The Detection Of Potein Of APRIL In The Human's Pancreatic Cancer And The Construction Of Double Strand RNA Expressive Plasmid Which Is Targeted APRIL Gene And Preparation Of Small Interfering RNA

1.The detection of the expression of APRIL in the human's pancreatic cancerObjective: For finding new tumor's marker in pancreatic cancer's early diagnosis and prognostic evaluation, we detected the expression of protein of APRIL in the human's pancreatic cancer and compared with normal pancreatic tissue , analyzed the relationship of expression of protein of APRIL with pancreatic cancer's biological characteristic.Methods: Detected the expression of protein of APRIL in the human's pancreatic cancer (20 cases) by En Vision with two steps of immunohisto chemistry, and compared with normal pancreatic tissue (10 cases).Results: The rate of positive is 90.0% (18/20) in pancreatic cancer, it is similar in different type of pancreatic cancers. There is none of normal pancreatic tissue show postive (0/10), and the expression of APRIL in pancreatic cancer have no relation with degree of cancer' s differentiation, nodal metastasis, distance metastasis, size of cancer. Conclusion: It indicated that APRIL is involved in the pancreatic cancer's developement and APRIL is a molecular target of treatment and diagnosis of pancreatic cancer.2 .Selection of the APRIL high-level expression of pancreatic cancer cell lineObjective: Select high-level expression of APRIL in three pancreatic cancer cell lines, it's foundation for further research treatment of pancreatic cancer with RNA interference technology and the mechanism of APRIL gene in the cancer's developing and metastasis in the pancreatic cancer and even in other system.Methods: Abstract mRNA from digestive cancer cell line, which is carefully chosen among human's three pancreatic cancer cell lines (PANC-1 CFPAC-1 BXPC-3) . And transfer it to cDNA, then APRIL fracture be copied by PCR with special primer and GAPDH as internal control. The expression level is calculated as relative figures by computer and those five cell lines are sequenced from high level to the low.Results: According to the detection which is created by APRIL expression level, the sequence of the result was presented as follows: CFPAC-1 >BXPC-3>PANC-1.Condusion: As it is shown different expression lever in different cancer cells, APRIL play a role in the process of cancer's developing and metastasis. 3. The construction of dsRNA expressive plasmid which is targeted pancreatic cancer cell line's APRIL gene and preparation of siRNAObjective: We prepared targeted APRIL genetic siRNA (anti-APRIL-siRNA) for knock-down APRIL gene's expression specificly in human pancreatic cancer cell line of APRIL high-lever expression-CFPAC-1 by RNA interference technology.Methods: Construct pET-22b-APRIL which is an expressive plasmid of targeted APRIL genetic dsRNA. The dsRNA of the gene was expressed in E. coli and purified by chromatography using CF 11 column, and dsRNA was digested by RNase III .Then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNase III from oligonucleotides, and Purify 21-bp siRNA with size exclusion chromatography.Results: Targeted APRIL genetic dsRNA which was expressed in E. coli by inducement of IPTG, was purified by CF 11 column. 15 % nondenaturing PAGE and 12% SDS-PAGE show that RNase III was removed from oligonucleotides and 21-bp siRNA was purified with size exclusion chromatography.Conclusion: We have prepared anti-APRIL-siRNA targered CFPAC-1 cell line with in vitro transcription, and it is an elementary process for the next step of our research.