Down-regulating The Expression Of CENP-E Gene Has An Influence On The Apoptosis Of PC12Cells Induced By Lactacystin

PART IRNA INTERFERENCE SUPPRESSION OF CENP-E GENEEXPRESSION IN PC-12CELLS1、objective：To construct the recombinant,which can inhibit the expression ofCENP-E gene, transfected recombinant expression vector into PC12cell;observethe expression of CENP-E gene in the cell.2、 Methods： Design and construct three Plasmid vctors containing shRNAtargeting CENP-E gene.The recombinant can Down-regulate the gene.Transfetedthe recombinant into PC12cell．Incubate for24hours.Fluorescence microscopewas used to evaluate the efficiency of transfection;the Western Bloting andimmumohistochemical stain were used to evaluate the expression of the CENP-E.3、Results： Three pshRNA-CENP-E Plasmid vctors were constructedsuccessfully. the efficiency of transfection is about72.5%. Western Bloting andimmumohistochemical stain show that: the expression of CENP-E in the celltransfected by the third recombinant is lower than others.4、Conclusion：the third recombinant can inhibited the expression of CENP-E in the PC12cell. PARTⅡNGF induced PC12cells neuron-like differentiation andlactacystin induced them to be cell model of Parkinson’sdisease1、objective：Induced PC12cells neuron-like differentiation and establishment ofcell model of Parkinson’s disease.2、 Methods： PC12cells were exposed to NGF whose concentration is50μg/L,incubated for24h. Devide the cells into five groups which were treaded withlactacystin of different concentrations. There are5、10,20and30μM·L-1lactacystin groups and control group which was added normal culture me-dium.,Incubate for24h. MTT was used to meansure the cell survival rate. stainedwith HE.Observe the structure under the microscope. Then devided the cells intothree groups:control gloup、treaded with double distilled water gloup、treadedwith lactacystin whose concentration can induce cells to apoptosis, stained cellswith immuneistochemistry.3、 Results:After incubating in the medium containing NGF， there are manysynapses on the surface of PC12cells.The cells look like neuron cells.Aftertreaded with lactacystin, the survival rate decreased significantly in10μM、20μM and30μM goulps，comparing with the control group.HE staining show:thesynapses in the control、5μM groups are long，the cell body is complete.nevertheless in groups of10μ M、20μ M and30μ M， the cell densitydecreased， the synapses reduced or fracture， nuclear condensate. Immuohistochemical stainin shows the α-synuclein increases in the goulp whichtreaded with lactacystin comparing with the control group and the doubledistilled water gloup.4、 Conclusion： The NGF of50μ g/L can induce PC12cells to neuro-likedifferentiation.Lactacystin can induce the cells apoptosis.The Lactacystin whoseconcentration is10μM can reduce survival rate of the cells significantly and itcan make the α-synuclein increase in the cell. So we can used lactacystin toestablish the cell model of Parkinson’s disease PARTⅢDown-regulating the expression of CENP-E gene has aninfluence on the apoptosis of PC12cells induced bylactacystin1、objective： To study the affection of inhibits expression of the centromereprotein E (CENP-E) to the PC12cells which were treated withlactacytin.Observed the relationship of the expression between the CENP-E andother enzyme in the cell. This experiment provide a theoretical basis for study ofthe pathogenesis of Parkinson’s disease.2、Methods: PC12cells were seeded in plates with6wells，nerve growth factor(NGF) was added， incubate for24hours，some of the cells were dealedwith lactacystin to make them for PD’s cell model. the cells were divided into6groulps: the control group (A)、 empty vector transfecting group (B)、ShRNA vector transfecting group (C)、PD’s cell model group (D)、emptyvector transfecting PD’s cells model group (E)、ShRNA vector transfecting PD’s cell model group (F). MTT was used to meansure the cell survival rate,TUNEL assay was used to detect apoptosis, stained with HE andimmuneistochemistry. The expression of tyrosine hydroxylase (TH) and acet--ylcholinesterase (AchE) are analysis by Westernbloting.3、Results:1、 MTT: survival rate of group C、 D、 E、 F had decreasedsignificantly (P <0.05) comparing with the control group.2、TUN EL: positivecells of apoptosis in group C、D、 E、F increased significantly comparing withthe control group (P <0.05).3、HE staining showed that there were obviousultrastructure changes related to apoptosis in group C、 D、 E、 F.4、immuneohistochemical appraisal found: the positive cells of CENP-E in groupC、 D、 E、 F decreased comparing with the control group(P <0.05). α-synuclein in group A、B、 C had no difference；Positive cells in group D、E、 F increased significantly (P <0.05) after lactacystin was added.5、Westernbolting show tyrosine hydroxylase (TH), acetylcholine ester (AchE) expressionis not affected by knocking out CENP-E,but it will decreased after lactacystinwas added and the immuneistochemistry experiment of TH、AchE comfirmthe result of western bolting.4、Conclusion:1The expression of CENP-E in PD’s cell model is lower than in thecontrol group.2、Inhiter the expression of CENP-E can significantly reducesurvival rate of the normal PC12cells and PD’s cell model; cells reduce byapoptosis.3、 Inhiter the expression of CENP-E does not increase the α-synuclein in the cells.4、Inhiter the expression of CENP-E has not decreasedthe expression TH and AchE, unless the nerve poison was added...