이를 후술하는 바와 같이 본 발명에 따른 중간엽줄기세포에 전달이입하여 사용할 수 있다. 세포에서 단백질을 과발현하는 방법은 공지되어 있다. sFlt-1 단백질 유전자를 적절한 진핵세포 발현벡터, 예를 들면, 렌티바이러스 또는 아데노바이러스 유래의 벡터 또는 플라즈미드에 발현 프로모터에 작동 가능하도록 연결되도록 클로닝하여, 발현 벡터를 구축한 후, 이를 본 발명에 따른 세포에 도입할 수 있다. 도입 방법은 공지되어 있으며 예를 들면 리포좀 매개 전달이입, 칼슘포스페이트법, DEAE-덱스트란 매개 전달이입, 양하전 지질 매개 전달이입법, 전기천공, 파아지 시스템을 이용한 트랜스덕션 또는 바이러스를 이용한 감염 방법 등을 포함하나 이로 제한하는 것은 아니다. The sFlt-1 protein gene of the present invention produces a primer that can specifically recognize it from the known sequence, amplifies the gene through polymerase chain amplification reaction, and expresses the gene as described above. After assembling a lentivirus by introducing it into a vector, it can be transferred to the mesenchymal stem cells according to the present invention and used as described later. Methods for overexpressing proteins in cells are known. The sFlt-1 protein gene is cloned so as to be operably linked to an expression promoter to an appropriate eukaryotic expression vector, for example, a lentivirus or adenovirus-derived vector or plasmid, to construct an expression vector, and then it is according to the present invention. Can be introduced into cells. The introduction method is known, for example, liposome-mediated transduction, calcium phosphate method, DEAE-dextran-mediated translocation, positively charged lipid mediated translocation, electroporation, transduction using a phage system, or infection using a virus. Includes, but is not limited to.
일 측면에서, 본 발명은 상기 바이러스 발현 벡터가 형질도입된 바이러스 (입자)에 관한 것이다.In one aspect, the present invention relates to a virus (particle) transduced with the virus expression vector.
일 구현예에서, 상기 바이러스 (입자)는 렌티바이러스일 수 있다.In one embodiment, the virus (particle) may be a lentivirus.
일 측면에서, 본 발명은 상기 바이러스 (입자)로 형질도입 (감염)된 중간엽줄기세포 "sFlt-1 hATMSCs"에 관한 것이다.In one aspect, the present invention relates to mesenchymal stem cells "sFlt-1 hATMSCs" transduced (infected) with the virus (particle).
일 구현예에서, 상기 중간엽줄기세포는 동물 모델 제작용일 수 있고, sFlt-1 유전자가 과발현될 수 있으며, 이를 도입한 동물의 태반 조직 내에 생착될 수 있다.In one embodiment, the mesenchymal stem cells may be used for making animal models, and the sFlt-1 gene may be overexpressed, and may be engrafted in placental tissues of animals into which the sFlt-1 gene is introduced.
일 구현예에서, 상기 중간엽줄기세포는 자가, 타가 또는 이종 유래일 수 있으며, 지방 조직 유래일 수 있다.In one embodiment, the mesenchymal stem cells may be autologous, taga, or heterogeneous, and may be derived from adipose tissue.
본 발명에서, "중간엽줄기세포(Mesenchymal Stem Cell; MSC)"는 이미 성인이 된 몸의 각 부위에서 얻어지는 성체줄기세포이며 제대혈, 지방, 골수(bone marrow), 혈액, 진피 또는 골막에서 분리되는 줄기세포로서, 다양한 세포 예컨대 지방세포 및 운동신경세포 등으로 분화할 수 있는 전능성(pluripotent) 또는 다능성(multipotent) 세포를 의미한다. 또한, 중간엽줄기세포는 면역억제제의 사용 없이도 동종 또는 이종 수혜자에서 효과적으로 생착될 수 있는 특징이 있다. 상기 중간엽줄기세포는 동물, 바람직하게는 포유동물, 보다 바람직하게는 인간의 중간엽줄기세포일 수 있다. 중간엽줄기세포는 지방 조직 유래의 줄기세포이며, 지방조직 유래의 중간엽줄기세포는 골수, 양수, 제대혈 줄기 세포와 달리 다량을 제공받을 수 있는 실용적인 장점을 가지고 있고, 지방 세포의 1% 정도가 줄기 세포로 추정되고 있으며, 최근 선진국에서 광범위하게 이루어지고 있는 성형수술이 지방흡입술(liposuction)임을 고려하다면 지방유래 줄기세포는 다량 확보할 수 그 유용성이 매우 높다. 중간엽줄기세포를 얻는 과정은 다음과 같다: 인간 또는 마우스를 포함하는 포유동물, 바람직하게는, 인간의 중간엽줄기세포 소스, 예컨대, 지방조직, 혈액 또는 골수로부터 중간엽줄기세포를 분리하여 적합한 배지에서 배양한다. 배양과정에서 부유 세포를 제거하고 배양 플레이트에 부착된 세포들을 계대 배양하여, 최종적으로 선별된 중간엽줄기세포를 수득한다. 상기 과정에서 이용되는 배지로는, 줄기세포의 배양에 이용되는 일반적인 어떠한 배지도 이용할 수 있다. 본 발명에서 이용될 수 있는 배지는, 예를 들어, RPMI 시리즈, Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), α-MEM (Stanner, C.P. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923(1978)), 199 배지(Morgan et al., Proc. Soc. Exp. Bio. Med., 73:1(1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199:519(1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53:288(1965)), F10 (Ham, R.G. Exp. Cell Res. 29:515(1963)), DMEM (Dulbecco's Modified Eagle's medium, Dulbecco, R. et al., VirProcy 8:396(1959)), DMEM과 F12의 혼합물(Barnes, D. et al., Anal. Biochem. 102:255(1980)), Way-mo, h's MB752/1 (Waymo, h, C. J. Natl. Cancer Inst. 22:1003(1959)), McCoy's 5A (McCoy, T.A., et al., Proc. Soc. Exp. Biol. Med. 100:115(1959)) 및 MCDB 시리즈(Ham, R.G. et al., In Vitro 14:11(1978))을 포함하나, 이에 한정되는 것은 아니다. 상기 배지에는, 다른 성분, 예를 들어, 항생제 또는 항진균제(예컨대, 페니실린, 스트렙토마이신) 및 글루타민 등이 포함될 수 있다. 배지 및 배양에 대한 일반적인 설명은 R. Ian Freshney, Culture of Animal Cells, Alan R. Liss, Inc., New York (1984)에 기재되어 있으며 본 명세서 참조로 삽입된다. 중간엽줄기세포의 검증은, 예컨대, 중간엽줄기세포의 특이한 표면 마커를 이용하여 유세포 분석을 통해 실시된다. 본 발명에 따른 일 구현예에서 본 발명의 중간엽줄기세포는 마커로서 CD29, CD44 및 CD90와 음성 표지마커인 CD34, CD45 및 HLA-DR의 표현형을 가진다. In the present invention, "Mesenchymal Stem Cell (MSC)" is an adult stem cell obtained from each part of the body that has already become an adult, and is separated from umbilical cord blood, fat, bone marrow, blood, dermis or periosteum. As stem cells, it refers to pluripotent or multipotent cells capable of differentiating into various cells such as adipocytes and motor neurons. In addition, mesenchymal stem cells have a feature that can be effectively engrafted in allogeneic or heterogeneous recipients without the use of immunosuppressants. The mesenchymal stem cell may be an animal, preferably a mammal, more preferably a human mesenchymal stem cell. Mesenchymal stem cells are stem cells derived from adipose tissue, and mesenchymal stem cells derived from adipose tissue have a practical advantage of being able to receive a large amount, unlike bone marrow, amniotic fluid, and cord blood stem cells. Stem cells are presumed to be stem cells, and considering that liposuction is a plastic surgery that is widely performed in developed countries, a large amount of adipose-derived stem cells can be secured and its usefulness is very high. The process of obtaining mesenchymal stem cells is as follows: A suitable mesenchymal stem cell is isolated from a source of mesenchymal stem cells, such as adipose tissue, blood or bone marrow, of a mammal, including a human or mouse, preferably. Incubate in medium. The suspended cells are removed during the culture process and the cells attached to the culture plate are subcultured to obtain finally selected mesenchymal stem cells. As the medium used in the above process, any general medium used for culturing stem cells may be used. The medium that can be used in the present invention is, for example, RPMI series, Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), α-MEM (Stanner, CP et al., Nat. New Biol. 230:52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923 (1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Exp. Bio. Med., 73:1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199:519 (1967)), F12 (Ham, Proc. Natl. Acad. USA 53:288 (1965)), F10 (Ham, RG Exp. Cell Res. 29:515 (1963)), DMEM (Dulbecco's Modified Eagle's medium, Dulbecco, R. et al., VirProcy 8:396 ( 1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102:255 (1980)), Way-mo, h's MB752/1 (Waymo, h, CJ Natl. Cancer Inst. 22 :1003(1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc. Exp. Biol. Med. 100:115(1959)) and MCDB series (Ham, RG et al., In Vitro 14: 11 (1978)), but is not limited thereto. The medium may contain other components such as antibiotics or antifungal agents (eg, penicillin, streptomycin), glutamine, and the like. A general description of media and cultures is described in R. Ian Freshney, Culture of Animal Cells, Alan R. Liss, Inc., New York (1984) and is incorporated herein by reference. Verification of mesenchymal stem cells is performed, for example, through flow cytometry using a specific surface marker of mesenchymal stem cells. In one embodiment according to the present invention, the mesenchymal stem cells of the present invention have a phenotype of CD29, CD44 and CD90 as markers and CD34, CD45 and HLA-DR as negative markers.
일 측면에서, 본 발명은 상기 중간엽줄기세포를 포함하는 임신성 고혈압 모델 제조용 조성물에 관한 것이다.In one aspect, the present invention relates to a composition for preparing a gestational hypertension model comprising the mesenchymal stem cells.
일 구현예에서, 상기 중간엽줄기세포를 포함하는 임신성 고혈압 모델 제조용 조성물은 동물에 복강 내 주사로 도입 (투여)될 수 있다.In one embodiment, the composition for preparing a gestational hypertension model comprising the mesenchymal stem cells may be introduced (administered) to an animal by intraperitoneal injection.
일 측면에서, 본 발명은 임신성 고혈압 모델 제조용 조성물이 투여된 임신성 고혈압 동물모델에 관한 것이다.In one aspect, the present invention relates to a gestational hypertension animal model administered with a composition for preparing a gestational hypertension model.
일 구현예에서, 상기 동물은 태반을 통해 태내의 태아를 기르는 동물일 수 있으며, 포유동물일 수 있고, 마우스, 래트, 원숭이, 개, 고양이, 토끼, 모르모트, 소, 양, 돼지 및 염소로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있고, 마우스인 것이 더욱 바람직하다.In one embodiment, the animal may be an animal that raises a fetus in the womb through the placenta, may be a mammal, and consists of a mouse, rat, monkey, dog, cat, rabbit, morpho, cow, sheep, pig, and goat. It may be any one or more selected from the group, more preferably a mouse.
본 발명에서, "동물모델"이란 인간의 질환과 아주 유사한 형태의 질환을 가진 동물모델을 말한다. 인간의 질환 연구에 있어 질환 동물모델이 의미를 갖는 것은 인간과 동물들 간의 생리적 또는 유전적인 유사성에 의한다. 질환 연구에 있어 생체의학 동물모델은 질환의 다양한 원인과 발병과정 및 진단에 대한 연구용 재료를 제공해주고, 질환 동물모델의 연구를 통해 질환과 관련된 발병기전을 이해할 수 있게 하고, 개발된 신약후보물질의 실제 효능 및 독성 검사를 통해 실용화 가능성의 여부를 판단하는 기초 자료를 얻을 수 있다.In the present invention, "animal model" refers to an animal model having a disease very similar to that of humans. In the study of human diseases, the significance of disease animal models is based on physiological or genetic similarities between humans and animals. In disease research, biomedical animal models provide materials for research on various causes, onset processes, and diagnosis of diseases, enable understanding of disease-related pathogenesis through research on disease animal models, and develop new drug candidates. Basic data for judging the possibility of practical use can be obtained through actual efficacy and toxicity tests.
본 발명에서 "도입"은 "주입", "주사" 또는 "감염"과 혼용되어 사용할 수 있다.In the present invention, "introduction" may be used interchangeably with "injection", "injection" or "infection".
본 발명에서, "동물" 또는 "실험동물"이란 인간 이외의 임의의 포유류 동물을 의미한다. 상기 동물은 배아, 태아, 신생아 및 성인을 포함하는 모든 연령의 동물을 포함한다. 본 발명에서 사용하기 위한 동물들은, 예를 들어, 상업용 소스로 부터 이용할 수 있다. 이런 동물들은 실험용 동물 또는 다른 동물, 토끼, 설치류 (예를 들어, 생쥐, 쥐, 햄스터, 게르빌루스 및 기니피그), 소, 양, 돼지, 염소, 말, 개, 고양이, 새 (예를 들어, 닭, 칠면조, 오리, 거위), 영장류 (예를 들어, 침팬지, 원숭이, 붉은털원숭이)를 포함할 수 있다.In the present invention, "animal" or "experimental animal" means any mammalian animal other than humans. The animal includes animals of all ages, including embryos, fetuses, newborns and adults. Animals for use in the present invention can be used, for example, from commercial sources. These animals are laboratory animals or other animals, rabbits, rodents (e.g. mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (e.g., Chickens, turkeys, ducks, geese), primates (eg chimpanzees, monkeys, rhesus monkeys).
일 측면에서, 본 발명은 본 발명의 임신성 고혈압 모델 제조용 바이러스 발현 벡터를 포함하는 바이러스를 중간엽줄기세포에 도입하고; 및 중간엽줄기세포를 인간을 제외한 포유동물에 투여하는 것을 포함하는, 임신성 고혈압 동물모델의 제조방법에 관한 것이다.In one aspect, the present invention introduces a virus containing the viral expression vector for preparing a gestational hypertension model of the present invention into mesenchymal stem cells; And it relates to a method for producing a gestational hypertension animal model comprising administering mesenchymal stem cells to mammals other than humans.
본 발명에서, "투여"란, 어떠한 적절한 방법으로 개체에게 특정한 조성물을 도입하는 것을 의미하며, In the present invention, "administering" means introducing a specific composition to an individual by any suitable method,
본 발명에서, sFlt-1 hATMSCs의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 예를 들어, 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여될 수 있으나, 이에 제한되지는 않는다. 본 발명에 따른 조성물은 정맥 투여 방식으로 투여될 수 있다. 본 발명의 실시예에서는 sFlt-1 hATMSCs는 복강 내 투여되었다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 포함하는 것이 중요하다. 예컨대, 본 발명의 조성물의 투여량은 2.0×107 내지 8.0×107 세포/kg(체중)일 수 있다. In the present invention, the route of administration of sFlt-1 hATMSCs can be administered through any general route as long as it can reach the target tissue. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration, but are not limited thereto. The composition according to the present invention can be administered by intravenous administration. In an example of the present invention, sFlt-1 hATMSCs was administered intraperitoneally. It is important to include an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors. For example, the dosage of the composition of the present invention may be 2.0×10 7 to 8.0×10 7 cells/kg (body weight).
일 측면에서, 본 발명은 본 발명의 임신성 고혈압 동물모델에 임신성 고혈압 치료 후보 물질을 투여하고; 및 상기 임신성 고혈압 동물모델에서 혈압을 측정하여 임신성 고혈압 치료 후보물질의 혈압 강하 효과를 확인하는 것을 포함하는 임신성 고혈압 치료제의 스크리닝 방법에 관한 것이다.In one aspect, the present invention administers a candidate substance for treatment of gestational hypertension to the gestational hypertension animal model of the present invention; And measuring blood pressure in the gestational hypertension animal model to determine the blood pressure lowering effect of a candidate substance for treating gestational hypertension.
본 발명에서 "스크리닝"이란, 여러 물질로 이루어진 후보군으로부터 목적으로 하는 어떤 특정한 성질을 갖는 물질을 특정한 조작 또는 평가 방법으로 선별하는 것이다. 본 발명의 목적상, 본 발명의 스크리닝은, 본 발명에 의한 임신성 고혈압 동물모델에 임신성 고혈압 예방용 또는 치료용 후보물질을 처리하고, 상기 후보물질에 의하여 임신성 고혈압가 예방되거나, 치료되거나, 대조군 물질에 비하여 예후가 증진된 경우에 후보물질을 임신성 고혈압 예방제 또는 치료제로 결정하는 것이다.In the present invention, "screening" refers to selecting a substance having a specific target property from a candidate group consisting of several substances by a specific operation or evaluation method. For the purposes of the present invention, the screening of the present invention is to treat a candidate for preventing or treating gestational hypertension in an animal model of gestational hypertension according to the present invention, and to prevent, treat or treat gestational hypertension by the candidate material. In contrast, when the prognosis is improved, the candidate substance is determined as a preventive or therapeutic agent for gestational hypertension.
본 발명에서 "치료" 또는 "경감"이란 시험 물질 또는 후보 물질의 투여로 임신성 고혈압의 증세를 치료, 호전시키거나 이롭게 변경하는 모든 행위를 의미한다. 구체적으로, "치료"는 이롭거나 바람직한 임상적 결과를 수득하기 위한 접근을 의미한다. 본 발명의 목적을 위해서, 이롭거나 바람직한 임상적 결과는 비제한적으로, 증상의 완화, 질병 범위의 감소, 질병 상태의 안정화 (즉, 악화되지 않음), 질병 진행의 지연 또는 속도의 감소, 질병 상태의 개선 또는 일시적 완화 및 경감 (부분적이거나 전체적으로), 검출가능 여부를 포함한다. "치료"는 치료학적 치료 및 예방적 또는 예방조치 방법 모두를 가리킨다. 상기 치료들은 예방되는 장애뿐만 아니라 이미 발생한 장애에 있어서 요구되는 치료를 포함한다. 질병을 "완화 (palliating)"하는 것은 치료를 하지 않은 경우와 비교하여, 질병상태의 범위 및/또는 바람직하지 않은 임상적 징후가 감소되거나 및/또는 진행의 시간적 추이(time course)가 늦춰지거나 길어지는 것을 의미한다.In the present invention, "treatment" or "reduction" refers to any action that treats, ameliorates, or beneficially alters the symptoms of gestational hypertension by administration of a test substance or a candidate substance. Specifically, “treatment” refers to an approach to obtaining beneficial or desirable clinical outcomes. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (i.e., not exacerbation), delay or decrease in disease progression, disease state Improvement or temporary alleviation and alleviation (partial or total) of, detectable or not. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include the disorder to be prevented as well as the treatment required for a disorder that has already occurred. “Palliating” the disease is a reduction in the extent of the disease state and/or undesirable clinical signs and/or a delayed or prolonged time course of progression compared to without treatment. Means to lose.
본 발명의 스크리닝 방법에 의해 분석되는 임신성 고혈압 치료용 후보 화합물 (또는 시험 물질)은 단일 화합물, 화합물들의 혼합물 (예컨대, 천연 추출물 또는 세포 또는 조직 배양물), 또는 바이오의약품 (예컨대, 단백질, 항체, 펩타이드, DNA, RNA, 안티센스 올리고뉴클레오티드, RNAi, 앱타머, RNAzyme 및 DNAzyme) 일 수 있다. 상기 시험 물질은 합성 또는 천연 화합물의 라이브러리로부터 얻을 수 있으며 이러한 화합물의 라이브러리를 얻는 방법은 당업계에 공지되어 있다.Candidate compounds (or test substances) for the treatment of gestational hypertension analyzed by the screening method of the present invention are a single compound, a mixture of compounds (e.g., natural extracts or cell or tissue culture), or biopharmaceuticals (e.g., proteins, antibodies, Peptide, DNA, RNA, antisense oligonucleotide, RNAi, aptamer, RNAzyme and DNAzyme). The test substance can be obtained from a library of synthetic or natural compounds, and methods of obtaining a library of such compounds are known in the art.
임신성 고혈압이 유발된 동물 모델에 시험 물질을 처리하는 방법은 당업계에 공지된 다양한 방법으로 실시할 수 있다. 예를 들어, 임신성 고혈압 동물 모델에 정맥 내 주입, 피하 주입, 근육 주입, 복강 주입, 국소 투여 또는 경피 투여 등의 방법이 포함될 수 있고, 사료 또는 음용수에 포함시켜 경구 투여 형태로 섭식시킬 수 있으나, 이에 제한되는 것은 아니다.The method of treating the test substance in an animal model in which gestational hypertension is induced can be carried out by various methods known in the art. For example, a method such as intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration or transdermal administration may be included in the gestational hypertension animal model, and may be included in feed or drinking water and fed in an oral dosage form. It is not limited thereto.
본 발명의 스크리닝 방법에서 상기 시험 물질이 임신성 고혈압의 경감 또는 치료에 효과가 있는지 평가하기 위해서 대조군을 이용한 스크리닝이 함께 수행될 수 있다. 대조군이란 시험 물질 처리 후 유의한 변화를 관찰하기 적합한 동물 모델 실험군을 의미하는 것으로, sFlt-1 유전자를 포함하지 않는 중간엽줄기세포를 주입 (투여)한 동물모델, 시험 물질로 처리되지 않은 동물모델, 기존의 알려진 임신성 고혈압 치료제로 처리한 동물모델, 다른 유전자 혹은 유도방법을 통해 임신성 고혈압이 유발된 동물모델 등을 들 수 있으나, 이에 제한되는 것은 아니다.In the screening method of the present invention, screening using a control may be performed together to evaluate whether the test substance is effective in reducing or treating gestational hypertension. Control refers to an animal model experimental group suitable for observing a significant change after treatment with the test substance, an animal model injected (administered) with mesenchymal stem cells that do not contain the sFlt-1 gene, and an animal model that has not been treated with the test substance. , Animal models treated with known gestational hypertension treatments, animal models in which gestational hypertension is induced through other genes or induction methods, etc. may be mentioned, but the present invention is not limited thereto.
이러한 측면에서 본 발명의 방법은 대조군 동물모델을 제조하는 단계, 대조군 샘플에 후보 화합물을 처리하는 단계 및 후보 화합물이 임신성 고혈압의 치료 또는 경감의 효과가 있는지를 대조군과 비교 평가하여 선정하는 단계를 추가로 포함할 수 있다.In this respect, the method of the present invention adds a step of preparing a control animal model, treating a candidate compound in a control sample, and comparing and evaluating whether the candidate compound has an effect of treating or reducing gestational hypertension with the control group. Can be included as.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention, and the present invention is not limited thereto.
실시예 1. sFlt-1 유전자 도입 렌티바이러스 제작Example 1. sFlt-1 gene transduction lentivirus production
1-1. sFlt-1-렌티바이러스 발현 벡터 제작1-1. Construction of sFlt-1-lentivirus expression vector
도 1에 나타낸 과정과 같이 sFlt-1 유전자 (서열번호 1)가 삽입된 렌티바이러스 발현 벡터를 제작하였다. 구체적으로, 제한효소(SpeI 또는 XhoI)가 표지된 특이적인 프라이머 세트 (표 1)를 제작하여 PCR을 통해 마우스 cDNA 라이브러리로부터 sFlt-1 유전자 (서열번호 2)를 증폭하였다. 증폭된 DNA를 전기영동한 뒤 젤 추출 키트(gel extraction kit)를 이용하여 순도 높은 sFlt-1 DNA를 회수하였다. 회수한 sFlt-1 DNA와 pGEM®Easy Vector Systems (Promega company)를 이용하여 TA 클로닝한 후 염기서열을 분석하여 확인하였다. 그 후, peI 및 XhoI으로 sFlt-1 유전자가 삽입된 상기 플라스미드를 이중 절단하여 sFlt-1 유전자 DNA를 확보하고, 같은 방법으로 처리하여 얻어진 렌티바이러스 벡터(lentiviral vector) (Invitrogen company)에 상기 sFlt-1 유전자를 삽입하였다 (도 1a). JM109 컴피턴트 세포(competent cell) (Takara company)에 트랜스포메이션(transformation)한 후 엠피실린이 첨가된 LB 한천배지에 배양함으로써 형질전환된 콜로니를 확보하였다. 확보한 콜로니에서 플라스미드를 추출하여 제한효소 절단 및 PCR 분석을 통해 sFlt-1 유전자 삽입되어 있음을 확인하였다 (도 1b). sFlt-1 유전자가 삽입되어 있는 상기 형질전환균주를 50ml LB 배지(broth)에서 대량 배양한 뒤, 미디프렙 키트(midiprep kit)를 이용하여 고농도 고순도의 플라스미드를 추출하여 sFlt-1-렌티바이러스 발현 벡터(sFlt-1-lentiviral expression vector)를 확보하였다.As shown in FIG. 1, a lentiviral expression vector into which the sFlt-1 gene (SEQ ID NO: 1) was inserted was prepared. Specifically, a specific primer set (Table 1) labeled with a restriction enzyme ( Spe I or Xho I) was prepared, and the sFlt-1 gene (SEQ ID NO: 2) was amplified from the mouse cDNA library through PCR. After electrophoresis of the amplified DNA, high purity sFlt-1 DNA was recovered using a gel extraction kit. After TA cloning using the recovered sFlt-1 DNA and pGEM® Easy Vector Systems (Promega company), the nucleotide sequence was analyzed and confirmed. Then, by double cutting the plasmid into which the sFlt-1 gene was inserted with pe I and Xho I, the sFlt-1 gene DNA was obtained, and the lentiviral vector (Invitrogen company) obtained by processing the same method described above. The sFlt-1 gene was inserted (Fig. 1A). Transformed colonies were obtained by transforming into JM109 competent cells (Takara company) and then culturing on LB agar medium to which Mpicillin was added. The plasmid was extracted from the obtained colony, and it was confirmed that the sFlt-1 gene was inserted through restriction enzyme digestion and PCR analysis (FIG. 1B). After culturing the transformant strain into which the sFlt-1 gene is inserted in 50 ml LB medium (broth), a high-concentration, high-purity plasmid was extracted using a midiprep kit, and the sFlt-1-lentivirus expression vector (sFlt-1-lentiviral expression vector) was obtained.
1-2. sFlt-1 발현 렌티바이러스 생산1-2. sFlt-1 expressing lentivirus production
렌티바이러스 입자를 ViraPower™ Lentiviral Expression Systems (invitrogen company)을 이용하여 생산하였다. 구체적으로, ViraPower Packaging mix 9㎍를 혼합하여 리포펙타민(lipofectamine)으로 293FT 세포주에 처리한 후 48시간 및 72시간 후의 상등액을 수거 및 원심분리하여 세포 데브리스(debris)를 제거한 상등액을 회수하였다. 상기 실시예 1-1의 sFlt-1-렌티바이러스 발현 벡터와 상기 packaging mix를 이용하여 293FT 세포를 co-트랜스펙션한 뒤, 이로부터 총 RNA을 추출하고 cDNA를 합성하여 RT-PCR 분석을 통해 sFlt-1 유전자의 과발현을 확인하였다 (도 1c). 회수한 sFlt-1-렌티바이러스 입자 상등액을 Fast-Trap virus Purification 및 Concentration Kit를 이용하여 100배 농축하고 sFlt-1-렌티바이러스 입자의 역가(titer)를 측정하였다 (도 1d). 회수한 sFlt-1-렌티바이러스 입자를 다양한 농도로 희석하여 종양세포주에 첨가하여 16시간 배양한 뒤 blastocidin 항생제 (5-10 ug/ml)를 첨가하여 1-3일간 배양하고 크리스탈 바이올렛(Crystal violet) 염색을 통해 바이러스 감염으로 인한 형질도입율을 산정하였다 (도 1d). Lentiviral particles were produced using ViraPower™ Lentiviral Expression Systems (invitrogen company). Specifically, 9 μg of ViraPower Packaging mix was mixed and treated with lipofectamine on the 293FT cell line, and the supernatant after 48 and 72 hours was collected and centrifuged to recover the supernatant from which cell debris was removed. After co-transfecting 293FT cells using the sFlt-1-lentivirus expression vector of Example 1-1 and the packaging mix, total RNA was extracted therefrom, and cDNA was synthesized through RT-PCR analysis. Overexpression of the sFlt-1 gene was confirmed (Fig. 1c). The recovered sFlt-1-lentivirus particle supernatant was concentrated 100 times using Fast-Trap virus Purification and Concentration Kit, and the titer of the sFlt-1-lentivirus particle was measured (FIG. 1D ). The recovered sFlt-1-lentivirus particles were diluted to various concentrations, added to the tumor cell line, incubated for 16 hours, blastocidin antibiotic (5-10 ug/ml) was added and incubated for 1-3 days, and crystal violet Through staining, the transduction rate due to viral infection was calculated (FIG. 1D).
실시예 2. sFlt-1 과발현 중간엽줄기세포 제작Example 2. Preparation of sFlt-1 overexpressing mesenchymal stem cells
K-STEMCELL IRB(Institutional review board)의 동의 및 양해하에 얻은 인간 지방유래 중간엽줄기세포(human adipose tissue-derived mesenchymal stem cells, hATMSCs)는 항생제가 첨가된 RKCM 배양배지 (10% FBS 첨가, K-STEMCELL에서 제공)에서 5% CO2 공급하에 37℃ 조건하에서 배양되었다. sFlt-1 단백질을 과발현하는 중간엽줄기세포를 제작하기 위해 sFlt-1-렌티바이러스 입자를 MOI (세포하나당 감염시킬 바이러스 수) 20으로 중간엽줄기세포에 처리한 후 16시간 배양하였다. Blastocidin 항생제 (5-10 ug/ml)를 상기 세포에 첨가한 후 1-3일간 배양하고 세포를 관찰하였다 (도 2A). 배양 증폭한 중간엽줄기세포를 회수한 후 RT-PCR을 통해 sFlt-1의 발현을 확인한 결과, 중간엽줄기세포에서 sFlt-1가 과발현되는 것을 확인할 수 있었다 (도 2B). 또한 유세포분석기를 이용한 면역 표현형(immunophenotyping) 분석 결과 중간엽줄기세포 표면 마커인 CD29, CD44, CD73, CD90 및 CD105의 발현이 양성으로 나타났고, CD31, CD34 및 CD45의 발현은 음성인 것으로 나타나 줄기세포성이 검증되었다 (도 2C). 이러한 결과들을 통해 sFlt-1-hATMSCs가 성공적으로 제작되었음을 알 수 있다.Human adipose tissue-derived mesenchymal stem cells (hATMSCs) obtained with the consent and understanding of K-STEMCELL IRB (Institutional Review Board) are RKCM culture medium supplemented with antibiotics (10% FBS added, K- STEMCELL) was incubated at 37° C. under 5% CO 2 supply. In order to produce mesenchymal stem cells overexpressing the sFlt-1 protein, sFlt-1-lentivirus particles were treated with MOI (number of viruses to be infected per cell) of 20 to the mesenchymal stem cells and cultured for 16 hours. Blastocidin antibiotics (5-10 ug/ml) were added to the cells, and then cultured for 1-3 days and the cells were observed (FIG. 2A). After recovering the culture-amplified mesenchymal stem cells, as a result of confirming the expression of sFlt-1 through RT-PCR, it was confirmed that sFlt-1 was overexpressed in the mesenchymal stem cells (FIG. 2B). In addition, as a result of immunophenotyping analysis using flow cytometry, the expression of the surface markers of mesenchymal stem cells CD29, CD44, CD73, CD90 and CD105 was positive, and the expression of CD31, CD34 and CD45 was negative. The sex was verified (Fig. 2C). Through these results, it can be seen that sFlt-1-hATMSCs were successfully produced.
실시예 3. 임신성 고혈압 마우스 모델 수립Example 3. Establishment of gestational hypertension mouse model
3-1. 중간엽줄기세포의 태반 생착능 분석 3-1. Analysis of placental engraftment of mesenchymal stem cells
GFP 렌티바이러스 발현 벡터를 이용하여 제작한 중간엽줄기세포 GFP-hATMSCs를 임신한 마우스모델에 복강 내 투여한 후 3일째 태반조직을 분리하여 GFP 항체를 이용한 면역조직화학요법을 실시하였다. 그 결과, hATMSCs가 태반조직 내에 전반적으로 분포되었음을 확인하였다 (도 3). 이와 같이, hATMSCs가 태반으로 이동하여 생착할 수 있으므로, sFlt-1-hATMSCs도 태반 조직 내에 생착하여 sFlt-1을 풍부하게 발현시킬 수 있음을 유추할 수 있다.After intraperitoneal administration of mesenchymal stem cells GFP-hATMSCs prepared using a GFP lentivirus expression vector to a pregnant mouse model, placenta tissue was isolated on the third day, and immunohistochemical therapy using a GFP antibody was performed. As a result, it was confirmed that hATMSCs were distributed throughout the placental tissue (FIG. 3). As described above, since hATMSCs can migrate to the placenta and engraft, it can be inferred that sFlt-1-hATMSCs can also engraft into placental tissues and express sFlt-1 abundantly.
3-2. 임신성 고혈압 마우스모델 제작 및 확인3-2. Construction and confirmation of gestational hypertension mouse model
sFlt-1-hATMSCs를 이용하여 임신성 고혈압 마우스 모델을 제작하기 위해, 상기 실시예에서 제작한 sFlt-1 hATMSCs(1 x 106 cells)을 임신 9-10일째 마우스 복강 내 주사하였다. 여기에서, 빈 벡터를 포함한 지방유래줄기세포를 주입한 마우스군 (hATMSc군)과 정상 임신 마우스군 (정상대조군)을 대조군으로 준비하였다. 그 후, 14일차에 마우스 군들의 혈압을 확인한 결과, hATMSc군 및 정상대조군에 비해 sFlt-1 hATMSCs 투여군에서 혈압이 통계적으로 유의하게 증가하는 것으로 나타났다 (도 4). 또한, 모체 마우스의 혈액을 채혈하여 다양한 혈관 생성 관련 인자들의 농도를 ELIZA 분석을 통해 측정한 결과, 모체 혈액 내 sFlt-1 농도가 sFlt-1 hATMSCs 투여군에서 대조군에 비해 통계적으로 유의하게 증가된 것으로 나타났고, sEng 및 VEGF의 농도는 대조군에 비해 sFlt-1 hATMSCs군에서 유의하게 감소된 것으로 나타났다 (도 5). 이를 통해, sFlt-1 hATMSCs의 투여로 인해 임신성 고혈압 마우스모델이 성공적으로 제작되었음을 알 수 있다.In order to construct a gestational hypertensive mouse model using sFlt-1-hATMSCs, sFlt-1 hATMSCs (1 x 10 6 cells) prepared in the above example were injected intraperitoneally at 9-10 days of pregnancy. Here, a group of mice injected with adipose-derived stem cells including an empty vector (hATMSc group) and a normal pregnant mouse group (normal control group) were prepared as controls. Thereafter, as a result of checking the blood pressure of the mouse groups on day 14, it was found that the blood pressure was statistically significantly increased in the sFlt-1 hATMSCs administration group compared to the hATMSc group and the normal control group (FIG. 4). In addition, as a result of measuring the concentrations of various angiogenesis-related factors by collecting blood from maternal mice through ELIZA analysis, it was found that the sFlt-1 concentration in the maternal blood was statistically significantly increased in the sFlt-1 hATMSCs administration group compared to the control group It was found that the concentration of sEng and VEGF was significantly decreased in the sFlt-1 hATMSCs group compared to the control group (Fig. 5). Through this, it can be seen that the gestational hypertension mouse model was successfully produced due to the administration of sFlt-1 hATMSCs.
실시예 4. 임신성 고혈압 마우스 모델의 병리적 특성 확인Example 4. Confirmation of pathological characteristics of gestational hypertension mouse model
모체 및 태아 뇌에서 신경교세포의 손상을 확인하기 위해 S100B의 변화를 sFlt-1 hATMSCs 투여군, hATMSCs 투여군 및 정상대조군에서 확인하였다. 그 결과, 모체의 뇌에서는 차이를 보이지 않았으나, S100B가 sFlt-1 hATMSCs 투여군의 태아 뇌에서 hATMSCs 투여군 및 정상대조군에 비해 통계적으로 유의하게 증가되어 있는 것을 확인 할 수 있었다 (도 6). 즉, sFlt-1 과발현이 태아 뇌의 신경교세포 손상을 야기하는 것을 알 수 있다. 또한, 모체 장기 내에서 산화스트레스를 확인하기 위해 산화스트레스 시 감소하는 글루타치온(glutathione)의 농도를 분석한 결과, 모체 뇌에서는 글루타치온 농도가 sFlt-1 hATMSCs 투여군에서 정상대조군에 비해 유의하게 높은 것으로 나타났다 (도 7). 반면, 간, 신장, 태반 및 태아의 뇌에서는 sFlt-1 hATMSCs 투여군과 정상대조군간의 차이가 나타나지 않았 (도 7). 아울러, 모체 장기 내에서 저산소 손상을 확인하기 위해 저산소 손상 시 증가하는 락테이트/피루베이트(lactate/pyruvate)의 농도를 분석한 결과, 모체의 간 및 뇌에서는 sFlt-1 hATMSCs군에서 락테이트/피루베이트의 비율이 정상대조군에 비해 유의하게 높은 것으로 나타났다 (도 8). 반면, 모체의 신장, 태반 및 태아의 뇌에서는 sFlt-1 hATMSCs 투여군과 정상대조군간의 차이가 나타나지 않았다 (도 8).To confirm the damage to glial cells in the maternal and fetal brain, changes in S100B were confirmed in the sFlt-1 hATMSCs administration group, the hATMSCs administration group, and the normal control group. As a result, there was no difference in the maternal brain, but it was confirmed that S100B was statistically significantly increased in the fetal brain of the sFlt-1 hATMSCs administration group compared to the hATMSCs administration group and the normal control group (Fig. 6). That is, it can be seen that sFlt-1 overexpression causes glial cell damage in the fetal brain. In addition, as a result of analyzing the concentration of glutathione that decreases during oxidative stress to confirm oxidative stress in the maternal organs, it was found that the concentration of glutathione in the maternal brain was significantly higher in the sFlt-1 hATMSCs group than in the normal control group ( Fig. 7). On the other hand, there was no difference between the sFlt-1 hATMSCs administration group and the normal control group in liver, kidney, placenta and fetal brain (FIG. 7 ). In addition, as a result of analyzing the concentration of lactate/pyruvate (lactate/pyruvate) that increases during hypoxic damage to confirm hypoxic damage in the maternal organs, lactate/pyruvate in the sFlt-1 hATMSCs group in the maternal liver and brain. It was found that the bait rate was significantly higher than that of the normal control group (FIG. 8). On the other hand, there was no difference between the sFlt-1 hATMSCs administration group and the normal control group in the maternal kidney, placenta, and fetal brain (FIG. 8).
<110> Korea University Research and Business Foundation <120> AN ANIMAL MODEL HAVING PREECLAMPSIA AND METHOD FOR PRODUCING THE SAME <130> PN1910-541 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 2079 <212> DNA <213> Mus musculus <400> 1 actagtgagg tcagctgctg ggacaccgcg gtcttgcctt acgcgctgct cgggtgtctg 60 cttctcacag gatatggctc agggtcgaag ttaaaagtgc ctgaactgag tttaaaaggc 120 acccagcatg tcatgcaagc aggccagact ctctttctca agtgcagagg ggaggcagcc 180 cactcatggt ctctgcccac gaccgtgagc caggaggaca aaaggctgag catcactccc 240 ccatcggcct gtgggaggga taacaggcaa ttctgcagca ccttgacctt ggacacggcg 300 caggccaacc acacgggcct ctacacctgt agatacctcc ctacatctac ttcgaagaaa 360 aagaaagcgg aatcttcaat ctacatattt gttagtgatg cagggagtcc tttcatagag 420 atgcacactg acatacccaa acttgtgcac atgacggaag gaagacagct catcatcccc 480 tgccgggtga cgtcacccaa cgtcacagtc accctaaaaa agtttccatt tgatactctt 540 acccctgatg ggcaaagaat aacatgggac agtaggagag gctttataat agcaaatgca 600 acgtacaaag agataggact gctgaactgc gaagccaccg tcaacgggca cctgtaccag 660 acaaactatc tgacccatcg gcagaccaat acaatcctag atgtccaaat acgcccgccg 720 agcccagtga gactgctcca cgggcagact cttgtcctca actgcaccgc caccacggag 780 ctcaatacga gggtgcaaat gagctggaat taccctggta aagcaactaa gagagcatct 840 ataaggcagc ggattgaccg gagccattcc cacaacaatg tgttccacag tgttcttaag 900 atcaacaatg tggagagccg agacaagggg ctctacacct gtcgcgtgaa gagtgggtcc 960 tcgttccagt ctttcaacac ctccgtgcat gtgtatgaaa aaggattcat cagtgtgaaa 1020 catcggaagc agccggtgca ggaaaccaca gcaggaagac ggtcctatcg gctgtccatg 1080 aaagtgaagg ccttcccctc cccagaaatc gtatggttaa aagatggctc gcctgcaaca 1140 ttgaagtctg ctcgctattt ggtacatggc tactcattaa ttatcaaaga tgtgacaacc 1200 gaggatgcag gggactatac gatcttgctg ggcataaagc agtcaaggct atttaaaaac 1260 ctcactgcca ctctcattgt aaacgtgaaa cctcagatct acgaaaagtc cgtgtcctcg 1320 cttccaagcc cacctctcta tccgctgggc agcagacaag tcctcacttg caccgtgtat 1380 ggcatccctc ggccaacaat cacgtggctc tggcacccct gtcaccacaa tcactccaaa 1440 gaaaggtatg acttctgcac tgagaatgaa gaatccttta tcctggatcc cagcagcaac 1500 ttaggaaaca gaattgagag catctctcag cgcatgacgg tcatagaagg aacaaataag 1560 acggttagca cattggtggt ggctgactct cagacccctg gaatctacag ctgccgggcc 1620 ttcaataaaa tagggactgt ggaaagaaac ataaaatttt acgtcacaga tgtgccgaat 1680 ggctttcacg tttccttgga aaagatgcca gccgaaggag aggacctgaa actgtcctgt 1740 gtggtcaata aattcctgta cagagacatt acctggattc tgctacggac agttaacaac 1800 agaaccatgc accatagtat cagcaagcaa aaaatggcca ccactcaaga ttactccatc 1860 actctgaacc ttgtcatcaa gaacgtgtct ctagaagact cgggcaccta tgcgtgcaga 1920 gccaggaaca tatacacagg ggaagacatc cttcggaaga cagaagttct cgttagaggt 1980 gagcactgcg gcaaaaaggc cattttctct cggatctcca aatttaaaag caggaggaat 2040 gattgtacca cacaaagtca tgtcaaacat taactcgag 2079 <210> 2 <211> 3394 <212> DNA <213> Mus musculus <400> 2 agcgcggagg cggacactcc cgggaggtag tgctagtggt ggtggctgct gctcggagcg 60 ggctccggga ctcaagcgca gcggctagcg gacgcgggac ggcgtggatc cccccacacc 120 acccccctcg gctgcaggcg cggagaaggg ctctcgcggc gccaagcaga agcaggaggg 180 gaccggctcg agcgtgccgc gtcggcctcg gagagcgcgg gcaccggcca acaggccgcg 240 tcttgctcac catggtcagc tgctgggaca ccgcggtctt gccttacgcg ctgctcgggt 300 gtctgcttct cacaggatat ggctcagggt cgaagttaaa agtgcctgaa ctgagtttaa 360 aaggcaccca gcatgtcatg caagcaggcc agactctctt tctcaagtgc agaggggagg 420 cagcccactc atggtctctg cccacgaccg tgagccagga ggacaaaagg ctgagcatca 480 ctcccccatc ggcctgtggg agggataaca ggcaattctg cagcaccttg accttggaca 540 cggcgcaggc caaccacacg ggcctctaca cctgtagata cctccctaca tctacttcga 600 agaaaaagaa agcggaatct tcaatctaca tatttgttag tgatgcaggg agtcctttca 660 tagagatgca cactgacata cccaaacttg tgcacatgac ggaaggaaga cagctcatca 720 tcccctgccg ggtgacgtca cccaacgtca cagtcaccct aaaaaagttt ccatttgata 780 ctcttacccc tgatgggcaa agaataacat gggacagtag gagaggcttt ataatagcaa 840 atgcaacgta caaagagata ggactgctga actgcgaagc caccgtcaac gggcacctgt 900 accagacaaa ctatctgacc catcggcaga ccaatacaat cctagatgtc caaatacgcc 960 cgccgagccc agtgagactg ctccacgggc agactcttgt cctcaactgc accgccacca 1020 cggagctcaa tacgagggtg caaatgagct ggaattaccc tggtaaagca actaagagag 1080 catctataag gcagcggatt gaccggagcc attcccacaa caatgtgttc cacagtgttc 1140 ttaagatcaa caatgtggag agccgagaca aggggctcta cacctgtcgc gtgaagagtg 1200 ggtcctcgtt ccagtctttc aacacctccg tgcatgtgta tgaaaaagga ttcatcagtg 1260 tgaaacatcg gaagcagccg gtgcaggaaa ccacagcagg aagacggtcc tatcggctgt 1320 ccatgaaagt gaaggccttc ccctccccag aaatcgtatg gttaaaagat ggctcgcctg 1380 caacattgaa gtctgctcgc tatttggtac atggctactc attaattatc aaagatgtga 1440 caaccgagga tgcaggggac tatacgatct tgctgggcat aaagcagtca aggctattta 1500 aaaacctcac tgccactctc attgtaaacg tgaaacctca gatctacgaa aagtccgtgt 1560 cctcgcttcc aagcccacct ctctatccgc tgggcagcag acaagtcctc acttgcaccg 1620 tgtatggcat ccctcggcca acaatcacgt ggctctggca cccctgtcac cacaatcact 1680 ccaaagaaag gtatgacttc tgcactgaga atgaagaatc ctttatcctg gatcccagca 1740 gcaacttagg aaacagaatt gagagcatct ctcagcgcat gacggtcata gaaggaacaa 1800 ataagacggt tagcacattg gtggtggctg actctcagac ccctggaatc tacagctgcc 1860 gggccttcaa taaaataggg actgtggaaa gaaacataaa attttacgtc acagatgtgc 1920 cgaatggctt tcacgtttcc ttggaaaaga tgccagccga aggagaggac ctgaaactgt 1980 cctgtgtggt caataaattc ctgtacagag acattacctg gattctgcta cggacagtta 2040 acaacagaac catgcaccat agtatcagca agcaaaaaat ggccaccact caagattact 2100 ccatcactct gaaccttgtc atcaagaacg tgtctctaga agactcgggc acctatgcgt 2160 gcagagccag gaacatatac acaggggaag acatccttcg gaagacagaa gttctcgtta 2220 gaggtgagca ctgcggcaaa aaggccattt tctctcggat ctccaaattt aaaagcagga 2280 ggaatgattg taccacacaa agtcatgtca aacattaaag gactcatttg aaaagtaaca 2340 gttgtctctt atcatctcag tttattgtta ctgttgctaa ctttcaggcc cagaggaaac 2400 gctcctccca aaatgagttt ggacatgata acgtaataag aaagcccagt gccctctgcc 2460 cggggtgccc gctggcccgg gggtgctctg tgggccgccc ggtgtgtgtt tggatttgaa 2520 gatccctgta ctctgtttct tttgtgtgtc tgctcttctg tcttctgctt catagcagca 2580 acctgggacg catgtttttc ttccactctg atgccaacct cttttgatat atatatatat 2640 ttttaagttg tgaagctgaa caaactgaat aatttaagca aatgctggtt tctgccaaag 2700 acggacatga ataagttaat tttttttcca gcacaggatg cgtacagttg aatttggaat 2760 ctgtgtcggg tgtctacctg gttttatttt ttactatttc attttttgct cttgatttgt 2820 aaatagttcc tggataacaa gttataatgc ttatttattt gaaacttggt tgttttgttg 2880 tttttttttt cttttcatga agtatattga tcttaaactg gagggttcta agatgggtcc 2940 caggggctca agatgttgat gtcattccga gagtaaagct atgtcccaat gtgaattatg 3000 aaggtccagc aggtctgctc caccctcctc tgtccaccca ggtaattaca cgtgtgtttc 3060 ctgctgtgtt agatgctgtt cctcattgtc cttggctgga ctgacagccc ctgactgacg 3120 gcaaaagtgc agcaagcctt cattataaac actcatggcc cctgggcact gttttaaagc 3180 ccttcaccaa gctttgatgg cattcaaaga tgtccacaac ccatgtatcc aggatataaa 3240 ggctattgtg agtggagatt taatgcaatc ttcttaatgt ctattgaaaa atctacccat 3300 gagagaaaga aaagtccacc ttctctatat gcaaatgttt tatggggatt aagaaattgc 3360 aaaagctaag aaattacaaa aaaaaaaaaa aaaa 3394 <110> Korea University Research and Business Foundation <120> ANIMAL MODEL HAVING PREECLAMPSIA AND METHOD FOR PRODUCING THE SAME <130> PN1910-541 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 2079 <212> DNA <213> Mus musculus <400> 1 actagtgagg tcagctgctg ggacaccgcg gtcttgcctt acgcgctgct cgggtgtctg 60 cttctcacag gatatggctc agggtcgaag ttaaaagtgc ctgaactgag tttaaaaggc 120 acccagcatg tcatgcaagc aggccagact ctctttctca agtgcagagg ggaggcagcc 180 cactcatggt ctctgcccac gaccgtgagc caggaggaca aaaggctgag catcactccc 240 ccatcggcct gtgggaggga taacaggcaa ttctgcagca ccttgacctt ggacacggcg 300 caggccaacc acacgggcct ctacacctgt agatacctcc ctacatctac ttcgaagaaa 360 aagaaagcgg aatcttcaat ctacatattt gttagtgatg cagggagtcc tttcatagag 420 atgcacactg acatacccaa acttgtgcac atgacggaag gaagacagct catcatcccc 480 tgccgggtga cgtcacccaa cgtcacagtc accctaaaaa agtttccatt tgatactctt 540 acccctgatg ggcaaagaat aacatgggac agtaggagag gctttataat agcaaatgca 600 acgtacaaag agataggact gctgaactgc gaagccaccg tcaacgggca cctgtaccag 660 acaaactatc tgacccatcg gcagaccaat acaatcctag atgtccaaat acgcccgccg 720 agcccagtga gactgctcca cgggcagact cttgtcctca actgcaccgc caccacggag 780 ctcaatacga gggtgcaaat gagctggaat taccctggta aagcaactaa gagagcatct 840 ataaggcagc ggattgaccg gagccattcc cacaacaatg tgttccacag tgttcttaag 900 atcaacaatg tggagagccg agacaagggg ctctacacct gtcgcgtgaa gagtgggtcc 960 tcgttccagt ctttcaacac ctccgtgcat gtgtatgaaa aaggattcat cagtgtgaaa 1020 catcggaagc agccggtgca ggaaaccaca gcaggaagac ggtcctatcg gctgtccatg 1080 aaagtgaagg ccttcccctc cccagaaatc gtatggttaa aagatggctc gcctgcaaca 1140 ttgaagtctg ctcgctattt ggtacatggc tactcattaa ttatcaaaga tgtgacaacc 1200 gaggatgcag gggactatac gatcttgctg ggcataaagc agtcaaggct atttaaaaac 1260 ctcactgcca ctctcattgt aaacgtgaaa cctcagatct acgaaaagtc cgtgtcctcg 1320 cttccaagcc cacctctcta tccgctgggc agcagacaag tcctcacttg caccgtgtat 1380 ggcatccctc ggccaacaat cacgtggctc tggcacccct gtcaccacaa tcactccaaa 1440 gaaaggtatg acttctgcac tgagaatgaa gaatccttta tcctggatcc cagcagcaac 1500 ttaggaaaca gaattgagag catctctcag cgcatgacgg tcatagaagg aacaaataag 1560 acggttagca cattggtggt ggctgactct cagacccctg gaatctacag ctgccgggcc 1620 ttcaataaaa tagggactgt ggaaagaaac ataaaatttt acgtcacaga tgtgccgaat 1680 ggctttcacg tttccttgga aaagatgcca gccgaaggag aggacctgaa actgtcctgt 1740 gtggtcaata aattcctgta cagagacatt acctggattc tgctacggac agttaacaac 1800 agaaccatgc accatagtat cagcaagcaa aaaatggcca ccactcaaga ttactccatc 1860 actctgaacc ttgtcatcaa gaacgtgtct ctagaagact cgggcaccta tgcgtgcaga 1920 gccaggaaca tatacacagg ggaagacatc cttcggaaga cagaagttct cgttagaggt 1980 gagcactgcg gcaaaaaggc cattttctct cggatctcca aatttaaaag caggaggaat 2040 gattgtacca cacaaagtca tgtcaaacat taactcgag 2079 <210> 2 <211> 3394 <212> DNA <213> Mus musculus <400> 2 agcgcggagg cggacactcc cgggaggtag tgctagtggt ggtggctgct gctcggagcg 60 ggctccggga ctcaagcgca gcggctagcg gacgcgggac ggcgtggatc cccccacacc 120 acccccctcg gctgcaggcg cggagaaggg ctctcgcggc gccaagcaga agcaggaggg 180 gaccggctcg agcgtgccgc gtcggcctcg gagagcgcgg gcaccggcca acaggccgcg 240 tcttgctcac catggtcagc tgctgggaca ccgcggtctt gccttacgcg ctgctcgggt 300 gtctgcttct cacaggatat ggctcagggt cgaagttaaa agtgcctgaa ctgagtttaa 360 aaggcaccca gcatgtcatg caagcaggcc agactctctt tctcaagtgc agaggggagg 420 cagcccactc atggtctctg cccacgaccg tgagccagga ggacaaaagg ctgagcatca 480 ctcccccatc ggcctgtggg agggataaca ggcaattctg cagcaccttg accttggaca 540 cggcgcaggc caaccacacg ggcctctaca cctgtagata cctccctaca tctacttcga 600 agaaaaagaa agcggaatct tcaatctaca tatttgttag tgatgcaggg agtcctttca 660 tagagatgca cactgacata cccaaacttg tgcacatgac ggaaggaaga cagctcatca 720 tcccctgccg ggtgacgtca cccaacgtca cagtcaccct aaaaaagttt ccatttgata 780 ctcttacccc tgatgggcaa agaataacat gggacagtag gagaggcttt ataatagcaa 840 atgcaacgta caaagagata ggactgctga actgcgaagc caccgtcaac gggcacctgt 900 accagacaaa ctatctgacc catcggcaga ccaatacaat cctagatgtc caaatacgcc 960 cgccgagccc agtgagactg ctccacgggc agactcttgt cctcaactgc accgccacca 1020 cggagctcaa tacgagggtg caaatgagct ggaattaccc tggtaaagca actaagagag 1080 catctataag gcagcggatt gaccggagcc attcccacaa caatgtgttc cacagtgttc 1140 ttaagatcaa caatgtggag agccgagaca aggggctcta cacctgtcgc gtgaagagtg 1200 ggtcctcgtt ccagtctttc aacacctccg tgcatgtgta tgaaaaagga ttcatcagtg 1260 tgaaacatcg gaagcagccg gtgcaggaaa ccacagcagg aagacggtcc tatcggctgt 1320 ccatgaaagt gaaggccttc ccctccccag aaatcgtatg gttaaaagat ggctcgcctg 1380 caacattgaa gtctgctcgc tatttggtac atggctactc attaattatc aaagatgtga 1440 caaccgagga tgcaggggac tatacgatct tgctgggcat aaagcagtca aggctattta 1500 aaaacctcac tgccactctc attgtaaacg tgaaacctca gatctacgaa aagtccgtgt 1560 cctcgcttcc aagcccacct ctctatccgc tgggcagcag acaagtcctc acttgcaccg 1620 tgtatggcat ccctcggcca acaatcacgt ggctctggca cccctgtcac cacaatcact 1680 ccaaagaaag gtatgacttc tgcactgaga atgaagaatc ctttatcctg gatcccagca 1740 gcaacttagg aaacagaatt gagagcatct ctcagcgcat gacggtcata gaaggaacaa 1800 ataagacggt tagcacattg gtggtggctg actctcagac ccctggaatc tacagctgcc 1860 gggccttcaa taaaataggg actgtggaaa gaaacataaa attttacgtc acagatgtgc 1920 cgaatggctt tcacgtttcc ttggaaaaga tgccagccga aggagaggac ctgaaactgt 1980 cctgtgtggt caataaattc ctgtacagag acattacctg gattctgcta cggacagtta 2040 acaacagaac catgcaccat agtatcagca agcaaaaaat ggccaccact caagattact 2100 ccatcactct gaaccttgtc atcaagaacg tgtctctaga agactcgggc acctatgcgt 2160 gcagagccag gaacatatac acaggggaag acatccttcg gaagacagaa gttctcgtta 2220 gaggtgagca ctgcggcaaa aaggccattt tctctcggat ctccaaattt aaaagcagga 2280 ggaatgattg taccacacaa agtcatgtca aacattaaag gactcatttg aaaagtaaca 2340 gttgtctctt atcatctcag tttattgtta ctgttgctaa ctttcaggcc cagaggaaac 2400 gctcctccca aaatgagttt ggacatgata acgtaataag aaagcccagt gccctctgcc 2460 cggggtgccc gctggcccgg gggtgctctg tgggccgccc ggtgtgtgtt tggatttgaa 2520 gatccctgta ctctgtttct tttgtgtgtc tgctcttctg tcttctgctt catagcagca 2580 acctgggacg catgtttttc ttccactctg atgccaacct cttttgatat atatatatat 2640 ttttaagttg tgaagctgaa caaactgaat aatttaagca aatgctggtt tctgccaaag 2700 acggacatga ataagttaat tttttttcca gcacaggatg cgtacagttg aatttggaat 2760 ctgtgtcggg tgtctacctg gttttatttt ttactatttc attttttgct cttgatttgt 2820 aaatagttcc tggataacaa gttataatgc ttatttattt gaaacttggt tgttttgttg 2880 tttttttttt cttttcatga agtatattga tcttaaactg gagggttcta agatgggtcc 2940 caggggctca agatgttgat gtcattccga gagtaaagct atgtcccaat gtgaattatg 3000 aaggtccagc aggtctgctc caccctcctc tgtccaccca ggtaattaca cgtgtgtttc 3060 ctgctgtgtt agatgctgtt cctcattgtc cttggctgga ctgacagccc ctgactgacg 3120 gcaaaagtgc agcaagcctt cattataaac actcatggcc cctgggcact gttttaaagc 3180 ccttcaccaa gctttgatgg cattcaaaga tgtccacaac ccatgtatcc aggatataaa 3240 ggctattgtg agtggagatt taatgcaatc ttcttaatgt ctattgaaaa atctacccat 3300 gagagaaaga aaagtccacc ttctctatat gcaaatgttt tatggggatt aagaaattgc 3360 aaaagctaag aaattacaaa aaaaaaaaaa aaaa 3394
Claims (16)
상기 중간엽 줄기세포는 자가, 타가 또는 이종 유래의 지방에서 유래된 것을 특징으로 하고,
상기 중간엽줄기세포는 서열번호 1의 염기서열을 포함하는 sFlt-1 유전자가 과발현되며 태반 조직 내에 생착되는 것을 특징으로 하는 중간엽줄기세포.As mesenchymal stem cells (sFlt-1 hATMSCs) transduced with the lentivirus of claim 5,
The mesenchymal stem cells are characterized in that it is derived from fat derived from autologous, taga, or heterogeneous,
The mesenchymal stem cells are mesenchymal stem cells, characterized in that the sFlt-1 gene including the nucleotide sequence of SEQ ID NO: 1 is overexpressed and engrafted in placental tissue.
상기 중간엽줄기세포는 자가, 타가 또는 이종 유래의 지방에서 유래되고,
상기 중간엽줄기세포에 서열번호 1의 염기서열을 포함하는 렌티바이러스 벡터를 도입하여 sFlt-1 유전자가 과발현되어 태반 조직 내에 생착되는 것인, 방법.A method for producing a gestational hypertension animal model comprising the step of introducing the lentiviral expression vector for preparing a gestational hypertension model of claim 1 into a mesenchymal stem cell and administering it to a mammal,
The mesenchymal stem cells are derived from fats derived from autologous, taga, or heterogeneous,
By introducing a lentiviral vector comprising the nucleotide sequence of SEQ ID NO: 1 into the mesenchymal stem cells, the sFlt-1 gene is overexpressed and engrafted in placental tissue.
2) 상기 임신성 고혈압 동물모델에서 혈압을 측정하여 임신성 고혈압 치료 후보물질의 혈압 강하 효과를 확인하는 것을 포함하는 임신성 고혈압 치료제의 스크리닝 방법.1) administering a candidate substance for treatment of gestational hypertension to the gestational hypertension animal model of claim 13; And
2) A method for screening a therapeutic agent for gestational hypertension comprising measuring blood pressure in the gestational hypertension animal model to determine the blood pressure lowering effect of a candidate substance for treating gestational hypertension.
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| EP2543729A1 (en) * | 2010-03-04 | 2013-01-09 | Okabe, Masaru | Model animal for pregnancy-induced hypertension syndrome, and treatment method therefor |
| WO2016151287A1 (en) * | 2015-03-20 | 2016-09-29 | Aston University | Preeclampsia |
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| EP2543729A1 (en) * | 2010-03-04 | 2013-01-09 | Okabe, Masaru | Model animal for pregnancy-induced hypertension syndrome, and treatment method therefor |
| WO2016151287A1 (en) * | 2015-03-20 | 2016-09-29 | Aston University | Preeclampsia |
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| GenBank QAccession D88690.1(2010.6.15.)* * |
| Mol Cell Biochem.,449(1-2):doi: 10.1007/s11010-018-3337-5(2018.3.1.) * |
| Molecular Medicine Report.,DOI:10.3892/mmr2017.7310(2017.)* * |
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