자궁경부 이형성증과 암의 진단을 위한 액상세포 검체에서 p16^INK4a/Ki-67 이중면역염색의 평가

성미희,이훈택,신민식,오서영,김욱연,Sung, Mi Hee,Lee, Hoon Taek,Shin, Min Shik,Oh, Seo Young,Kim, Wook Youn 대한임상검사과학회 2015 대한임상검사과학회지(KJCLS) Vol.47 No.3

Recently, $p16^{INK4a}$/Ki-67 dual immunostaining has been introduced as a new biomarker protocol for early detection of uterine cervical dysplasia and cancer in liquid-based cytology (LBC). We performed the $p16^{INK4a}$/Ki-67 dual immunostaining using a CINtec$^{(R)}$ PLUS kit in a total of 109 LBC cases of cervicovaginal smear and compared its results with those from LBC, HPV hybrid capture II (HC II) test and histological diagnosis. Expression of $p16^{INK4a}$ and Ki-67 was significantly associated with cases of LSIL or higher in cytological diagnosis and cases of cervical intraepithelial neoplasia (CIN) 1 or higher in histological diagnosis (p<0.001 and p<0.001, respectively). Among forty-six cases of atypical squamous cells of undetermined significance (ASCUS) in LBC, $p16^{INK4a}$ and Ki-67 was expressed in 31 (67.4%), which were positively associated with cases of CIN I lesion or higher in histology. The sensitivity of $p16^{INK4a}$/Ki-67 dual immunostaining for finding lesions of CIN 1 or higher was 89.0%, which was higher than LBC. The specificity was 73.5%, which was higher than that of the HC II test. Based on these results, the $p16^{INK4a}$/Ki-67 dual immunostaining method can be a useful diagnostic marker for improving the sensitivity of LBC and the specificity of HC II test. 최근 $p16^{INK4a}$/Ki-67 이중면역염색은 액상세포검사에서 자궁경부 이형성증과 암을 조기에 발견하기 위한 새로운 생체 표지자로 대두되고 있다. 저자들은 자궁경부질도말의 액상세포검체 총 109례에서 CINtec$^{(R)}$ PLUS kit를 사용하여 $p16^{INK4a}$/Ki-67 이중면역염색을 시행하였고, 그 결과를 액상세포검사, HPV hybrid capture II (HC II) 검사 그리고 조직학적 진단과 서로 비교하였다. $p16^{INK4a}$/Ki-67 양성 발현은 세포학적 진단에서 저등급 편평상피내병변 이상의 증례 그리고 조직학적 진단에서 자궁경부 상피내종양 1등급 이상의 증례에서 유의미하게 높았다. 액상세포검사상 비정형 편평 상피세포 소견을 보이는 46례 중, 31례 (67.4%)가 $p16^{INK4a}$과 Ki-67 양성 소견을 보였고, 이러한 양성 증례들은 조직검사에서도 대부분 자궁경부 상피내종양 1등급 이상의 병변에 해당하였다. 자궁경부 상피내종양 1등급 이상의 병변을 발견하기 위한 $p16^{INK4a}$/Ki-67 이중면역염색의 민감도는 액상세포검사보다 높은 89.0%였고, 특이도는 73.5%로 HC II 검사보다 높게 분석되었다. 따라서, $p16^{INK4a}$/Ki-67 이중면역염색 방법은 액상세포검사법의 민감도와 HC II 검사법의 특이도를 보완하기 위한 진단적 검사로 유용하다고 할 수 있다.

국내 돼지에서 분리한 내인성 레트로 바이러스 gag 유전자의 분자 생물학적 특성 분석

이정은(Jungeun Lee),이동희(Donghee Lee),유재영(Jae-Young Yoo),김계웅(Gye-Woong Kim),박홍양(Hong-Yang Park),이훈택(Hoon-Taek Lee),김영봉(Young Bong Kim) 대한미생물학회 2006 Journal of Bacteriology and Virology Vol.36 No.3

미세 절제에 의한 폐 선암 세포 검체에서 EGFR 분석

한정연 ( Jeong Yeon Han ),이훈택 ( Hoon Taek Lee ),오서영 ( Seo Young Oh ) 대한임상검사과학회 2015 대한임상검사과학회지(KJCLS) Vol.47 No.3

The discovery of activating mutations in EGFR in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Cytologic specimen procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used microdissection to isolate small numbers of tumor cells to assess for EGFR mutations from 76 cytological smear slides of patients with lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient’s initial clinical work-up. Not only were we able to detect the identical EGFR mutation through the pyrosequencing, but we were also able to consistently detect the mutation from as few as 25 microdissected tumor cells. Furthermore, isolating a purer population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from microdissection-enriched cases. Therefore, microdissection can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.

자궁경부 이형성증과 암의 진단을 위한 액상세포 검체에서 p16INK4a/Ki-67 이중면역염색의 평가

성미희 ( Mi Hee Sung ),이훈택 ( Hoon Taek Lee ),신민식 ( Min Shik Shin ),오서영 ( Seo Young Oh ),김욱연 ( Wook Youn Kim ) 대한임상검사과학회 2015 대한임상검사과학회지(KJCLS) Vol.47 No.3

Recently, p16INK4a/Ki-67 dual immunostaining has been introduced as a new biomarker protocol for early detection of uterine cervical dysplasia and cancer in liquid-based cytology (LBC). We performed the p16INK4a/Ki-67 dual immunostaining using a CINtec® PLUS kit in a total of 109 LBC cases of cervicovaginal smear and compared its results with those from LBC, HPV hybrid capture II (HC II) test and histological diagnosis. Expression of p16INK4a and Ki-67 was significantly associated with cases of LSIL or higher in cytological diagnosis and cases of cervical intraepithelial neoplasia (CIN) 1 or higher in histological diagnosis (p<0.001 and p<0.001, respectively). Among forty-six cases of atypical squamous cells of undetermined significance (ASCUS) in LBC, p16INK4a and Ki-67 was expressed in 31 (67.4%), which were positively associated with cases of CIN I lesion or higher in histology. The sensitivity of p16INK4a/Ki-67 dual immunostaining for finding lesions of CIN 1 or higher was 89.0%, which was higher than LBC. The specificity was 73.5%, which was higher than that of the HC II test. Based on these results, the p16INK4a/Ki-67 dual immunostaining method can be a useful diagnostic marker for improving the sensitivity of LBC and the specificity of HC II test.

Cryopreservation of Oocytes and Embryos by Vitrification

무케쉬 쿠마르 굽타,이훈택,Gupta, Mukesh Kumar,Lee, Hoon-Taek The Korean Society for Reproductive Medicine 2010 Clinical and Experimental Reproductive Medicine Vol.37 No.4

최근 동결기술이 발달하면서 다양한 목적에 따라 초기 발생단계, 특히 수정 전후의 난자나 수정란의 생명을 연장하는 것이 가능해졌다. 이러한 난자나 수정란의 보존기술은 인간의 수정능력을 배가시키거나 임신조절에서 응용되고 있으며, 동물에서는 우수한 유전자원의 보존과 운영, 저렴한 국제간 운송수단, 그리고 생식보조기술과 유전공학 등의 연구에 필요한 생식세포의 공급하는 데서도 중요하게 활용되고 있다. 최근 개발된 완만동결과 유리화 동결방법은 난자와 수정란을 장기간 동결하여 보존하는데 활용하는 주요 기술이다. 이러한 방법들은 각각 장점과 단점을 가지고 있지만, 상당한 수준의 효율성이 입증되어 실용화되어 있는 실정이다. 무엇보다도 유리화 방법은 완만동결 방법보다 13년이나 늦게 개발되었으나 보다 우수한 기술로 인정을 받고 있다. 비록 유리화 동결은 아직 대한 상반된 의견과 오염문제가 있지만 인간과 동물의 생식보조기술로 활용되는 빈도가 점차 많아지고 있는 실정이다. 따라서 본 원고에서는 먼저 난자와 수정란의 동결보존에 대한 기초적인 기술에 대해서 고찰한 다음, 유리화 동결에 관 한 최근의 연구동향에 대해서 종합적으로 검토하고자 한다. Life can be kept in suspended animation either before fertilization at oocyte stage or after fertilization at different stages of embryonic development for a variety of reasons. It not only has potential applications in fertility preservation and management in human but also has important roles in the preservation and management of animal genetic resources, low-cost international movement of selected genetics, and rapid dissemination of germplasm through assisted reproductive technologies (ART) and genetic engineering. Currently, slow-freezing and vitrification are the two approaches by which oocytes and embryos can be cryopreserved for long-term storage. Both of these methods have their own advantages and disadvantages but allow the cryopreservation of oocytes and embryos with comparable efficiency. Vitrification of oocyte and embryos, although proven successful just 13 years after slow-freezing, is generally considered an emerging technology and appears to slow gain acceptance in both animal and human ART despite having controversial storage and contamination issues. In this manuscript, we discuss the basic techniques of oocyt/embryo cryopreservation and review the current status and recent developments in vitrification.

신경성장촉진 인자가 인간 배아줄기세포 유래 도파민 분비 신경세포형성에 미치는 영향

이금실,김은영,신현아,조황윤,왕규창,김용식,이훈택,정길생,이원돈,박세필,임진호,Lee, Keum-Sil,Kim, Eun-Young,Shin, Hyun-Ah,Cho, Hwang-Yoon,Wang, Kyu-Chang,Kim, Yong-Sik,Lee, Hoon-Taek,Chung, Kil-Saeng,Lee, Won-Don,Park, Se-Pill,Lim, Jin 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.1

Objective: This study was to examine the in vitro neural cell differentiation patterns of human embryonic stem (hES) cells following treatment of various neurotrophic factors [basic fibroblast growth factor (bFGF), retinoic acid (RA), brain derived neurotrophic factor (BDNF) and transforming growth factor (TGF)-$\alpha$], particulary in dopaminergic neuron formation. Methods: The hES cells were induced to differentiate by bFGF and RA. Group I) In bFGF induction method, embryoid bodies (EBs, for 4 days) derived from hES were plated onto gelatin dish, selected for 8 days in ITSFn medium and expanded at the presence of bFGF (10 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14 and 21 days. Group II) For RA induction, EBs were exposed of RA ($10^{-6}M$) for 4 days and allowed to differentiate in N2 medium for 7, 14 and 21 days. Group III) To examine the effects of additional neurotrophic factors, bFGF or RA induced cells were exposed to either BDNF (10 ng/ml) or TGF-$\alpha$ (10 ng/ml) during the 21 days of final differentiation. Neuron differentiation and dopamine secretion were examined by indirect immunocytochemistry and HPLC, respectively. Results: The bFGF or RA treated hES cells were resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with BDNF or TGF-$\alpha$ during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression of a dopaminergic neuron marker, compared to control (p<0.05). In contrast, no effect was observed on the rate of mature neuron (NF-200) or glutamic acid decarboxylase-positive neurons. Immunocytochemistry and HPLC analyses revealed the higher levels of TH expression (20.3%) and dopamine secretion (265.5 $\pm$ 62.8 pmol/mg) in bFGF and TGF-sequentially treated hES cells than those in $\alpha$ RA or BDNF treated hES cells. Conclusion: These results indicate that the generation of dopamine secretory neurons from in vitro differentiated hES cells can be improved by TGF-$\alpha$ addition in the bFGF induction protocol.

체외수정 유래 생쥐 배아줄기세포와 유사한 특성을 보유한 단위발생 유래 생쥐 배아줄기세포

박세필,김은영,이금실,이영재,신현아,민현정,이훈택,정길생,임진호,Park, Se-Pill,Kim, Eun-Young,Lee, Keum-Si,Lee, Young-Jae,Shin, Hyun-Ah,Min, Hyun-Jung,Lee, Hoon-Taek,Chung, Kil-Saeng,Lim, Jin-Ho 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.2

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

단위발생 유래 생쥐 배아줄기세포의 기능성 심근세포 형성

신현아,김은영,이영재,이금실,박은미,이훈택,정길생,박세필,임진호,Shin, Hyun-Ah,Kim, Eun-Young,Lee, Young-Jae,Lee, Keum-Sil,Park, Eun-Mi,Lee, Hoon-Taek,Chung, Kil-Saeng,Park, Se-Pill,Lim, Jin-Ho 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.2

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

pHOxsFV벡터와 배아주간세포를 이용한 형질전환생쥐 생산 기초연구

이훈택,이봄이,정길생,김진회 건국대학교 동물자원연구센터 1998 動物資源硏究誌 Vol.19 No.-

pHook™-1 hapten 4-ethoxy-methylene-2-phenyl-2-oxazolin-5one(phOX)의 단일 항체 sFV를 암호화 하고 있으며, murine의 Ig k-chain V-J2-C 영역유래의 signal peptide에 의하여 항체를 세포 표면에 배열시키도록 고안되어 있다. 또한, 항체를 세포막 바깥쪽에 부착되어 있도록 하기 위해 PDGFR 유래의 transembrane domain의 C 말단에 결합되어 있다. 이렇게 고안된 vector을 발현하는 세포는 세포막에 sFV을 발현함으로, phOX로 코팅된 자석베드를 이용하여 배양체로부터 목적의 유전자를 발현하는 세포만을 분리할 수 있을 것이다. 따라서, 본 연구는 pHook™-1 유전자를 co-transfection함으로써 목적의 유전자를 가진 배아주간세포를 단시간 내에 효율적으로 선발하기 위하여 실시하였다. 또한, 배아주간세포에서 목적의 DNA 발현 또는 존재를 검증하기 위해 DNA 발현 또는 존재를 검증하기 위해 PCR 방법과 조직화학적 방법을 사용하였다. 형질전환유전자 발현을 transfection(유전자 전이) 후 4∼14일 사이에 모든 배아주간세포에서 확인되었다. Magnetic bead를 이용하여 선발된 세포에서 co-transfected DNA는 배아주간세포에서 효율절으로 삽입되었으며, 선발된 세포의 약 90%는 co-transfected 유전자를 발현하였다. 이 결과는 세포생리학에서 특이 유전자의 급성변이와 만성변이를 연구하거나, 또는 형질전환동물을 생산하기 위해 pHook™-1 목적유전자와 함께 전이함으로서 효율적으로 목적의 유전자를 가진 세포를 선발 가능함으로써 보다 간편하게 형질전환도 동물의 생산에 이용 가능하다는 사실을 확인하였다. pHook™-1 encodes a single-chain antibody(sFv) directed toward the hapten 4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one(phOx): the signal peptide from the murine Igk-chain V-J2-C region is fused in front of coding region of the sFv to direct the antibody to the plasma membrane. The antibody is fused at the C-termius to the transmembrane domain from the platelet derived growth factor receptor(PDGFR), allowing the antibody to be anchored and displayed on the extracellular side of theemmbrane. Transfected cells expression sFv can be isolated from whole cultures by using magnetic coated with phOx and a strong magnetic strand. Thus, the present study was designed to apply the embryonic stem cells by using pHook™-1 . Cell-transduction efficiency was measured by morphometric analysis. Polymerase chain reaction and histochemistry were used to detect the presence and/or expression of objective DNA in embryonic stem cells. Transgene expression was detected in all cases between 4 and 14 day after transfection. In selected cells using magnetic bead, co-transfected DNA was also incorporated efficiently in embryonic stem cells and approximately 90% of the selected cells expressed co-transfected gene. This result suggested that this selection system can be used as a feasible tool, when pHook™-1 is cotransfected with objective gene, to isolate and study for acute and chronic changes of a specific gene in cellular physiology.