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Icariin Regulate The Expression Of Glucocorticoid Receptor GR

Posted on:2008-10-16Degree:MasterType:Thesis
Country:ChinaCandidate:X Y LiFull Text:PDF
GTID:2144360218459113Subject:Pharmacology
Abstract/Summary:PDF Full Text Request
Objective:Inducible suppression of GR using a combination of the RNA interference and the Cre-LoxP system to validate the inhibition by the constructed RNAi vector in mouse macrophage RAW264.7 cells.Then used traditional Chinese drug Yinyanghuo's monomer ingredient icariin to cultivate the cell, observed the change of GR to validate the relationship of GR and tranditional Chinese medicine warming kidney to invigorate yang.Methods:Establish eukaryotic expression vector of glucocorticoid receptor GR, the recombinant were designed and established by targeted gene GR and plasmid pBS/U6-Neo based on GR mRNA sequence.Two pairs of oligonucleotides were synthesized and inserted into plasmid pBS/U6-Neo to generate siRNA eukaryotic expression vectors. the recombinant sequences were identified by PCR and sequenced.The mouse macrophage RAW264.7 cells stably transfected with Cre-ERT2 plasmid and GR-siRNA recombined expression plasmids, used 4-HT to induce the expression of Cre-ERT2, reduced GR expression as measured on mRNA and protein level by RT-PCR and Western blot. Used the icariin to cultivate the cell whose GR expression is lower than the normal cell, reduced GR expression as measured on mRNA and protein level by RT-PCR and Western blot, to validate the regulation of GR expression by icariin.Results: Established GR-siRNA recombined expression plasmids , which significantly suppressed target gene GR expression. But the icariin almostly has any capability to change the GR expression. Statistical analysis shows that GR mRNA expression of ecombined expression plasmid E and F has significant deviation(p<0.01), GR mRNA expression has been cut down 62% and 73%.Conclusion: Succeedingly constructed GR-siRNA expression plasmids to use in further study the relationship of GR and deficiency of kidney YANG and cultivate transgenic mouse, but the icariin has not the capability to change the GR expression. It is possible that monomer ingredient of warming kidney to invigorate yang traditional Chinese drug has not the capability to cure deficiency of kidney YANG.
Keywords/Search Tags:Glucocorticoid receptor (GR), RNA interference, Cre-LoxP system, mouse macrophage RAW264.7 cells, icariin
PDF Full Text Request
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