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Stress Response Gene Himpl Interference

Posted on:2014-07-22Degree:MasterType:Thesis
Country:ChinaCandidate:H H HanFull Text:PDF
GTID:2250330425452799Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
Himp1is a kind of anti-stress gene and its protein is conserved highly,asubstantial increase in hypoglycemia and hypoxia caused by stress.Over expression ofHimp1in cells protects the cells from apoptosis induced by several stimuli andprolongs their survival.But in case of insufficient Himp1expression level, what theperformance of histiocytes has not been reported.This study is to use two differentRNA interference methods to reduce Himp1expression,and to study the resultingtarget cell response.In order to verify Himp1overexpression effect,we designedcloning primers,and cloned Himp1gene by RT-PCR method,and constructed theeukaryotic expression recombinant plasmid pcDNA3.1(+)-Himp1. After transfectedthe Hela and293Rb cell,Himp1gene mRNA expression levels detected byquantitative PCR.The results showed as follows the cells transfected gene Himp1gene that mRNA levels expression was significantly increased (p<0.01).Determinedby MTT method is used to detect Himp1genetic relationship with cell proliferation,result shows that cells the Himp1gene group proliferation higher than the controlcells. At the same time,DNA Ladder method is used to test in the10%ethanol,Himp1genetic relationship with apoptosis,results show that the transfection Himp1gene,apoptosis is less than the control group. Demonstrated that the Himp1gene expressioncan not only resist of apoptosis, and promote the role of cell proliferation.In order to study the effect of insufficient Himp1gene expression of cell, thisstudy adopts two interference methods of Himp1gene:one is dsRNA method, theother is a method of siRNA.dsRNA method:used in vitro transcription to synthetizeHimp1dsRNA,control green fluorescent protein EGFP dsRNA.PEGFP-N1and EGFPdsRNA transient co-transfection Hela and293Rb cells,using a fluorescencemicroscope, real-time fluorescent quantitative PCR detected the interference effect ofthe level of EGFP gene expression.The results show:the the Helaand293Rb cellsco-transfection with pEGFP-N1+dsRNA cells holes,the fluorescence reducesignificantly by about50%than single transfected pEGFP-N1cells holes,real-timePCR analysis of mRNA expression level of EGFP.The analysis results: the cellstransfected dsRNA can inhibit EGFP genes (p<0.01).Himp1and Himp1dsRNA transient co-transfection Hela and293Rb cells,using real-time fluorescent quantitativePCR detection the interference effect of the level of Himp1gene expression.Theanalysis results:the cells transfected dsRNA can inhibit Himp1genes (p<0.05).DsRNA interference experiment summary: When use pEGFP-N1plasmid transfectedHela and293cells,green fluorescence was detected in both cells.When pEGFP-N1plasmid with in vitro transcription to get EGFP dsRNA co-transfected and greenfluorescence weaken or disappear. We are using Himp1plasmid and Himp1dsRNAtransient co-transfected Hela and293Rb cells, using real-time fluorescent quantitativePCR detection the level of Himp1gene expression. Than the single transfected Himp1gene reduce by50%. The cell apoptosis after disturbance.In order to further study the Himp1gene of large animals, we have cloned pigHimp1gene by electronic clone,and constructed the eukaryotic expressionrecombinant plasmid pcDNA3.1(+)-Himp1. Used the methods of chemical synthesis,syntheticed siRNA1、 siRNA2、 siRNA3、 siRNA4、 non-sliencing, respectivelytransfected Hela cell,using real-time fluorescent quantitative PCR detected theinterfered level of Himp1gene expression. Results:transfected siRNA1caneffectively inhibit endogenous Himp1gene expression (p<0.05). Successful screenedeffective siRNA sequences.For the next step use pig Himp1gene specificityinterference, explore Himp1gene function laid a foundation.
Keywords/Search Tags:Himp1, anti-stress, overexpression, gene silencing, cell apoptosis
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