알츠하이머 형질전환 마우스의 출생 후 뇌에서 유전자 발현

지승완(Seung Wan Jee),송연숙(Youn Suk Song),오재호(Jae Ho Oh),김용규(Yong Kyu Kim),심선보(Sun Bo Shim),황대연(Dae Youn Hwang),이수해(Su Hae Lee),서수진(Su Jin Seo),조준용(Jun Yong Cho),조정식(Jung Sik Cho) 한국실험동물학회 2005 Laboratory Animal Research Vol.21 No.3

DNA microarrays are a well-established technology for measuring gene expression levels. The aim in the study was to gain insight into potential over-expressed effect of amyloid precursor protein sw (APPsw) on modulation of genes for Alzheimer's Disease (AD). APPsw-transgenic mice, which has been produced by us previously, provide an important resource for identifying differentially expressed genes, since this transgenic line was shown cognitive deficits along with Aβ-42 depositions at 12 months of age. Using cDNA microarray, we compared mRNA levels in 7.4K cDNA clones from postnatal brain of age-matched wild-type mice and Alzheimer's diseased transgenic mice. A total of 44 differentially expressed genes with a 15 up-regulated and with a 29 down-regulated were found in brains from moderatively transgenic mice compared to non-transgenic littermates. Thus, the results allow to the future studies in the functions of each gene, which may be targeted for developing drugs.

γ-Secretase 활성억제단백질인 TMP21의 과발현이 신경세포주에서 NGF 수용체 신호전달과 정에 미치는 영향

Sun Il Choi(최선일),Seung Wan Jee(지승완),Youn Kyung Her(허윤경),Ji Eun Kim(김지은),So Hee Nam(남소희),In Sik Hwang(황인식),Hye Ryun Lee(이혜련),Jun Seo Goo(구준서),Young Ju Lee(이영주),Eon Pil Lee(이언필),Hae Wook Choi(최해욱),Hong 한국생명과학회 2011 생명과학회지 Vol.21 No.8

TMP21은 AD의 원인으로 작용하는 Aβ-42 펩타이드 생성에 중요한 γ-secretase 활성을 억제하는 p24 family에 속하는 type I 막 단백질이다. 본 연구에서는 TMP21이 세포의 성장과 분화에 중요한 NGF 수용체 신호전달과정에 미치는 영향을 분석하고자 인간의 TMP21 cDNA를 합성하고, CMV promoter 조절 하에 hTMP21를 클로닝하여, CMV/hTMP21 벡터를 제조하였다. 그리고 이들 벡터를 B35 neuroblastoma에서 과발현시킨 후 γ-secretase 구성단백질과 NGF 수용체 연관 단백질의 변화를 관찰하였다. 그 결과, 4종류의 γ-secretase 구성단백질의 발현은 vehicle transfectants보다 CMV/hTMP21 transfectants에서 유의적으로 감소하였다. 또한 NGF low affinity 수용체인 p75<SUP>NTR</SUP>과 downstream 단백질인 RhoA의 양은 NGF를 처리하지 않은 TMP21 transfectants에서 유의적으로 증가하였으나 NGF 처리에 의해 감소되었다. High affinity NGF 수용체인 TrkA의 인산화도 NGF 처리가 없는 경우 유의적으로 감소하였으나 NGF 처리에 의해 증가되었다. 또한 downstream 신호전달 과정 중에서 ERK의 인산화는 TrkA와 유사한 발현변화를 나타내었으나 Akt 인산화는 NGF의 처리에 의해 더욱 증가하였다. 이러한 결과는 TMP21이 neuroblastoma에서 NGF 수용체 신호전달과정를 조절하는 중요한 단백질로서 작용함을 제시하며, AD의 작용기전 연구에 중요한 기초자료를 제공할 것으로 사료된다. Transmembrane protein 21 (TMP21) is a member of the p24 cargo protein family and has been shown to modulate γ-secretase-mediated Aβ production which was specifically observed in the brains of subjects with Alzheimer’s disease (AD). In order to investigate whether TMP21 could affect nerve growth factor (NGF) receptor signaling pathway, the alteration of NGF receptors and their downstream proteins were detected in TMP21 over-expressed cells. CMV/hTMP21 vector used in this study was successfully expressed into TMP21 proteins in B35 cells after lipofectamin transfection. Expressed TMP21 proteins induced the down-regulation of γ-secretase complex components including Presenlin-1 (PS-1), PS-2, Nicastrin (NST), Pen-2 and APH-1. Also, the expression level of NGF receptor p75<SUP>NTR</SUP> and RhoA were significantly higher in CMV/hTMP21 transfectants than vehicle transfectants, while their levels returned to vehicle levels after NGF treatment. However, the phosphorylation of NGF receptor TrkA was dramtically decreased in NGF No-treated CMV/hTMP21 transfectants compared with vehicle transfectants, and increased in NGF treated CMV/hTMP21 transfectants. In TrkA downstream signaling pathway, the phosphorylation level of ERK was also decreased in CMV/hTMP21 transfectants, while the phosphorylation of Akt was increased in the same transfectants. Furthermore, NGF treatment induced the increase of phosphorylation level of Akt and ERK in CMV/hTMP21 transfectants. Therefore, these results suggested that over-expression of TMP21 may simultaneously induce the up-regulation of p75<SUP>NTR</SUP>/RhoA expression and the down-regulation of TrkA/ERK phosphorylation through the inhibition of γ-secretase activity.

마우스 피부암 발생과정에 있어서 2,3,7,8-Tetrachlorodibenzo-p-Dioxin(TCDD)처리에 의한 유전자발현 변화 연구

염태경(Tai Kyung Ryeom),김옥희(Ok Hee Kim),강미경(Mi Kyung Kang),박미선(Misun Park),지승완(Seung Wan Jee),엄미옥(Mi Ok Eom),강호일(Hoil Kang) 한국환경성돌연변이발암원학회 2005 한국환경성돌연변이·발암원학회지 Vol.25 No.1

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) displays high toxicity in animals and has been implicated in human carcinogenesis. Although the mechanism of carcinogenesis by TCDD is unclear, it is considered to be a non-genotoxic compound and tumor promoter. In our experiment, we investigated the effects of TCDD on gene expression in mouse skin carcinogenesis. We used cDNA microarray to detect the differential gene expression in tumors induced in hairless mouse skin by MNNG plus TCDD protocol. We found that erb-2, c-ets2 and p27^(kip1) were significantly up-regulated, but TNFR2, AKT-1, integrin β1, maspin, IGF-1, c-raf-1, Rb were significantly down-regulated, in tumor region, respectively. We also found that the expression of 53 genes involved in cell cycle, signal transduction, apoptosis, adhesion molecule, angiogenesis, and invasion, were changed two fold more, in tumor surrounding region, These data suggest that TCDD alters the expression of a large array of genes involved in apoptosis, cytokine production and angiogenesis in mouse skin carcinogenesis.

사람 백혈구 및 위 조직중의 8-Hydroxy-2'-Deoxyguanosine 측정에 관한 연구

강호일(Hoil Kang),엄미옥(Mi Ok Eom),박미선(Misun Park),염태경(Tai Kyung Ryeom),지승완(Seung Wan Jee),전해명(Hea Myung Jeon),김옥희(Ok Hee Kim) 한국환경성돌연변이발암원학회 2005 한국환경성돌연변이·발암원학회지 Vol.25 No.1

In the present study, we have measured 8-hydroxy-2'-deoxyguanosine in DNA of stomach cancers, adjacent stomach cancer tissues, normal stomach tissues and peripheral blood leukocytes of the same stomach cancer patients (n=48) to investigate their etiological association with gastric cancer and possibility whether peripheral blood leukocytes can use surrogate marker for early stomach cancer diagnosis by HPLC/ECD system. In normal stomach tissues, we found that 8-hydroxy-2'-deoxyguanosine levels in tissues infected with Helicobacter pylori were 1.4 fold higher than those in tissues without infected with Helicobacter pylori. However, in adjacent stomach cancer tissues, we found that 8-hydroxy-2'-deoxyguanosine levels in tissues infected with Helicobacter pylori were 1.5 fold lower than those in tissues without infected with Helicobacter pylori. In stomach cancer tissues, 8-hydroxy-2'-deoxyguanosine levels in tissues infected with Helicobacter pylori were not significantly different from those in tissues without infected with Helicobacter pylori. In Helicobacter pylori-negative specimens, 8-hydroxy-2'-deoxyguanosine levels of adjacent stomach cancer tissues were found to be significantly higher than those of normal stomach and cancer tissues. The 8-hydroxy-2'-deoxygnanosine levels of female were 1.7 fold higher than those of male in peripheral blood leukocytes of the same stomach cancer patients. The 8-hydroxy-2'-deoxyguanosine levels in Helicobacter pylori-negative specimens among adjacent stomach cancer tissues were found to be reversely correlated with those in peripheral blood leukocytes, suggesting that 8-hydroxy-2'-deoxyguanosine in peripheral blood leukocytes may not use as surrogate marker for the early diagnosis of human stomach cancer.

pT7T7을 이용한 인간 세포에서의 외래성 유전자 발현

지승완,유병제 大邱大學校附設 基礎科學硏究所 1998 基礎科學硏究 Vol.14 No.2

The viral cell culture is essential for the research of hepatitis C virus, but the cell culture of HCV has been not established recently. Since the cell culture by the RNA transfection has the problems in the quantifiction of transfected RNA, the cell culture by the DNA transfection has been tried. The expression of CAT as reporting gene by pT7T7 transfection in Huh7 cells was performed as the preexperiment of the HCV cell culture by the DNA transfection. As increasing of the concentration of target gene DNA, the expression of CAT was increased. The CAT was more expressed in the cells transfected with EMCV-CAT than in the cells with HCV-CAT. The difference between EMCV and HCV was resulted from the ability of enhancement of translation in untranslational region used for regulatory site of virus.

C형 간염 바이러스의 세포배양에 관한 연구

지승완,유병제 大邱大學校附設 基礎科學硏究所 1998 基礎科學硏究 Vol.15 No.1

The cells transfected with defective HCV RNAs alone, were harvested at various days after transfection. The replication of various defective HCV RNAs was checked by the quantification of HCV RNAs in transfected cell. The amount of HCV RNAs were estimated by the chiron bDNA kit. All the case, the amounts of HCV RNAs in transfected cells were decreased.

Anti-sense RNA에 의한 C형 간염 바이러스의 번역의 억제

지승완,유병제 大邱大學校附設 基礎科學硏究所 1998 基礎科學硏究 Vol.14 No.2

The gene therpys have been investigated as the new therpy of the desease, the inhibition of the specific gene expressin by anti-sense RNA is an one method of the them. The inhibition of viral translation or viral replication by anti-sense RNA causes the viral proliferation. Since hepatitis C virus is RNA virus, HCV may be the target virus of anti-sense RNA as anti-viral reagent. The inhibition of the translation of the defective virus having CAT as the reporting gene by anti-sense RNA was performed. The inhibition of CAT translation did not occure in cells pretransfected with anti-sense RNA, the inhibition of CAT tranalation occured in cells co-transfected with anti-sense RNA. It seems that the difference between pretransfection and co-transfection was resulted from the cellular delivery system of anti-sense.

긴 cDNA를 이용한 C형 간염 바이러스의 검색

지승완,유병제 大邱大學校附設 基礎科學硏究所 1998 基礎科學硏究 Vol.14 No.2

Generally, the detection of virus was performed at the level of viral proteins and viral genome. The detection of virus by the viral genome more directly identifies the virus and estimates the concentration of virus. Because the genome of hepatitis C virus is RNA, the detection of the HCV genome was done by RT-PCR. But, PCR has the problems in contamination and false positive result. To deduce the upper problems, we developed the RT-PCR of the two ends of the HCV genome by long range cDNA. It seems that this method deduce the problems of contamination and false positive result, identify the full length genome of HCV and estimate the accurate concentration of HCV, and the fidelity and sensitivity of this method are good.

C형 간염 바이러스의 복제에 관한 연구

지승완,유병제 大邱大學校附設 基礎科學硏究所 1998 基礎科學硏究 Vol.15 No.1

Hepatitis C virus (HCV) is the major causative agent of posttransfusion and sporadic non-A, non-B hepatitis. To time, many research for HCV have been performed, but the molecular mechanism of HCV replication is still not obvious. To define the essential region of HCV genome for replication and develop the RNA-dependent RNA polymerase(RdRp) of HCV assay system in vivo. I constructed defective HCV cDNA that contained 5'Untranslate reagion,(UTR), Core(C), Enveloper 1(4 amino adds), Nonstructure(NS)5b, and various 3'UTR.

인간 간세포에서 C형 간염 바이러스의 replicase발현

지승완,유병제 대구대학교 기초과학연구소 1998 基礎科學硏究 Vol.15 No.1

To analyze the RdRp in eukaryotic HuH7 cells transfected defective HCV RNAs, cotransfection of defective HCV RNAs and defective HCV-CAT negative RNAs that had the essential portion of HCV for replication and translation, and CAT as reporting gene was performed. In cells transfected with defective HCV RNA and negative HCV-CAT RNA, the amount of CAT was quantified by Chloramphenicol acetyl transferase(CAT) enzyme-linked immunosorbent assay(ELISA). The NS5b of HCV was expressed from defective HCV 14H RNA and had a activity of RdRp. And, the defective HCV RNA and defective negative HCV-CAT RNA were replicated without other viral factors.