微米晶片在细胞生物学上的应用.ppt

微米晶片在細胞生物學上的應用,細胞研究:細胞研究的方向。細胞生長環境。細胞微環境的因素。生物科技的劣勢。演講者:陳俊男07.27.2005,生物技術,DNA:agarose電泳(McDonell,1977),Southernblot,clone,PCR(1971).RNA:RT-PCR(1988),Northernblot(1994).Protein:SDSpage(1960),westernblot(1979)Proteomics:2DElectrophoresis(1970s).,ProteomicsProcesses,生物科技的缺點:1.耗時2.費人力3.高成本4.無法在微米尺度細操控細胞,細菌5.無法及時觀徹細胞生理狀況(realtime),微米級晶片的新工具-微流道,微流道晶片(lab-On-aChip)陣列式晶片晶片的應用,55C.,72C,95C.,cBLOOD-ON-A-CHIP,C.rightpole:MitotrackerGreenFM;leftpole:MitotrackerRedCM-H2XRos.entirecell:DNA-bindingdyeHoechst33342.D.2.5hrslaterE.Blueregion:latrunculin=causethedisruptionofactinfilamentF.PhalloidinAlexa594-labelled,and10minlatter,treatmentwithlatrunculinA.,Figure1Chemotaxis.,Figure2ElastomericMembranes,(Bovineadrenalcapillaryendothelialcell),Figure3.(a)Acellspreadonasquare30um30umisland;thecellwasstimulatedwithplatelet-derivedgrowthfactor(PDGF)andstainedforF-actinwithfluorophore-labeledphalloidin.(b)Cellsconfinedtovariousshapes(allwithareasof900um2)werestimulatedwithPDGFandstainedwithphalloidinand4-6-diamidino-2-phenylindoletovisualizeF-actin(green)andnuclei(blue).Imagesobtainedbyfluorescencemicroscopy.,18minofalivingfibroblastafterstimulationwithPDGF.fibroblast,coatedwithfibronectin,stainedwithfluoresceinatedphalloidin(C)Endothelialcell,coatedwithfibronectin,stainedwithfluoresceinatedphalloidin(D)skeletalC2C12myoblast,coatedthrombospondin-1,stainedwithanti-fascinantibodies.A,B)3030misland;C,D)4040mislands.,Figure4.Elastomericsiliconemicroposts-asubstratetocellularcontractions.(a)cellsadheretothetipsofanarrayofcloselyspaced.(b)SEMshowingacellattachedtotheentiremicropostsubstrate(c)illustrationshowingtheprocessformicroprintingadhesiveproteinsonthemicroposts.(d)Differentialinterferencecontrast(top)andimmunofluorescence(bottom)micrographs:2x2arrayofpostsprintedwithfibronectin.(e),cellsonlyattachonthetips.Scalebarsindicate10m.,Figure5.Bovinecapillaryendothelialcellswereinitiallyonrectangularpatternsusing(SAMs)monolayerssurroundedbyethylene-glycolterminated(EG-terminated)SAMs.Applicationofacathodicvoltagepulse(1.2Vfor30s)releasedthecellsfromtheconstraintsofthemicroislandsbydesorbingtheEG-terminatedSAMsandenablingproteinstoadsorbfromtheculturemediumthatallowedtheattachmentandmovementofcells.Thenumbersindicatethetimeelapsed(inminutes)afterthevoltagepulse.,Figure6.(a)Agaroseiswickedintochannelsformedbyapoly(dimethylsiloxane)stampsealedagainstaglasscoverslipandallowedtogelbeforethestampispeeledoff.(b)Whencellsareseededontothesesubstratescontainingbowtie-shapedwells,cellsattachandcultureaseithersinglecellsorpairs.(c)Thesinglecell-to-cellcontactformedinthesepairscanbeblockedbyfabricatingsubstratesinwhichtheagaroseformsathinwa