Tissue Microarray를 이용한 원발 신장암 조직과 전이 조직 간의 조직생물학적 표지자(Tissue-BasedBiomarker)발현의 상관성에 관한 연구

김성한(Sung Han Kim),박원서(Weon Seo Park),박은영(Eun Young Park),박보람(Boram Park),주정남(Jungnam Joo),정재영(Jae Young Joung),서호경(Ho Kyung Seo),이강현(Kang Hyun Lee),정진수(Jinsoo Chung) 대한비뇨기종양학회 2016 대한비뇨기종양학회지 Vol.14 No.3

Purpose: The study was aimed to determine the correlations of tissue-based biomarker expressions between primary and metastatic specimens of renal cell carcinoma and with several well-known prognostic clinicopathological parameters. Materials and Methods: The immunohistochemistry (IHC) was used to determine the expression levels of 9 tissue-based markers calculated in H-score expressed by percentage of expression multiplied by the intensity score (0, 1, 2, and 3 points). Using 17 patients’ 38 specimens paired with primary renal lesion and its metastatic lesions collected between 2004 and 2015, Tissue microarray with IHC was performed with BAP1, PBRM1, pS6, PTEN, TGase2, PD-L1, CA9, PSMA, and Ki-67 on formalin-fixed paraffin-embedded sections. Pearson correlation and accuracy test were performed to analyze the correlation between primary and metastatic tissues. Results: The 17 patients’ mean age was 56.9 years old, mean tumor size was 7.9 cm, and the male to female ratio was 13:4 (76.5%:23.5%), respectively. Three patients had 2, 3, and 3 metastatic tissues, and the rest of 14 patients had only one metastatic tissue. The H-score (PSMA and Ki67) and intensity score (pS6 and PSMA) showed that some differential significant markers were identified which had statistical correlations of expression levels between primary and metastatic lesions among 9 markers. However, no real correlation of PSMA, Ki67, and pS6 markers were found their expressions of between primary and metastatic tissues because of their skewed expressions. Conclusions: Tissue markers failed to correlate their expression levels in primary lesions with those of metastatic lesions.

Molecular Cloning of Adipose Tissue-specific Genes by cDNA Microarray

Kim, Kee-Hong,Moon, Yang Soo Asian Australasian Association of Animal Productio 2003 Animal Bioscience Vol.16 No.12

In an attempt to isolate novel molecules that may play a regulatory role in adipocyte differentiation, we devised an experimental strategy to identify adipose tissue-specific genes by modifying cDNA microarray technique. We used genefilter membranes containing approximately 15,000 rat non-redundant EST clones of which 4,000 EST were representative clones of known genes and 11,000 ESTs were uncharacterized clones. A series of hybridization of genefilter membranes with cDNA probes prepared from various rat tissues and nucleic acids sequence analysis allowed us to identify two adipose-tissue specific genes, adipocyte-specific secretory factor (ADSF) and H-rev107. Verification of tissue-specific expression patterns of these two genes by Northern blot analysis showed that ADSF mRNA is exclusive expressed in adipose tissue and the H-rev107 mRNA is predominantly expressed in adipose tissue. Further analysis of gene expression of ADSF and H-rev107 during 3T3-L1 adipocyte differentiation revealed that the ADSF and H-rev107 gene expression patterns are closely associated with the adipocyte differentiation program, indicating their possible role in the regulation of adipose tissue development. Overall, we demonstrated an application of modified cDNA microarray technique in molecular cloning, resulting in identification of two novel adipose tissue-specific genes. This technique will also be used as a useful tool in identifying novel genes expressed in a tissue-specific manner.

Expression of Tiam1 in Lung Cancer and its Clinical Significance

Wang, Hong-Ming,Wang, Jing Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.2

The aim of this study was to ana1yze T-cell lymphoma invasion and metastasis-inducing factor 1 (Tiam1) expression in 1ung cancer patients. A total of 204 patients with lung cancer tissue lesions were enrolled in the present study, along with 40 cases of normal lung tissue and 40 of normal fetal lung tissue. Tiam1 protein expression level was determined using intensity quantitative analysis, for comparison in lung cancer, metastatic, normal lung, and fetal lung tissue. The positive unit (PU) of Tiam1 was $13.5{\pm}5.42$ in lung cancer,$5.67{\pm}1.56$ in norma1 epithelial cells, and $5.89{\pm}1.45$ in fetal lung epithelial cells. The value in the lung cancer tissue was significantly higher than that in the normal lung tissue and the fetal lung tissue (P<0.01). The Tiam1 PU values with lymph node metastasis and without 1ymph node metastasis were $15.2{\pm}4.34$ and $12.5{\pm}4.23$, respectively, and the difference was statistically significant (P<0.05). The Tiam1 PU values in different tumor, nodes, metastasis (TNM) stages, III-IV period, and I-II phase were $14.7{\pm}4.14$ and $11.0{\pm}5.34$ (P<0.05). A correlation was found between Tiam1 expression and the age of patient, tumor size, tumor type, and tumor differentiation. Tiam1 protein expression in the lung tumor tissue is significantly higher than that in the normal lung tissue and fetal lung tissue. Tiam1 expression may be closely related to lung cancer development and metastasis.

Construction of Ovine Customer cDNA Chip and Analysis of Gene Expression Patterns in the Muscle and Fat Tissues of Native Korean Cattle

Kyung Ho Han(한경호),Eun Young Choi(최은영),Yeon-Hee Hong(홍연희),Jae Yeong Kim(김재영),In Soon Choi(최인순),Sang-Suk Lee(이상석),Yun Jaie Choi(최윤재),Kwang Keun Cho(조광근) 한국생명과학회 2015 생명과학회지 Vol.25 No.4

소의 질을 평가하기 위해서는 중요한 인자인 근육내 지방(또는 마블링)을 조절하는 분자를 연구해야 한다. cDNA microarray를 사용하여 등지방 조직과 최장근의 유전자발현 차이를 비교하였다. 이 연구를 통해, 우리는 한우의 지방조직에 1211개, 근육조직에서 1346개의 특이 유전자를 확인하였다. bovine chip은 지방조직의 920개 유전자와 근육조직의 760개 유전자로 이루어진 1680개의 특이 유전자로 구성되어있다. 이 실험에서 Microarray 분석은 등지방조직(Cy3)과 최장근(Cy5)의 유전자 발현에 있어서 큰 차이를 보여준다. 차이를 보이는 많은 특이유전자 중에서, 12-리폭시게나아제 유전자와 프로스타글란딘 D 합성효소는 근육내 지방의 축적을 조절하는 중요한 효소이다. 본 연구에서, 일반적으로 발현되지만 한우의 근육과 지방 조직에서 차이를 보이는 많은 유전자를 hybridization 분석을 통해 발견하였다. 선택된 유전자의 발현 수준은 반정량적 RT-PCR을 통해 확인하였고, 그 결과는 cDNA microarray와 유사하였다. To investigate the molecular events of controlling intramuscular fat (or marbling), which is an important factor in the evaluation of beef quality, we performed cDNA microarray analyses using the longissimus dorsi muscle and back fat tissues. For this study, we constructed normalized cDNA libraries: fat tissues in native Korean cattle (displaying 1,211 specific genes), and muscle tissues in native Korean cattle (displaying 1,346 specific genes). A bovine cDNA chip was constructed with 1,680 specific genes, consisting of 760 genes from muscle tissues and 920 genes from fat tissues. The microarray analysis in this experiment showed a number of differentially expressed genes, which compared the longissimus dorsi muscle (Cy5) with back fat tissue (Cy3). Among many specific differentially expressed genes, 12-lipoxygenase (oxidizing esterified fatty acids) and prostaglandin D synthase (differentiation of fibroblasts to adipocytes) are the key candidate enzymes that should be involved in controlling the accumulation of intramuscular fat. In this study, differentially and commonly expressed genes in the muscle and fat tissues of native Korean cattle were found in large numbers, using the hybridization assay. The expression levels of the selected genes were confirmed by semi-quantitative RT-PCR, and the results were similar to those of the cDNA microarray.

Tissue microarray를 이용한 사이모신 베타4(Thymosin β4)와 vascular endothelial cell growth factor (VEGF)의 정상 인간 조직 발현 양상 연구

옥미선(Mee Sun Ock),차희재(Hee-Jae Cha) 한국생명과학회 2009 생명과학회지 Vol.19 No.12

사이모신 베타 4와 VEGF의 발현을 여러 인간 조직에서 tis sue microarray를 사용하여 조사하였다. 사이모신 베타 4는 간, 이자, 침샘의 관상피, 심장에서 강한 발현을 보였으며 피부, 폐, 이자, 림프절, 갑상선, 요관, 폐와 부신의 혈관 내피세포 등에서 중간 수준의 발현 양상을 보였다. VEGF의 발현 양상은 대체적으로 사이모신 베타 4와 동일하였으며 이자, 요관, 유선, 간, 식도, 신장, 폐, 부신 등의 혈관 내피세포에서 강하게 발현되었다. 이러한 결과를 통해 사이모신 베타 4는 간, 이자, 침샘의 관상피, 심장에서 중요한 역할을 담당하며 VEGF와 같은 발현 양상을 보여 혈관 신생작용에 관여함을 확인하였다. Thymosin β4, a small protein containing 43 amino acids, has multi-functional roles in cell physiology. It was first identified as a thymic maturation factor and recently has been shown to accelerate wound healing, hair growth, angiogenesis, tumor growth, and metastasis. It was also reported to play a key role in developing organs, including the nervous system and heart. Thymosin β4 induces the expression of vascular endothelial cell growth factor (VEGF), laminin-5, and other important biologically active genes. Using tissue microarray analysis, we investigated the expression patterns of thymosin β4 and VEGF in various normal human adult tissues. Thymosin β4 was highly expressed in the liver, pancreas, ductal epithelium of the salivary gland, and heart, and moderately expressed in the skin, lung, spleen, lymph node, thymus, ureter, and blood endothelial cells in both the lung and adrenal gland. The expression of VEGF generally co-localized with thymosin β4 and VEGF was highly expressed in the pancreas, ureter, mammary gland, liver, esophagus, and blood endothelial cells in both the lung and adrenal gland. These results suggest that thymosin β4 plays an important role in the function of various organs and since the expression pattern of thymosin β4 co-localized with VEGF, part of that function may be to induce or maintain angiogenesis.

Tissue microarray를 이용한 여러 암에서의 thymosin β4, vascular endothelial growth factor, 및 hypoxia-inducible factor-1α 발현양상 연구

Bo-Young Lee(이보영),Seung-Hyun Lee(이승현),Byung-Kwon Ahn(안병권),Mee Sun Ock(옥미선),Hee-Jae Cha(차희재) 한국생명과학회 2011 생명과학회지 Vol.21 No.3

사이모신 베타 4와 관련 단백질인 HIF-1α 및 VEGF의 발현을 여러 인간 암 조직에서 tissue microarray를 사용하여 조사하였다. 사이모신 베타 4는 골육중, 대장 선암, 식도 편평세포암, 신장 및 방광의 이행세포암, 폐암 및 간암에서 많이 발현되었으며 HIF-1α은 비강 역위성 유두종, 폐암 및 식도 편평세포암에서 강한 발현을 보였으며 대체로 발현되는 양상이나 위치가 사이모신 베타 4와 일치하는 것으로 관찰되었다. VEGF는 암 조직에서보다 암조직에 분포된 혈관내피에서 강하게 발현되는 양상을 나타내었으며 암세포에서는 사이모신 베타 4나 HIF-1α에 비해 강하게 발현되지 않았다. 위암, 간 혈관육종, 담낭 선암과 자궁 내막 선암에서 적당 수준의 VEGF 발현이 관찰되었으며 VEGF의 발현 양상 및 위치는 위암, 골육종, 지방종, 폐암, 간암, 담낭 선암, 식도 편평세포암, 대장 및 직장암, 신세포암을 포함하는 특정 암에서 사이모신 베타 4 및 HIF-1α와 일치하는 것으로 관찰되었다 Thymosin β4 (TB-4) has been reported to play a key role in tumor growth, metastasis and angiogenesis. In addition, TB-4 induced the expression of vascular endothelial growth factor (VEGF) and stabilized the hypoxia-inducible factor (HIF)-1α in melanoma cells. Although the importance of thymosin β4 in angiogenesis and metastasis has been proven, there are few studies that show the expression patterns of TB-4, VEGF and HIF-1α. This study was conducted to analyze the relationship among these proteins in various tumors. Using tissue microarray analysis, we investigated the expression patterns of TB-4, VEGF and HIF-1α in various tumors to identify the expression patterns and relationships of these proteins in certain tumors. TB-4 was highly expressed in osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. HIF-1α was highly expressed in nasal cavity inverted papilloma, lung cancer, and esophageal squamous cell carcinoma. The expression patterns of TB-4 and HIF-1α were almost similar and co-localized. VEGF expression was high in the blood vessels in tumors, but usually not high in the tumors themselves. VEGF was moderately expressed in stomach cancer, liver angiosarcoma, gall bladder adenocarcinoma, and uterus endometrial adenocarcinoma. The expression patterns of VEGF shows similarities in certain tumors including stomach cancer, osteosarcoma, liposarcoma, lung cancer, liver cancer, gall bladder adenocarcinoma, esophageal squamous cell carcinoma, stomach cancer, colorectal carcinoma and renal cell carcinoma. These results suggest that the expression patterns of TB-4, HIF-1α and VEGF were co-localized and related to tumorigenesis and angiogenesis of certain tumors.

Tissue Microarrays in Biomedical Research

Chung, Joon-Yong,Kim, Nari,Joo, Hyun,Youm, Jae-Boum,Park, Won-Sun,Lee, Sang-Kyoung,Warda, Mohamad,Han, Jin Korean Society for Bioinformatics and Systems Biol 2006 Bioinformatics and Biosystems Vol.1 No.1

Recent studies in molecular biology and proteomics have identified a significant number of novel diagnostic, prognostic, and therapeutic disease markers. However, validation of these markers in clinical specimens with traditional histopathological techniques involves low throughput and is time consuming and labor intensive. Tissue microarrays (TMAs) offer a means of combining tens to hundreds of specimens of tissue onto a single slide for simultaneous analysis. This capability is particularly pertinent in the field of cancer for target verification of data obtained from cDNA micro arrays and protein expression profiling of tissues, as well as in epidemiology-based investigations using histochemical/immunohistochemical staining or in situ hybridization. In combination with automated image analysis, TMA technology can be used in the global cellular network analysis of tissues. In particular, this potential has generated much excitement in cardiovascular disease research. The following review discusses recent advances in the construction and application of TMAs and the opportunity for developing novel, highly sensitive diagnostic tools for the early detection of cardiovascular disease.

Gene Microarray Analysis for Porcine Adipose Tissue: Comparison of Gene Expression between Chinese Xiang Pig and Large White

Guo, W.,Wang, S.H.,Cao, H.J.,Xu, K.,Zhang, J.,Du, Z.L.,Lu, W.,Feng, J.D.,Li, N.,Wu, C.H.,Zhang, L. Asian Australasian Association of Animal Productio 2008 Animal Bioscience Vol.21 No.1

We created a cDNA microarray representing approximately 3,500 pig genes for functional genomic studies. The array elements were selected from 6,494 cDNA clones identified in a large-scale expressed sequence tag (EST) project. These cDNA clones came from normalized and subtracted porcine adipose tissue cDNA libraries. Sequence similarity searches of the 3,426 ESTs represented on the array using BLASTN identified 2,790 (81.4%) as putative human orthologs, with the remainder consisting of "novel" genes or highly divergent orthologs. We used the gene microarray to profile transcripts expressed by adipose tissue of fatty Chinese Xiang pig (XP) and muscley Large White (LW). Microarray analysis of RNA extracted from adipose tissue of fatty XP and muscley LW identified 81 genes that were differently expressed two fold or more. Transcriptional differences of four of these genes, adipocyte fatty acid binding protein (aP2), stearyl-CoA desaturase (SCD), sterol regulatory element binding transcription factor 1 (SREBF1) and lipoprotein lipase (LPL) were confirmed using SYBR Green quantitative RT-PCR technology. Our results showed that high expression of SCD and SREBF1 may be one of the reasons that larger fat deposits are observed in the XP. In addition, our findings also illustrate the potential power of microarrays for understanding the molecular mechanisms of porcine development, disease resistance, nutrition, fertility and production traits.

Genetic Profiling in Human Adipose Tissue-Derived Mesenchymal Stromal Cells from the Idiopathic and Familial Parkin-Deficient Patients of Parkinson`s Disease in Comparison with non-PD patients

( Hyo Eun Moon ),( Hyung Woo Park ),( Hye Young Shin ),( Seung Leal Paek ),( Dong Gyu Kim ),( Jin H Son ),( Sun Ha Paek ) 한국조직공학과 재생의학회 2010 조직공학과 재생의학 Vol.7 No.2

Purpose: With an approach of carrying out a transcriptome microarray analysis using early-passage adipose tissue-derived mesenchymal stromal cells from human adult patients with early-onset hereditary parkin deficient Parkinson`s disease (PD) as well as late-onset idiopathic PD, we aimed to understand brain pathology in PD patients. Methods: Here, we isolated human adipose tissue-derived mesenchymal stromal cells (hAD-MSCs) of patients with idiopathic PD and parkin deficient PD in comparison with non-PD patients and profiled their gene expression using Affymetrix cDNA microarray analysis. Human adipose tissue is a rich source of MSCs, providing an abundant and accessible source of adult stem cells. Results: The hAD-MSCs of patients with idiopathic PD were named as "PD", with parkin deficient PD as "Parkin" and with pituitary adenoma as "non-PD" shortly. Initially, Differentially Expressed Genes (DEGs, total 413 genes) were classified and summarized among non-PD, PD and Parkin. Moreover, using K-mean clustering analysis, we grouped DEGs into 7 clusters and gene names and genebank accession numbers between 2 and 6 were arranged. Additionally, the functional groups of human biomarker candidates were organized and compared between non-PD vs. PD and non-PD vs. Parkin. Finally, PD-related differentially regulated genes by oxidative stress were categorized among non-PD, PD and Parkin. Conclusions: This study showed that knowledge of selective gene expression profile derived from PD patients might potentially lead to better understanding of PD pathology and development of early diagnosis and effective therapy targeted their human biomarkers.

유방암조직의 Tissue Microarrays에서의 면역화학염색법을 이용한 Apoptosis 관련단백질의 발현

김정수(Jeong Soo Kim),김기환(Kee Hwan Kim),안창혁(Chang Hyeok Ahn),전해명(Hae Myung Jeon),정상설(Sang Seol Jung),임석아(Seock Ah Im) 대한외과학회 2001 Annals of Surgical Treatment and Research(ASRT) Vol.60 No.6