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Construction Of Eukaryotic Expressive Vector Of Ri Gene And The Effects Of The Transfected Pcdna3.1-RI On Huvec Cells

Posted on:2013-06-10Degree:MasterType:Thesis
Country:ChinaCandidate:H Y LiFull Text:PDF
GTID:2234330374977852Subject:Cell biology
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Objective To clone and construct an eukaryotic expressive vector ofribonuclease inhibitor(RI)gene, as well as to observe the effects of thetransfected PCdna3.1-RI on the growth of HUVEC cells and the relationbetween RI and ANG.Methods A segment of RI gene augmented by RT-PCR was digestedby special enzyme and cloned into pcDNA3.1–. The vectors ofpcDNA3.1–RI and pcDNA3.1–were transfected into HUVEC cellsrespectively. The mRNA and protein expression level of the RI, ANG,MMP-2and MMP-9gene were tested by RT-polymerase chain reaction(PCR), Western blotting the relationship of ANG mRNA was detected byCO-IP method. HUVEC cells proliferation ability were detected by MTTand cell flow cytometry.Results Enzyme digestion and DNA sequencing verified theconstructed vectors were correct. In the control group (PCdna3.1-groupand the comparison group, no plasmid) mRNA and protein expression ofRI of the experimental group were significantly increased (P<0.05), the levels of ANG mRNA and protein expression was not obvious, the levels ofMMP-2, MMP-9protein expression were lowered (P<0.05), and the cellproliferation activity was significantly reduced (P<0.05), inhibited in theG1-S phase. ANG and RI were combined in internal cells detected byCO-IP method.Conclusion The eukaryotic expressive vector of RI gene wasconstructed successfully, which can increase levels of RI mRNA andprotein expression. The ANG gene can be directly decreased at thetranscriptional level by RI, and RI inhibited HUVEC cell proliferationability significantly.
Keywords/Search Tags:ribonuclease inhibitor, human umbilical vein endothelialcells, angiogenin, eukaryotic expression
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