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Study on the Potentiality of Malignant Transformation of the C-kit Oncogene Mutation in Human Gastrointestinal Stromal Tumor

Author: XieQiang
Tutor: MaDaLie
School: Second Military Medical University
Course: Pathology and pathophysiology
Keywords: Gastrointestinal stromal tumors C-kit proto-oncogene Gene mutation Polymerase chain reaction ( PCR ) DNA sequencing Artificial mutation Gene transfection Cell proliferative activity Cell cycle Malignant transformation. Cell culture
CLC: R735
Type: Master's thesis
Year: 2003
Downloads: 67
Quote: 0
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Abstract


Objective: PCR method combined with DNA sequencing analysis of C-kit gene mutation type, trying to find a new mutation sites; construct the C-kit gene cDNA eukaryotic expression vector containing the mutation, transfected human embryonic kidney cell line and through the observation of stable recombinant plasmid transfected human embryonic kidney cells by observing the mutant C-kit gene on cell proliferation and cell cycle analysis of their functional status; reveal its vicious human cells tumorigenic in nude mice. Transformation. Method: 1 extracted from eight cases of GIST specimens containing C-kit gene mutation detected by PCR-SSCP prompt genomic DNA screening C-kit gene mutation sequences using DNA sequencing technology. By RT-PCR technology wild-type C-kit gene cDNA was cloned from a human fetal brain tissue. 3 according to the detected sequencing results, the in vitro mutation in the wild-type C-Kit cDNA mutant C-kitcDNA; obtain sexual function C-kit gene mutation according to the literature has been reported at the same time sequence, using in vitro mutagenesis methods to give the corresponding mutant C -kit cDNA, as a positive control. 4 of the mutant and wild-type C-kit gene cDNA was constructed on the cutting body pcDNA3 eukaryotic expression plasmid. 5 will be constructed in pcDNA3 mutant C-kit cDNA and wild-type C-kit cDNA (as a negative control) recombinant plasmid and empty pcDNA3 plasmid by liposome stable transfected HEK cells. C-kit protein expression was detected by Western blot, observing the growth curve of the transfected cells, cell proliferation was detected by MTT activity changes, and the detection of C-kit gene mutant of the cell cycle by flow cytometry. 6 observed stably transfected with C-kit gene mutations in HEK cells of the body in the tumorigenic in nude mice. Results: 1 C-kit gene mutation detection: 8 cases of GIST C-kit gene exon11 sequencing results show three cases with mutations: one cases of insertional mutagenesis, white codon Codon 579 insertion 12bp;, Codon 557 cases of mutation in -559 between the missing 6bp, resulting in codons 557 and 559 deletion mutation; were two point mutations, Codon560 aspartic acid valine mutation Codon Second Military Medical University Thesis Chinese shoot to 563 by the exclusive leucine mutation A acids. 2 reverse transcriptase cloned from fetal brain tissue in the area of ??the base the trapped GDNA encoding wild-type C-kit mutation sequencing results showed that it cloned cDNA fragment and Genbank Accession inch C gene cDNA coding region sequence; artificial mutation wild it cDM, type C \The 3 by downshear identified confirmed gene of wild and mutant C \4 stably transfected with mutant C - kit cDNA fragment HEK cells transfected with wild-type C-kit gene and empty vector HEK fine to do than, cell growth was significantly faster cell proliferation activity was significantly enhanced Cell cycle analysis showed: The the transfected C \control group, the proportion of cells in the proliferative phase of each group as follows: 48.34% (experimental group), 48.24% (positive control group), 42.03% (42.16% of the negative control group of people (blank control group Peng K). 5 stable transfection the plasmid and PCDNA3KIT State W species of of HRK fine touch tumor formation in nude mice, and transfection PCDNA3-KITW the blank PCDNA3 A body cells not a tumor. Conclusion: 14j1Ak, three cases outside the the detection to CAlt gene exon door mutations, including two cases of mutation sites are located in the position of the the Man Chau Man Road mutation hot, prompted the Chinese C \GI ST C ten it gene mutations parts of different complex type of mutation. fetal brain tissue RNA as a template RTPCR method seven wild-type C on CDNA cloning and use it as a template with the the CAn Codon579 insertion mutations in vitro mutations Codon559-560 mutation in the corresponding cDNA, the three BU the kit cD back A subcloned to pcDNA3 the BamHI and XhOI double digestion confirmed three C the \my body PCDNA3, lay the foundation for the C-kit gene function studies. 3 observation the gene mutant GDM C a t see K fine jump reproductive activity and cell cycle, the results show that the stably transfected C seven it mutant transfected blank PGD * 3 vector and transfected with wild-type C inch it CDNA 'Digest group HEK cell lines compared to cell growth faster, stronger proliferative activity in the proliferation of cells ratio was significantly higher in the Second Military Medical University, master's thesis Liu sexual function mutations described in the cases of GI ST C inch it gene mutation, prompted C-kit gene mutation proliferative activity of human cells can be significantly enhanced, and can make more cells by both the period of proliferation; gene expression of wild-type C with disabilities it on cell function had no significant effect. 4 with estern blot Western blot detection of plasmid pcDNA3 pcDNA3-KITw, pcDNA3-KIT dish, pcDM3KIT0W expression at XING K cells, results showed that transfection pc0M3 plasmid blank control group did not display strip, while the rest of the group are Ck it protein expression, thus confirmed that each of the recombinant plasmid has been transfected stably in HEK cells and expression of .5 percent mutant recombinant plasmid in HEK cells were injected A # mice, induction courses mouse tumor generated to be successful, to illustrate this example, seven C it gene mutation of fine Peng strong conversion effect, suggesting that the gene mutation C seven it may shut off the system caused by the malignant transformation of the GI ST a.

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