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Construction of Eukaryotic Expression Vector of siRNA Specific for STAT1 Gene and Its Influence on Expression of STAT1 Protein in 16HBE Cells

Author: JiaXiaoQin
Tutor: XiongZuo
School: Luzhou Medical College
Course: Internal Medicine
Keywords: STAT1 gene Plasmid Construction siRNA Transfection RNAi Airway epithelial cells (16HBE)
CLC: R562.25
Type: Master's thesis
Year: 2009
Downloads: 89
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Abstract


Purpose: signal transduction and transcription-activating factor 1 (STAT1) is the STAT family found a transcription factor, involved in signal transduction of IFN essential for innate immunity. Cytokines such as IFN-γ activation of STAT1 through the JAK-STAT pathway to induce the expression of the target gene, involved in cytokine-mediated signal transduction process, leading to inflammation and airway hyperresponsiveness, and bronchial airway inflammation in asthma is often associated with airway STAT1 signaling pathway in epithelial cell abnormalities. In this study, by design for STAT1 hairpin-like RNA interference recombinant IFN-γ stimulation of airway epithelial cells (16HBE liposomal transfection) study of the STAT1 protein expression in 16HBE cells detected interference activity, to lay the foundation to explore gene therapy for bronchial asthma and other inflammatory diseases. Method: The study is divided into two parts: STAT1-specific siRNA eukaryotic expression vector construction: According to the co-nucleotide sequence the GeneBank database provides the STAT1 gene of two variants, Tuschl design principles to select the design of double-stranded siRNA , the final choice of the two fragments as STAT1 gene interference fragment; another design of a random rearrangement by either a fragment from a negative control; then are converted to complementary DNA sequences capable of expressing the shRNA, to form double-stranded by annealing, to connect it eukaryotic expression vectors with kanamycin resistance and enhanced green fluorescent protein pGenesil-1.1, transformed into E. coli strain DH5α was amplified, extracted plasmid restriction enzyme digestion with SacI and do sequencing analysis. The recombinant plasmids were named pGSTAT1-1, pGSTAT1-2, and negative control pG-HK. STAT1 siRNA eukaryotic expression vector of STAT1 protein expression in 16HBE cells: the recombinant plasmid pGSTAT1-1 with liposomes 16HBE cells were transfected with a ratio of 1:1 to 1:5, and blank control group, a total of six groups, 24,48,72 h after transfection observed changes in cell morphology and fluorescence expression identified the transfection efficiency under different conditions; divided into four groups 16HBE cells stimulated with IFN-γ, respectively, after stimulation 0min , 30min, 12h, 24h mention of total cellular protein, Western blotting was detected after stimulation with IFN-γ STAT1 protein in the cell relative content; selection pGSTAT1-1 quality liposome volume 1μg: 2μl conditions divided into four groups turn dyed 30min, IFN-γ stimulate the 16HBE cell, respectively: empty plasmid group, pGSTAT1-1 group, pGSTAT1-2 group and negative control the pG-HK group 48 h after transfection, total protein was extracted and detected by Western blotting techniques the relative content of the STAT1 protein in each group, in order to identify RNA interference with the activity of the recombinant. Experimental data SPSS13.0 software for statistical analysis, ANOVA analysis and randomized block design analysis of variance between multiple number of samples for multiple comparisons using LSD method, P lt; 0.05, said the difference was statistically significance. Results: confirmed by restriction analysis and sequencing the recombinant of two STAT1 targeted RNA interference pGSTAT1, pGSTAT1-2, and a negative control pG-HK is properly connected, and successfully transfected into 16HBE cells. The pGSTAT1-1 with liposomes in different proportions transfected 16HBE cells 48 h after transfection 1:2 transfection group highest transfection efficiency of 58.21% (P lt; 0.01) by Western blotting analysis: discovery IFN -γ stimulated the 30min group STAT1 protein content was significantly higher than that stimulate the 24h group (P = 0.01), IFN-γ stimulated 30min group was not statistically significant (P = 0.531) compared with the stimulation of a the 12h group of cells STAT1 protein expression of IFN-γ stimulation 0min group difference was statistically significant (P = 0.000), with the remaining three groups of cells STAT1 protein expression. Reorganization interference plasmid transfection IFN-γ stimulation 30min 16HBE cells can reduce cell STAT1 protein levels after transfection 48h, pGSTAT1-2 inhibition rate of 57.62%, STAT1 protein content was significantly lower than the empty plasmid group and pG-HK group (P = 0.01), as well as significantly lower than pGSTAT1-1 group (P = 0.04); pGSTAT1-1 inhibition rate of 30.64%, the STAT1 protein relative content was significantly lower than the empty plasmid group the pG-HK group (P lt; 0.05), and empty plasmid group the pG-HK group difference was not statistically significant (P = 0.713). Conclusion: of STAT1 targeting of RNA interference reorganization body pGSTAT1 pGSTAT1-2 was successfully constructed, and able to efficiently transfected into 16HBE cells. IFN-γ stimulated 16HBE cells can increase the expression of STAT1 protein in the cells, and reached a peak and continue until 24h 30min. The STAT1 siRNA eukaryotic expression vector can inhibit the expression of STAT1 protein in 16HBE cells, while the choice of target sites to construct plasmid disrupting activity. 4 the STAT1 target RNA interference technology is expected to become the new method of asthma and other airway inflammatory disease gene therapy.

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CLC: > Medicine, health > Internal Medicine > Respiratory system and chest diseases > Trachea and bronchial disease > Bronchial disease > Bronchial asthma
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