环孢菌素A对NK细胞免疫球蛋白样转录子4的表达和杀伤功能的影响
배포균소A대NK세포면역구단백양전록자4적표체화살상공능적영향
Regulatory effect of CsA on the expression of NK cell inhibitory receptor ILT4 and cytotoxicity of NK cells
  • 万方数据
    中华检验医学杂志 중화검험의학잡지
    CHINESE JOURNAL OF LABORATORY MEDICINE
    2011年 11期 1029-1033 ,共5页

    司元全%张义%卞晓琨%陆楠%郏雁飞

    사원전%장의%변효곤%륙남%겹안비

    受体,免疫%环孢菌素%杀伤细胞,天然%HLA抗原 受體,免疫%環孢菌素%殺傷細胞,天然%HLA抗原
    수체,면역%배포균소%살상세포,천연%HLA항원
    Receptors,immunologic%Cyclosporine%Killer cells,natural%HLA antigens
    目的 探讨CsA对NK细胞抑制性受体ILT4的表达及NK细胞杀伤功能的影响.方法 以10 mg/L CsA分别处理NK细胞12、24、36 h作为3个实验组,以DMSO分别作用NK细胞12、24、36 h作为3个对照组.通过RT-qPCR和流式细胞术分别检测各组中NK细胞ILT4 mRNA和蛋白表达变化,同时测定人胃癌细胞株BGC-823、绒毛膜癌细胞株JEG-3 HLA-G的表达,采用MTT法检测NK细胞经CsA处理36 h前后对肿瘤细胞杀伤活性的变化.用药处理不同时间段ILT4表达数据比较先使用单因素方差分析,再进行各均数间两两比较Dunnett t检验,而JEG-3、BGC-823细胞HLA-G的表达差异及NK细胞对JEG-3、BGC-823细胞的杀伤活性比较采用t检验.结果 用RT-qPCR检测经CsA处理12、24、36 h组的NK细胞ILT4 mRNA相对表达水平依次为0.99±0.27、1.79±0.29、6.79±0.64,经DMSO处理的对照组分别为0.86±0.11、0.94±0.12、1.06±0.17；CsA处理NK细胞24、36 h组的ILT4表达较对照组明显增加(t值分别为4.69、14.99,P均＜0.05),而作用12 h前后组间的差异无统计学意义(t=0.78,P＞0.05).用流式细胞术检测经CsA处理12、24、36 h组的NK细胞ILT4蛋白表达阳性率分别为(5.16±0.42)％、(6.23±0.48)％、(23.8±1.5)％,分别高于经DMSO处理12、24、36 h组[(3.08±0.19)％、(3.35±0.12)％、(3.36±0.21)％],且不同处理组在不同处理时间的阳性率比较差异有统计学意义(t值分别为7.70、10.06、20.72,P均＜0.01)；CsA处理NK细胞36 h组的ILT4 mRNA和蛋白表达均显著高于处理12 h和24h组(t值分别为16.38、14.12、21.81、20.56；P均＜0.01).同时,当经CsA处理前后且效靶比为10∶1时,NK细胞对低表达HLA-G的肿瘤细胞BGC-823的杀伤率分别为(81.96±2.80)％、(60.23±1.57)％,CsA对NK细胞杀伤活性的抑制率为(26.48±2.42)％；而NK细胞对高表达HLA-G的JEG-3细胞的杀伤率分别是(53.46±2.21)％、(28.30±1.85)％,抑制率为(47.10±1.59)％.经CsA处理前后NK细胞对肿瘤细胞杀伤活性的变化表明CsA可抑制NK细胞对BGC-823和JEG-3细胞的杀伤(t值分别为11.74、15.16,P均＜0.01),对JEG-3细胞杀伤抑制率显著高于对BGC-823细胞的杀伤抑制率(t=12.31,P＜0.01).结论 CsA可上调NK细胞抑制性受体ILT4的表达,并通过ILT4与HLA-G相互作用影响NK对肿瘤细胞的杀伤.
    목적 탐토CsA대NK세포억제성수체ILT4적표체급NK세포살상공능적영향.방법 이10 mg/L CsA분별처리NK세포12、24、36 h작위3개실험조,이DMSO분별작용NK세포12、24、36 h작위3개대조조.통과RT-qPCR화류식세포술분별검측각조중NK세포ILT4 mRNA화단백표체변화,동시측정인위암세포주BGC-823、융모막암세포주JEG-3 HLA-G적표체,채용MTT법검측NK세포경CsA처리36 h전후대종류세포살상활성적변화.용약처리불동시간단ILT4표체수거비교선사용단인소방차분석,재진행각균수간량량비교Dunnett t검험,이JEG-3、BGC-823세포HLA-G적표체차이급NK세포대JEG-3、BGC-823세포적살상활성비교채용t검험.결과 용RT-qPCR검측경CsA처리12、24、36 h조적NK세포ILT4 mRNA상대표체수평의차위0.99±0.27、1.79±0.29、6.79±0.64,경DMSO처리적대조조분별위0.86±0.11、0.94±0.12、1.06±0.17；CsA처리NK세포24、36 h조적ILT4표체교대조조명현증가(t치분별위4.69、14.99,P균＜0.05),이작용12 h전후조간적차이무통계학의의(t=0.78,P＞0.05).용류식세포술검측경CsA처리12、24、36 h조적NK세포ILT4단백표체양성솔분별위(5.16±0.42)％、(6.23±0.48)％、(23.8±1.5)％,분별고우경DMSO처리12、24、36 h조[(3.08±0.19)％、(3.35±0.12)％、(3.36±0.21)％],차불동처리조재불동처리시간적양성솔비교차이유통계학의의(t치분별위7.70、10.06、20.72,P균＜0.01)；CsA처리NK세포36 h조적ILT4 mRNA화단백표체균현저고우처리12 h화24h조(t치분별위16.38、14.12、21.81、20.56；P균＜0.01).동시,당경CsA처리전후차효파비위10∶1시,NK세포대저표체HLA-G적종류세포BGC-823적살상솔분별위(81.96±2.80)％、(60.23±1.57)％,CsA대NK세포살상활성적억제솔위(26.48±2.42)％；이NK세포대고표체HLA-G적JEG-3세포적살상솔분별시(53.46±2.21)％、(28.30±1.85)％,억제솔위(47.10±1.59)％.경CsA처리전후NK세포대종류세포살상활성적변화표명CsA가억제NK세포대BGC-823화JEG-3세포적살상(t치분별위11.74、15.16,P균＜0.01),대JEG-3세포살상억제솔현저고우대BGC-823세포적살상억제솔(t=12.31,P＜0.01).결론 CsA가상조NK세포억제성수체ILT4적표체,병통과ILT4여HLA-G상호작용영향NK대종류세포적살상.
    Objective To investigate the regulatory effect of CsA on the expression of NK cell inhibitory receptor ILT4 and cytotoxicity of NK cells.Methods NK cells treated with CsA ( 10 mg/L) or DMSO for 12,24 and 36 h were chosen as three experimental groups and control groups respectively.RTqPCR and flow cytometry were performed to detect the alteration of ILT4 at the mRNA and protein level respectively.The expression of HLA-G in human gastric cancer cell line BGC-823 and human placental choriocarcinoma cell line JEG-3 were measured at the same time,and then the cytolytic activity of the untreated NK cells and NK cells treated with CsA for 36 h against BGC-823 and JEG-3 cells was determined with MTT.One-way analysis of variance was employed to compare the different ILT4 expression at different time points after medication; Dunnett test was performed to carry out the pairwise comparison between each mean.The difference of HLA-G expression between JEG-3 cells and BGC-823 cells,and the difference of NK cell cytolytic activity against JEG-3 cells and BGC-823 cells were analyzed by student's t-test.Results RT-qPCR assay indicated that the relative levels of ILT4 mRNA in NK cells treated with CsA for 12,24 and 36 h in turn were 0.99 ± 0.27,1.79 ± 0.29,6.79 ± 0.64,and those of their contrast groups treated with DMSO were 0.86 ±0.11,0.94 ±0.12,1.06 ±0.17.The expression of ILT4 in NK cells treated with CsA for 24 h or 36 h was higher than that in NK cells of their contrast groups respectively ( t value of 4.69,14.99,P ＜0.05,respectively),but there was no significant difference between the two groups of NK cells treated for 12 h ( t =0.78,P ＞0.05 ).Through flow cytometry,the positive rates of ILT4 protein expression in NK cells treated with CsA for 12,24 and 36 h [(5.16 ± 0.42 ) ％,( 6.23 ± 0.48 ) ％,( 23.8 ± 1.5 ) ％]were higher than those in NK cells after treatment with DMSO for 12,24 and 36 h respectively[(3.08 ±0.19)％,(3.35 ±0.12)％,(3.36 ±0.21 )％ ;t value of 7.70,10.06,20.72,P＜0.01,respectively].The expression of ILT4 in NK cells treated for 36 h was much higher than that in NK cells for 12 and 24 h at the mRNA and protein level (t value of 16.38,14.12 ;21.81,20.56,P ＜ 0.01,respectively).Meanwhile the killing rates of NK cells treated with 10∶1 effector-target ratio CsA on BGC-823 cells (low HLA-G expression) were ( 8 1.96 ± 2.80 ) ％ ( before treatment) and ( 60.23 ± 1.57 ) ％ ( after treatment),which were higher than those on JEG-3 cells (HLA-G-overexpression) [(53.46 ±2.21 )％ ( before treatment),(28.30 ± 1.85 ) ％ ( after treatment)].The changes of cytotoxicity of NK cells treated with CsA against target cells showed that CsA inhibited the killing activity of NK cells to BGC-823 and JEG-3 cells (t value of 11.74,15.16,P＜0.01,respectively),and the inhibitory rates were (26.48 ±2.42)％ and (47.10 ±1.59 ) ％ respectively.CsA had a higher killing rate inhibition on JEG-3 than on BGC-823 ( t =12.31,P ＜0.01 ).Conclusion CsA induces upregulation of ILT4 in NK cells,and the cytotoxicity of NK cells to tumor cells can be affected by interaction of ILT4 and HLA-G.
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