中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2015年
8期
1070-1073
,共4页
李登瑞%杨永辉%李辉%郭素敏%朱桂云%李秀武%耿书军%赵荣娣%任雪飞%高莉%辛欣
李登瑞%楊永輝%李輝%郭素敏%硃桂雲%李秀武%耿書軍%趙榮娣%任雪飛%高莉%辛訢
리등서%양영휘%리휘%곽소민%주계운%리수무%경서군%조영제%임설비%고리%신흔
重组K562%IL-6%自然杀伤细胞%扩增%51Cr释放实验
重組K562%IL-6%自然殺傷細胞%擴增%51Cr釋放實驗
중조K562%IL-6%자연살상세포%확증%51Cr석방실험
Genetic modified K562 cell%Interleukin-6%Nature killer cells%Expansion%51Cr release assay
目的：：为了研究表达IL-6的重组K562细胞对自然杀伤细胞( NK）细胞的扩增数量、表型和功能的影响。方法：根据人类IL-6 cDNA的5′端序列，设计PCR引物以K562细胞cDNA文库的DNA为模板进行扩增，表达，转染，在K562细胞上表达IL-6基因，构建重组的K562工程细胞作为刺激细胞，以人外周血单个核细胞( PBMC）为扩增培养对象，使NK细胞在体外培养条件下得到大量的扩增。流式细胞仪分析NK细胞表型。将NK细胞作用到K562细胞，对其杀伤性进行功能分析，51 Cr释放实验检测NK细胞对K562细胞杀伤水平的影响。结果：成功构建了表达IL-6的重组K562细胞，和PBMC共同孵育后，培养体系经过21 d的刺激后，IL-6重组K562细胞诱导PBMC扩增，CD56＋CD16＋CD3-细胞数量比诱导前扩增了(760±18）倍，CD56＋CD16＋CD3-细胞的纯度从培养前占PBMC的6％±0.4％，扩增后第3组结果比未扩增的多了91％±2％。细胞毒实验表明，在NK效应细胞∶K562靶细胞为5∶1时，扩增的NK细胞的杀伤率达到了92％±2％。结论：本方法以重组K562为刺激细胞，能够实现NK细胞体外的大规模制备，建立了优化的NK细胞体外扩增方法，且扩增的细胞杀伤K562细胞的活性较好。对于NK的大量扩增和应用于临床具有重要的指导意义。
目的：：為瞭研究錶達IL-6的重組K562細胞對自然殺傷細胞( NK）細胞的擴增數量、錶型和功能的影響。方法：根據人類IL-6 cDNA的5′耑序列，設計PCR引物以K562細胞cDNA文庫的DNA為模闆進行擴增，錶達，轉染，在K562細胞上錶達IL-6基因，構建重組的K562工程細胞作為刺激細胞，以人外週血單箇覈細胞( PBMC）為擴增培養對象，使NK細胞在體外培養條件下得到大量的擴增。流式細胞儀分析NK細胞錶型。將NK細胞作用到K562細胞，對其殺傷性進行功能分析，51 Cr釋放實驗檢測NK細胞對K562細胞殺傷水平的影響。結果：成功構建瞭錶達IL-6的重組K562細胞，和PBMC共同孵育後，培養體繫經過21 d的刺激後，IL-6重組K562細胞誘導PBMC擴增，CD56＋CD16＋CD3-細胞數量比誘導前擴增瞭(760±18）倍，CD56＋CD16＋CD3-細胞的純度從培養前佔PBMC的6％±0.4％，擴增後第3組結果比未擴增的多瞭91％±2％。細胞毒實驗錶明，在NK效應細胞∶K562靶細胞為5∶1時，擴增的NK細胞的殺傷率達到瞭92％±2％。結論：本方法以重組K562為刺激細胞，能夠實現NK細胞體外的大規模製備，建立瞭優化的NK細胞體外擴增方法，且擴增的細胞殺傷K562細胞的活性較好。對于NK的大量擴增和應用于臨床具有重要的指導意義。
목적：：위료연구표체IL-6적중조K562세포대자연살상세포( NK）세포적확증수량、표형화공능적영향。방법：근거인류IL-6 cDNA적5′단서렬，설계PCR인물이K562세포cDNA문고적DNA위모판진행확증，표체，전염，재K562세포상표체IL-6기인，구건중조적K562공정세포작위자격세포，이인외주혈단개핵세포( PBMC）위확증배양대상，사NK세포재체외배양조건하득도대량적확증。류식세포의분석NK세포표형。장NK세포작용도K562세포，대기살상성진행공능분석，51 Cr석방실험검측NK세포대K562세포살상수평적영향。결과：성공구건료표체IL-6적중조K562세포，화PBMC공동부육후，배양체계경과21 d적자격후，IL-6중조K562세포유도PBMC확증，CD56＋CD16＋CD3-세포수량비유도전확증료(760±18）배，CD56＋CD16＋CD3-세포적순도종배양전점PBMC적6％±0.4％，확증후제3조결과비미확증적다료91％±2％。세포독실험표명，재NK효응세포∶K562파세포위5∶1시，확증적NK세포적살상솔체도료92％±2％。결론：본방법이중조K562위자격세포，능구실현NK세포체외적대규모제비，건립료우화적NK세포체외확증방법，차확증적세포살상K562세포적활성교호。대우NK적대량확증화응용우림상구유중요적지도의의。
Objective:To study the influence of different culture conditions on charcic and inhibition activity of nature killer cells ( NK) ,whether to join the modified K562 cells with IL-6 cytokine.Methods:According to the 5′end of the human IL-6 cDNA sequence,PCR primers designed to amplificate,express and transfect K562 cells cDNA library as a template for DNA.Genetic modified K562 cells as stimulating cells were prepared by expressing IL-6.To extract peripheral blood mononuclear cells( PBMC) from human peripheral blood.PBMC were explanted by genetic modified K562 stimulated.The expansion was initiated by CO-culture of PBMC and irradiate genetic modified K562 cell.The number of NK cell increased by directed induced generation of genetic modified K562 cell.Immunophenotypic analysis of NK cell surface markers was performed by flow cytometry (FCM).51Cr release assay was employed to measure the specific lysis skilling of NK cell target K562 cells.Results:We have constracted genetic modified K562 cells by genetic engineering.As stimulated cell added into the PBMC,an average of 760 ±18 fold expansion of CD56+CD16+CD3-cells was observed after 3 weeks of co-culture system.The NK cells population could proliferated more 91%±2% after expansion comparing with 6%± 0.4%in PBMC before expansion by FCM.The cytotoxical activity of NK cells which was induced by genetic modified K562 cell was the strongest than induced by IL-6 cytokine alone.The expanded NK cells lysed 92%±2% of K562 targets in a 5∶1 effector to target ratio.In this case,the NK cells induced by genetic modified K562 cells against tumor cells was more lethal.Conclusion:PBMC based in vitro expansion of natural killer cells was set up by genetic modified K562 cells.The cytotoxicity of NK cells was the strongest induced by genetic modified K562 cell treated.These results had important guiding significance for the the NK large number of amplification and used in clinical.
