Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

송견지,석경호,김재홍,김종헌,송승은,박하나,Zhong-Yin Zhang 한국뇌신경과학회 2016 Experimental Neurobiology Vol.25 No.5

Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases.

생쥐 초기배아의 Glucose Transporter유전자 발현 양상에 관한 연구

염혜원,변혜경,송견지,김해권,이호준,Youm, Hye-Won,Byun, Hye-Kyung,Song, Gyun-Ji,Kim, Hae-Kwon,Lee, Ho-Joon 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.1

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

한국 남성 불임환자에서 Y 염색체상의 AZF Gene에 대한 분석 및 DAZ Gene의 발현 양상

이호준,이형송,송견지,변혜경,서주태,김종현,이유식,Lee, Ho-Joon,Lee, Hyoung-Song,Song, Gyun-Jee,Byun, Hye-Kyung,Seo, Ju-Tae,Kim, Jong-Hyun,Lee, You-Sik 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.1

Cytogenetic observations of loss of the distal portion of the Y chromosome long arm were found to be associated with disrupted spermatogenesis. The existence of a gene involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was postulated. In this study, we screened the AZF region including DAZ and DAZH genes and observed the expression pattern of DAZ and DAZH transcript in infertile men with azoospermia and oligospermia by using a sequence-tagged site (STS)-based PCR method. PCR primers were synthesized for 11 STSs that span Yq interval 6, SRY, DAZ, and DAZH, functional DAZ homologue on chromosome 3. Microdeletions were detected in 4/32 (12.5%) azoospermic men and 1/11 (9%) severe oligospermic men. Only 2 of 5 patients had microdeletions of Yq that contained the DAZ gene, whereas the other 3 patients had deletions extending from intervals 5L-6F proximal to the DAZ gene on Yq. Testis biopsies of the azoospermic patients revealed a variety from Sertoli cell-only syndrome to testicular maturation arrest. Of 4 men with clinical data available, average testis size was R: 13.8 cc, L: 13.8 cc, serum T was $4.0{\pm}1.25$ ng/ml, LH was $3.63{\pm}1.90$ mIU/ml, and FSH was $8.85{\pm}5.13$ mIU/ml. These values did not differ significantly from the remainder of the patients tested. We could not observed the DAZ transcript in 2 patients, who have no mature spermatozoa. In 11.6% of patients microdeletions of the AZF could be detected. These deletions in the AZF region seem to be involved causing spermatogenic failure. But the frequency of microdeletions proximal to DAZ suggests that DAZ is not the only gene associated with spermatogenic failure.

Analysis of Gene Mutation and Expression Level of Follicle Stimulating Hormone Receptor in Premature Ovarian Failure(POE) Patients

김정욱,염혜원,이형송,송견지,천강우,박용석,김계현 한국발생생물학회 2000 발생과 생식 Vol.4 No.1

본 연구에서는 조기 난소 부전증 환자를 대상으로 난포 자극 호르몬 수용체 유전자의 돌연변이와 발현 양상을 분석하였다. 돌연변이 분석을 위해 환자의 말초혈액에서 genomic DNA를 분리하고 nucleotide 566을 포함하고 있는 exon 7에 특이적인 primer쌍을 이용하여 중합효소 연쇄 반응을 시행하였다. 전기 영동으로 반응산물을 확인한 다음, 돌연변이 여부를 조사하기 위하여 제한효소 절단분석 (Restriction Fragment Length This study was investigated to analyze the inactivating point mutation and expression level of follicle-stimulating hormone(FSH) receptor mRNA. In first experiment, we analyzed the point mutation. Peripheral blood was collected from each patient. To screen individuals for the C566T mutation, PCR was performed for exon 7 of the FSH receptor gene in 10 patients. No inactivating point mutation of FSH receptor gene was identified in women with premature ovarian failure. To analyze the expression level of FSH receptor, mRNA expressions were examined by RT-PCR method using specific primers for the FSH receptor. The amount of FSH receptor mRNA expressed in POF patients was lower than that in the control group. But it was not significantly different. These finding suggests that lower expression of FSH receptor in premature ovarian failure patients might be the cause of the low response to the gonadotropin during the hyperstimulation in IVF-ET cycles.

동결보존이 생쥐 난소 조직 내 Heat Shock Protein 90의 발현에 미치는 영향

이선희,박용석,염혜원,송견지,한상철,배인하,Lee, Sun-Hee,Park, Yong-Seog,Yeum, Hye-Won,Song, Gyun-Jee,Han, Sang-Chul,Bae, In-Ha 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.1

Objective : Heat shock protein family is related to protective mechanism of cells by environmental changes. This study was performed to evaluate the effect of cryopreservation on the heat shock protein 90 (Hsp90) expression in mouse ovarian tissue. Methods : Cryopreservation of mouse ovarian tissue was carried out by slow freezing method. The mRNA level of Hsp90 expression in both fresh and cryopreserved mouse ovarian tissue was analyzed by RT-PCR. The protein expression of Hsp90 was evaluated by Western blot analysis and immunohistochemistry. Results: The mRNA and protein of Hsp90 were expressed in both fresh and cryopreserved mouse ovarian tissue. The amount of Hsp90 mRNA was increased in cryopreserved ovarian tissue after 60 and 90 minutes after thawing and incubation. The amount of Hsp90 protein was increased in the cryopreserved ovarian tissue after 6 hours of the incubation in Western blot analysis. In immunohistochemical study, Hsp90 protein was localized in cytoplasm of oocytes and granulosa cells. Significant level of immunoreactive Hsp90 protein was detected in theca cells contrast to the weak expression in ovarian epithelial cells. Conclusion: This results showed the increase of Hsp90 expression in both mRNA and protein level in the cryopreserved mouse ovarian tissue. It can be suggested that Hsp90 may play a role in the protective or recovery mechanism against the cell damage during cryopreservaion.

균형 전좌 또는 Robertsonian 전좌 보인자의 체외수정 및 배아이식술에서 형광직접보합법을 이용한 착상전 유전자진단의 임상적 적용

전진현(Jin Hyun Jun),송견지(Gyun Jee Song),김정욱(Jeong Wook Kim),박소연(So Yeon Park),김계현(Kye Hyun Kim),최범채(Bum Chae Choi),궁미경(Mi Kyoung Koong),강인수(Inn Soo Kang),임천규(Chun Kyu Lim),한미현(Mi Hyun Han) 대한산부인과학회 2000 Obstetrics & Gynecology Science Vol.43 No.7

목적 : 본 연구는 균형 전좌 또는 Robertsonian 전좌 보인자의 체외수정 및 배아이식술에서 형광직접보합법을 이용한 착상전 유전자진단을 시행하여 그 결과와 효율을 알아보고자 시행하였다. 연구방법 : 본원에서 착상전 유전자진단을 시행한 15쌍, 25주기의 체외수정 및 배아이식술을 연구대상으로 하였으며, 3주기에서 54개의 극체를 이용하여, 22주기에서 234개의 할구를 이용하여 형광직접보합법으로 염색체의 숫적 이상을 분석하였다. 형광직접보합법을 이용하여 분석한 후 정상의 형광직접보합법 signal을 나타내는 배아만을 모체에 이식하였다. 임신이 확인된 경우 양수천자의 방법으로 태아의 핵형을 확인하였다. 결과 : 극체를 이용한 형광직접보합법 분석에서, 18개의 극체가 정상이었고, 3주기 모두에서 배아를 이식하였으며, 형광직접보합법의 효율은 95.0%였다. 할구를 이용한 형광직접보합법 분석에서는 49개의 배아가 정상으로 확인되었다. 정상의 배아를 확인할 수 없었던 1주기를 제외한 21주기에서 배아이식을 시행하였으며, 형광직접보합법의 효율은 92.7%였다. 3주기에서 임신이 되었고, 2주기에서 건강한 균형 전좌 보인자인 남아와 Robertsonian 전좌 보인자인 여아가 태어났다. 1주기는 임신이 진행중이며, 양수천자에서 정상의 핵형으로 확인되었다. 결론 : 형광직접보합법을 이용한 착상전 유전자진단은 균형 전좌 또는 Robertsonian 전좌 보인자의 체외수정 및 배아이식술에 성공적으로 적용되어 태아의 염색체 이상으로 인하여 발생되는 여러가지 문제를 해결할 수 있는 효과적인 방법으로 사료된다. Objective : This study was performed to evaluate the efficiency of preimplantation genetic diagnosis (PGD) using fluorescence in-situ hybridization (FISH) in Robertsonian or balanced reciprocal translocation carriers in human IVF-ET programm.Method : FISH was carried out in 25 cycles of 15 couples. Two-color FISH analysis was performed on 54 polar bodies in 3 cycles and 234 blastomeres in 22 cycles. After FISH analysis, the embryos with normal FISH signals were transferred into mother's uterus.Results : In FISH analysis of polar bodies, 18 nuclei of polar bodies were normal and 12 embryos were transferred in 3 cycles. FISH efficiency per oocyte was 95.0% in cases using polar bodies. In FISH analysis of blastomeres, 49 embryos were normal and transferred in 21 cycles. FISH efficiency per embryo was 92.7% using blastomeres. At present, three pregnancies were achieved. A girl and a boy were delivered. Both of them were translocation carriers. The other conceptus showed normal karyotype.Conclusion : According to this study, PGD using FISH can be successfully applied for the patients with translocations of chromosomes.

일반연제 발표 : 생식내분비 2 ; 염색체 전좌 및 역위를 가진 습관성 유산 환자에서 착상전 유전진단의 결과

김진영,임천규,민동미,양광문,한국선,송견지,박용석,송지홍,궁미경,강인수 대한산부인과학회 2001 대한산부인과학회 학술대회 Vol.87 No.-

일반연제 발표 : 산과학 2, 유전학 ; 염색체전좌(chromosomal translocation)를 가진 습관성유산 환자에 있어서 telomere probe를 이용한 착상전 유전 진단의 임상적 적용

강인수,김계현,한미현,임천규,박소연,김영미,김진우,송견지,전진현,김정욱,송지홍,송인옥,유근재,백은찬,최범채,궁미경,손일표,전종영 대한산부인과학회 1999 대한산부인과학회 학술대회 Vol.84 No.-