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1、醫(yī)學(xué)論文■體外擴(kuò)增高純度的人外周血來(lái)源的NK細(xì)胞的研究作者李曉紅馬健汪菲菲竇立萍李猛高春記【摘要】本研究探討從人外周血中分選及擴(kuò)增高純度NK細(xì)胞的優(yōu)化技術(shù)。先用miniMACS及陰性免疫磁珠分選方法從外周血單個(gè)核細(xì)胞PBMNC得到純化的NK細(xì)胞隨后在干細(xì)胞培液基條件下通過(guò)、和等細(xì)胞因了的不同組合將培養(yǎng)體系分為組、IL2IL12組、IL2IL15組、IL2IL15IL12組和對(duì)照組不加任何細(xì)胞因子分別培養(yǎng)15天每3天半量換液并補(bǔ)充細(xì)胞因子檢測(cè)分選及擴(kuò)增前后CD3-CD56NK細(xì)胞含量、擴(kuò)增倍數(shù)及擴(kuò)增

2、前后各組在不同效靶比下的殺傷率。結(jié)果顯示經(jīng)miniMACS陰性免疫磁珠分選后CD3-CD56NK細(xì)胞含量由分選前的11.19±5.25捉高到94.23±3.50。培養(yǎng)15天后除對(duì)照組CD3-CD56NK細(xì)胞純度略下降外其余4組與擴(kuò)增前無(wú)顯著性差異P0.05o在、IL2IL12、IL2IL15、IL2IL15IL12培養(yǎng)體系中NK細(xì)胞擴(kuò)增倍數(shù)分別為15.43±1.0819.87±3.8750.46±4.31和52.35±6.72均顯著高于對(duì)照組6.14±1.0P0.01但I(xiàn)L2IL15與IL2IL1

3、5IL12組間未見(jiàn)顯著差異P0.05。各組擴(kuò)增的NK細(xì)胞對(duì)K562細(xì)胞的殺傷率均較擴(kuò)增前增強(qiáng)在不同效靶比TIL2IL15UL2IL15IL12組對(duì)K562細(xì)胞的殺傷率顯著高于其他組但兩組間無(wú)顯著性差異P0.05。結(jié)論經(jīng)miniMACS免疫磁珠陰性分選得少fiNK細(xì)胞后應(yīng)用IL2IL15培養(yǎng)條件是獲得高純度NK細(xì)胞的簡(jiǎn)單有效方法?！娟P(guān)鍵詞】NK細(xì)胞miniMACS白介素2白介素12白介素15ExVivoExpansionofHighlyPurifiedNKCellsfromHumanPeripher

4、alBloodAbstractAdoptiveimmunotherapyusingallogeneicnaturalkillerNKcellsprovidestobeusefulinrecipientsafteritsapplicationhasbeenlimitedbytheinabilitytoobtainsufficientnumbersofpureNKcells?ThisstudywasaimedtooptimizetheexpansionofhighpurityNKcellsfromhu

5、manperipheralblood?FirsttheNKcellswereisolatedfromPBMNCbyusingminiMACSThentheisolatedcellswereculturedinSCEMStemlineHematopoieticStemCellExpansionMediumSigmasupplementedwith10humanABscrumCultureswerefedwithfreshmediaandcytokinesevery3daysandwereevalua

6、tedforcellexpansionphenotypeandcytotoxicityattheendofthecultureperiod?TheresultsshowedthatingroupIL2IL15andIL2IL15IL12cellswereexpanded50?46±4.31andhigherthanothersPO.OlbutnosignificantdifferencebetweenthemselvesP0.05?AndthepurityofCD3-CD56NKcellswaso

7、ver94inallgroupsexceptthecontrol.ThecytotoxicityofexpandedNKcellsculturedwithcytokineswassignificantlyhigherthanthestartingpopulationatdifferentE:TratioPO.OlalthoughthecytotoxicityofIL2IL15IL12groupwasslightlyhigherthanthatofIL2IL15groupbutnosignifica

8、ntdifferencebetweenthemselvesP0.05?ItisconcludedthathighpurityofNKcellscanbeefficientlyexpandedinculturewithIL2IL15.KeywordsNK細(xì)胞作為機(jī)體固有免疫的重耍組成部分是機(jī)體抗腫瘤、抗感染的第一道天然防線。隨著對(duì)NK細(xì)胞牛物功能及活化機(jī)制的了解NK細(xì)胞在造血干細(xì)胞/骨髓移植的輔助治療及腫瘤過(guò)繼免疫治療方面的作用倍受關(guān)注。最近在異基因造血干細(xì)胞移植屮發(fā)現(xiàn)供者異源反應(yīng)性