A Study Of Oncogenes In Human Pancreatic Carcinomas

For clarifying the relationship between oncogene over expression and gene point mutations in human pancreatic carcinoma, 4 cell lines and 34 cases of formalin fixed and paraffine embedded pancreatic adenocacinoma tissues were studied The human pancreatic carcinoma cell lines, PC-1, PC-2, PC-3 and PC-4, were established in our department.The normal pancreatic tissue was taken from the resected specimen of a patient with carcinoma of Ampulla of Vater. The total cellular RNA were extracted and bloted onto nitrocellulose membranes .After drying in vacuum, the membranes were hybridized with ³²PdCTP labelled oncogene probes (c-myc,v-fos,c-N-ras,c-Ki-ras,v-H-ras). Hybridization in situ technique was performed. PC-1,PC-2, PC-3,and PC-4 cells were collected after 0.04%EDTA digestion. Two or 3 drops of cell suspension were put onto the surface of poly-1-lysine coated slides, and fixed in 3:1 ethanol:acetic acid for 20 minutes. After washing and PK digestion, the slides were hybridized with ³⁵-S labelled oncogene probes. The PCR technique was performed as follows: the primers and probes were synthesized by Institute of Microbiology, Chinese Academy of Sciences. The volume of PCR mixture was 100 ul. One handred ng each primers were added. The final concentration of the buffer was 10 mM Tris-CL PH8.3, 50mM KCI,3mM MgCl₂,, 0.01% BSA, 200uM each dATP, dCTP, dGTP and dTTP. After denaturing at 97℃ for 5 minutes, 2U Taq DNA polymerase were added to the mixture. The amplification was performed in GTC-1 amplifier for 30 cycles. The temperature and time for denaturing, annealing. enlongation were 94℃, 30 seconds, 50℃,45 seconds, and 70℃, 45 seconds respectively. After amplification. 5ul PCR products mixture were electrophoresesed in 2.5% Agarose gels for examining the amplifing effects. The products of PCR is 116 bp. Fifty ng each oligoneocloetide probes were end-labelled with -P-ATP by polynuocleotide kinase. After 2.5%agarose gel electrophoresis for examining PCR effects, 5ul PCR products were spoted onto Duralose-UV membranes . After drying the membranes were hybridized with labelled probes in 3M tetramethyl amminum chloride (TMAC) at 56℃ and washed at 59℃.