| Objective:The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are important multiple tumor suppression (MTS) genes and frequently homozygously deleted in a variety of tumor cell lines and primary tumors. The frequent mutation especially related to the bile duct carcinoma and pancreas carcinoma. P16INK4A and p15INK4B are very similar in the structure, but have obviously different function. It should be clarified whether p15 and p16 have the same effect on the cell cycle and cell proliferation or which one is the dominant effector. This study detected the expression of pl6INK4A and p15INK4B in the bile duct carcinoma with the immunohistology to explore their roles in the development of the bile duct carcinoma.The recombined p16INK4A and p15INK4B palsmid transfected the QBC939 to compare the difference in the suppression of cell proliferation. Our study may provide new insight for the bile duct carcinoma screen and find a new anti-oncogene in the treatment of bile duct carcinoma.Materials and methods:1. Ten bile duct carcinoma, 30 gallbladder carcinoma, 10 chronic cholecystitis and 10 cholesterol polyp (all from the First Affiliated Hospital of Nanjing Medical University)were parafilmed and detected the expression of p16INK4A, p15INK4B with the SP method.2. The construction and identification of the p16INK4A,p 15INK4B eukaryon expression system:(l)The construction of the p16INK4A, p15INK4B eukaryon expression system: Design primer according to the pl6INK4A,p15INK4B cDNA sequence and add the restriction enzyme site at two end of the cDNA, then amplify it with PCR method ,the PCR product were purified with the Kits. The enzymed vector and insert were ligated with T4-ligase.Recombinated pcDNA3-_ p15 and pcDNA3-p16 transformed the competent JM109 Ecoli. bacteria and ;i Itured in the aminobenzylpenicil -lin positive LB plate.(2)Cut with enzyme to identify the positive clone: selected the clones and cultured in LB medium overnight and extract the plasmid with the kits, then cut them with restriction enzyme.(3)Sequencing the recombination plasmids. 3.Cell culture and stable transfection: QBC939 cell line cultured with 10%FCS DMEM medium in 37 ℃ ,5%CO2 incubator. P16INK4A and p15INK4B plasmid transfected QBC939 by lipofectin and passagedafter 48 ~ 72 hours. The G-418 was added and the stable expression cell line was selected out after 28 ~ 35 days. The protein expression was detected with Western-Blot. The cell growth curve was assayed by MTT method and the cell cycle was analyzed with flow cytometry.Results:l.The positive expression rates of p15INK4B, p16INK4A in gallbladder carcinoma were 26.7%,20.0% and in bile duct carcinoma were 30.0%,20.0% respectively.lt had no relationship with the tumor stages of pathology(p>0.05). The expression of p15INK4B, p16INK4A had positive relationship with gallbladder carcinoma(p>0.05 ,r=0.452). 2.The pcDNA3-p15 positive cloning could show a 464bp band and the pcDNA3-p16 positive cloning showed a 478bp band with PCR methods. After cutting with restriction enzyme, the positive recombination plasmid showed two bands (464bp and 478bp) when running in the gel electrophoresis. The sequence result verified the reading frame was correct. 3.The Western-blot result showed that the p16INK4A and p15INK4B expression in transfected cell line are significantly higher than that of the control group. The growth rate of the p16INK4A or p15INK4B overexpression cell line are significantly decreased when compared with the control group. The S phase cells of these two overexpression cell lines are alsoless than that of the control and there are also significantly difference between the p16INK4A and p15INK4B overexpression cell line. Conclusion:1.The decreased expression level of anti-oncogene pl6INK4A,p15INK4B in bile duct carcinoma, gallbladder carcinoma parafilm sample,and the expression of p15INK4B, p16INK4A had positive relationship with gallbladder carcinoma both indicate that p16INK4A and p15INK4B have an intim...
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