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1、 Medical Cell Biology Chapter 3,18 Research methods of Cell biology and Cell engineering Microscope technology Cell isolation and cell culture Isolation and purification of cellular componentCytochemistry and labeling in alive cells Techniques in genomics Structure determination of biomacromoleculeC

2、ell engineering 一一、Microscope technology 人眼：人眼：100m;光學光學顯微微鏡：0.2m；電子顯微鏡：；電子顯微鏡：0.2nm（一）（一）Light microscope technique Resolution(分辨率分辨率)：區(qū)分相近兩點的最小距離：區(qū)分相近兩點的最小距離 0.2m 1.Light microscope Fixation embedding section staining 2.Fluorescence microscope吖啶橙熒光染色（血涂片）吖啶橙熒光染色（血涂片）吖啶橙吖啶橙/溴化乙錠雙熒光染色溴化乙錠雙熒光染色 acrid

3、ine orange/ethidium bromide(AO/EB)3.Phase contrast microscope(相差顯微鏡相差顯微鏡)4.Dark-field microscope（暗視野顯微鏡）（暗視野顯微鏡）5.Microcinematography（顯微電影攝像術）（顯微電影攝像術）6.Confocal laser scanning microscope （共聚焦顯微鏡）（共聚焦顯微鏡）（二）（二）Electron microscope technique 1.Transmission electron microscope(透射電子顯微鏡透射電子顯微鏡)電子顯微鏡：電子顯微

4、鏡：0.2nm2.Scanning electron microscope(掃描電子顯微鏡掃描電子顯微鏡)3.Metal shadowing (金屬投影法金屬投影法)冰凍斷裂法（冰凍斷裂法（freeze-fracture)冰凍蝕刻冰凍蝕刻(freeze-etching)（三）（三）Scanning probe microscope(掃描探針顯微鏡掃描探針顯微鏡)Scanning tuneling microscope(掃描隧道顯微鏡掃描隧道顯微鏡)Atomic force microscope(原子力顯微鏡原子力顯微鏡)（四）（四）Super-resolution microscope(超分辨

5、顯微鏡超分辨顯微鏡)二、二、Cell isolation and cell culture(一一)Cell isolation 1.Physical and biological property 2.Fluorescence activated cell sorter(FACS,熒光熒光激活細胞分選儀激活細胞分選儀)3.Magnetic-activated cell sorting(MACS,免疫磁珠分免疫磁珠分選技術選技術)4.Laser capture microdissection(激光捕獲顯微切割激光捕獲顯微切割技術技術)(二二)Cell culture 三、三、Isolation

6、and purification of cellular component1.Differential centrifugation(差速離心法差速離心法)2.Velocity sedimentation(速度沉降速度沉降)3.Equilibrium sedimentation（平衡沉降）（平衡沉降）4.Column chromatography(柱層析柱層析)5.SDS-PAGE(SDS聚丙烯酰聚丙烯酰胺凝膠電泳胺凝膠電泳)6.Isoelectric focusing(等點聚集電泳等點聚集電泳)7.Two-dimensional electrophoresis(雙向電泳雙向電泳)8.Iso

7、lation,purification and identification of nuclear acidDifferential centrifugation(差速離心法差速離心法)Electrophoresis(電泳電泳)Agarose gel electrophoresis picture四、四、Cytochemistry and labeling in alive cells 1.Enzyme cytochemistry(酶細胞化學技術酶細胞化學技術)2.Immunocytochemistry(免疫細胞化學技術免疫細胞化學技術)一抗一抗：rb,mouse二抗：羊抗兔，羊抗鼠二抗：羊抗

8、兔，羊抗鼠用兔子用兔子IgG去免疫綿羊去免疫綿羊標記熒光素標記熒光素(一一)Cytochemistry3.Autoradiography(放射自顯影技術放射自顯影技術)放射性標記的有絲分裂標記法放射性標記的有絲分裂標記法放射性標記的有絲分裂標記法放射性標記的有絲分裂標記法（percentage labeled mitosespercentage labeled mitoses，PLMPLM）：原理是對測定細胞進：原理是對測定細胞進行脈沖標記、定時取材、利用放行脈沖標記、定時取材、利用放射自顯影技術顯示標記細胞，通射自顯影技術顯示標記細胞，通過統(tǒng)計標記有絲分裂細胞百分數(shù)過統(tǒng)計標記有絲分裂細胞百分

9、數(shù)(PLM)的辦法來測定細胞周期的辦法來測定細胞周期.(二二)Labeling in alive cells用鈣離子探針觀察細胞內鈣離子的動態(tài)變化用鈣離子探針觀察細胞內鈣離子的動態(tài)變化1.Ion microprobe 2.Green fluorescent protein(GFP,綠色熒光蛋白綠色熒光蛋白)238 AA450-490nm520nmAdv Rem was used to infect Hep G2 cells Hep G2controlHep G2Infection Adv RemCy3-cy5,Alexa546-Alexa647;CFP-YEP,BFP-EGFP3.Fluore

10、scence resonance energy transfer,FRET(熒熒光共振能量轉移光共振能量轉移)4.Single molecular fluorescence imaging 五五.Techniques in genomics 1.Blotting （印跡雜交）（印跡雜交）(一一)quantitative analysis the gene expression Western blot 2.In situ hybridization（ISH,原位雜交原位雜交)3.Polymerase chain reaction（PCR,聚合酶鏈式反應）聚合酶鏈式反應）(二二)Over-exp

11、ression and knock down of gene expression 1.Over-expression 2.RNA interference(RNA干擾干擾)(三三)Interaction between proteinsImmunoprecipitation (免疫沉淀免疫沉淀)1.Regular 2.Yeast two-hybridizaiton(酵母雙雜交酵母雙雜交)3.Phage display(噬菌體展示噬菌體展示)(四四)Interaction between protein and nuclear acid 1.Chromatin immunoprecipitat

12、ion(ChIP,染色質免疫染色質免疫沉淀技術沉淀技術)2.Ultraviolet crosslinking and immunoprecipiation(CLIP,紫外交聯(lián)免疫沉淀技術紫外交聯(lián)免疫沉淀技術)(五五)Biochip(生物芯片生物芯片)Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail.(六六)Proteome(蛋白質組蛋白質組)(七七)Deep sequencing (深度測序深度測序)DNA序列測定的條件序列測定的條件：1.能分

13、辨一個堿基差別的單鏈能分辨一個堿基差別的單鏈DNA2.形成一系列的單鏈形成一系列的單鏈DNA(DNA polymerase)英國人英國人 Sanger 雙脫氧璉末端合成終止法雙脫氧璉末端合成終止法ATCGGGCCAATTCCTAATCGGGCCAATTCCTATCGGGCCAATTCCATCGGGCCAATTC ATCGGGCCAATTATCGGGCCAATATCGGGCCAAATCGGGCCAATTCCTAATCGGGCCA引物引物模板模板(八八)Animal Model (模式動物模式動物)Transgenic mice Gene knock-out mice 六六.Structure d

14、etermination of biomacromoleculenuclear magnetic resonance spectroscopy（NMR,核磁共振光譜儀核磁共振光譜儀)X-ray diffraction(X-射線衍射技術射線衍射技術)七七.Cell engineering（一）（一）Techniques in cell engineering 1.Bulk culture(大規(guī)模培養(yǎng)大規(guī)模培養(yǎng))Suspension culture Immobilization culture Microcarriers culture Three-dimensional cell culture

15、 2.Cell fusion(細胞融合細胞融合)3.Nuclear transfer(細胞核移植細胞核移植)4.Gene transfer (基因轉移基因轉移)（二）（二）Application of cell engineering 1.Monoclonal antibody 2.Pharmaceutical protein3.Cell therapy Characterization Cell proliferation(BrdU,PCNA)Cell viability/death(Live/Dead staining)Cell differentiation -Neurogenesis

16、Neuronal generation(TuJ1,MAP-2,RT-PCR)Ion channels/receptors(Ca2+imagimg)Synaptic activity(FM1-43)-Gliogenesis Glial generation(GFAP,O4,RT-PCR)TransplantationMechanical dissociation Stem cellsLVIIIIVSCccRat Embryos Cell-Collagen Constructs Type I Collagen Cell-Hydrogel ConstructsProliferativeNeural Stem and Progenitor CellsDifferentiatedNeuronal NetworksBrain-likeTissues SyntheticNanofibers 100mmRotary Wall Vessel BioreactorBGFAP(red)/BrdU(green)ControlTransplanted