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Laser Captured Microdissection을 이용한 유전자 발현에 대한 연구 (I): RT-PCR을 위한 난자의 RNA 추출 및 증폭을 위한 최소한도의 확립
박창은,고정재,차광렬,이경아,Park, Chang-Eun,Ko, Jung-Jae,Cha, Kwang-Yul,Lee, Kyung-Ah 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.3
Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.
윤세진,전은현,박창은,고정재,최동희,차광열,김세년,이경아,Yoon, Se-Jin,Jeon, Eun-Hyun,Park, Chang-Eun,Ko, Jung-Jae,Choi, Dong-Hee,Cha, Kwang-Yul,Kim, Se-Nyun,Lee, Kyung-Ah 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4
Object: The present study was accomplished to obtain a gene expression profile of the luminal epithelium during embryo apposition in comparison of implantation (1M) and interimplantation (INTER) sites. Material and Method: The mouse uterine luminal epithelium from IM and INTER sites were sampled on day 4.5 (Day of vaginal plug = day 0.5) by Laser Captured Microdissection (LCM). RNA was extracted from LCM captured epithelium, amplified, labeled and hybridized to microarrays. Results from microarray hybridization were analyzed by Significance Analysis of Microarrays (SAM) method. Differential expression of some genes was confirmed by LCM followed by RT-PCR. Results: Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were EST/unknown function, and the remain 53 were categorized to the structural, cell cycle, gene/protein expression, immune reaction, invasion, metabolism, oxidative stress, and signal transduction. Of the 24 structural genes, 14 were related especially to extracellular matrix and tissue remodeling. Meanwhile, among 13 genes up-regulated at INTER, 8 genes were EST/unknown function, and the rest 5 were related to metabolism, signal transduction, and gene/protein expression. Among these 58 (53+5) genes with known functions, 13 genes (22.4%) were related with $Ca^{2+}$ for their function. Conclusions: Results of the present study suggest that 1) active tissue remodeling is occurring at the IM sites during embryo apposition, 2) the INTER sites are relatively quiescent than IM sites, and 3) the $Ca^{2+}$ may be a crucial for apposition. Search for human homologue of those genes expressed in the mouse luminal epithelium during apposition will help to understand the implantation process and/or implantation failure in humans.
생쥐 Preantral 난포의 체외배양: FSH의 종류와 농도 및 초자화 냉동보존의 영향
이숙현,신창숙,정형민,고정재,차광렬,이경아,Lee, Sook-Hyun,Shin, Chang-Sook,Chung, Hyung-Min,Ko, Jung-Jae,Cha, Kwang-Yul,Lee, Kyung-Ah 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.4
Objectives: 1) To compare the efficacy of urofollitropin (Follimon) to that of recombinant human FSH (rhFSH) on the growth and maturation of mouse early preantral follicles in vitro, and 2) effect of vitrification on the growth and maturation of preantral follicles and oocytes . Methods: Isolated early preantra1 follicles (100-130 ${\mu}m$ diameter) were cultured for 12 days in 20 ${\mu}l$ ${\alpha}$-MEM media drop under the mineral oil. Follimon or rhFSH was added to the culture medium at various concentrations (0, 10, 100, and 1000 mIU/ml). Results: With Follimon, the dose of 10 mIU/ml showed the best follicle survival, growth, and MIl rate of oocyte than the other concentrations. Whereas the optimal dose of rhFSH was 100 mIU/ml. Despite the different optimal doses, the efficacy of two different FSHs on the follicle growth and maturation was similar. Isolated mouse preantral follicles were cryopreserved by vitrification and cultured in vitro for 12 days with 100 mIU/ml rhFSH. Despite the decreased follicular survival rate after thawing, the follicular growth and maturation rate of its oocyte were comparable to those of the fresh follicle. Conclusion: Results from the present study revealed that 1) the optimal doses of Follimon and rhFSH for in-vitro culture of mouse follicles are different, and 2) the frozen-thawed follicles develop normally after vitrification.
김은경,손원영,지희준,고정재,윤태기,차광열,Kim, Eun-Kyung,Son, Weon-Young,Chi, Hee-June,Ko, Jung-Jae,Yoon, Tae-Ki,Cha, Kwang-Yul 대한생식의학회 1992 Clinical and Experimental Reproductive Medicine Vol.19 No.2
This study was carried out to set up the ovum bank for ovum donation and to determine the best freezing method for human immature oocytes. Human immature follicular oocytes were cryopreserved by slow freezing and rapid thawing method. Immature follicular oocytes were treated by propanediol(PROH) solution by 2 and 4 step method in protocols A & B, respectively. In protocol C, immature oocytes were exposed to sucrose prior to treatment of PROH by 4 step method. We compared survival rate, maturation rate, and fertilization rate of immature oocytes among three protocols. Results were as follows. 1. Oocytes treated by the protocol C showed the highest survival rate( 70.3 %) and maturation rate(34.6%) after thawing. 2. Survival rate of oocytes treated by the protocol C was significantly higher than that of the protocol B after thawing(p<0.05). In conclusion, treatment of oocytes with sucrose prior to expose PROH was the best freezing method. Sucrose may have reduced the toxic effect of cryoprotectant to oocytes. We failed to induce fertilization of oocytes, which were treated by any protocols, by conventional insemination method, but obtained 28.8% fertilization rate by using partial zona dissection(PZD) method. This result suggests that micromanipulation(PZD) of the thawed oocytes before insemination will improve the fertilization rate.
불임환자에 있어서 Partial Zona Dissection(PZD) 의한 임상적인 결과
박성은,최동희,노환철,고정재,박종영,차광열,Park, Sung-Eun,Choi, Dong-Hee,Rho, Hwan-Cheol,Ko, Jung-Jae,Park, Jong-Young,Cha, Kwang-Yul 대한생식의학회 1993 Clinical and Experimental Reproductive Medicine Vol.20 No.1
Micromanipulation procedures have been used to improve fertilization rates in patients with male factor or with unexplained infertility. Partial zona disseetion(PZD), a method using mechanical force to open the zona pellucida increase the chances of fertilization. The purpose of this study is to increase rates of fertilization and pregnacy in the ART program by using PZD. The influence of PZD on the fertilization rate was investigated in 57 couples with semen defects, antisperm antibodies(ASA), or unknown factors. PZD directly performed in 35 couples with a history of fertilization failure in previous cycle (Group 1), and PZD applied in 22 couples with the failure of initial fertilization in the same cycle (Group 2). The fertilization rates of the male facor, ASA positive factor and unknown factor in Group 1 were 37.6%, 20.0% and 59.2%, respectively. The rates of fertilization of male factor, ASA positive factor and unknown factor in Group 2 were 34.8%, 20.0% and 26.5%, respectively. The incidences of polyspermy in Group 1 and Group 2 were 5.9% and 9.0%, respectively. Among 35 patients of Group 1, one patient was pregnant and successfully delivered, whereas 1 of 22 patients of Group 2 became pregnant, but aborted at 7 weeks.
김현규,엄기붕,김현주,고정재,이숙환,윤태기,차광열,Kim, Hyun-Kyoo,Oum, Ki-Boong,Kim, Hyun-Joo,Ko, Jung-Jae,Lee, Sook-Hwan,Yoon, Tae-Ki,Cha, Kwang-Yul The Korean Society for Reproductive Medicine 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.2
폐색성 혹은 비폐색성 무정자증에서 부정소 정자채취법 등이 부적절하다고 여겨질때는 정소 조직을 일부 절제하여 그 조직으로부터 정자를 직접 채취하게 되는데 일반적으로 이렇게 정소로부터 추출한 정소정자는 운동성이 전혀 없거나 매우 약한 운동성을 보이는 경우가 많다. 본 연구의 목적은 이러한 정소정자를 Vero cell과 공배양을 시킴으로써 운동성을 획득시키거나 향상시키고 이를 수정시키는 시기까지 지속시킴으로써 정소정자추출술 (TESE)을 시행하는 환자나 의료진들에게 보다 편안하고 융통성있는 시간대를 부여하고, 아울러 정자직접주입술 (ICSI)을 보다 용이하게 하여 성공적인 수정률과 임신율을 얻음에 있다. 또한 ICSI를 시행한 후, 운동성이 향상된 잉여의 정소정자를 냉동보존함으로써 차후에 TESE을 다시 시행치않고도 시험관 아기 시술을 시도할 수 있는 부가적인 잇점도 있다고 할 수 있다. 대상환자군은 정관폐색증(n=11) 혹은 비정관폐색증(n=2)을 보이는 13명의 무정자증의 남성불임환자였으며 난자회수예정일 3일전에 TESE를 시행하여 정소정자를 얻은 후 이를 정자직접주입술이 시행되는 당일까지 Vero cell과 공배양을 실시하였다. Vero cell과의 공배양에 의하여 운동성이 있는 정소정자의 수는 공배양전과 비교하여 평균 3.3배가 증가하였으며, 특히 공배양전에 운동성이 있는 정소정자의 수가 50,000/ml이하의 미약한 운동성만을 보였던 경우 (n=5)에는 공배양 후에 운동성이 있는 정소정자 수의 평균증가율이 7.7배였다. 공배양전 정자운동성이 전혀 없었던 2례의 비정관폐색증환자중 3일간의 공배양을 통하여 1례에서 운동성을 획득한 정소정자를 얻을 수 있었으며 (14,300/ml), 정자직접주입술을 통하여 성공적인 수정 및 임신에 도달할 수 있었다. Vero cell과 공배양을 하고 ICSI했던 결과, 평균 수정률은 75.0% 이었으며 임신율은 61.5%였다.
시험관아기 시술시 미세조작에 의한 임신율의 증진에 관한 연구
노환철,김은경,구정진,고정재,윤태기,차광열,Rho, Hwan-Cheol,Kim, Eun-Kyung,Koo, Jung-Jin,Ko, Jung-Jae,Yoon, Tae-Ki,Cha, Kwang-Yul 대한생식의학회 1993 Clinical and Experimental Reproductive Medicine Vol.20 No.2
This study was carried out to improve pregnancy rate in IVF-ET program through Assisted Hatching (AH) by the use of micromanipulation technique. Among 72 IVF patient, randomized 29 IVF patients were performed for AH by Partial Zona Dissection(PZD). Two to eight cell embryos were micromanipulated just before uterine transfer. The results were as follows: 1. The implantation rates of embryos between PZD group and control group were 10.0%, 4.9%, respectively. 2. The clincal pregnancy rates of both groups were 34.5%,20.9%, respectively. 3. Among 131 PZD embrys, only 2 embryos were damaged mechanically. Although there were no statistical difference in the rates of implantation and pregnancy between PZD group and control group due to small sample size, the PZD group had increasing trend in the rates of implantation and pregnancy. In conclusion, it would be thought that PZD could be adequately used to improve implantation rate and pregnancy rate in IVF-ET program as an assisted technique if much more studies were done. Also the risks resulting from this study can be reduced because of technical stability, which showed the low rate of damaged embryos.
김남근,정형민,이유진,남윤성,최동희,손태종,이숙환,고정재,차광열,Kim, Nam-Keun,Chung, Hyung-Min,Lee, Eu-Gene,Nam, Yoon-Sung,Choi, Dong-Hee,Sohn, Tae-Jong,Lee, Sook-Hwan,Ko, Jung-Jae,Cha, Kwang-Yul The Korean Society for Reproductive Medicine 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.2
연구목적: 한국여성의 다낭성 난포증후군 환자에서 황체형성 호르몬 exon 2 유전자의 변이를 탐색하여 이들 변이와 질환과의 관련성 여부를 밝히고자 하였다. 연구재료 및 방법: 21명의 다낭성 난포증후군 환자를 대상으로 황체형성 호르몬 exon 2(Trp8Arg;TGG to CGG and Ile15Thr; ATC to ACC)의 변이를 탐색하였다. 혈액에서 Genomic DNA를 추출하여 PCR로 증폭한 후 RFLP 방법으로 변이형을 구분하였다. 결과: 황체형성 호르몬 exon 2의 변이형이 다낭성 난포증후군 환자에서 28.6%로 이미 조사된 바 있는 대조군의 16.7% 보다 약간 높게 나타났으나 통계적으로 유의한 차이는 없었다(p>0.05). 결론: 황체형성 호르몬 exon 2의 변이가 한국인의 다낭성 난포증후군 발병과 관련이 있는지를 밝히기 위해 더 많은 개체에 대한 연구가 요구된다.
김남근,이유진,남윤성,이상희,정기화,고정재,이숙환,차광열,Kim, Nam-Keun,Lee, Eu-Gene,Nam, Yoon-Sung,Lee, Sang-Hee,Chung, Ki-Wha,Ko, Jung-Jae,Lee, Sook-Hwan,Cha, Kwang-Yul The Korean Society for Reproductive Medicine 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.1
연구목적: 본 연구는 자궁내막증과 무월경 불임환자들을 대상으로 $LH{\beta}$ exon 2 유전자의 돌연변이를 탐색하고자 시도하였다. 연구재료 및 방법: 그 대상으로 22명의 자궁내막증 환자와 12명의 무월경 환자 그리고, 54명의 건강한 비임신 여성을 대조군으로 사용하였다. 이들을 대상으로 한 돌연변이 탐색은 PCR-RFLP(polymerase chain reaction-restriction fragment polymorphism) 방법으로 수행되었다. 결과: 그 결과 자궁내막증과 무월경증 환자에서 그 변이의 비율이 각각 18.2%, 16.7% 그리고, 대조군에서 역시 16.7%의 빈도를 나타냈다. 결론: 따라서, 자궁내막증과 무월경증 환자는 $LH{\beta}$ exon 2 돌연변이와는 서로 관련이 없거나 매우 적음을 알 수 있었다.
동결 보존된 고환 정자로 ICSI 시술 후 수정된 수정란의 이식에 의한 임신 1례
이우식,김종식,김현규,김영찬,박찬,김시영,고정재,차광열,Lee, Woo-Sik,Kim, Jong-Sik,Kim, Hyun-Kyoo,Kim, Young-Chan,Park, Chan,Kim, Si-Young,Ko, Jung-Jae,Cha, Kwang-Yul 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1
This case report describes the pregnancy following the transfer of embryos generated from intracytoplasmic sperm injection (ICSI) using frozen-thawed sperms obtained by testicular sperm extraction (TESE) in patient with hypoplasia of vas deferens.