The Construction Of Regulating Expression Vector And Treating Vector Targeting The AFP Positive Hepatoma

Objective:Liver cancer is the most common malignant tumors . The most advanced found is that liver cancer is associated with chronic hepatitis B and liver cirrhosis closely. It is not sensitive to radiotherapy and chemotherapy, resulting poor prognosis of patients with hepatocellular carcinoma. Gene therapy is expected to ease the patients'symptoms and to prolong life. But, how to make the treatment of specific gene products only to anti-liver cancer cells without damaging normal cells of the liver and other normal cells and how to improve the therapeutic effect and avoid drug side effects of gene therapy are universal difficult problems. Alpha-fetoprotein is a kind of mammals'protein in the process of development subjected to strict regulation and control. AFP level will rise with the AFP gene expression when HCC happen. This imply that there are some mechanisms of regulating this gene expression in the liver.At the upstream of AFP'5 end is 7 kb regulatory sequence. In this region ,there is a tissue-specific promoter, three independent enhancers, and a silencer. General transcription factors and tissue-specific transcription factors jointly regulate AFP expression. AFP expression is strict to specificial time and space. We ligated the most remote AFP enhancerⅢwith the promoter to construct a 1.2 kb sequence of gene expression regulation and cloned it into pEGFP-N1 plasmid. We replaced the CMV promoter of PEGFP-N1 plasmid with AFP promoter.In AFP positive hepatoma cells we proved its relative specificity. Meanwhile, in order to validate these gene sequences'possibility in the treatment of the liver cancer.At the regulatory sequence 1.2 kb downstream we connected it with the tumor suppressor gene P53 and proved it relative specificity of the cell cycle arrest and apoptosis in different cells.Methods:1 Construction of pEGFP-N1 vector without CMV promoter.1.1 Linking steps are as follows: The CMV promoter of pEGFP -N1 plasmid is removed by AseⅠand NheⅠ.1.2 Reclaim the 4100 bp fragment from agarose gel after electro- phoresis, ligate the two ends of 4100 bp fragment with the DNA -blunting kit.1.3 Transform the ligated product into E. coli in DH5αand selected monoclone.2 The construction of pAFP-EGFP plasmid2.1 Extract the DNA from SD rat liver cells . the promoter 900 bp and enhancer 300 bp fragments are amplified by PCR. At the same time, we design a pair of primers which introduce SmaⅠand EcoRⅠrestriction sites at the two ends of 900 bp fragment.2.2 Reclaim the 900bp and 300bp fragments of PCR product from agarose gel, purify it, digest the purified product with SmaⅠ,purify the digested product, ligase the two fragments in 16 ℃。2.3 Amplify the ligated product of 300bp and 900bp fragment by PCR; purify and digest them with Xhol and EcoRⅠrestriction enzyme; ligate it with the pEGFP-N1 plasmid without CMV promoter digested by Xhol and EcoRⅠrestriction enzyme. 1.4 Transform the ligated product into E. coli in DH5αand sele- ct monoclone.3 Transfect pAFP-EGFP into HepG2, SMMC7721, HeLa cells, under the fluorescence microscopy to observe fluorescent protein expression.4 The construction of pAFP-P53-EGFP plasmid4.1 P53 fragment is amplified by PCR ,the template is pCMV-P53.Through designing primers we introduce EcoRⅠand BamHⅠrestriction sites at the two ends of p53 fragment.4.2 Digest p53 PCR product and the pAFP-EGFP plasmid with EcoRⅠand BamHⅠrestriction enzyme at the same time .Liagate the digested PCR fragment with the digested vector. Construct the pAFP-P53-EGFP plasmid.5 Transfect pAFP- P53-EGFP into HepG2, SMMC7721, HeLa cells, the non-transfected HepG2, SMMC7721, HeLa cells as control groups, the difference of p53 protein expression is detected with Western blot method.6 Wash the HepG2,SMMC7721,HeLa cells transfected with pAFP-P53-EGFP plasmid with PBS two times; fix the cells with 70% ethanol;The percent of G1 phase cells and the rate of apoptosis are examined by Flow cytometry. Results:1 The pEGFP-N1 vector without CMV promoter and pAFP -EGFP vector identified by PCR and restriction enzyme digestion validated that the construction was successful.2 The pAFP-EGFP plasmid was transfected into HepG2, SMMC7721, HeLa cells, under fluorescence microscopy to analysis fluorescent protein expression. The expression of green fluorescent protein was significant different. (Fig.8) indicated.3 The pAFP-P53-EGFP recombinant plasmid identified by restriction enzyme digestion was constructed successfully.4 The transfected HepG2, SMMC7721, HeLa cells with pAFP -P53-EGFP were detected by western blot method. The P53 protein expression was significant different. The gray value of HepG2 was higher than the other kinds of cells. We also detected the P53 protein of non-transfected HepG2, SMMC7721, HeLa cells. The gray value of HepG2 transfected with pAFP -P53-EGFP was the highest than all the other tansfected and non-transfected cells.The log of grey in HepG2, SMMC7721, HeLa transfected and non-transfected cells were 7.20±0.05,6.77±0.16, 6.81±0.12, 6.67±0.15,6.65±0.25,6.69±0.13.This imply that by the drive of AFP promoter the P53 fusion protein expression is relative specific in different cells.5 The HepG2, HeLa ,SMMC7721cells transfected with pAFP -P53-EGFP plasmid was examined by Flow cytometry.The percent of G1 phase cells and apopotosis rate were diferent. group HepG2 %gate = 2.65±0.08, %G1 = 66.7±0.25, %G2 = 11.8±0.23,%S = 20.1±0.22; group HeLa gate% = 0.42±0.025, %G1 = 50.5±0.18, %G2 = 20.5±0.19, %S = 29.8±0.18; group SMMC7721 %gate = 0.39±0.018, %G1 = 51.0±0.20, %G2 = 10.6±0.13, %S = 37.8±0.21. So the rate of apoptosis and the percent in G1 phase cells of HepG2 cells transfected with the recombinant plasmid pAFP-P53-EGFP was higher than SMMC7721, HeLa cells transfected with pAFP-P53-EGFP recombinant plasmid. The %S of HepG2 cells transfected with pAFP-P53-EGFP plasmid was significantly lower than SMMC7721,HeLa cells transfected with pAFP- P53-EGFP recombinant plasmid.Conclusions:1 The expression of pAFP-EGFP expression is relative specific in different kind of cells.The expression of green fluorescent protein in HepG2 cells was significantly higher than in the SMMC7721,HeLa cells.2 P53 protein was examined by western blot . HepG2,SMMC7721,HeLa cells transfected and non-transfected all expressed the P53 protein. The P53 protein expression was not significantly different in the non-transfected HepG2,SMMC7721,HeLa cells. The P53 protein expression in HepG2 cells transfected with pAFP-P53-EGFP was higher than SMMC7721,HeLa cells transfected with pAFP-P53-EGFP plasmid. The P53 protein expression of HepG2 cells transfected with pAFP-P53-EGFP was also higher than the non-transfected HepG2,SMMC7721,HeLa cells. All this imply that the pAFP-P53-EGFP plasmid driven by AFP promoter expression is specific .3 Flow cytometry analysis cell cycle phase and apoptosis rate of HepG2, SMMC7721, HeLa transfected with pAFP-P53-EGFP plasmid. The percent of G1 phase cells and apoptosis rate in HepG2 were significantly higher than in SMMC7721, HeLa cells.This imply that G1 phase arrest and apoptosis of P53 driven by AFP promoter is specific in different kind of cells.