CAJ | 학술논문

目的研究在Jurkat细胞上敲减Itk蛋白的表达对细胞增殖及炎症相关的细胞因子产生的影响,为Itk作为小分子药物靶标提供可行性实验依据.方法针对Itk基因设计合成三个shRNAs,通过与pEGFP-C1-hItk质粒共转染,观察其抑制Itk-GFP融合蛋白的表达情况,筛选有效序列包装成慢病毒颗粒.将慢病毒颗粒感染Jurkat细胞,观测Itk蛋白的表达水平、细胞增殖情况及细胞因子的变化.结果 Itk-shRNA1感染Jurkat细胞后Itk基因的mRNA水平与细胞对照组及感染shRNAnon的对照组相比,敲减率约55%,差异有统计学意义P＜0.05.Itk蛋白的敲减导致Jurkat细胞在受刺激时增殖降低,以未感染病毒的Jurkat细胞受刺激后酶标仪检测的A值为1,Itk-shRNA1感染组平均比值为0.54,shRNAnon对照组平均比值为0.83,两组差异有统计学意义,P＜0.05.Itk-shRNA1感染组中IL-2、IL-5、IL-10及IFN-γ等细胞因子水平均较shRNAnon对照组低,差异有统计学意义,提示Itk蛋白的表达减少导致Jurkat细胞产生细胞因子减少.结论抑制Itk表达能有效降低淋巴细胞的增殖,同时减少与炎症相关细胞因子分泌.
목적 연구재Jurkat세포상고감Itk단백적표체대세포증식급염증상관적세포인자산생적영향,위Itk작위소분자약물파표제공가행성실험의거.방법 침대Itk기인설계합성삼개shRNAs,통과여pEGFP-C1-hItk질립공전염,관찰기억제Itk-GFP융합단백적표체정황,사선유효서렬포장성만병독과립.장만병독과립감염Jurkat세포,관측Itk단백적표체수평、세포증식정황급세포인자적변화.결과 Itk-shRNA1감염Jurkat세포후Itk기인적mRNA수평여세포대조조급감염shRNAnon적대조조상비,고감솔약55%,차이유통계학의의P＜0.05.Itk단백적고감도치Jurkat세포재수자격시증식강저,이미감염병독적Jurkat세포수자격후매표의검측적A치위1,Itk-shRNA1감염조평균비치위0.54,shRNAnon대조조평균비치위0.83,량조차이유통계학의의,P＜0.05.Itk-shRNA1감염조중IL-2、IL-5、IL-10급IFN-γ등세포인자수평균교shRNAnon대조조저,차이유통계학의의,제시Itk단백적표체감소도치Jurkat세포산생세포인자감소.결론 억제Itk표체능유효강저림파세포적증식,동시감소여염증상관세포인자분비.
Objective To study the effect of Itk down regulation on Jurkat cell proliferation and inflammatory cytokines production, and provide useful data for Itk as an attractive target for potential drugs.Methods Three shRNAs against different region of Itk were constructed and cotransfected with pEGFP-C1-hItk. The shRNA, which can knock down Itk, was selected and packed into lentivirus. After Jurkat cells were transfected with shRNA lentivirus, the change of Itk protein expression, cell proliferation and cytokines production was observed. Results Itk mRNA was reduced about 55% in Jurkat cells transfected with ItkshRNA1, compared with that in control cells shRNAnon (P ＜ 0. 05 ). Knocking down Itk expression had a profound inhibitory effect on Jurkat cell proliferation. In addition, there was a substantial decrease in level of cytokines, such as IL-2, IL-5, IL-10 and IFN-γ, produced by cell transfected with Itk-shRNA1.Conclusion Knocking down Itk expression can inhibit Jurkat cell proliferation and inflammatory cytokines production.