The Study Of Abnormal Activating Mechanism Of C-fms Oncogene And Antisense C-fms Bearing AFP Enhancer With Hepatocellular Carcinoma

Background Colony stimulating factor I receptor(CSF-1R) is coded by c-fins oncogene, it was over-expressed in hepatocellular carcinogenesis. However, the molecular mechanism remained obscure. CSF-IR had tyrosine kinase activity, it influenced the occurrence and deterioration of hepatoellular carcinoma(HCC) through multi-signal transduction mode. There was a activatiI~g mutation site at 3Ol~ codon of human CSF-1R, when the transition of normal Leu(TTG)-4Ser(TCG) occurred, CSF- 1 R could mimic the conformational change induced by CSF-l, therefore it leaded to the self- activation of CSF-IR tyrosine kinase, through a series of signal transduction mechanism produced the abnormality of cellular growth and differentiation, eventually resulted in tumor occurrence. The 969th tyrosine and the neighboring amino acids negatively regulated tyrosine kinase activity of human CSF-1R, when there were mutations or deletions at this site, tyrosine kinase activity of CSF-IR would be up-regulated, through the signal transduction mechanism it participated in tumor occurrence. It was generally reckoned that hypomethylation of oncogene was a kind of important molecular mechanism that leaded to its over-expression. Encoded products of partial oncogenes were growth factors or their receptors. Oncogenes expressed large amounts of growth factors or their receptors with the mechanism of hypomethylation, they promoted occurrence and development of HCC by stimulating ways of autocrine/paracrine. The 571st tyrosine of human CSF-IR was necessary for the activation of CSF-IR tyrosine kianse and the signal transduction. As a kind of biological therapy method, antisense gene acquired delectable effect recently. AFP enhancer could control target- gene to be highly and specifically expressed in AFP-positive HCC cells. To induce apoptosis was a kind of method for HCC therapy. The data showed that mono-antibodies against CSF-l/CSF-1R could inhibit the growth of HCC cells, and demonstrated that CSF-1/CSF-IR was a pair of autocrine/paracrine system in HCC occurrence and development. Therefore, there was practical significance for HCC therapy to block the abnormal expression of CSF-l/CSF-IR. About the abnormal activation and high expression mechanism of c-fins oncogene in HCC was still in exploration, there were no reports about antisense c-fins on the biological behavior of HCC cells so far. Objective To observe whether there were deletions or mutations of c-fins oncogene in HCC carcinogenesis. To clarify the relationship between c-fins oncogene and HCC, to provide fundamentals for the mechanism of HCC occurrence. To detect the methylation status of c-fins oncogene in HCC and clarify its abnormal high expression mechanism. To explore a new gene therapy method for HCC, constructed human antisense c-fins eukaryotic expressing vector and antisense c-fins eukaryotic expressing vector bearing AFP enhancer, and observed the effect of the two vectors on biological behavior of HCC cells. Methods I. Mutation and clinical significance of c-fins oncogene in HCC 1.1 Materials: to extract DNA of HCC and matching para-cancer tissues, the tissues were verified with patholog); 25 males and 5 females, 30 cases in total, age range from 32y to 76y, mean 55y. Control group was HBV negative normal hepatic tissue from renal-transplantation donor. 1.2 Primers design: two pairs of primers were designed to analyse 301~ and...