Dissertation > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors

Anti-tumor Effect of Recombinant Retroviral Vector Mediated Human ANGPTL4 Gene Transfect

Author LiKeQiang
Tutor PengShuZuo;LiWenLin;ShiXiaoYu;LiuYingBin
School Zhejiang University
Course Surgery
Keywords ANGPTL4 HL-7702 cells HepG2 cells Retroviral vectors
CLC R735.7
Type PhD thesis
Year 2005
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I. Background and Purpose human ANGPTL4 gene is a newly discovered angiogenesis-related genes. The protein from the 406 amino acids, molecular weight about 60,000. Recent studies have shown that human ANGPTL4 in human tissues as follows: normal liver tissue adjacent liver tissue ≥ gt; hepatocellular carcinoma and human ANGPTL4 gene is highly expressed in the placenta, heart, liver, muscle, pancreas, lung moderate expression in the brain , kidney weak expression, human ANGPTL4 inhibit the growth of HCC. Previous studies, mainly through human ANGPTL4 gene plasmid liposome mediated into target cells, human ANGPTL4 gene expression in target cells is instantaneous, only last a few days, and liposome-mediated human ANGPTL4 gene transfection efficiency is low. Therefore, this study in order to further understanding of the human ANGPTL4 gene antitumor effect and explore an effective liver cancer treatment modalities by retroviral vector-mediated gene transfer of human ANGPTL4, stable, efficient expression of the gene in human hepatoma cell line HepG 2 -ANGPTL4. Second, the method to human liver cell line HL-7702 total RNA using RT-PCR method in vitro amplification of human hepatocellular carcinoma related genes ANGPTL4 cDNA, subcloned into retroviral vector plasmid pMSCV, identified and sequenced. Liposome were given high-titer recombinant virus pMSCV-ANGPTL4 and empty virus pMSCV. With the recombinant virus pMSCV-ANGPTL4 and empty pMSCV virus were infected with human hepatoma cell line HepG 2 . Flow cytometry and fluorescence microscopy were used to detect each group HepG 2 cells GFP protein. RT-PCR method was used to detect the HepG 2 Cell ANGPTL4 mRNA expression. Nude mice were detected by MTT assay experiments and each group HepG 2 cell growth in vivo. Zhejiang University PhD thesis a cell culture 293 EBNA cells, NIH3T3 cells and HePG: cells in DMEM containing 10% fetal bovine serum cell culture medium. With 10% fetal bovine serum in RPMI one a reward O cell culture medium human HL-7702 cells. These cells were cultured at 37 ℃, 5% CO: in. 2 gene amplification HL-7702 cells from the extraction of total RNA, reverse a female general. RNA synthesis of single-stranded cDNA (si nglestrand cDNA, 55 a eDNA). As the human ANGPTL4 gene cl) NA longer, about 1 .2 kb, will be divided into A, B two fragments were amplified. 50 Lin 1 PCR reaction system including 2.sng 55 a cDNA,, 5 Forest 1 10/pCR buffer buffer, 1 .5 Lin 1 10mM dNTp, 3 Lin Lin 110 M primers and 0.5 l Order buffeting DNA polymerase. 94oC denaturation 1 omin, then 94oC, TS 305; 6 twisted; oC, refolding extension 905; circulation amplified 30 times. Finally a loop 68 ℃ extension 5 min. Gel extraction of the PCR amplification of A, B gene fragment, EcoRI digested respectively, digestion product with T4 DNA ligase to obtain the complete human body connection ANoPTL4 eDNA, by manually fitting the primers, the 5 'and 3' ends, respectively, comprising Bgl 11 and Hapl restriction sites. The primer sequences as follows: A fragment: FSGGAAGATCTATGAGCGGTGCTCCGACGGC3 434bP Bgl 11 R SCTTTGCAGATGCTGAATTCGCAGGTGCTG3 EeoRI B fragment: FSACCTGCGAATTCAGCATCTGCAAAGCCAG3 830bP EeoRI R SGGGGTTAACCTAGGAGGCTGCCTCTGCTG3 HPal3. Recombinant retroviral vector plasmid containing the retroviral vector plasmid pMscv gene encoding green fluorescent protein GFP. GFP gene is a reporter gene, the foreign gene has prompted successful transduction and expression. Restriction endonuclease BglH and Hapl were digested vector plasmid pMSCV and human ANGPT14 cDNA, gel digestion products recycling, T4 DNA ligase in vitro ligation, recombinant retrovirus vector plasmid. ANGPTL4cDNA containing human recombinant retrovirus vector plasmid was named pMSCV-ANGPTL4. This recombinant plasmid by PCR, restriction endonucleases and DNA sequencing. Zhejiang University PhD thesis 4. Retrovirus packaging liposome retroviral vector plasmid pMSCV A NGf, TL4/pMSCV, pVSV (plasmidof vesieular stomatitis virusG), pGAG a pOL of 293 EBNA transfected packaging cells, 24h after transfection, with DMEM containing 10% fetal bovine serum culture medium for the transfection was terminated, 293 EBNA packaging cells cultured. 24h after each collection retrovirus containing the recombinant cell culture supernatant, collected three times, the recombinant retrovirus pMscv-ANGPTL4 and reverse empty virus pMScv. After dilution of viral supernatant infected NIH3T3 cells, NIH3T3 cells by flow cytometry for expression of GFP positive cells are infected and to calculate the virus titer. Virus titer is calculated as follows: the virus titer (infectious viral particles / ml) = MH3T3 total number of cells and positive cells / viral supernatant volume (ml). Virus collected a 70 ℃ preservation. 5 retroviral infection inoculation HepG: cells in 96-well plates (1 x 1 04 / well) for 24h a 4sh. When the cells fused to 60% serum-free DMEM cells rinsed twice added to each well and the virus supernatant containing 25 8 Chuan Chuan poly gel (100mg / L) in serum-free DMEM medium 100 HS l. After floating sterile sh Township, containing 100,0 FBS in DMEM medium was changed to terminate the infection. Continue to foster 2d, the fluorescence microscope after infection HepG: GFP-expressing cells, the selection of high expression HePG: cultured cells. lw, the flow cytometry positive cells are infected and the average fluorescence intensity. 6 weeks after infection gene expression, were extracted from infected with recombinant virus HePGZ a factory state GPTL4 cells infected with the virus HepGZ an empty pMSCV cells uninfected HepG: total cellular RNA, to p a aetin as an internal reference, RT-PCR assay ANGPTL4 mRNA expression. PCR parameters: 94 ℃ denaturation, Zmin, 94 ℃ denaturation, 30 5; 61.5 ℃ refolding, 305; 72 ℃ extension 605 cycles amplified 34 times. Finally a cycle 72'C extension 5 min. PCR products electrophoresis, photographed. CMIAS an 8 electrophoresis image analysis system, to detect the actin ANGPTL4 and p an integral optical density value, ANGPTL4 with p an internal reference actin integrated optical density divided, resulting figure is the bite of each group ANGPTL4m Rent A expression, two were proportional. Primer sequences bad, one as follows: ANGPTL4: F SGGCGAGTTCTGGCT'3GGTCT3 329bP R STGGCCGTTGAGGTT [Fen GAATG3 is a aetin: F SAGCGGGAAATCGTG (: GTGAC3 5 1 lbP R SAAGCATTTGCGGTG (owned ACGAT3

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