Screening And Identification Of Human Hepatocelluar Carcinoma Embryonic Antigen

Objective: To screen and identify antigenic genes of human HCC from cDNA expression libraries of human embryonic stem cells, to explore methods modified of SEREX ,and to find out cells and tissue mRNA expression of Cap2 which is one of HCC associated antigens.Methods: The best concentration gradient and volume gradient of Top-agar were tested, to make sure Top-agar least stick on NC memberane. Serum was pretreated by two methods of memberane absorption and coupling affinity chromatography respectively for eliminating the false positive. The serum of 38 primary liver cancer patients were pretreated. The recombinant cDNA expression library was screened with HCC serum pretreated by the methods of SEREX and Western-blot. The positive antigens were screened again to exclude false positive antigens. Positive clones were digested with two enzymes after inside deleted, and got their DNA sequence. All positive antigens were analyzed its information by bioinformatics. Cap2 mRNA was checked in liver cancer cell, stomach cancer cell, lung cancer cell, breast cancer cell and normal liver cell lines by RT-PCR, and identified tissue expression by tissue microarrays in situ RT–PCR. Results: The optimal condition for film off Top-agar is 15ml Top-agar in 150mm petri dish with 0.83% concentration and 4℃refrigeration for 20 minutes. Serum pretreated with coupling affinity chromatography is better than membrane absorption. In the first screening using methods of SEREX, we got 176 phage clones,and each clone was screened again to exclude false positive. Finally, 31 positive clones were identified. 31 plasmids were got after phage insert excised from the -ZAP vector. The DNA inserted in 31 plasmids were sequenced using T3, T7 as primers. All the antigen genes were known except one plasmid no sequence signal. The proteins for 23 antigens are known, and 7 antigens had not found their homologic protein, so they are unknown protein. Cap2 (144 calponin2) mRNA expressed strongly by RT-PCR in cell lines of liver cancer, stomach cancer and lung cancer,but weakly expressed in breast cancer cell lines and not expressed in normal liver cell lines. Furthermore, using tissue microarrays and method of in situ RT - PCR, we found that there were 89 cases Cap2 mRNA expression in 167 cases of liver cancer tissue and 4 positive cases in 50 cases of normal tissue, Chi-square test is P < 0.05. The results showed Cap2 mRNA mainly expression in the liver cancer tissue but not in normal liver tissue.Conclusion: For the first time,HCC antigens were screened from human embryonic stem cells cDNA expression librarie.Base on the commonness of human ESC and HCC, both of them can specifically express in different differentiation periods. At first,in the protein level with SEREX technology, we detected higher expression antigens in serum and analyzed the characteristics and distribution of each specific antigen by bioinformatics; Next, in cell level, we judged Cap2 mRNA expression in different cell lines by RT- PCR; Finally, we returned to tissue level using the methods of in situ RT-PCR with tissue microarray to find out Cap2 mRNA differences expression in tissues. The research shows that the antigen selected for hepatocellular carcinoma is concert with liver cancer and can be used in diagnosis and treatment liver cancer in the future.