CationicliposomesaretrADItionallyusedforthedeliveryofgeneticmaterialssuchasvarioustypesofDNA(pDNA,CDNA,CpGDNA,oligonucleotide,antisenseoligonucleotide,etc.),varioustypesofRNAsuchas(siRNA,mRNA,etc.)andnucleicacidmimics(NAMs). TheencapsulationofDNAintotheconventionalneutralchargedPCbasedliposomescanbeatechnicalproblemmainlyduetotheplasmidsize.Duetothisprobleminlate80s,theliposomescomposedofcationiclipidsandPEhavebeendeveloped.TheideawastoneutralizethenegativechargeofpDNAwithpositivechargeofcationiclipidsinordertocapturemoreplasmidefficientlymainlyduetoelectrostaticinteractionanddeliverthemintothecells.Generally,theprocedureissimplybasedonmixingthecationicliposomeswithDNAorRNAandaddingthemtothecells.Thisresultsintheformulationsofaggregates.
Inordertodesignapropercationiclipidforgenedelivery,twoapproacheshavebeenusedforthecationiclipidsynthesis:1)cholesterol-baseddesignsuchasDC-CholesterolandGL-67lipids,and2)non-cholesterol-baseddesignssuchasDOTAB,DDABandDOTMA. Tosuccessfullytransferthegeneinvitrousingliposomes,someconsiderationshouldbetakenintoaccount: i)theABIlityofbindingandpackingDNA/RNAinliposomes; ii)theinteractionofthepackagedDNA/RNAtothecellsurface; iii)theefficiencyoftheinternalizationofDNA/RNA; iv)theintracellularDNA-releasefromendosomesincaseofendocytosisinvolvement; v)thetransgenicexpressionlevelincellnuclei. pH-sensitiveliposomeshavebeendesignedbasedontheirtendencytoreleasetheircontentintheacidiccondition.TheprimaryconceptwasbasedonvirusesthatfusewiththeendosomalmembranebymeansofaproteinatpH5-6,deliveringtheirgeneticmaterialtothecytosolbeforereachingthelysosomes. Typically,apH-sensitiveliposomeconsistsofdioleoylphosphatidylethanolamine(DOPE).Sincephosphatidylethanolamine(PE)changesinacidicconditions,itisbelievedtoactasamembranefusionpromoter. Theeffectivenessoftheinteractionbetweenliposomesandcellsishighlydependentontheliposomecompositions.Liposomesarecapturedbyvariousendocytosicprocesses,andtheefficiencydependsonthecelltypeandliposomesize.Liposomesofvarioussizesandchargescanattachtothemacrophagesandneutrophilsthroughactivephagocytosis.Afterattachmentoftheliposometothecellsurface,theinternalizationintotheendosomesoccursduetoamoreacidicpH(6.50)atearlyendosomes.TheliposomesaretransferredtothelastendosomewithmoreacidicpH(5.5-6.0)bymaturationorvesicularfusion,whichtakes10-15min.Twentyminutes(ormore)afteruptake,thecontentsaredeliveredtothelysosomewithpH5.0orless.Lysosomesarethemaindegradingandlastendocytoticsectionintheendocytoticpathway,inwherepH-insensitiveliposomesareaccumulatedanddegraded.However,afterpenetrationofpH-sensitiveliposomesintocells,theaccumulationanddegradationdonotoccur.
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Bone Mesenchymal Stem Cells 作为一个细胞群体，还没有发现有特定细胞表面marker. 对于那些可以代表自我更新和分化的marker, 也不清楚到底要发现哪一个的表达才能确定该细胞就是BMSC。
目前常用的方法，就是采用培养，colony-forming unit-fibroblasts (CFU-F)这个方法。一般BMSC可以24-48小时贴壁。
流式细胞计数，比如STRO-1，但是一般认为STRO-1阳性的细胞更趋向于造血干细胞，和BMSC简单区别还不是很清楚。
这里有个培养分化的产品
http://www.rndsystems.com/pdf/SC020.pdf
GlucosestarvationcausestranslocationofAMPKβ2tothelysosomeinHEK-293cellsthatisdependentonN-myristoylation.Theexperimentwasperformedinβ2KOcellsasinFig.1c,exceptthatthelysosomalMarkerLAMP1(taggedwithRFP)wasco-expressedwiththewild-typeormutantAMPKβ2.Upperpanelsshowmergedimagesstainedblue(4′,6-diamidino-2-phenylindole(DAPI),nuclei),red(LAMP1,lysosomes)andgreen(AMPKβ2,detectedusingantibodyvalidatedine),incellsincubatedwithorwithoutglucosefor20 min.Lowersmallpanelsaremagnificationsoftheareasindicatedbydashedboxesintheupperpanels,showing(LtoR)redandgreenchannelsandmergedimages.
下面的这段话是图注，图注的意思我明白，但是我想知道merge后的图看什么颜色的荧光，蓝色是细胞核，红色是lysosome(位于胞质），绿色是AMPKβ2，该实验是想观察AMPKβ2是否转位到lysosome上了，如果确实发生了AMPKβ2转位到lysosome上，那么merge后是红色与绿色融合在一起，是吗？融合在一起发什么颜色的光了？
