The Study On The Cell Apoptosis Induction,Growth Suppression And Gene Expression Profile Regulation Of Human Gallbladder Carcinoma GBC-SD Cell Lines After Transfection Of Wild-Type P53 Gene

Part Ⅰ:p53 gene mutation in human gallbladder carcinoma GBC-SD cell linesObjective To use immunocytochemistry SP staining and PCR products direct sequencing methods to investigate p53 gene status in human gallbladder carcinoma GBC-SD cell lines in vitro. To explore its effect on the biological characteristics of GBC-SD cell lines.Methods To culture GBC-SD cell lines using the cell culture technique in vitro. Immunocytochemistry SP staining was used to detect P53 protein expression of GBC-SD cell lines. Genomic DNA was isolated from GBC-SD cell pellets, exons 4~9 of the p53 gene were amplified using PCR technique. After identified by 2% agar gel electrophoresis, the amplification products were direct sequencing analysed. The sequencing results were compared with the human p53 gene sequence of Genebank using Blast software in NCBI website.Results The gallbladder carcinoma GBC-SD cell lines were maintained in RPMI-1640 supplemented with 10% inactivation NBS. The cells grown on glass slide were fixed and stained by immunocytochemistry staining, the result showed P53 protein overexpression in GBC-SD cell lines. Exons 4~9 were successfully amplified by PCR technique, and the purified products were detected by forward and reverse direct sequencing analysis. A transversion of TAC→AAC at codon 126 of exon 5 was confirmed, which brought out missense mutation , Tyr→Asn of expressed amino acid.Conclusion Human gallbladder carcinoma GBC-SD cell lines express mutant-type P53 protein, and point mutation of 126 codon may be the main reason for function inactivation of p53 gene.Part Ⅱ:Experimental study on the cell apoptosis induced by wild-type p53 gene in human gallbladder carcinoma GBC-SD cell linesObjective To transfect exogenous wtp53 gene into human gallbladder carcinoma GBC-SD cell lines and observe the effect of transient expression on cell proliferation and apoptosis.Methods Recombinant eukaryotic expressing plasmid pCMV-p53 containing wtp53 gene or pCMV-p53mt135 containing mtp53 gene was introduced by lipofectamine-mediated gene transfection into human gallbladder carcinoma GBC-SD cell lines which containing mutant-type p53 gene, respectively. The cell growth was observed before and after transfection by MTT assay. The morphologic character was observed under the light microscope and the transmission electron microscope, chromosome DNA ladder was ascertained by agarose gel electrophoresis, thepercentage of the cell apoptosis and the alteration of cell cycle were detected by flow cytometry on 241k 48h after transfection.Results The GBC-SD cell growth was obviously inhibited through transfected pCMV-p53, the cell growth inhibition ratio were 52.8% and 77.5% on 24h and 48h, respectively. However, the control group which transfected pCMV-p53mt135 were 3.8% and 6.3%, it was significantly different between two groups (p<0.01) . Typical apoptotic character was observed after 48h under the light microscope and the transmission electron microscope in gallbladder carcinoma GBC-SD cell lines which transfected pCMV-p53 , including cell shrinkage, chromatin condensation and formation of cytoplasmic blebs and apoptotic bodies. It was also demonstrated on DNA ladder by agarose gel electrophoresis. The cell apoptosis rates were tested by the flow cytometry were 12.98% on 48h after transfected pCMV-p53, it was significantly higher than that of the control group(3.7%) (p<0.01) .The cell cycle distribution had been changed yet, the G0/G1 ratio greatly increased, the S and the G2/M ratio significantly decreased in GBC-SD cell after transfected pCMV-p53 (p<0.01) .Conclusion The transient expression of wild-type p53 gene by lipofectamine-mediated could obviously inhibits the growth and induced the apoptosis of human gallbladder carcinoma GBC-SD cell lines in vitro.Part Ⅲ:The study on the cell growth suppression and gene expressionprofile regulation of human gallbladder carcinoma GBC-SDcell lines after transfection of wild-type p53 geneObjective To investigate the effect on inhibition of the cell growth of humangallbladder carcinoma GBC-SD cell lines in vitro and in vivo with exogenous wild-type p53(wtp53) gene transferred by lipofectamine-mediated and to analysis the difference of the gene expression profile after transfection of wild-type p53 in GBC-SD cell lines by cDNA microarray.Methods (1) Recombinant eukaryotic expressing plasmid pCMV-p53 containing human wtp53 gene or pCMV-p53mtl35 containing mtp53 gene was transferred by lipofectamine-mediated gene transfection into human gallbladder carcinoma GBC-SD cell lines with p53 gene mutation, respectively. Growing transfected cells was cloned by method of G418 selection.The presence of exogenous p53 gene was confirmed by PCR , the expression of exogenous p53 mRNA was identified by RT-PCR, the expression of P53 protein in GBC-SD cells before and after transfection wild-type p53 gene was detected by Western blot. The cellular proliferating ability was assessed using the cell growth curve and cloning assay; the change of the cell cycle was analyzed by flow cytometry, the xenograft in nude mice was performed to examine the effect of tumorigenicity in vivo. (2) The cDNA probes were synthesized from total RNA of study group (GBC-SD-wtp53) and control group (GBC-SD) ,which were labeled by fluorescence dyes respectively, hybridized with cDNA microarray which have 447 known genes and EST genes , then obtained the gene expression profile. To analysis the scan results with Quantarray software, and proofread with Normalization software. Different expression genes were then screened out in GBC-SD cell lines after stable transfection by wild-type p53 gene.Results (1)After transfected pCMV-p53 or pCMV-p53mt135 and selected by G418 , the cell lines GBC-SD-wtp53 and GBC-SD-mtp53 were successfully established. No obvious morphologic difference in the cell lines before and after transfection p53 gene was observed by light microscope. PCR and RT-PCR test showed that exogenous p53 gene had successfully transfected into GBC-SD cells and obtained its expression. The expression of high level P53 protein was detected by Western blot after transfection . The cell growth rates of GBC-SD-wtp53 were greatly decreased, compared to that of GBC-SD-mtp53 and GBC-SD. Cloning efficiency for GBC-SD-wtp53 was 3.2%, which obviously lower than that for GBC-SD-mtp53(28%)and GBC-SD (29%) (P < 0.01) .The G0/G1 ratio was greatly increased, S ratio was significantly decreased and G2/M ratio was not changed in cell cycle distribution of GBC-SD-wtp53, compared to that of GBC-SD. There was no difference in cell cycle distribution between GBC-SD-mtp53 and GBC-SD cell lines. The tumorigenicity in nude mices showed that two of eight mices after injection GBC-SD-wtp53 failed to form tumor and six of them delayed to form tumor comparing with GBC-SD-mtp53 and GBC-SD cells, the mean volume and weight of subcutaneous tumors of injection GBC-SD-wtp53 cells group were lower than that of control GBC-SD-mtp53 group and parent GBC-SD group (P < 0.05 ). (2) The results of cDNA microarray showed that GBC-SD cell gene expression profile had been changed after stable transfection of wild-type p53 gene. 100 of 447 genes expressed differently ,17 genes were up-regulated, while 83 genes down-regulated. These genes related to signal pathway, cell metabolism, oncogene and antioncogene, metastasis,cell factor receptor, and apoptosis.Conclusion (1) The method of using lipofectamine-mediated gene transfection could effectively transfect human gallbladder carcinoma GBC-SD cell and obtain high expression of exogenous aim gene; the stable expression of exogenous wild-type p53 gene could obviously inhibit the growth ability and tumorigenicity of human gallbladder cancer GBC-SD cell lines in vitro and in vivo, and arrest the cell cycle in G0/G1 phase (2) cDNA microarray showed that gene expression profile of gallbladder carcinoma GBC-SD cell was significantly regulated after transfection of wild-type p53 gene, it is a useful method in understanding the effect mechanism of wild-type p53 gene by analyzing the differential gene expression.