KR20150110894A - Anti-aging Cellular Therapeutic Agent Comprising Adult Stem Cells under Hypoxic Conditions - Google Patents
Anti-aging Cellular Therapeutic Agent Comprising Adult Stem Cells under Hypoxic Conditions Download PDFInfo
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Abstract
Description
본 발명은 저산소 조건에서 배양된 항노화 인자를 발현하는 성체줄기세포의 제조방법 및 상기 성체줄기세포를 유효성분으로 함유하는 항노화용 세포치료제에 관한 것이다.
The present invention relates to a method for producing adult stem cells expressing anti-aging factors cultured under hypoxic conditions and an anti-aging cell therapy agent containing the adult stem cells as an active ingredient.
최근 배아줄기세포, 성체줄기세포 등의 줄기세포를 다양한 세포로 분화시킴으로써, 세포치료법(cell therapy)으로서의 활용 가능성에 관한 연구가 다양하게 진행되고 있다. 다분화능이 있는 배아줄기세포는 다양한 세포로 분화됨으로써 세포치료제로 주목되었지만 배아줄기세포를 활용함에 있어 윤리적인 문제점 때문에 실질적으로 세포치료로 사용하기에 어려움을 가지고 있다. 이러한 윤리적인 문제점을 회피하기 위하여 성체줄기세포를 이용한 연구가 활발히 진행되고 있다. Recently, there have been various studies on the possibility of utilization of stem cells such as embryonic stem cells and adult stem cells as cell therapy by differentiating into various cells. Although embryonic stem cells with pluripotency are differentiated into various cells, they have attracted attention as a cell therapy agent. However, because of ethical problems in using embryonic stem cells, they are difficult to use as cell therapy. In order to avoid such ethical problems, research using adult stem cells has been actively conducted.
성체줄기세포는 세포수를 많이 얻을 수는 있으나 타인에게 이식할 때 감염의 위험이나 분화능력이 상대적으로 떨어지는 단점이 있지만, 의학적으로 적용하기에 대단히 안전하다는 특징이 있다. 구체적으로, 장기재생을 위해 몸 안에 이식하여도 암이 발생하지 않으며, 성인의 몸속에 있었기 때문에 면역거부반응이 발생하지 않아 자기 자신의 세포를 사용하는 자가 이식(autologous transplantation)이 가능하다. 또한, 주변조직의 특성에 자신을 맞추어 분화하는 조직 특이적 분화능력(site-specific differentiation)이 있고, 미분화 상태에서 주입하여도 암을 유발하지 않기 때문에 이식된 이후 당장 필요한 세포를 만들어내는 것 이외에도 나중에 필요한 미분화 상태의 줄기세포를 다시 만들어서 저장하는 자가 재생산(self-renewal) 능력이 있는 장점이 있다.Although adult stem cells can obtain a large number of cells, they have a disadvantage in that the risk of infection or differentiation is relatively low when they are transplanted into other people, but they are characterized as being very safe for medical application. Specifically, cancer does not occur even when transplanted into the body for long-term regeneration. Since the immune rejection does not occur because it is in the adult body, autologous transplantation using its own cells is possible. In addition, there is a site-specific differentiation that differentiates itself according to the characteristics of the surrounding tissues. Since it does not cause cancer even if it is injected in undifferentiated state, besides producing cells necessary immediately after transplantation, It has the advantage of self-renewal ability to regenerate and store necessary undifferentiated stem cells.
성체줄기세포로는 대표적으로 골수, 지방, 제대혈 등으로부터 유래된 줄기세포가 알려져 있으며, 이들 세포에 대한 특정 세포로의 분화방법에 대한 연구가 다양하게 진행되고 있다. 성체줄기세포는 분화 유도에 있어서도 줄기세포의 종류에 따라 분화되어 생성되는 세포의 종류가 전혀 상이하게 나타나기도 한다. 일 예로, 지방전구세포(preadipocyte)를 지방세포로의 분화를 유도하는 호르몬에 노출시켰을 때 지방세포로 분화되나, 저산소 조건(hypoxia condition)에서는 지방전구세포가 지방세포의 분화를 유도하는 호르몬에 노출시켜도 지방세포로 분화되지 않는다는 것이 보고된 바 있다(Lin et al ., J Biol Chem . 13;281(41):30678-83, 2006). 그러나, 골수-유래의 중간엽 줄기세포를 저산소 조건(hypoxia condition)에서 배양할 경우 지방세포와 조골세포로의 분화능이 증가된다는 것이 또한 보고된 바 있다(Grayson et al ., J Cell Physiol 207:331-339, 2006; Basciano et al ., BMC Cell Biology 12:12, 2011)As adult stem cells, stem cells derived from bone marrow, fat, umbilical cord blood and the like are known, and studies on the differentiation of these cells into specific cells have been conducted variously. Adult stem cells are also differentiated according to the type of stem cells in the induction of differentiation. For example, when preadipocytes are exposed to hormones that induce differentiation into adipocytes, they are differentiated into adipocytes. Under hypoxia conditions, adipose precursors are exposed to hormones that induce the differentiation of adipocytes And not to differentiate into adipocytes (Linmeat get ., J Biol Chem . 13; 281 (41): 30678-83, 2006). However, it has also been reported that when bone marrow-derived mesenchymal stem cells are cultured under hypoxia conditions, their ability to differentiate into adipocytes and osteoblasts is increased (Graysonmeat get ., J Cell Physiol 207: 331-339, 2006; Basciano meat get ., BMC Cell Biology 12:12, 2011)
따라서, 성체줄기세포를 이용한 세포치료제의 개발을 위해서는, 세포의 줄기세포성(stemness)를 유지하면서 효과적으로 증식시킬 수 있는 방법을 확립하는 것이 필수적이다.Therefore, in order to develop a cell therapy agent using adult stem cells, it is essential to establish a method capable of effectively growing stem cells while maintaining the stemness of the cells.
이에, 본 발명자들은 윤리적으로 안전한 성체줄기세포를 이용한 항노화 또는 노화에 의한 인지기능 저하 억제용 세포치료제를 개발하고자 예의 노력한 결과, 저산소 조건에서 배양된 성체줄기세포가 항노화 인자를 발현하고, 동시에 줄기세포성을 유지하면서 효과적으로 노화를 억제하는 것을 확인하고, 본 발명을 완성하게 되었다.
Therefore, the present inventors have made intensive efforts to develop a cell therapy agent for inhibiting cognitive dysfunction by anti-aging or senescence using ethically safe adult stem cells. As a result, adult stem cells cultured under hypoxic condition express anti- Confirming that aging is effectively inhibited while maintaining stem cell characteristics, and the present invention has been completed.
본 발명의 목적은 성체줄기세포를 저산소 조건에서 배양하는 것을 특징으로 하는 AIMP3의 발현이 감소되고 TET1, SIRT1, SIRT6 및 HIF1의 발현이 증가된 줄기세포의 제조방법을 제공하는데 있다. It is an object of the present invention to provide a method for producing stem cells in which the expression of AIMP3 is decreased and the expression of TET1, SIRT1, SIRT6 and HIF1 is increased, by culturing adult stem cells under hypoxic conditions.
본 발명의 다른 목적은 상기 성체줄기세포를 유효성분으로 함유하는 항노화 또는 노화에 의한 인지기능 저하 억제용 세포치료제를 제공하는데 있다.
It is another object of the present invention to provide a cell therapy agent for inhibiting the deterioration of cognitive function by anti-aging or aging comprising adult stem cells as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 성체줄기세포를 1~8% 산소분압을 갖는 저산소 조건에서 배양하는 것을 특징으로 하는 AIMP3의 발현이 감소되고 TET1, SIRT1, SIRT6 및 HIF1의 발현이 증가된 성체줄기세포의 제조방법을 제공한다.In order to achieve the above object, the present invention provides a method for culturing adult stem cells, which comprises culturing adult stem cells under a hypoxic condition with an oxygen partial pressure of 1 to 8%, wherein the expression of AIMP3 is decreased and the expression of TET1, SIRT1, SIRT6 and HIF1 is increased Thereby producing a stem cell.
본 발명은 또한, 상기 성체줄기세포를 유효성분으로 함유하는 항노화 또는 노화에 의한 인지기능 저하 억제용 세포치료제를 제공한다.
The present invention also provides a cell therapy agent for inhibiting the deterioration of cognitive function by anti-aging or aging comprising the adult stem cell as an active ingredient.
본 발명에 따른 저산소 조건에서 배양되어 AIMP3의 발현이 감소되고 TET1, SIRT1, SIRT6 및 HIF1의 발현이 증가된 성체줄기세포를 유효성분으로 함유하는 세포치료제는, 항노화 또는 노화에 의한 인지기능 저하 억제용 세포치료제로 사용에 유용하다.
The cell therapy agent containing the adult stem cell, which is cultured under the hypoxic condition according to the present invention and has decreased expression of AIMP3 and increased expression of TET1, SIRT1, SIRT6 and HIF1, is useful as an anti- It is useful for cell therapy.
도 1A은 정상산소 및 저산소 조건에서 배양된 태반 유래 줄기세포에서의 노화 관련 인자들의 발현을 나타낸 것이다.
도 1B, 1C는 저산소 조건에서 노화관련 인자들의 과발현 및 녹다운(knock-down)에 따른 노화관련 인자간의 상호작용을 나타낸 것이다.
도 1D은 HIF1α의 녹다운(knock-down)에 따른 줄기세포 마커들의 변화를 나타낸것이다.
도 2는 저산소 조건에서 배양된 성체줄기세포의 RNA sequencing을 통한 DEG(differentially expressed genes) 분석을 나타낸다.
도 3은 중간엽줄기세포(MSCs) 및 중간엽유래 신경세포전구체(MSC-NPCs)에서의 TET1의 mRNA 수준을 비교한 것이다.
도 4는 TET1 및 HIF1α의 과발현 및 녹다운(knock-down)에 따른 줄기세포 마커들의 변화를 나타낸 것이다.
도 5는 TET1와 HIF1α의 상호작용을 나타낸것이다.
도 6은 저산소 조건에서 배양된 태반 유래 줄기세포를 투여한 쥐의 생존률을 보여준 것이다.
도 7은 저산소 조건에서 배양된 태반 유래 줄기세포를 투여한 쥐의 인지능력을 보여준 것이다.Figure 1A shows the expression of senescence-related factors in placenta-derived stem cells cultured under normoxic and hypoxic conditions.
Figures 1B and 1C show the interaction between aging-related factors with overexpression of aging-related factors and knock-down under hypoxic conditions.
FIG. 1D shows changes in stem cell markers due to knock-down of HIF1α.
Figure 2 shows DEG (differentially expressed genes) analysis through RNA sequencing of adult stem cells cultured under hypoxic conditions.
Figure 3 compares mRNA levels of TET1 in mesenchymal stem cells (MSCs) and mesenchymal-derived neuronal precursors (MSC-NPCs).
Figure 4 shows changes in stem cell markers due to overexpression and knock-down of TET1 and HIF1a.
Figure 5 shows the interaction of TET1 with HIF1a.
Fig. 6 shows the survival rate of rats receiving placenta-derived stem cells cultured under hypoxic conditions.
FIG. 7 shows the cognitive abilities of rats receiving placenta-derived stem cells cultured under hypoxic conditions.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명은 일 관점에서, 성체줄기세포를 1~8% 산소분압을 갖는 저산소 조건에서 배양하는 것을 특징으로 하는 AIMP3의 발현이 감소되고 TET1, SIRT1, SIRT6 및 HIF1α의 발현이 증가된 줄기세포의 제조방법에 관한 것이다.In one aspect, the present invention provides a method for producing stem cells in which the expression of AIMP3 is decreased and the expression of TET1, SIRT1, SIRT6 and HIF1? Are increased, wherein adult stem cells are cultured under hypoxic conditions with an oxygen partial pressure of 1 to 8% ≪ / RTI >
본 발명에 있어서, 상기 줄기세포의 제조방법은 c-Myc, Sox2, Nanog 및 Oct4로 구성된 군에서 선택된 어느 하나 이상의 유전자가 추가로 발현되는 것을 특징으로 한다.In the present invention, the method for producing a stem cell according to the present invention is characterized in that at least one gene selected from the group consisting of c-Myc, Sox2, Nanog and Oct4 is further expressed.
본 발명의 AIMP3는 고등 진핵세포 시스템에서 Aminoacyl-tRNA synthetases (ARSs)와 ARS-interacting multifunctional proteins (AIMPs)의 복합체를 구성하는, 복합체의 안정성에 중요한 역활을 하는 가장 작은 구성요소이다. DNA 손상이나 발암성 자극이 있으면 활성화되고 핵으로 옮겨져 p53의 상위 upstream kinases인 ATM이나 ATR을 활성화 한다. p53을 활성화하는 AIMP3의 능력을 불활성화 하거나 줄이는 돌연변이(point mutation)나 대립유전자 결실 현상이 암 환자에게서 관찰되었으며, AIMP3가 종양억제 능력이 있음이 밝혀진 바 있다. 그러나 AIMP3의 항노화 활성에 대해서는 연구가 미비한 실정이다.The AIMP3 of the present invention is the smallest component that plays a crucial role in the stability of the complex, which is a complex of Aminoacyl-tRNA synthetases (ARSs) and ARS-interacting multifunctional proteins (AIMPs) in higher eukaryotic systems. If DNA damage or carcinogenic stimulation is present, it is activated and transferred to the nucleus, activating ATM or ATR, the upper upstream kinases of p53. A mutation (point mutation) or allele deletion that inactivates or diminishes the ability of AIMP3 to activate p53 has been observed in cancer patients, and AIMP3 has been shown to be capable of tumor suppression. However, the anti-aging activity of AIMP3 has not yet been studied.
또한, 본 발명의 TET1은 주로 배아줄기세포에서 발현되는 유전자로 특정 유전자들의 크로마틴(chromatin)을 조절하여 세포를 분화를 막는 것으로 알려져 있으며, 노화와 관련된 역할에 대해서는 알려진 바가 없다.In addition, TET1 of the present invention is mainly expressed in embryonic stem cells. It is known that chromatin of specific genes is regulated to prevent differentiation of cells, and its role related to aging is unknown.
SIRT1 및 SIRT6은 만성염증,암,노화를 조절하는 유전자로 HIF1을 저해하는 단백질을 만들어 내는 유전자이다. 이 단백질은 서투인(Sirtuin)으로 단백질로 DNA에서 아세틸그룹을 제거하는 역할을 하는 탈아세틸화 효소이다. 아세틸그룹이 있는 유전자는 쉽게 발현되며, 이 아세틸그룹이 대부분 노화를 촉진하는 유전자에 주로 존재하는데, 시투인 단백질에 의해 아세틸그룹니 제거되면 노화촉진 유전자 발현이 억제된다.SIRT1 and SIRT6 are genes that regulate chronic inflammation, cancer, and senescence, and produce genes that inhibit HIF1. This protein is Sirtuin, a protein that acts as a deacetylating enzyme to remove acetyl groups from DNA. The gene with acetyl group is easily expressed, and most of the acetyl group exists mainly in the gene promoting senescence. When the acetyl group is removed by the protein, the senescence promoting gene expression is suppressed.
본 발명의 c-Myc, Sox2, Nanog 및 Oct4 유전자는 배아줄기세포에서 많이 발현하는 줄기세포의 전분화능, 즉 줄기세포성을 유지하는데 중요한 역할을 하는 유전자이다.The c-Myc, Sox2, Nanog, and Oct4 genes of the present invention are genes that play important roles in maintaining the pluripotency of stem cells, that is, stem cells, which are highly expressed in embryonic stem cells.
본 발명의 배양은 10%의 소태아혈청, 1%의 페니실린-스트렙토마이신, 1g/ml의 헤파린, 25ng/ml의 섬유아세포성장인자-4, 1㎍/ml의 토코페롤, 1㎍/ml의 토코페롤 아세테이트가 첨가된 alpha-MEM 배지에서 10 내지 20 계대까지 계대배양 하는 것이 바람직하지만, 이에 한정되는 것은 아니다.The cultures of the present invention contained 10% fetal bovine serum, 1% penicillin-streptomycin, 1 g / ml heparin, 25 ng / ml fibroblast growth factor-4, 1 ug / ml tocopherol, 1 ug / ml tocopherol It is preferable to subculture to 10 to 20 passages in an alpha-MEM medium supplemented with acetate, but it is not limited thereto.
본 발명에 있어서, 상기 성체줄기세포는 태반 유래 줄기세포, 골수 유래 줄기세포, 지방 유래 줄기세포 및 말초혈 유래 줄기세포인 것이 바람직하며, 인간 양막 유래 중간엽 줄기세포 또는 인간 양막 상피 세포인 것이 더욱 바람직하지만, 이에 한정되는 것은 아니다.In the present invention, the adult stem cells are preferably placenta-derived stem cells, bone marrow-derived stem cells, adipose-derived stem cells and peripheral blood-derived stem cells, and human amniotic membrane-derived mesenchymal stem cells or human amniotic epithelial cells But is not limited thereto.
또한, 본 발명에 있어서, 상기 줄기세포는 노화에 의한 인지기능 저하를 억제하는 것을 특징으로 한다.In the present invention, the stem cells are characterized by inhibiting the deterioration of cognitive function by aging.
본 발명은 다른 관점에서, 상기 방법에 의해 제조된, 성체줄기세포를 유효성분으로 함유하는 항노화 또는 노화에 의한 인지기능 저하 억제용 세포치료제에 관한 것이다.
In another aspect, the present invention relates to a cell therapy agent for inhibiting the deterioration of cognitive function by anti-aging or aging comprising adult stem cells as an active ingredient produced by the above method.
실시예Example
이하 본 발명을 실시예에 의하여 더욱 상세히 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 이에 의해 본 발명의 기술적 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명 할 것이다.
Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.
실시예Example 1: One: 성체줄기세포의Adult stem cells 배양 culture
중간엽 줄기세포를 정상산소 조건(37℃, 5% CO2) 및 저산소 조건(37℃, 5% CO2, 1~8% O2)에서 배양하였다. 증식을 위한 배지로는 10%의 소태아혈청, 1%의 페니실린-스트렙토마이신, 1g/ml의 헤파린, 25ng/ml의 섬유아세포성장인자-4, 1㎍/ml의 토코페롤, 1㎍/ml의 토코페롤 아세테이트가 첨가된 alpha-MEM 배지를 사용하였다. 중간엽 줄기세포는 cm2당 6 x 103~7 x 103 세포를 계수하여 접종하고, 이틀에 한 번씩 배지를 교체하면서 배양 후 약 3일 내지 4일간 플라스크에 80%이상 자랐으면(confluent), 배양된 세포를 인산완충액 (phosphate-buffered saline, PBS)을 사용하여 세척한 후, 0.25%의 트립신/EDTA를 이용해 2분간 효소 처리한 다음, 소 태아혈청을 가하여 효소반응을 정지시키고, 1000rpm에서 약 5분 동안 원심분리한 다음, 상층액을 제거하여 세포들을 수확하였다. 수확된 세포는 다시 cm2당 6 x 103~7 x 103 세포로 계수하여 동일한 방법으로 제15~20계대까지 계대배양을 수행하였다.
Mesenchymal stem cells were cultured under normal oxygen conditions (37 ° C, 5% CO 2 ) and hypoxic conditions (37 ° C, 5% CO 2 , 1-8% O 2 ). As the culture medium for proliferation, 10% fetal bovine serum, 1% penicillin-streptomycin, 1 g / ml heparin, 25 ng / ml fibroblast growth factor-4, 1 g / ml tocopherol, Alpha-MEM medium supplemented with tocopherol acetate was used. The mesenchymal stem cells were seeded at 6 × 10 3 to 7 × 10 3 cells per cm 2 , inoculated, and cultured for two to three days. After incubation for about 3 to 4 days, the cells were confluent (80% The cultured cells were washed with phosphate buffered saline (PBS), treated with 0.25% trypsin / EDTA for 2 minutes, and then the fetal bovine serum was added to stop the enzyme reaction. After centrifugation for about 5 minutes, the supernatant was removed and cells were harvested. The harvested cells were counted again at 6 × 10 3 to 7 × 10 3 cells per cm 2 and subcultured to the 15th to 20th passages in the same manner.
실시예Example 2: 노화 관련 인자들의 발현 분석 2: Expression analysis of aging-related factors
본 실시예에서는 저산소 조건에서 배양된 성체줄기세포의 항노화 인자 발현을 분석하였다.In this example, the expression of anti-aging factors of adult stem cells cultured under hypoxic condition was analyzed.
실시예 1에 따른 정산산소 조건 및 저산소 조건의 성체줄기세포를 각각 제1계대, 제5계대, 제10계대까지 계대배양하여 수확한 세포에 대하여, 항노화 관련 유전자와 줄기세포 마커 유전자의 전사수준을 RT-PCR을 통해, 단백질 발현 수준을 웨스턴블롯팅을 통해 분석하였다.The cells harvested by passive oxygenation and hypoxic conditions according to Example 1 were subcultured to
그 결과, HIF1α가 저산소 조건에서 활성화되는 것을 확인하였으며, 항노화 관련 유전자인 SIRT1 및 SIRT6 역시 발현이 증가되었다(도 1A). 또한, AIMP3의 발현이 저산소 조건에서 감소하는 것을 확인하고(도 1A), HIF1α를 녹다운(knock-down)하여 AIMP3의 항노화 관련을 분석하였다. AIMP3는 HIF1α가 억제됨에 따라 발현이 증가(도 1C)하므로, 저산소 조건의 성체줄기세포에서 AIMP3 발현 감소는 저산소 조건에 의한 HIF1α 발현 증가에 따른 것임을 확인하였으며, 이로써 AIMP3가 저산소 배양 줄기세포의 항노화 효과와 연관있음을 알 수 있다.As a result, it was confirmed that HIF1α was activated under hypoxic conditions, and SIRT1 and SIRT6, which are anti-senescence related genes, also increased expression (FIG. 1A). Furthermore, it was confirmed that the expression of AIMP3 decreased under hypoxic condition (Fig. 1A), and the anti-aging of AIMP3 was analyzed by knocking down HIF1α. AIMP3 was found to be associated with an increase in HIF1α expression under hypoxic conditions in adult stem cells with hypoxic conditions, as HIF1α was suppressed (FIG. 1C). As a result, AIMP3 inhibited the anti-aging of hypoxic cultured stem cells Effect is associated with the effect.
또한, HIF1α의 녹다운(knock-down)에 의한 줄기세포 마커들의 발현을 확인하였다. 그 결과, c-Myc, Sox2, Nanog 및 Oct4의 줄기세포 마커들은 HIF1α의 녹다운(knock-down)에 영향을 받지 않음을 확인하였다(도 1D).
In addition, expression of stem cell markers by knock-down of HIF1α was confirmed. As a result, it was confirmed that the stem cell markers of c-Myc, Sox2, Nanog, and Oct4 were not affected by the knock-down of HIF1α (FIG. 1D).
실시예Example 3: 항노화 관련 인자 3: anti-aging-related factor TET1TET1 의 분석Analysis of
실시예 1에 따른 정상산소 및 저산소 조건 성체줄기세포에서 RNA를 추출한 후, Ion ProtonTM System for Next-Generation Sequencing(Life Technologies, USA)를 사용한 RNA 서열분석을 통하여 DEG(differentially expression genes)을 비교하였다(도 2).Carried out after the extraction of the top oxygen and low oxygen conditions, RNA from the adult stem cells according to Example 1 was compared to Ion Proton TM System for Next-Generation Sequencing DEG (differentially expression genes) through RNA sequence analysis using (Life Technologies, USA) (Fig. 2).
그 결과, 정산산소 조건의 줄기세포에서는 제5계대 이하(early passage)에 비해 제15계대 이상(late passage)에서 히스톤 관련 유전자들이 모두 발현이 감소하였으며, 특히 TET1(methylcytosine dioxygenase I) 유전자의 발현은 거의 사라졌다. 반대로 저산소 조건의 줄기세포에서는 제5계대 이하(early passage)에 비해 제15계대 이상(late passage)에서 히스톤 관련 유전자들이 모두 발현이 증가하였으며, 특히 TET1(methylcytosine dioxygenase I) 유전자의 발현은 현저히 증가하였다. 이는 TET1 유전자가 저산소 조건에서 항노화 활성을 나타냄을 알 수 있다.As a result, the expression of TET1 (methylcytosine dioxygenase I) gene was decreased in the late passage of the stem cells of the oxygenated condition compared to the early passage of the fifth passage. Almost disappeared. On the other hand, the expression of TET1 (methylcytosine dioxygenase I) gene was significantly increased in the hypoxic condition of stem cells in the late passage than in the passage of the fifth passage . This indicates that the TET1 gene exhibits anti-aging activity under hypoxic condition.
또한, 중간엽줄기세포가 신경전구세포체로 분화되기 전, 후의 TET1의 발현을 비교하여, TET1이 분화된 세포에서는 거의 발현되지 않는 것을 확인하였다(도 3).
In addition, the expression of TET1 before and after differentiation of mesenchymal stem cells into neural progenitor cell bodies was compared and it was confirmed that TET1 was hardly expressed in cells differentiated with TET1 (FIG. 3).
실시예Example 4: 4: HIF1HIF1 α와 alpha and TET1TET1 의 상호작용 Interaction of
본 실시예에서는 HIF1α와 TET1의 상호작용을 분석하기 위하여, HIF1α와 TET1를 각각 녹다운(knock-down) 및 과발현하였다.In this example, HIF1α and TET1 were knocked down and overexpressed in order to analyze the interaction between HIF1α and TET1.
그 결과, HIF1α의 녹다운(knock-down)에 의해 TET1의 발현은 감소하고, HIF1α의 과발현에 의해 TET1의 발현은 증가하였으며, c-Myc, Sox2, Nanog 및 Oct4의 줄기세포 마커들의 발현 또한 증가하였다. 그러나, TET1의 녹다운(knock-down) 및 과발현에 의해 HIF1α의 발현은 변화가 없었지만, 줄기세포 마커들의 발현은 증가하였다(도 4). 이는 HIF1α와 TET1가 상호작용에 의해 항노화 효과를 나타낸다는 것을 알 수 있다. 이에, TET1 유전자의 프로모터에 HIF1α 결합부위가 존재함을 확인하고, 제3계대 저산소 배양 줄기세포를 이용하여 CHIP 분석을 수행하여 HIF1α가 TET1 유전자의 프로모터에 결합한다는 것을 확인하였다(도 5).
As a result, expression of TET1 was decreased by knock-down of HIF1α, expression of TET1 was increased by overexpression of HIF1α, and expression of stem cell markers of c-Myc, Sox2, Nanog and Oct4 was also increased . However, the knock-down and overexpression of TET1 did not change the expression of HIF1α, but the expression of stem cell markers was increased (FIG. 4). It can be seen that HIF1α and TET1 exhibit an anti-aging effect by interaction. Thus, it was confirmed that HIF1α binding site was present in the promoter of TET1 gene, and CHIP analysis was performed using third-line hypoxic culture stem cells to confirm that HIF1α binds to the promoter of TET1 gene (FIG. 5).
실시예Example 5: 5: 저산소Hypoxia 배양 culture 성체줄기세포의Adult stem cells 항노화 효과 확인 Confirmation of anti-aging effect
본 실시예에서는 실시예 1에 따른 저산소 조건 배양 성체줄기세포 1x105개를 18~20개월령의 노화된 쥐(C57BL/6)의 꼬리에 6주 간격으로 총5회 정맥 투여한 후, 투여시점으로부터 6개월 후인 24~25개월령 쥐(C57BL/6)의 생존률을 비교하였다(도 8). 또한, 저산소 조건 배양 성체줄기세포를 1회 투여 후, 2주 후에 MWM(모리스 수중 미로) 테스트를 실시하였으며, 6주 간격으로 2, 3 회 반복 투여 후 MWM 테스트를 실시하여 인지능력을 비교하였다.In this Example, 1x10 5 cultured adult stem cells of the hypoxic condition according to Example 1 were intravenously administered to the tail of aged rats (C57BL / 6) at 18 to 20 months of age at intervals of 6 weeks in total five times, Survival rates of 24-to 25-month-old mice (C57BL / 6) after 6 months were compared (Fig. 8). In addition, MWM (Morris water maze) test was performed after one dose of hypoxic condition cultured adult stem cells, and MWM test was performed 2 or 3 times at 6 week intervals to compare cognitive ability.
그 결과, 저산소 조건 배양 성체줄기세포를 투여한 군에서 높은 생존률을 나타냈다(도 6). 인지능력 테스트에서는 18~20개월령 쥐에 1회 줄기세포를 투여한 경우에는 차이가 나타나지 않았으나, 2회 및 3회 반복투여 후에는 2배 이상 인지능력의 차이를 확인할 수 있었다(도 7). 이는 투여된 저산소 조건에서 배양된 성체줄기세포에 의한 항노화 효과를 보여주는 것이다.
As a result, high survival rate was shown in the group administered with hypoxic condition cultured adult stem cells (FIG. 6). In the cognitive ability test, no difference was observed when the stem cells were administered once to the 18-20 month old rats, but the difference in the cognitive ability was confirmed twice or more after the second and third repeated administrations (FIG. 7). This shows the effect of anti-aging by adult stem cells cultured under the hypoxic conditions administered.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (7)
Wherein the expression of AIMP3 is decreased and the expression of TET1, SIRT1, SIRT6, and HIF1? Is increased, wherein adult stem cells are cultured under hypoxic conditions with an oxygen partial pressure of 1 to 8%.
The method according to claim 1, wherein at least one gene selected from the group consisting of c-Myc, Sox2, Nanog and Oct4 is further expressed.
The pharmaceutical composition according to claim 1, comprising 10% fetal bovine serum, 1% penicillin-streptomycin, 1 g / ml heparin, 25 ng / ml fibroblast growth factor-4, 1 g / ml tocopherol, Wherein the cells are cultured in an alpha-MEM medium supplemented with tocopherol acetate to 10 to 15 passages.
The method of claim 1, wherein the adult stem cells are placenta-derived stem cells, bone marrow-derived stem cells, adipose-derived stem cells, and peripheral blood-derived stem cells.
[Claim 5] The method according to claim 4, wherein the placenta-derived stem cells are human amniotic membrane-derived mesenchymal stem cells or human amniotic epithelial cells.
The method according to claim 1, wherein the stem cell inhibits deterioration of cognitive function by aging.
A cell treatment agent for inhibiting the deterioration of cognitive function by anti-aging or aging comprising adult stem cells as an active ingredient, which is produced by the method of any one of claims 1 to 6.
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| WO2021206402A1 (en) * | 2020-04-08 | 2021-10-14 | 이엔셀 주식회사 | Stem cell culturing method for promoting initial yield of stem cel.ls |
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| WO2021206402A1 (en) * | 2020-04-08 | 2021-10-14 | 이엔셀 주식회사 | Stem cell culturing method for promoting initial yield of stem cel.ls |
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