• Clinical and Experimental Reproductive Medicine
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• 제45권3호
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• pp.143-148
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• 2018

Objective: The favored method of preserving fertility in young female cancer survivors is cryopreservation and autotransplantation of ovarian tissue. Reducing hypoxia until angiogenesis takes place is essential for the survival of transplanted ovarian tissue. The aim of this study was to investigate the role of angiopoietin-1 (Angpt-1), angiopoietin-2 (Angpt-2), and vascular endothelial growth factor (VEGF) in ovarian tissue grafts that were cryopreserved using two methods. Methods: Ovarian tissues harvested from ICR mice were divided into three groups: group I (control), no cryopreservation; group II, vitrification in EFS (ethylene-glycol, ficoll, and sucrose solution)-40; and group III, slow freezing in dimethyl sulfoxide. We extracted mRNA for VEGF, Angpt-1, and Angpt-2 from ovarian tissue 1 week following cryopreservation and again 2 weeks after autotransplantation. We used reverse transcriptase-polymerase chain reaction to quantify the levels of VEGF, Angpt-1, and Angpt-2 in the tissue. Results: Angpt-1 and Angpt-2 expression decreased after cryopreservation in groups II and III. After autotransplantation, Angpt-1 and Angpt-2 expression in ovarian tissue showed different trends. Angpt-1 expression in groups II and III was lower than in group I, but Angpt-2 in groups II and III showed no significant difference from group I. The vitrified ovarian tissues had higher expression of VEGF and Angpt-2 than the slow-frozen ovarian tissues, but the difference was not statistically significant. Conclusion: Our results indicate that Angpt-2 may play an important role in ovarian tissue transplantation after cryopreservation although further studies are needed to understand its exact function.

• Clinical and Experimental Reproductive Medicine
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• 제28권3호
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• pp.183-190
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• 2001

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.

• Journal of Periodontal and Implant Science
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• 제28권4호
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• pp.659-678
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• 1998

본 연구에서는 한국인 성인성 치주염의 관련세균 중에서 구강 스피로헤타의 분포를 조사하기 위하여 배양하지 않고도 구강 스피로헤타를 분리할 수 있는 16S rRNA에 의거한 올리고뉴클레오타이드 소식자를 사용하였다. 성인성 치주염 환자 29명을 대상으로 한 사람당 6mm 이상의 탐침 깊이를 보이는 부위 4곳(실험군)과 3mm 이하의 탐침 깊이를 보이는 건강한 부위 1곳(대조 1군), 건강한 치주조직을 가진 학생 20명을 대상으로 한 사람당 5부위로부터 치은연하 치태를 채취한 뒤(대조 2군) 중합효소연쇄반응과 점상블롯보합결합법을 시행하였다. 소식자로는 구강 스피로헤타의보편 소식자 및 현재 배양이 되는 구강 스피로헤타중에서 T. denticola, T. pectinovorum, T. socranskii, T. vincentii, T. maltophilum에 대한 종 특이 소식자 TDEN, TPEC, TSOC, TVIN, TMAL과 현재 배양이 되지않은 구강스피로헤타중에서 I-VII군에 대한 군 특이 소식자 TRE I-TRE VII을 이용하여 다음과 같은 결론을 얻었다. 1. 위상차 현미경으로 본 결과는 실험군, 대조 1군에서 각기 91.37%, 14.28%의 구강스피로헤타가 관찰되었으며 대조 2군에서는 관찰되지 않았다. 2. 보편 소식자를 사용한 경우는 실험군, 대조 1군, 대조 2군에서 각기 98.27%, 46.42%, 22.0%의 구강 스피로헤타가 관찰되었다. 3. 특이 소식자를 사용한 경우는 실험군, 대조 1군, 대조 2군에서 각기 95.68%, 35.71%, 19.0%의 구강 스피로헤타가 관찰되었다. 4. 종 특이 소식자를 사용한 경우는 T. socranskii가 가장 많이 관찰되었으며 (81.89%), 그다음이 T. maltophilum(50.0%), T. vincentii(36.20%), T. denticola(13.79%)순이었고, 군 특이 소식자를 사용한 경우는 TREIV(85.34%), TRE II(77.58%), TREI(56.89%), TRE III(25.86%), TREVI(5.17%), TRE V(2.58%) 순이었다. 5. T. vincentii는 치주염에 이환된 부위에서만 관찰되었고, 건강한 부위에서는 관찰되지 않았다. 6. T. pectinovorum과 VII군에 속하는 구강스피로헤타는 어느 표본에서도 관찰되지 않았다. 이상의 결과에서 16S rRNA에 의거한 올리고뉴클레오타이드 소식자로 구강 스피로헤타의 성인성 치주염과의 연관성과 분리되지 않은 구강 스피로헤타를 확인하였으며, 인종 및 치주염의 형태에 따른 구강 스피로헤타의 분포 차이, T. vincentii의 병원성, 치료 전후의 구강 스피로헤타의 분포 변화등의 보다 세분화된 연구가 필요하다고 생각된다.

• Advances in pediatric surgery
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• 제15권1호
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• pp.1-10
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• 2009

최근에 양수에 존재하는 세포들은 다양한 세포분화의 능력을 가지고 있으며 세포 치료와 조직공학의 세포원으로서의 가능성이 높다는 연구 결과가 많이 보고되고 있다. 그러나 양수 내에 어떤 종류의 세포가 정상적으로 존재하는 지에 대해서는 제한적으로 알려져 있다. 본 연구에서는 사람의 양수 내에 호흡기세포를 분리 배양하는 것을 목표로 하였다. 임신 17주에서 20주 사이의 산모 10명에서 각각 5 mL의 양수를 획득하여 small airway growth medium (SAGM) 배양액에서 계대 배양을 시행하였으며 type II alveolar cells이 존재하는 지에 대하여 면역형광염색 및 RT-PCR을 시행하였다. 배양된 세포는 광학현미경 상 상피세포와 유사한 다면체의 모양이었으며 계대 배양 시에 변화 없이 동일한 형체를 보였다. 배양된 세포는 면역형광염색 상 type II alveolar cell의 특이표지자인 surfactant protein C (SPC) 및 TTF-1 protein에 대해서는 양성이었으나 CD 31 및 vimentin에 대해서는 음성이었으며, RT-PCR 상 SPC mRNA 가 발현되었다. 이상의 결과로 사람의 양수를 특이 배양액에 배양하면 호흡기세포를 분리, 확인할 수 있음을 알았으며 이러한 결과가 향후 양수세포의 추가적인 연구에 활용될 수 있다고 생각된다.

• 한국균학회지
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• 제26권4호통권87호
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• pp.466-477
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• 1998

진흙버섯류 22개 균주를 균총의 형태와 PCR 기법을 사용하여 종간의 구분 방법을 찾고자 하였다. PDA등 4가지 배지에서 균사생장 및 배지의 변색여부 등을 기준으로 특성을 구분할 때 목질진흙버섯의 균총 색깔은 진한 황색으로 균사생장이 늦고 배지를 푸르게 변색시켰다. rDNA 분석 결과 $ITSI{\sim}II$ 부위는 목질진흙버섯이 약 800 bp, 말똥진흙버섯은 약 700 bp였고, IGRI 부위는 목질진흙버섯은 약 700 bp, 말똥진흙버섯은 균주에 따라 약 500, 600, 700, 800 bp에서 4가지 각기 다른 밴드를 보였다. $ITSI{\sim}II$와 IGRI부위의 증폭된 DNA를 6개의 제한효소로 절단하여 다형성을 비교해 본 결과 $ITSI{\sim}II$의 HaeIII 절단으로 목질진흙버섯과 말똥진흙버섯을 구분할 수 있었으며 이들 밴드를 이용하여 유연관계를 조사한 결과 목질진흙버섯은 95%의 유사도를 보였으며, 말똥진흙버섯은 89%의 유사도로 complex를 형성하였다. 목질 진흙버섯은 RAPD 분석과 AP-PCR에 의한 밴드양상으로도 확실한 구분이 가능하였으며, $ITSI{\sim}II$ 부위의 HaeIII 제한효소 처리로 나타난 벤드는 이종의 특이적인 marker로 사용할 수 있을 것으로 본다.

• Tuberculosis and Respiratory Diseases
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• 제82권2호
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• pp.133-142
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• 2019

Background: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. Methods: Mouse AE II cell line MLE-12 were exposed to $1-10{\mu}g/mL$ BLM and $0.01-100{\mu}M$ baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. Results: BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor ${\alpha}$, and transforming growth factor ${\beta}1$. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. Conclusion: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

• 미생물학회지
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• 제28권1호
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• pp.19-26
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• 1990

To examine the effects of sequence variations near the transcriptional start site on the rate of formation of the open complexes at bacteriophage $\lambda P_{R}$ promoter, two mutant promoters were created by site-specific mutagenesis using synthetic oligonucleotides. Mutant I coatains changes at positions -3 and -4 from TT to CC, thus having a 6-bp long G/C stretch between -10 region and transciptional start site (+1). Mutant II has changes at positions -5 and -6 from GG to AA, thereby having a 9-bp long A/T stretch between positions -11 and -3. Selective filter binding assays were performed to measure the rate of formation of the open complexes between the wild-type or two mutant $P_{R}$ promoters on 664 bp fragments and E. coli RNA polymerase at two temperatures. At 37.deg.C, the wild-type and two mutants showed similar rates for the formation of open complex. The second order rate constant $k_{a}$ and $\tau _{int}$, as determined from the .tau.-plot analysis, were $(6.0\pm0.4)\times10^{6}M^{-1}sec^{-1}$ and $11\pm5$sec, respectively. At 18.deg.C, however, the wild-type and two mutant promoters showed differences in the kinetic parameters. k for the wild-type promoter was (2.2$\pm$0.1)\times 10^{6}M^{-1}sec^{-1}$ and $\tau _{int}$ was 76$\pm$sec. Mutant I and II exhibited differences mainly in the rate of isomerization ($\tau_{int,I}=91\pm$10 sec, int,II=34$\pm$ sec), whereas the second order rate constant $k_{a}$ was similar to the wild type value. This result implies that at $18^{\circ}C$, the isomerization rate is determined by both protein conformational change and DNA melting, which are separable kinetically according to the 3-step mechanism of Roe et al.(1984,1985), and that the base changes affected mainly the rate of DNA melting as predicted.lting as predicted.

• 대한의생명과학회지
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• 제17권3호
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• Molecules and Cells
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• 제44권9호
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• pp.658-669
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• 2021

Enhancers have been conventionally perceived as cis-acting elements that provide binding sites for trans-acting factors. However, recent studies have shown that enhancers are transcribed and that these transcripts, called enhancer RNAs (eRNAs), have a regulatory function. Here, we identified putative eRNAs by profiling and determining the overlap between noncoding RNA expression loci and eRNA-associated histone marks such as H3K27ac and H3K4me1 in hepatocellular carcinoma (HCC) cell lines. Of the 132 HCC-derived noncoding RNAs, 74 overlapped with the eRNA loci defined by the FANTOM consortium, and 65 were located in the proximal regions of genes differentially expressed between normal and tumor tissues in TCGA dataset. Interestingly, knockdown of two selected putative eRNAs, THUMPD3-AS1 and LINC01572, led to downregulation of their target mRNAs and to a reduction in the proliferation and migration of HCC cells. Additionally, the expression of these two noncoding RNAs and target mRNAs was elevated in tumor samples in the TCGA dataset, and high expression was associated with poor survival of patients. Collectively, our study suggests that noncoding RNAs such as THUMPD3-AS1 and LINC01572 (i.e., putative eRNAs) can promote the transcription of genes involved in cell proliferation and differentiation and that the dysregulation of these noncoding RNAs can cause cancers such as HCC.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2006년도 Proceedings of The Convention
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• pp.103-115
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• 2006

$\beta$-Glucan is a glucose polymer that has linkage of $\beta$-(1,3), -(1,4) and -(1,6). As exclusively found in fungal and bacterial cell wall, not in animal, $\beta$-glucans are recognized by innate immune system. Dendritic cells (DC) or macrophages possesses pattern recognition molecule (PRM) for binding $\beta$-glucan as pathogen-associated molecular pattern (PAMP). Recently $\beta$-glucan receptor was cloned from DC and named as dectin-l which belongs to type II C-type lectin family. Human dectin-1 is consisted of 7 exons and 6 introns. The polypeptide of dectin-1 has 247 amino acids and has cytoplasmic, transmembrane, stalk and carbohydrate recognition domains. Dectin-1 could recognize variety of beta-1,3 and/or beta-1,6 glucan linkages, but not alpha-glucans. In our macrophage cell line culture system, dectin-1 mRNA was detected in RA W264.7 cells by reverse transcription-polymerase chain reaction (RT-PCR). Dectin-1 was also detected in the murine organs of spleen, thymus, lung and intestines. Treatment of RA W264.7 cells with $\beta$-glucans of Ganoderma lucidum (GLG) resulted in increased expression of IL-6 and TNF-$\alpha$ in the presence of LPS. However, GLG alone did not increase IL-6 nor TNF-$\alpha$. These results suggest that receptor dectin-1 cooperate with CD14 to activate signal transduction that is very critical in immunoresponse.