RNA Interference Gene Silencing Bax Gene Suppression Of Chondrocyte Apoptosis And Its Possible Mechanism

Objectives一、In this study using neonatal rat chondrocytes as a research object,Construction of Bax siRNA (small interfereing RNA, siRNA), Transfected ratchondrocytes cultured in vitro, By RT-PCR and Western blot analysis of itsprotein expression level.二、The application of siRNA technology, silence Bax gene expression invitro cultured chondrocytes, the purpose of the Bax gene and cartilage cellapoptosis and its regulatory mechanism.MethodsBorn5-7days SD rats of clean grade,either male or female rats weresacrificed,cut knee,hip cartilage in vitto were separated and cultured for1week or so,you can be subcultured.Passage2siRNA transfection experimentwas divided into a control group of Bax siRNA transfection on group andnegative control transfection group.Replacement of serum-free and dualanti-DMEM medium,siRNA and the lipofectarnine2000mixture,in a CO2incubator and incubated4-6h,and then continue to foster a culture mediumreplaced by fetal bovine serum.Collection of ex perimental cells in eachgroup,prepared a cell suspension of5×104/mL,seeded in96-well cultureplates,cells of a group of eight holes.Divided into control group,Modelgroup(join2mmol/L sodium nitroprusside SNP,the role of24h),the Model+BaxsiRNA group and the Model+Control siRNA group.Was detected by MTT cellviability,enzyme-linked immunosorbent detector detection at570nm at the absorbance value of each hole.Annexin V-FITC/PI double labeling method todetect apoptosis rate;Western blot analysis of Bax and Bcl-2,of cytochrome Cprotein expression.ResultsFirst, interfere wih detection of the Bax gene and protein expression levelsofTransfected after24h Bax mRNA expression was1.03±0.153in thecontrol group was1.836±0.164;the expression of the protein0.359±0.089,control group was0.766±0.082,the experimental group and controlgroup was signgicantly lowered.(P<0.01).Second,Bax gene intergerence on cell viabilityControl group,Model group,Mdel+Bax siRNA group and Model+ControlsiRNA group A570values were0.85±0.13,0.41±0.02,0.67±0.04and0.38±0.03.Compared with the Control group,Model group cell A570valuesdecreased(P<0.01)；compared with the Model group,Model+Bax siRNA groupincreased cell A570values (P<0.01);Model of Bax siRNA group compared toModel+Control siRNA group cells A570values decreased(P<0.01).Third, flow cytometry to detect apoptosisThe flow cytometry results showed that the control group,Model group,theModel+Bax siRNA group and the Model+Control siRNA group cell apoptosisrate:2.87%,20.07%,9.21%and21.19%respectively.Model of cell apoptpsisrate far higher than that of the control group (P<0.01);Model+BAX siRNAapoptosis rate was significantly lower than the Model group(P<0.01).Fouth,the Bcl-2and Cytochrome C protein expression level of detectionBcl-2protein is highly expressed in the Control group and Model+Bax siRNAgroup,the degree of Bcl-2protein expression in the Model and Model+contronlsiRNA group,significantly lower(P<0.01).Of cytochrome C protein expressionin the Control group and the Model+Bax siRNA group is weak,while in theexpression level of the Model group and Model+Control siRNA group significantly enhanced(P<0.01).Conclusions1. Using RNA interference technology to silence Bax gene by RT-PCRand Western blot analysis of Bax mRNA and protein levels.Of Bax siRNAtransfection24h can effectively silence Bax gene and protein expression.2. RNA interference silencing Bax gene can inhibit apoptosis in ratchondrocytes and promote their survival,the mechanism may be related to themitochondrial pathway.