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사람 방광 종양 세포내 c-H-ras 종양 유전자 돌연변이의 검색

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 Identification of c-H-ras gene mutations in human bladder cancer 
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본 연구는 사람 방광 이행 상피 세포 종양에서 c-H-ras 유전자의 돌연 변이와 암의 예후와의 연관성을 알아보기 위하여 파라핀에 포매되어 보관된 사람 방광 이행 상피 세포암조직으로 부터 DNA를 추출하고 c-H-ras 유전자의 exon I 및 exon ll에 특이한 primer를 이용하여 polymerase chain reaction(PCR) 법으로 c-H-ras 유전자를 증폭하고 이를 direct sequencing 법 또는 PCR/single stand conformation Polymorphim(SSCP) 분석법을 이용하여 유전자내의 돌연 변이 여부를 검색한 결과 대상 총 39예중 3예에서 각각 exon I의 codon 11번의 첫 번째 염기가 G에서 C로 transveraion된 돌연 변이, codon 15번의 세 번째 영기에 두개의 염기 C 및 A가 삽입된 frame shift돌연 변이 및 exon Ⅱ의 codon 42번의 두 번째 염기가 G에서 C로 transversion된 돌연 변이가 검출되었다. 이들 부위의 돌연 변이는 아직까지 생체 조직을 이용한 실험에서는 발견되지 않은 새로운 부위에서 발견된 돌

연 변이로서 이들 부위의 돌연 변이가 사랑 방광 이행 상피 세포암 형성 과정과 연관성을 갖으리라는 새로운 사실을 시사하는 결과로 사료된다.


This experiment was designed to elucidate the role of c-H-ras mutation in the development of human bladder carcinoma and the association of it's mutation with clinical prognosis.

Thirty nine cases histologically diagnosed as transitional cell carcinoma were graded according to WHO classification. Mutations of c-H-ras gene for each case were analyzed by PCR-SSCP or PCR/direct sequencing. DNAs extracted from each paraffin-embedded block were amplified using a set of c-H-ras exon specific primer,

and then exon Ⅰ and exon Ⅱ were amplified using (32)**P-end labelled nested set of the specific primer for exon Ⅰ or exon Ⅱ, respectively. Each PCR product was denatured and electrophoresed on 6% non-denaturing polyacrylamide gel at 4℃. A

definite polymorphic band was observed in one case which had two nucleotide insertion(C and A) at codon 15 of exon I identified by sequence analysis. And one of three cases that had suspicious polymorphism, had G->C transversion mutation at codon 11, while the another two cases had normal sequences. Using PCR/direct

sequencing, we also found G->C transversion mutation at codon 42 of exon Ⅱ in only one case. All the other cases tested showed normal sequences. Our findings suggest that the different new type of mutations at the sites other than codons 12, 13, and

61 reported by other investigators, may be involved in the development of bladder cancer, although the incidence of c-H-ras mutation in bladder cancer was much less frequent than in other malignancies. Further study will be needed to evaluate the importance of mutations found in the present experiment on the development of bladder cancer.
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