KR101943189B1 - Biomarkers composition for detecting damage of cell and uses thereof - Google Patents
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Abstract
본 발명은 인산완충생리식염수(PBS)에 보관된 세포 손상 확인용 바이오마커 조성물에 관한 것으로, 상기 PBS에 보관된 중간엽줄기세포는 보관시간 경과에 따라, 세포 내에서 높은 과산화 지방질 생성이 나타났으며, 상기 과산화 지방질 생성과 관련된 유전자의 발현 수준 변화가 확인됨에 따라, 상기 발현 수준 변화가 확인된 유전자를 중간엽줄기세포의 손상도 확인 및 이식 적합성 판단을 위한 효과적인 생물학적 지표로 제공하여, PBS 보관에 따른 세포 손상 정도를 확인하여 이식에 적합한 중간엽줄기세포를 선별함으로써 생체 내 세포이식 효율성을 증가시킬 수 있다.The present invention relates to a biomarker composition for confirming cell damage stored in phosphate buffered physiological saline (PBS). The mesenchymal stem cells stored in the PBS exhibit a high peroxide lipid production in the cells over time As a result of confirming the expression level of the gene related to the production of peroxidized lipids, the gene having the expression level change was provided as an effective biological indicator for confirming the damage of the mesenchymal stem cells and determining the transplantability, And the selection of mesenchymal stem cells suitable for transplantation can increase the efficiency of in vivo cell transplantation.
Description
본 발명은 인산완충생리식염수(Phosphate bufferd saline; PBS)에 보관된 중간엽줄기세포의 손상 확인용 바이오마커 조성물에 관한 것이다.The present invention relates to a biomarker composition for confirming damage of mesenchymal stem cells stored in a phosphate buffered saline (PBS).
인간중간엽줄기세포(hMSCs)는 세포의 분화와 발생 및 여러 가지 면역조절작용 효과를 가지므로(Hidvegi, Raso et al. 1999; Felka, Schafer et al. 2009; Chen, Lee et al. 2012), 난치성 질환의 치료와 여러 가지 임상실험에 사용되고 있는 줄기세포로, 보고된 바에 따르면, hMSCs은 다 분화 가능성을 이용한 세포 치료와 조직공학에 이용되며, 신경, 혈관 및 간세포 등으로 분화하여 임상실험에 많이 적용되고 있다(Spadaccio, Rainer et al. 2009). Since human mesenchymal stem cells (hMSCs) have cell differentiation and development and various immunomodulatory effects (Hidvegi, Raso et al. 1999; Felka, Schafer et al. 2009; Chen, Lee et al. 2012) It has been reported that hMSCs are used for cell therapy and tissue engineering using the possibility of differentiation, and they are differentiated into nerve, blood vessel and hepatocyte, (Spadaccio, Rainer et al. 2009).
hMSCs 세포는 조직 내에 침투된 후 비교적 조직특이적으로 분화되고, 신체 내에서 안전하다는 장점을 가지므로, 다양한 의학 분야에서의 세포치료를 이용하여 장기 재생을 시도하는 재생의학의 영역에 사용되고 있으며, 세포증식억제 및 혈관생성 촉진, 항염증, 면역조절작용에 효과가 있어 조직이나 기관을 이식할 때 사용 되어지기도 한다(Zacharek, Chen et al. 2007; Comsa, Ciuculescu et al. 2011).hMSCs cells have been used in the field of regenerative medicine which attempts to regenerate by using cell therapy in various medical fields because they are relatively tissue specific and differentiated after penetration into tissues and are safe in the body, (Zacharek, Chen et al., 2007; Comsa, Ciuculescu et al., 2011), which are effective for the proliferation inhibition and promotion of angiogenesis, antiinflammation and immunomodulation.
또한, 발생과정이 끝난 이후에도 신체의 한 부분이 손상을 받았을 때 이들 세포를 재생할 수 있는 분화의 유연성 기능도 가지고 있다. It also has the flexibility of differentiation to regenerate these cells when a part of the body is damaged even after the development.
이와 같이 다양한 기능을 가지는 hMSCs는 현재 심근경색, 뇌졸중, 퇴행성 질환 그리고 다계통위축증(multiple system atrophy; MAS) 환자들에게 임상실험치료 도구로 사용되고 있는 데(Lai, Arslan et al. 2010), hMSCs를 환자에게 적용하기 위해서 사람의 골수에서 추출한 후 혈구계 줄기세포를 제거하고, 증식과정인 세포 배양을 거친 후 생리식염수에 보관되어 환자에게 주입된다.These hMSCs with various functions are now being used as clinical treatment tools for patients with myocardial infarction, stroke, degenerative diseases and multiple system atrophy (Lai, Arslan et al. 2010) In order to apply it to the patient, it is extracted from the human bone marrow, and the hematopoietic stem cells are removed. After the cell culture as the proliferation process, the cells are stored in physiological saline and injected into the patient.
그러나 상기 보관 시간이 경과할수록 hMSCs의 세포사멸(apoptosis)이 증가되고, 세포의 형태 변화가 나타남에 따라, 환자에게 이식할 수 있는 세포 수가 감소되고 hMSCs 세포 이식 후에도 향상된 이식효과를 기대하기 어렵다.However, as the storage time increases, apoptosis of hMSCs is increased and cell morphology changes. Therefore, the number of cells that can be transplanted into a patient is decreased and an improved transplantation effect is not expected even after hMSCs cell transplantation.
따라서 본 발명은 세포 이식 효율성을 향상시키기 위해, 개체로부터 분리된 후 PBS에 보관된 중간엽줄기세포의 손상도를 확인하고, 보관된 세포의 이식 적합성을 평가하기 위한 생물학적 지표를 제시하고자 한다.Therefore, in order to improve the efficiency of cell transplantation, the present invention provides a biological indicator for assessing the degree of damage of mesenchymal stem cells stored in PBS after isolation from an individual, and evaluating the transplantation suitability of the stored cells.
본 발명은 SOD2(superoxide dismutase 2; NCBI accession number: BC016934.1), CYP4A11(cytochrome P450, family 4 subfamily A polypeptide 11, NCBI accession number: BC041158.1), TTPA(tocopherol α transfer protein; NCBI accession number: BC058000.1), PTGER4(prostaglandin E receptor 4; NCBI accession number: BC101534.1) 및 PRKAA2(5'-AMP-activated protein kinase catalytic subunit alpha-2; NCBI accession number: BC069823.1)로 이루어진 군에서 선택된 하나 이상의 유전자를 포함하는 세포 손상 확인용 바이오마커 조성물을 제공할 수 있다.The present invention relates to a nucleic acid encoding a superoxide dismutase (SOD2) (NCBI accession number: BC016934.1), CYP4A11 (cytochrome P450, family 4 subfamily A polypeptide 11, NCBI accession number: BC041158.1), tocopherol alpha transfer protein (BCA58000.1), PTGER4 (prostaglandin E receptor 4, NCBI accession number: BC101534.1) and PRKAA2 (5'-AMP-activated protein kinase catalytic subunit alpha-2; NCBI accession number BC069823.1) It is possible to provide a biomarker composition for identifying a cell damage containing one or more genes.
또한, 본 발명은 SOD2(superoxide dismutase 2; NCBI accession number: BC016934.1), CYP4A11(cytochrome P450, family 4 subfamily A polypeptide 11, NCBI accession number: BC041158.1), TTPA(tocopherol α transfer protein; NCBI accession number: BC058000.1), PTGER4(prostaglandin E receptor 4; NCBI accession number: BC101534.1) 및 PRKAA2(5'-AMP-activated protein kinase catalytic subunit alpha-2; NCBI accession number: BC069823.1)로 이루어진 군에서 선택된 하나 이상의 유전자 발현 수준을 확인하는 단계; 및 상기 유전자의 발현 정도를 대조군과 비교하는 단계를 포함하는 세포의 이식 적합성 평가를 위한 정보를 제공하는 방법을 제공할 수 있다.The present invention also relates to the use of a nucleic acid encoding a superoxide dismutase such as SOD2 (NCBI accession number: BC016934.1), CYP4A11 (cytochrome P450, family 4 subfamily A polypeptide 11, NCBI accession number: BC041158.1), tocopherol alpha transfer protein number: BC058000.1), prostaglandin E receptor 4 (NCBI accession number: BC101534.1) and PRKAA2 (5'-AMP-activated protein kinase catalytic subunit alpha-2; NCBI accession number: BC069823.1) Identifying one or more gene expression levels selected from < RTI ID = 0.0 > And comparing the degree of expression of the gene with a control group, to provide information for evaluating the transplantation suitability of a cell.
본 발명에 따르면, 개체로부터 분리되어 PBS에 보관된 중간엽줄기세포가 보관시간 경과에 따라, 세포 내에서 높은 과산화 지방질 생성이 확인되었으며, 상기 과산화 지방질 생성과 관련된 유전자의 발현 수준 변화가 확인됨에 따라, 상기 발현 수준 변화가 확인된 유전자는 중간엽줄기세포의 손상도 확인 및 이식 적합성 판단을 위한 생물학적 지표로 사용될 수 있으므로, 보관된 세포 중 이식에 적합한 중간엽줄기세포를 선별하여 생체 내 세포이식 효율성을 증가시킬 수 있다.According to the present invention, the generation of high peroxidized lipids in the cells was confirmed as the storage time of the mesenchymal stem cells separated from the individual and stored in the PBS, and the expression levels of the genes related to the peroxide lipid production were confirmed , The gene whose expression level has been confirmed to change can be used as a biological indicator for confirming the damage of the mesenchymal stem cells and determining the suitability of the transplantation. Therefore, the mesenchymal stem cells suitable for transplantation in the stored cells are selected, Can be increased.
도 1은 12시간 동안 PBS에 보관된 hBM-MSCs의 마이크로 어레이 및 아미노산 프로필에 대한 생물 정보학 분석결과로, 도 1(A)는 IPA를 이용한 알고리즘으로 확인된 과산화 지질 관련 유전자 및 아미노산 네트워크를 나타낸 모식도로, 적색 및 녹색은 각각 증가 및 감소된 유전자를 나타내며, 도 1(B)는 PBS에 6 및 12 시간동안 보관된 hBM-MSCs세포에서 각각 지질 과산화관련 유전자 발현 수준을 확인한 qPCR 분석결과이다 *P < 0.05, **P < 0.001 vs. 0 h (대조군).
도 2는 PBS에 6 및 12 시간동안 보관된 hBM-MSCs의 NAC 처리 또는 미처리에 따른 과산화 지방질의 양을 측정한 결과이다.Fig. 1 shows the result of bioinformatics analysis of microarray and amino acid profile of hBM-MSCs stored in PBS for 12 hours. Fig. 1 (A) is a schematic diagram showing a gene for peroxidized lipid and an amino acid network confirmed by an algorithm using IPA 1 (B) shows the results of qPCR analysis of lipid peroxidation-related gene expression levels in hBM-MSCs cells stored in PBS for 6 and 12 hours, respectively. * P ≪ 0.05, ** P < 0 h (control group).
FIG. 2 shows the results of measurement of the amount of peroxidized lipids according to NAC treatment or non-treatment of hBM-MSCs stored in PBS for 6 and 12 hours.
본 발명은 SOD2(superoxide dismutase 2; NCBI accession number: BC016934.1), CYP4A11(cytochrome P450, family 4 subfamily A polypeptide 11, NCBI accession number: BC041158.1), TTPA(tocopherol α transfer protein; NCBI accession number: BC058000.1), PTGER4(prostaglandin E receptor 4; NCBI accession number: BC101534.1) 및 PRKAA2(5'-AMP-activated protein kinase catalytic subunit alpha-2; NCBI accession number: BC069823.1)로 이루어진 군에서 선택된 하나 이상의 유전자를 포함하는 세포 손상 확인용 바이오마커 조성물을 제공할 수 있다.The present invention relates to a nucleic acid encoding a superoxide dismutase (SOD2) (NCBI accession number: BC016934.1), CYP4A11 (cytochrome P450, family 4 subfamily A polypeptide 11, NCBI accession number: BC041158.1), tocopherol alpha transfer protein (BCA58000.1), PTGER4 (prostaglandin E receptor 4, NCBI accession number: BC101534.1) and PRKAA2 (5'-AMP-activated protein kinase catalytic subunit alpha-2; NCBI accession number BC069823.1) It is possible to provide a biomarker composition for identifying a cell damage containing one or more genes.
상기 SOD2(superoxide dismutase 2) 및 CYP4A11(cytochrome P450, family 4 subfamily A polypeptide 11) 유전자는 PBS 보관시간 증가에 의해 발현이 감소될 수 있다.The expression of SOD2 (superoxide dismutase 2) and CYP4A11 (cytochrome P450, family 4 subfamily A polypeptide 11) can be decreased by increasing the PBS storage time.
상기 TTPA(tocopherol α transfer protein), PTGER4(prostaglandin E receptor 4) 및 PRKAA2(5'-AMP-activated protein kinase catalytic subunit alpha-2) 유전자는 PBS 보관시간 증가에 의해 발현이 증가될 수 있다.Expression of the TTPA, PTGER4, and PRKAA2 (5'-AMP-activated protein kinase catalytic subunit alpha-2) genes may be increased by increasing the PBS storage time.
상기 세포는 인간으로부터 분리된 중간엽줄기세포일 수 있으며, 상기 세포는 인산완충생리식염수(PBS)에 보관된 것일 수 있으나, 이에 한정되지 않는다.The cells may be mesenchymal stem cells isolated from humans, and the cells may be stored in phosphate buffered saline (PBS), but are not limited thereto.
보다 상세하게는 상기 유전자들은 과산화 지방질과 관련된 유전자들로, 본 발명의 일실시예에 따르면, 인간으로부터 분리된 hBM-MSCs 세포를 PBS에서 6 및 12 시간 동안 보관된 후 각 보관 시간별 hBM-MSCs 세포에서 마이크로어레이 및 아미노산 프로파일링 데이터를 분석을 진행한 결과, 도 2와 같이 12시간 PBS에 보관된 세포에서 6시간 보관된 세포보다 과산화 지방질이 증가된 것을 확인할 수 있었다.More specifically, the genes are genes associated with peroxidative lipids. According to one embodiment of the present invention, hBM-MSCs cells isolated from human are stored in PBS for 6 and 12 hours, As a result of the analysis of microarray and amino acid profiling data, it was confirmed that the lipid peroxidation was increased in the cells stored in the PBS for 12 hours as compared with the cells stored for 6 hours in the PBS as shown in Fig.
상기 결과를 통하여, 과산화 지방질 관련 유전자의 발현 수준을 확인한 결과, 도 1b와 같이 0시간 보관된 실험군보다 12시간 보관된 중간엽줄기세포군에서 수퍼 옥사이드 디스뮤타제(superoxide dismutase 2; SOD2), 사이토크롬 P450(family 4 subfamily A polypeptide 11; CYP4A11)의 발현이 상대적으로 감소한 반면, 토코페롤 α 수송 단백질(tocopherol α transfer protein; TTPA), 프로스타클란딘 E 수용체 4(prostaglandin E receptor 4; PTGER4), PRKAA2(5'-AMP-activated protein kinase catalytic subunit alpha-2)는 12시간 보관된 세포군에서 0시간 보관된 세포군보다 상대적으로 발현이 증가된 것을 확인할 수 있었다.As a result of confirming the expression level of the peroxidase-related gene through the above results, superoxide dismutase 2 (SOD2), cytochrome P450 (family 4 subfamily A polypeptide 11, CYP4A11), while tocopherol α transfer protein (TTPA), prostaglandin E receptor 4 (PTGER4), PRKAA2 The 5'-AMP-activated protein kinase catalytic subunit alpha-2) was found to be more expressed in the 12 hour storage group than in the 0 hour storage group.
보고에 따르면, 생쥐 동물모델에서 주요 항산화 효소인 SOD2의 결핍은 아포지질단백질 B(apolipoprotein B) 활성을 통하여 과산화 지방질을 유도하고, 미토콘드리아에 존재하는 CYP4A 단백질은 항산화 경로를 조절하는 것으로 알려졌으며, α-토코페롤 수송 단백질(TTPA) 및 프로스타클란딘 E 수용체 4(PTGER4)의 발현 변화는 과산화 지방질과 관련된 세포 반응에 영향을 미치는 것으로 보고되어졌다.According to reports, deficiency of SOD2, a major antioxidant enzyme in mouse animal models, induces peroxylated lipids through apolipoprotein B activity, and CYP4A protein in mitochondria regulates the antioxidant pathway. Changes in the expression of tocopherol transport protein (TTPA) and prostaglandin E receptor 4 (PTGER4) have been reported to affect cellular responses associated with peroxide lipid.
세포 내 과산화 지방질 증가는 세포막의 유연성을 감소시키고, 이에 따라 세포변형률을 감소시키는 원인으로, 상기 결과와 같이 분리된 중간엽줄기세포의 PBS 보관 시간 경과에 따른 상기 유전자들의 발현 변화는 hBM-MSC 세포 내 과산화 지방질을 증가시켜 세포막의 유연성 및 세포변형률을 감소시키고, 줄기세포의 생체 내 이식 효율성 감소시키는 원인이 될 수 있다.The increase of peroxidic lipids in the cells decreases the flexibility of the cell membrane and thus decreases the cell strain. As a result, the expression of the genes of the isolated mesenchymal stem cells after the PBS storage time has been changed to hBM-MSC cells It may increase the peripherial lipid, decrease cell membrane flexibility and cell deformation rate, and cause decrease in the transplantation efficiency of stem cells in vivo.
또한, 본 발명은 SOD2(superoxide dismutase 2), CYP4A11(cytochrome P450, family 4 subfamily A polypeptide 11), TTPA(tocopherol α transfer protein), PTGER4(prostaglandin E receptor 4) 및 PRKAA2(5'-AMP-activated protein kinase catalytic subunit alpha-2)로 이루어진 군에서 선택된 하나 이상의 유전자 발현 수준을 확인하는 단계; 및 상기 유전자의 발현 정도를 대조군과 비교하는 단계를 포함하는 세포의 이식 적합성 평가를 위한 정보를 제공하는 방법을 제공할 수 있다.The present invention also relates to a method for the treatment and / or prophylaxis of superoxide dismutase 2 (SOD2), CYP4A11 (cytochrome P450, family 4 subfamily A polypeptide 11), tocopherol alpha transfer protein, PTGER4 (prostaglandin E receptor 4) and PRKAA2 kinase catalytic subunit alpha-2); And comparing the degree of expression of the gene with a control group, to provide information for evaluating the transplantation suitability of a cell.
상기 세포는 인간으로부터 분리된 중간엽줄기세포일 수 있으나, 이에 한정되지 않는다.The cell may be a mesenchymal stem cell isolated from a human, but is not limited thereto.
또한, 상기 세포는 인산완충생리식염수(PBS)에 보관된 세포일 수 있으나, 이에 한정되지 않는다.In addition, the cells may be cells stored in phosphate buffered saline (PBS), but are not limited thereto.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<< 실시예Example 1> PBS에 보관된 1 > stored in PBS 중간엽줄기세포의Of mesenchymal stem cells 이식 적합성 확인을 위한 For transplant compliance check 바이오마커Biomarker 확인 Confirm
1. 세포배양1. Cell culture
65세 백인 남성에게서 분리된 hBM-MSCs 세포를 PromoCell (Heidelberg, Germany)에서 구입하였다.Isolated hBM-MSCs cells from a 65 year-old white male were purchased from PromoCell (Heidelberg, Germany).
상기 세포를 PBS로 세척하고 10% 태아소혈청 및 1% 페니실린/스트렙토마이신이 포함된 글루코스가 낮은 DMEM(Dulbecco’s modified Eagle’s medium)에 부유시켰다. The cells were washed with PBS and glucose containing 10% fetal bovine serum and 1% penicillin / streptomycin was suspended in low DMEM (Dulbecco's modified Eagle's medium).
6번 계대배양한 세포를 수집하여 CD105 및 CD73을 포함하여 분화와 관련된 hBM-MSC 표면 마커 클러스터의 발현을 유세포 분석기로 확인하였다.6 cells were harvested and the expression of hBM-MSC surface marker clusters associated with differentiation including CD105 and CD73 was confirmed by flow cytometry.
그 결과, hBM-MSCs에서 상기 양성 마커의 높은 발현(~99%)이 확인되었으며, 음성 마커인 CD34 및 CD45는 낮은 발현(~1%)이 확인되었다.As a result, high expression (~ 99%) of the positive markers was confirmed in hBM-MSCs, and low expression (~ 1%) in CD34 and CD45 negative markers were confirmed.
각 분획의 106 세포를 500 × g에서 5분간 원심분리하고 PBS로 세 번 세척한 후 PBS에서 6 또는 12 시간 동안 인큐베이션하였다.10 6 cells of each fraction were centrifuged at 500 xg for 5 minutes, washed three times with PBS, and incubated in PBS for 6 or 12 hours.
2. 가스 크로마토그래피 (2. Gas Chromatography ( GCGC -MS) 분석-MS) analysis
각각의 아미노산 함량은 GC-MS 분석으로 확인하였다.Each amino acid content was confirmed by GC-MS analysis.
5975B mass-selective detector (70 eV, electron impact ionization mode; Agilent Technologies) 장비를 갖춘 model 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA)를 이용하여 각 스캔 및 SIM 모드에서 GC-MS 분석을 수행하였다.GC-MS analysis was performed in each scan and SIM mode using a model 6890N gas chromatograph (Agilent Technologies, Santa Clara, Calif., USA) equipped with a 5975B mass-selective detector (70 eV; electron impact ionization mode; Respectively.
3. 3. 마이크로어레이Microarray 및 아미노산 프로파일링 And amino acid profiling
54,675 프로브를 이용하여 Human U133 Plus 2.0 50K 마이크로어레이(microarray)와 연결된 Affymetrix system(Istech, Ilsan, Korea)으로 hBM-MSCs 세포 내 유전자 발현 변화를 확인하였다.The gene expression in hBM-MSCs was confirmed by Affymetrix system (Istech, Ilsan, Korea) connected to Human U133 Plus 2.0 50K microarray using 54,675 probes.
GenPlex 3.0 소프트웨어로 데이터 분포의 차이를 분석하였으며, IPA(Ingenuity pathway analysis) web-based bioinformatics software (Qiagen)를 이용하여 생물학적 경로 및 기능을 확인하였다.Data distribution differences were analyzed using GenPlex 3.0 software, and biological pathways and functions were confirmed using ingenuity pathway analysis (IPA) web-based bioinformatics software (Qiagen).
0시간 대조군과 비교하여 6 및 12시간 보관된 hBM-MSCs 세포에서 3배 발현 차이를 나타내는 유전자를 컷-오프 값으로 사용하였다.The cut-off values of genes expressing the 3-fold difference in expression in hBM-MSCs cells stored for 6 and 12 hours compared with 0 hour control group were used.
4. 정량적 실시간 중합효소 연쇄반응(quantitative polymerase chain reaction; 4. Quantitative real-time polymerase chain reaction (PCR). qPCRqPCR ))
과산화 지방질(lipid peroxidation)관련 유전자 분석을 위해, RealMOD SYBR Green real-time PCR kit (Intron)와 유전자 특이적 프라이머 쌍을 이용하여 Rotor Gene-Q system (Qiagen, Valencia, CA, USA)으로 qPCR을 수행하였다. 반응 조건은 95℃에서 5분 반응 후, 95℃에서 5초, 60℃에서 30초를 50 사이클 반복하였다. QPCR was performed with a Rotor Gene-Q system (Qiagen, Valencia, CA, USA) using RealMOD SYBR Green real-time PCR kit (Intron) and a gene-specific primer pair for analysis of lipid peroxidation related genes Respectively. The reaction conditions were 95 ° C for 5 minutes, 50 cycles of 95 ° C for 5 seconds and 60 ° C for 30 seconds.
통계적으로 백그라운드 수준보다 높게 검출된 형광 포인트를 Ct/Cq(threshold/quantification cycle) 값으로 결정하였으며, Rotor-Gene 1.7 software (Qiagen)를 이용하여 분석된 용융 곡선을 기초로 PCR 생산물을 분석하였다.Ct / Cq (threshold / quantification cycle) values were determined for fluorescence points that were statistically higher than the background level, and PCR products were analyzed based on the melting curve analyzed using Rotor-Gene 1.7 software (Qiagen).
상기 과정으로 PCR 반응을 독립적인 시료로 세 번 반복수행하고, 2- ΔΔCt 방법을 이용하여 목적 유전자 발현의 상대적 정량값을 얻었다.In the above procedure, the PCR reaction was repeated three times as an independent sample, and 2 - ΔΔCt Method was used to obtain the relative quantification of the expression of the target gene.
5. 5. hBMhBM -- MSCs세포에서In MSCs cells 유전자 동시 발현 네트워크 및 아미노산 프로파일 확인 Simultaneous gene expression network and amino acid profile identification
IPA를 이용하여 상기 실험과정으로 얻은 마이크로어레이 및 아미노산 프로파일링 데이터를 분석하여 유전자 동시 발현 네트워크를 확인하고, 도 1a와 같이 발현이 증가 및 감소된 유전자를 각각 적색 및 녹색으로 표시하였다.IPA was used to analyze the microarray and amino acid profiling data obtained by the above-described experiment procedure to confirm the gene coexpression network, and the genes whose expression was increased or decreased as shown in FIG. 1A were displayed in red and green, respectively.
상기 결과에 따라, qPCR를 수행하여 과산화 지방질 관련 유전자의 발현 수준을 확인하였다.Based on the above results, qPCR was performed to confirm the expression level of the peroxidative lipolytic gene.
그 결과, 도 1b와 같이 0시간 보관된 실험군과 비교하여 12시간 보관된 실험군에서는 수퍼 옥사이드 디스뮤타제(superoxide dismutase 2; SOD2), CYP4A11(cytochrome P450, family 4 subfamily A polypeptide 11)의 발현이 감소된 반면, 토코페롤 α 수송 단백질(tocopherol α transfer protein; TTPA), 프로스타클란딘 E 수용체 4(prostaglandin E receptor 4; PTGER4), PRKAA2(5'-AMP-activated protein kinase catalytic subunit alpha-2)는 0시간 실험군과 비교하여 12시간 보관된 실험군에서 상대적으로 발현이 증가된 것을 확인할 수 있었다.As a result, the expression of superoxide dismutase 2 (SOD2) and CYP4A11 (cytochrome P450, family 4 subfamily A polypeptide 11) was decreased in the experimental group which was stored for 12 hours compared with the group kept for 0 hours as shown in FIG. (TTPA), prostaglandin E receptor 4 (PTGER4), and PRKAA2 (5'-AMP-activated protein kinase catalytic subunit alpha-2) Compared with the time - treated group, the expression level was increased in the experimental group which was stored for 12 hours.
또한, 상기 결과를 바탕으로 hBM-MSCs 세포막의 과산화 지방질 양을 앞서 보고된 문헌(Lee, S. et al., An ultra-sensitive nanoarray chip based on single-molecule sandwich immunoassay and TIRFM for protein detection in biologic fluids. Analyst 134, 933-938 (2009))에 개재된 방법으로, 티오시안산제일철(ferrous thiocyanate)을 세포에 처리한 후 세포들의 형광강도를 측정하여 확인하였다.Based on the above results, the amount of lipid peroxidation in hBM-MSCs cell membranes was measured by the method described in Lee, S. et al., An ultra-sensitive nanoarray chip based on single-molecule sandwich immunoassay and TIRFM for protein detection in biologic fluids Analyst 134, 933-938 (2009)), the fluorescence intensity of the cells was measured after ferrous thiocyanate fermentation.
그 결과, 도 2에 나타난 바와 같이, 6시간 보관된 세포와 비교하여 12시간 보관된 세포에서 과산화 지방질이 증가된 것을 확인할 수 있었다.As a result, as shown in Fig. 2, it was confirmed that the lipid peroxidation was increased in the cells stored for 12 hours as compared with the cells stored for 6 hours.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 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 thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
Claims (8)
상기 유전자의 발현 정도를 정상대조군과 비교하는 단계를 포함하는 인산완충생리식염수에 보관된 인간으로부터 분리된 중간엽줄기세포의 이식 적합성 평가를 위한 정보를 제공하는 방법.(SOD2) (superoxide dismutase 2 (NCBI accession number: BC016934.1), CYP4A11 (cytochrome P450, family 4 subfamily A polypeptide 11, NCBI accession number: (NCBI accession number: BC058000.1), PTGER4 (prostaglandin E receptor 4: NCBI accession number: BC101534.1) and PRKAA2 (5'-AMP-activated protein kinase catalytic subunit alpha -2; NCBI accession number: BC069823.1) confirming the expression level of the gene using an agent for detecting expression of the gene; And
And comparing the degree of expression of the gene with a normal control, the method comprising the steps of:
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| 최기령, "인간 중간엽 줄기세포의 세포 변형율 분석", 이학 석사학위논문, 아주대학교 대학원 분자과학기술학과 (2013) |
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