醫(yī)學(xué)細(xì)胞生物學(xué)：細(xì)胞生物學(xué)技術(shù)

1、細(xì)胞生物學(xué)的研究方法細(xì)胞生物學(xué)研究的方法細(xì)胞的顯微結(jié)構(gòu)細(xì)胞的亞顯微結(jié)構(gòu)分子生物學(xué)上觀察結(jié)構(gòu)與功能的統(tǒng)一結(jié)構(gòu)與功能細(xì)胞的生命活動與現(xiàn)象（本質(zhì)是不同基因在不同時間與空間的表達(dá)與動態(tài)變化）觀察細(xì)胞的手段與方法細(xì)胞組分分離的方法蛋白質(zhì)分離純化與鑒定的方法(柱層析，電泳，等）（屬于生物化學(xué)）分子生物學(xué)的方法DNA 操作（PCR， Southern blot，in situ hybridization)RNA 應(yīng)用(RT-PCR, Northern blot, siRNA)Protein over-expression in a cell細(xì)胞培養(yǎng)及細(xì)胞分離transformationTransfectio

2、ns(3種）infection要求了解的技術(shù)與方法Protein down-expression in a cell (siRNA)從大到小蛋白質(zhì)相互作用的研究技術(shù)Co-IPYeast two-hybridization其它生物芯片技術(shù)cDNA microarray蛋白質(zhì)組學(xué)Mass spectrometry 細(xì)胞內(nèi)分子定位：酶細(xì)胞化學(xué)技術(shù)免疫化學(xué)技術(shù)放射自顯影細(xì)胞內(nèi)分子示蹤離子探針GFPFRETDNA 文庫，cDNA文庫轉(zhuǎn)基因動物報告基因技術(shù)及應(yīng)用細(xì)胞生物學(xué)的一些常用的特殊方法蛋白質(zhì)與染色質(zhì)的相互作用（ChIP assay)觀察細(xì)胞的技術(shù)1. 什么是顯微鏡的分辨率？2.下述公式含義何在？R=

3、0.61/n*sin能分辨出兩點(diǎn)間的最小距離。3. 光學(xué)顯微鏡的最大放大倍數(shù)？最大放大倍數(shù)=人眼的分辨率（100um)顯微鏡的分辨率（0.2um)=5003. 為什么相差顯微鏡可我們觀察的圖像更清晰 ？相差顯微鏡常用于觀察活細(xì)胞4. 暗視野顯微鏡通過散射光成像原理是什么？5. 熒光顯微鏡跟普通光學(xué)顯微鏡有何異同？6. 熒光顯微鏡有何用途？抗體可用于檢測特異的分子Primary antibody: rabbit antibodySecondary antibody: goat anti-rabbit IgG免疫綿羊得到抗體純化后標(biāo)記吖啶橙熒光染色（血涂片） 吖啶橙/溴化乙錠雙熒光染色 acrid

4、ine orange/ethidium bromide (AO/EB) 6. 什么是共聚焦顯微鏡？ 跟熒光顯微鏡相比它有何優(yōu)越性？為什么用共聚焦顯微鏡可顯示一個立體的畫面？共聚焦顯微鏡可通過慮出不聚集的光從而提供清晰圖像7. 電子顯微鏡的成像原理跟光學(xué)顯微鏡有何異同？為什么電子顯微鏡的分辨率比光鏡的高？R=0.61/n*sin可見光波長=500nm電子束的波長？10萬V的電子波長=0.04nm理論分辨率=0.02nm8. 你知道哪些電子顯微鏡應(yīng)用相關(guān)的技術(shù)？Specific Macromolecules can be localized by immunogold electron micri

5、scopyMetal shadowing allows surface features to be examined at high resolution by Transmission electron microscopy投射電鏡所用的一些方法金屬投影法（metal shadowing)冰凍斷裂法（freeze-fracture)冰凍蝕刻(freeze-etching)9. 為什么掃描電子顯微鏡常用于觀察生物樣品表明的立體結(jié)構(gòu)？10. 電子顯微鏡的3維重構(gòu)是怎樣操作的？Different views of a single object can be combined to give a

6、 three-dimensional reconstruction CT (computed tomography)Electron-microscopy (EM) tomography（EMT)細(xì)胞培養(yǎng)需要哪些基本的條件？操作：嚴(yán)格無菌（ 細(xì)胞的培養(yǎng)基、培養(yǎng)液、及培養(yǎng)器皿都嚴(yán)格無菌）細(xì)胞生長需要營養(yǎng)及生長因子（培養(yǎng)基，血清), 經(jīng)常需要加抗生素細(xì)胞培養(yǎng)的外界條件：CO2 5%， 37C， 一定的相對濕度什么是原代培養(yǎng)，什么是傳代培養(yǎng)？什么是細(xì)胞系（cell line)? 什么是細(xì)胞株（cell strain）怎樣分離細(xì)胞？ 怎樣純化細(xì)胞？分離細(xì)胞：用胰蛋白酶，EDTA去處理小組織塊1.差速離

7、心或密度梯度離心（粗分離）2.流式細(xì)胞技術(shù)細(xì)胞分離純化流式細(xì)胞儀的其它應(yīng)用（分析）細(xì)胞周期分析細(xì)胞凋亡分析原理？細(xì)胞周期2n4n G1G2+M細(xì)胞凋亡3.根據(jù)細(xì)胞表面抗原的情況進(jìn)行細(xì)胞純化(磁珠分離法）4.激光捕獲顯微切割技術(shù)什么是細(xì)胞融合技術(shù)， 單克隆抗體是怎樣制備的？什么是胚胎干細(xì)胞，它有什么應(yīng)用前景？胚胎干細(xì)胞的概念 Embryonic stem cell, ESC 構(gòu)建基因敲除動物（knockout animal）擴(kuò)大培養(yǎng)用作基因敲除動物Positive and negative selectionNeoTKXXXneoTK(thymidine kinase)ganciclovir d

8、erivativeKill the cell+-什么是IPS 細(xì)胞，它有什么應(yīng)用前景？iPS,Induced Pluripotent Stem Cells Yamanaka at Kyoto University Oct3/4, Sox2, c-Myc, and Klf4 Junying Yu, James Thomson at University of WisconsinMadison Oct4, Sox2, Nanog and Lin28 如何對細(xì)胞的組分進(jìn)行分離？差速離心分離體積、質(zhì)量差別較大的顆粒速度沉降分離沉降系數(shù)不同的顆粒平衡沉降分離密度不同 的顆粒Velocity sedime

9、ntationEquilibrium sedimentation 層析法分離蛋白質(zhì)蛋白電泳SDS聚丙烯酰胺凝膠電泳依據(jù)分子量的不同分離蛋白質(zhì)等點(diǎn)聚集電泳依據(jù)等電點(diǎn)不同分離蛋白質(zhì)雙向電泳可一次分離數(shù)千種蛋白鑒定蛋白質(zhì)在細(xì)胞內(nèi)表達(dá)的技術(shù)稱作什么？是怎樣實(shí)現(xiàn)的？Western blottingSteps for Western blot1. Prepare samples, extract protein solution form tissue or culture cells2. Resolve the proteins by SDS (SDS- polyacrylamide gel elect

10、rophoresis) 3.Transfer the resolved protein into a membrane (PDVF, Nitrocellulose) 4. Blocking 2% BSA (Bovine serum albumin ) or no fat milkTo block the unbinding site of the protein . To eliminate the non specific Binding .5. Detection 1)Primary antibody to recognize the specific protein 2)Secondar

11、y antibody labeled with Enzymes, isotope, Fluorescent Most popular used method is Enzyme labeling Alkaline phosphatase and horseradish peroxidase Chemiluminescence Substrate Light expose to a photograph filmDevelop into a bandAvian Leukosis Virus 什么是 放射自顯影技術(shù)，有何用途？放射自顯影技術(shù)（autoradiography)3H-亮氨酸，32S-蛋

12、氨酸標(biāo)記蛋白3H-胸腺嘧啶脫氧核苷、 3H-胸腺、 3H-尿苷標(biāo)記核酸放射性標(biāo)記的有絲分裂標(biāo)記法（percentage labeled mitoses，PLM）：原理是對測定細(xì)胞進(jìn)行脈沖標(biāo)記、定時取材、利用放射自顯影技術(shù)顯示標(biāo)記細(xì)胞，通過統(tǒng)計(jì)標(biāo)記有絲分裂細(xì)胞百分?jǐn)?shù)(PLM)的辦法來測定細(xì)胞周期.什么是離子探針？ 有何應(yīng)用價值？離子探針進(jìn)行細(xì)胞內(nèi)離子的實(shí)時檢測用鈣離子探針觀察細(xì)胞內(nèi)鈣離子的動態(tài)變化什么是報告基因，在細(xì)胞生物學(xué)中用什么用途？某個基因的產(chǎn)物易被檢測螢火蟲酶（luciferase）GFP （綠色熒光蛋白）綠色熒光蛋白用于顯示特定蛋白在細(xì)胞內(nèi)的定位綠色熒光蛋白（green fluores

13、cent protein, GFP)238 AA450-490nm520nmAdv Rem was used to infect Hep G2 cells Hep G2controlHep G2Infection Adv RemGFP， a report geneMonitor plamid existence, therefore, monitor the transfection rate. Monitor gene expressionDetecting the location of a protein by making GFP fusion proteinGFP fusion pr

14、oteinExpression of H2B-GFP before and after UV-B-induced apoptosis. A431 cells were stably transfected with H2B-GFP fusion protein (K/H2B-GFP). H2B-GFP is expressed only within the nuclei. In the right panel an apoptotic K/H2B-GFP cell is shown after UV-B irradiation. The “Blebs and bodies forming”

15、phase is shown. Noticeably, redistribution of SLE self antigens are among the remarkable changes that occur during apoptosis. Images were collected in real time by laser confocal microscopy.GFP expression in haematopoietic colonies from transgenic mice carrying the green fluorescent protein (GFP)-re

16、porter gene under the control of Kit (stem cell factor receptor) regulatory elements. These mouse lines provide useful means to mark rare adult multipotential cells which, upon transplantation, can be monitored for their ability to engraft, differentiate and regenerate different tissues. 什么是lucifera

17、se assay， 它有何用途？Using protein-protein interaction based transaction method.Luciferase assay C. Using protein-protein interaction based transaction method.Luciferase assay Luciferasefirefly螢火蟲B. Using protein-protein interaction based transaction method.Luciferase assay Luciferasefirefly螢火蟲Biolumines

18、cence (Chemiluminescence)Luciferase is the enzyme that catalyses the oxidation of luciferin, the basic substrate in bioluminescent reactions, producing a yellow- green light (560nm).JakGHRGHMembraneJakGHRGHmembraneinactiveactiveJakGHRGHmembraneinactiveactive+1OPENLightmRNA0f LucProteinProtein or pro

19、teins working on the promoterProtein 1Protein 2Protein or proteins working on the promoterProtein 1Protein 2Protein or proteins working on the promoterProtein 1Protein 2Protein or proteins working on the promoterProtein 1Protein 2Protein or proteins working on the promoterProtein 1Protein 2Protein o

20、r proteins working on the promoterProtein 1Protein 2觀察兩個蛋白質(zhì)之間相互作用的方法有哪些？最常用的是哪一種？免疫共沉淀熒光共振能量轉(zhuǎn)移（fluorescence resonance energy transfer, FRET）酵母雙雜交免疫共沉淀可驗(yàn)證蛋白-蛋白間的相互作用Immunoprecipitation (IP)RegularWestern blotCo-immunoprecipitation (co-IP)熒光共振能量轉(zhuǎn)移可實(shí)時觀察細(xì)胞內(nèi)蛋白與蛋白的相互作用熒光共振能量轉(zhuǎn)移（florescence resonance energy

21、 transfer, FRET)Cy3-cy5, Alexa546-Alexa647; CFP-YEP, BFP-EGFPExcitation:402nmemission 475nmExcitation: max395nm和min470nm Emission:509nm What is FRET？熒光共振能量轉(zhuǎn)移可實(shí)時觀察細(xì)胞內(nèi)蛋白與蛋白的相互作用熒光共振能量轉(zhuǎn)移（florescence resonance energy transfer, FRET)Cy3-cy5, Alexa546-Alexa647; CFP-YEP, BFP-EGFPExcitation:402nmemission 47

22、5nmExcitation: max395nm和min470nm Emission:509nm 酵母雙雜交用于篩選相互作用的蛋白質(zhì)Yeast Two-Hybrid Protein B and Protein P interactionProtein B Protein P 什么是small RNA interference 技術(shù)？ 這個技術(shù)有何用途？RNA 干擾技術(shù)Small interfering RNAsiRNA: small interfering RNAshRNA : small hairpin RNARNAi: RNA interferenceSmall interfering RN

23、A Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology .Most notably, it is involved in the RNA interference(RNAi) pathway where the siRNA interferes with t

24、he expression of a specific gene. StructureDiscoveryDiscoveryDavid Baulcombes group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants, and published their findings in Science in a paper titled A species of small antisense RNA in posttranscriptional gene silencing i

25、n plants Thomas Tuschl and colleagues , synthetic siRNAs were shown to be able to induce RNAi in mammalian cells, published in Nature How to design a siRNAAnnealing 什么是原位雜交（in situ hybridization, ISH)? 有何用途？什么是實(shí)時定量PCR技術(shù)，此技術(shù)有何用途？熒光實(shí)時PCR技術(shù)室檢測基因表達(dá)變化的常規(guī)技術(shù)mRNAcDNARTPCR模板基因表達(dá)的上調(diào)與下調(diào)技術(shù)用轉(zhuǎn)基因技術(shù)讓細(xì)胞某基因表達(dá)升高用轉(zhuǎn)基因技術(shù)

26、讓細(xì)胞內(nèi)某基因表達(dá)降低Knock outKnock down基因敲除敲出什么是cDNA 文庫，什么是DNA文庫？DNA文庫和基因克隆是實(shí)驗(yàn)室常用的工具與方法Genomic DNA libraryPlasmid, cosmid cDNA library什么是cDNA microarray？ 有何應(yīng)用前景？Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. 蛋白組學(xué)用的一般方法是什么？蛋白質(zhì)組學(xué)研究技術(shù)Mass spectrometry（質(zhì)譜） 鑒定某個蛋白質(zhì)與某段特殊的染色質(zhì)或DNA片段相互作用的方法是什么?是怎樣操作的？ChIP: chromatin immunopreci