The Clinical & Experimental Research On The K-ras Gene Point Mutations And Monoclonal Antibodies Of The Gene Expression Products P21 Protein In Pancreatic Adenocarcinoma

Recent research discovered that the incidence of K-ras gene point mutations at codon 12 in pancreatic adenocarcinoma tissue was 71%-100% , in pancreatic juice 67%-100%, in plasma 27%-81% and in duodenal juice was 60.9%-66%. The mutant types were nearly from the wild type GGT to CGT, GTT or GAT, but no mutant K-ras gene was found in normal pancreas ,insulinoma ,benign pancreatic cyst and other kinds of pancreatic tumors. Few reports showed low incidence of K-ras gene mutations(9.1%-33.3%) in chronic pancreatitis. Detection of K-ras gene mutation is helpful to distinguish benign lesions from malignant ones and to make early definitive diagnosis of pancreatic adenocarcinoma. There are six kinds of methods to detect K-ras gene point mutations at codon 12.(1)PCR-RMCM, (2)PCR-ASOH, (3)PCR-SSCP, (4)PCR-RFLP, (5)PCR-DSM and (6)PCR-MASA. According to the K-ras gene sequence and the mutant styles of CGT,GTT and GAT, three kinds of sequence-special primers (SSP) were designed & used for polymerase chain reaction to detect the mutations in paraffin-embedded tissues, frozen tissues, fine needle aspirates and pancreatic juice of pancreatic adenocarcinoma since 1994.The mutation rate was 74.2%, 95.1%, 91.4% and 94.1% respectively. No false positive results were found. The method of PCR-MASA is rapid, convenient, specific and sensitive. Only the routine methods of acrylamide gel electropheresis and ethidium bromide stain are needed for observing the amplification products. Restriction enzyme digestion, mutation specific oligonucleotide probe hybridization, radioisotopic and nonradioisotopic imaging were not necessary . On the basis of these results we started to study specimens of peripheral blood, ascites and duodenal juice of pancreatic adenocarcinoma since 1999 and the K-ras gene point mutation rate was 38.1%(8/21), 31.6%(6/19)and 17.4%(4/23) respectively. No mutant K-ras gene was noted in specimens of acute pancreatitis, chronic pancreatitis, insulinoma, ampullary carcinoma, duodenal papillary adenocarcinoma, stomach cancer, liver cancer and cholelithiasis.Many research reported that pancreatic carcinoma tissues had high expression of K-ras gene products P21 protein. The positive rate detected by immunohistochemical stain ranged from 61% to 88.7%. We began to prepare the monoclonal antibodies by hybridoma techniqure using the artificially composed polypeptide which specially directed to the sequence of k-ras gene as the antigen in 1999. The human pancreatic adenocarcinoma tissue sections were examined with the immunohistochemistry staining by the monoclonal antibodies indicated the positive rate was 88.1%(37/42). It failed to react with all normal pancreas tissues. On the basis of these findings, we used McAb, 5-FU, E-ADM, MMC and DDP to react with BxPC-3 and AsPC-1 cell lines of pancreatic carcinoma in vitro. The results of MTT discovered that inhibition rates of BxPC-3 and AsPC-1 cell lines gradually increased with the increasing of reaction time and drug concentration. We found that P21 McAb had direct anti-tumor effect and hold synergistic effect with each of the four kinds of chemothrapy agents.Flow cytometry(FCM) results revealed that P21 McAb had function of directly inducing apoptosis of BxPC-3 and AsPC-1 cell lines . At the same time we also found that combination use of P21 McAb and 5-FU or E-ADM could increase the apoptosis rate in these two cell lines.Pancreatic carcinoma(PC) is one of the common malignant tumors and the incidence of PC is increasing in recent years. It is very hard to make early definitive diagnosis and radical resection rate was only 15%-20%. The prognosis of PC was also very bad. Definitive diagnosis of pancreatic mass preoperatively was sometimes very difficult. The current different kinds of examinations could not meet the need of early diagnosis. Our research indicated that (1)Detecting K-ras gene mutations at codon 12 in specimens of peripheral blood, ascites and duodenal juice for pancreatic diseases by PCR-MASA may serve as a practical new method for distinguishing...