포스타 : 부인종양학 ; 자궁경부 이형증에서 Polyphenol E의 항암화학(Chemoprevention)효과

신봉영,조성대,안웅식,한구택,이준모,남궁성은,이헌영,김승조 대한산부인과학회 1998 대한산부인과학회 학술대회 Vol.82 No.-

자궁경부암 유전자 치료를 위한 재조합 p53 아데노 연관 바이러스 플라스미드 ( pAAVCMVp53 )의 제조

신봉영(Bong Young Shin),한유진(You Jin Han),조규남(Kyou Nam Cho),안웅식(Woong Shick Ahn),김진우(Jin Woo Kim),이준모(Jun Mo Lee),남궁성은(Sung Eun Namkoong),김수평(Soo Pyung Kim),이헌영(Hun Young Lee),김승조(Seung Jo Kim),김종국(Chong 대한산부인과학회 1998 Obstetrics & Gynecology Science Vol.41 No.11

N/A Objective: Human papillomavirus (HPV) has been identified in the majority of invasive cervical cancer patient and has been found to contribute in a significant way to the genesis of human cervical cancer. HPV has two transforming genes that encode the oncoproteins E6 and E7, E6 can form complexes with p53 and promote p53 degradation, E7 inhibit retinoblastoma protein (RB). The p53 protein is as a phosphoprotein which co-immunoprecipitated with the SV40 T-Antigen. The wild type p53 protein is capable of suppressing the tumorigenic phenotype and regulating cell cycle. Adeno-associated virus(AAV) is a linear single stranded DNA parvovirus which is dependent upon cotransfection by a second unrelated virus to undergo productive infection. It has been well documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through the termini. In order to introduce wild type p53 through AAV virus into a cervical cancer patient for gene therapy, we had constructed recombinant p53 adeno associated virus plasmid (pAAVCMVp53). Methods: pAAVCMVp53 was created new AAV-vector system, pRc/CMVp53 including p53 cDNA and AAV-derivative vector, pASPA-AAV-CMV-polyA were made to HindIII/blunt fragments. Eluated 1.8 kb fragment of wild type p53 cDNA was ligated to pAAV-CMV-polyA, 4.9 kb fragment deprived hASPA cDNA. Result: Recombinant AAVCMVp53 was constructed by using pRc/CMVp53 andpASPA-AAV-CMV-polyA. This pAAVCMVp53 was confirmed by various restriction enzyme-digestions and Southern-blotting. This new vector system will be studied on expression, stability in cervical cancer cell lines and animals. Conclusion: This system will be one of the useful vector system for cervical cancer gene therapy.

자궁경부암에서 Glyceraldehyde-3-phosphate Dehydrogenase 유전자의 발현 증가

김진우,남궁성은,박태철,이준모,신진웅,최은아,김기범,신봉영 대한산부인과학회 1998 Obstetrics & Gynecology Science Vol.41 No.10

목적: 종양세포에서는 특징적으로 해당작용(glycolysis)이 정상세포에 비해 증가되어 있다. 현재까지의 연구 결과를 보면 GAPDH 유전자 발현이 여러 종류의 종양들과 암세포주들에서 증가되어 있는 것으로 보고되고 있다. 그러나, 자궁경부암조직에서는 현재까지 GAPDH 유전자 발현 정도에 대한 보고가 없다. 본 연구에서는 자궁경부암 조직과 자궁경부암 세포주들을 대상으로 GAPDH 유전자의 발현 정도와 발현 빈도를 정상 자궁경부 대조군들과 비교하여 그 결과를 임상 및 병리학적 소인들과 연관시켜 GAPDH 발현의 정도와 악성도와의 상관관계, GAPDH 유전자와 종양의 성장과의 관계를 밝히고자 시도되었다. 연구 방법: 본 연구는 25예의 정상 자궁경부 조직, 35예의 치료받지 않은 원발성 자궁경부암 조직, CUMC-3 및 CUMC-6 자궁경부암 세포주, 누드마우스 유래 자궁경부암 세포주 (CUMC-3N 및 CUMC-6N)들에서 RNA들을 채취하여 GAPDH 유전자 발현검색을 위한 Northern blot 분석에 사용하였다. 결과: Northern blot 분석에 의하면 GAPDH 유전자 발현의 정도는 35예의 자궁경부암 조직 중 34예(97%)와 네종류의 자궁경부암 세포주들 모두에서 정상 자궁경부 조직들에 비하여 매우 높게 증가되어 있는 소견이 관찰되었다. GAPDH 유전자 발현 정도는 성장속도가 빠른 누드마우스 유래 자궁경부암 세포주들에서 가장 높게 나타났다. GAPDH 유전자 발현정도는 임상 및 병리학적 소인들인 임상적 병기, 종양의 조직학적 형태, 분화정도들과는 상관관계가 없었다. 결론: 이러한 연구 결과 자궁경부암에서 GAPDH 유전자 발현이 매우 증가되어 있음이 판명되었고 향후 자궁경부암의 성장에 중요하게 이용되는 GAPDH 효소 유전자에 대한 antisense olgonucleotide를 제조하여 자궁경부암의 성장을 억제시키는 유전자 요법의 수단으로 이용될 수 있는 가능성을 제시하여 주었다. Objective: Tumor cells characteristically exhibit an increased rate of glicolysis. There has been several studies showing an increase of GAPDH gene expression in a variety of human tumors and cancer cell lines. But, in cervical carcinoma, there has not been reported about the level of GAPDH gene expression still now. So, we tried to investigate the GAPDH gene expression patterns in cervical carcinoma tissues and cancer-derived cell lines compard to normal cervical tissues, and the relationships between the level of this gene and conventional clinicopathological parameters were evaluated. Materials and Methods: In this study, RNAs from 25 normal exocervical tissues, 35 primary untreated cervical cancer tissues, 2 cervical cancer cell lines (CUMC-3 and CUMC-6), and 2 postnude mice derived cervical cancer cell lines (CUMC-3N and CUMC-6N) were subjected to Northern blot analysis for GAPDH gene expression. Results: Northern blot analyses revealed that the levels of GAPDH gene expression were markedly elevated in 34 of 35 (97%) cervical carcinoma tissues and all of the four cancer cell lines compared to normal cervical tissues. The level of GAPDH gene expression was highest in rapidly growing postnude mice derived cervical cancer cell lines. And the level of the GAPDH gene expression was not associated with conventional clinicopathological parameters including clinical stage, histological type of the tumor, and degree of differentiation. Conclusion: This result suggests that increased GAPDH gene expression is characteristic of human cervical carcinomas, and antisense oligonucleotide directed against GAPDH mRNA might play a another tool for future gene therapy against cervical carcinoma.

자궁경부암 세포주들에서 리포좀을 이용한 종양억제 유전자 [pRcCMVp53/liposome]의 직접 이입법에 의한 세포 성장 억제 효과

김승조,이헌영,남궁성은,김수평,이준모,안웅식,김종국,박순희,박용석,김진철,한유진,양재명,신봉영,서상용,김동재 대한산부인과학회 1999 Obstetrics & Gynecology Science Vol.42 No.4

목적: 자궁경부암 유전자 치료를 위하여 자궁경부암 세포주들에 리포좀을 이용한 종양억제 유전자[wild-type p53 gene]의 직접 이입을 통하여 세포성장 억제효과를 보고자 하였다. 방법: 자궁암세포주들 [CaSki, SiHa, HeLa, HeLaS3, C33A, HT3]을 12 well에 104, 96 well에 5×103의 수로 triplicate를 형성한 뒤 대조군, pRcCMVp53, lipofectin, pRcCMV/lipofectin, pRcCMVp53/lipofetin이 함유되있는 것 을 각기 12 well에 20 ㎕씩 96 well에는 10 ㎕씩을 투여한 다음 7일간 배양하였다. 세포수 측정, ELISA [neutral red] assay, MTT등을 이용하여 세포성장과 cell viability를 측정하고 이를 one-way ANOVA F-test, multiple comparison Tukey method에 의해 날짜별 서로 군의 차이를 비교 하였다. 결과: p53 유전자에 의한 자궁경부암 세포주의 성장억제를 확인하기 위하여 세포 수 측정, ELISA assay, MTT assay를 시행하였으며 각각의 assay에 대한 측정 마지막날인 6일째를 기준으로 정상대조군 세포와 비교한 결과 세 포수 측정에서는 CaSki[89%], SiHa[59.2%], HeLa[86%], HeLaS3[78%], C33A[91%] 그리고 HT3[74%] 성장억제를 나타냈다. 또한 Neutral red를 이용한 세포 viability assay 에서는 CaSki[70%], SiHa[93%], HeLa[73%], HeLaS3[50%], C33A[67%] 그리고 HT3[52%]에서 는 52%의 세포감소를 보여주었다. MTT assay를 통한 각각의 자궁경부암 세포주의 세포성장억제 결과는 CaSki[38%], SiHa[72%], HeLa[53%], HeLaS3[28%], C33A[33%]그리고 HT3[53%]였다. 결론: 자궁경부암 세포주에 직접 이입법을 통한 유전자 전달 후 세포성장 억제는 매우 유익하게 감소되었으며 이와같은 결과는 향후 유전자 직접 이입법에 의한 자궁경부암 환자에 있어서 유전자 치료를 실시할 수 있는 새로운 장으 로 생각이 되어진다. Purpose: We investigated cell growth inhibitory effect of the wild type p53 gene into the cervical cancer cells via the recombinant p53 plasmid, pRcCMVp53 with lipofectin. Method: Inhibition of the growth of cervical cells as determined by a cell count assay. The cells were inoculated at density of 104 cells/well in each 12 well plate, 24 hrs before infection. At each indicated point, cells in three wells on each well plate were trypsinized and counted. The mean cell counts of triplicated well after transfection at day 1~6 was checked. Inhibition of the growth of cervical cells were checked by ELISA assay, MTT assay. The cells inoculated at densities of 5×103/well in each 96 plate 24 hrs before infection. At each indicated point cells of three wells were transfected for 6 days, harvested and counted by liquid scintillation counter the mean cpm per triplicate wells were plated for ELISA and MTT assay. One-way ANOVA F-test and multiple comparison Tukey method was used for statistical analysis. Result: Inhibition of the growth of cervical cells in cell count showed CaSki[89%], SiHa[59.2%], HeLa[86%], HeLaS3[78%], C33A[91%] and HT3[74%]. ELISA assay showed CaSki[70%], SiHa[53%], HeLa[73%], HeLaS3[50%], C33A[67%] and HT3[73%]. MTT assay showed HeLa[33%], HeLaS3[28%], SiHa[75%], CaSki[38%], C33A[33%] and HT3[53%]. Conclusion: These results indicate that transfection of cervical cancer cells with the wild type p53 gene via pRcCMVp53 with lipofectin is a potential novel approach to the gene therapy of cervical cancer.

정상 임신과 비정상 임신의 초기 혈중 CA - 125 수치에 관한 연구

노덕영(Duck Yeong Ro),김도강(Do Kang Kim),김수평(Soo Pyung Kim),문희봉(Hee Bong Moon),강규섭(Gyu Sub Kang),황지영(Jee Young Hwang),신봉영(Bong Young Shin),장병우(Byeung Woo Jang) 대한산부인과학회 1998 Obstetrics & Gynecology Science Vol.41 No.11

N/A A prospective study was initiated to compare maternal serum concentration of CA-125 during the first trimester of normal and abnormal pregnancies. Serum specimens were obtained from 87 women with a normal intrauterine pregnancy and 47 women with abnormal pregnancies which were ended in spontaneo abortion or pathologically confirmed to be missed abortion. In normal pregnancies, the mean serum CA-125 concentrations were increased significantly from amenorhea 6 weeks (139.838.7 IU/ml), and were higher statistically than the values tested in the same weeks of abnormal pregnancies. In abnormal pregnancies serum CA-125 concentations were relatively lower than those of normal pregnancies. But these differences were not statistically significant except the values tested in amenorhea 6weeks. So serum levels of CA-125 may not be proved useful in monitoring of early pregnancies outcome.