活化性杀伤细胞免疫球蛋白样受体阳性的NK细胞对靶细胞的杀伤作用

Objective To study the killing effects of the killer cell immunoglobutin-like receptors (KIR) KIR2DS1-positive natural killer (NK) cells against acute, myeloid leukemia (AML) target cells, and to explore the KIR, human leucocyte antigen-Cw, and Cw loci (HLA-Cw) of NK cells, and, HLA-Cw of target cells mismatches destruction mechanism. Methods High-purified NK cells separated by DNYAL bead negative selection from healthy donor peripheral blood were taken as effector cells, and the freshly isolated bone marrow mononuclear cells from newly diagnosed AML patients as target cells. The anti-CD158a, CD158b monoclonal antibody was used to block inhibitory KLR receptors of NK cells (such as: KIR2DL1, KIR2DL2, KIR2DL3). The NK cells cytotoxicities against target cells before and after KIR blockades were detected by MTT reduction assay in vitro. Polymerase chain reaction and sequence specific primers (PCR-SSP) genotyping techniques were used to detect HLA-Cw, KIR gene of the healthy donors and patients. NK cells and target cells were divided into group C1 (expressing HLA-Cw 01, 03, 07, 08, 12, 14, 16 alleles), C2 group (expressing HLA-Cw 02, 04, 05, 06, 15, 17, 18 alleles) and the C1/C2 group (co-expressing the alleles in C1 group and C2 group) based on HLA-Cw. Results The purity of NK cells analyzed by flow cytometry was (90.8±6.08)%, and the killing effects of NK cells were significantly enhanced after inhibitory KIR blockades (t=-3.00, P=0.005). The cell lysis in C1 group, KIR2DS1+ NK cells against C2 group target cells (57.37±1.40)% was significantly higher than that against C1 group (44.19±4.67) % and C1/C2 group (36.77±6.56)% target cells (F= 11.87, P = 0.021), furthermore the differences in cell lysis among the above three groups become more evident after the inhibitory KIR blockades (F = 18. 72, P=0. 009). Conclusions The activity of NK cells was increased after the inhibitory KIR blockades, and the killing activities of KIR2DS1+ NK cells were higher than KIR2DS1 NK cells. The incompatibility between KIR, HLA-Cw of NK cells and HLA-Cw of target cells mediated NK alloactivation, realizing "missing self" recognition, and enabling NK cells cytotoxieity against targets.     

肾母细胞瘤基因衍生肽诱导细胞毒性T淋巴细胞对白血病患者骨髓CD34+细胞的体外杀伤效应

目的 探讨肾母细胞瘤基因(WT1)衍生肽负载树突状细胞(DC)诱导细胞毒性T淋巴细胞(CTL)对白血病CD34+细胞的体外清除效应.方法 合成一段针对HLA-A*0201锚位的WT19聚肽,体外负载来源于HLA-A*0201*健康人的DC后,诱导产生WT1肽特异性CTL(A组),以噻唑盐(MTT)比色法观察其对WT1表达阳性白血病患者(HLA-A* 0201+者3例,HLA-A*0201-者3例)骨髓CD34+细胞、健康人(HLA-A*0201+者2例,HLA-A*0201-者1例)外周血CD34+细胞和白血病NB4、K562及U937细胞株的体外杀伤效应,粒细胞-巨噬细胞系集落形成试验观察其对白血病患者骨髓CD34+细胞和健康人外周血CD34+细胞粒细胞-巨噬细胞系集落形成单位(CFU-GM)形成的影响.设立单独DC诱导CTL(B组)和IL-2诱导T细胞(C组)作为对照.结果 在效靶比为20:1时,A组CTL对3例HLA-A*0201+白血病患者骨髓CD34+细胞和NB4细胞的杀伤活性(分别为55.3%±2.8%,67.1%±3.2%、49.4%±3.8%和55.0%±3.7%)明显高于对3例HLA-A*0201-白血病患者骨髓CD34+细胞、健康人外周血CD34+细胞及K562、U937细胞的杀伤活性(均<20%),并明显高于B组和C组CTL(均P＜0.01).2例HLA-A*0201+白血病患者骨髓CD34+细胞经A组CTL处理后CFU-GM集落相对形成率分别为17.8%±4.0%和20.8%±3.4%,明显低于经B组CTL处理后(分别为88.9%±3.4%和91.8%±5.7%,均P＜0.01);HLA-A*0201-白血病患者骨髓CD34+细胞、健康人外周血CD34+细胞经A组和B组CTL处理后CFU-GM集落相对形成率差异尤统计学意义.结论 WT1肽特异性CTL能够以HLA-1类抗原限制方式杀伤高表达WT1基因的白血病CD34+细胞,且能特异性抑制其CFU-GM集落形成,WT1基因的表达产物可以作为清除白血病CD34+细胞靶点.     

不同年龄成年人急性白血病形态学、免疫学及细胞遗传学分型比较

Objective To investigate the characters of morphology,immunology and cytogenetics of adult acute leukemia (AL) in different ages. Methods 172 cases of newly diagnosed adult AL were divided into two groups:the non-aged group (age＜60 years) and the aged group ( age≥60 years). Morphology,immunology and cytogenetics between the two groups were compared. Results The incidence of M3 in aged AL was significantly lower than that in non-aged AL[6.0 %(3/50) vs 18.9 %(23/122),P ＜0.05]. The incidence of hypo-or extremely hypo-cellular AL in aged AL was significantly higher than that in non-aged AL[14.0 %(7/50) vs 4.1 %(5/122),P ＜0.05],but the incidence of hyper-or extremely hyper-cellular was significantly lower than that in non-aged AL[52.0 %(26/50) vs 73.8 %(90/122),P ＜0.05]. Among aged acute myeloid leukemia (AML),the incidence of lymphoid antigen positive AML (Ly+AML) was significantly higher than that in non-aged AML[63.4 %(26/41) vs 41.3 %(38/92),P ＜0.05]. The incidence of adverse karyotypes in aged AML was significantly higher than that in non-aged AML[33.3 %(11/33) vs 14.1 %(10/71),P ＜0.05].Conclusion Age is an important prognostic factor in AL. Generally,aged AL has poorer prognosis than nonaged AL.     

多参数分析多发性骨髓瘤患者外周血中CD4~+CD25~+调节性T细胞的表达

目的 通过分析CD4+ CD25+ 调节性T细胞(CD4+ CD25+ regulatory T cells,CD4+ CD25+ Tress)在不同病程阶段多发性骨髓瘤(multiple myeloma,MM)患者外周血中的变化和临床意义,初步探讨MM患者的免疫抑制机制.方法 流式细胞术检测56例MM患者及30例健康志愿者外周血CD4+ CD25+、CD4+ CD25high、CD4+ CD25+ CD127low、CD4+ CD25high CD127low T细胞的比例并分析比较.结果 1)56例MM患者:化疗后组CD4+ CD25+、CD4+ CD25high、CD4+ CD25+ CD127low、CD4+ CD25high CD127low T 细胞的比例均高于正常组,差异具有显著性;初诊组的CD4+ CD25+ CD127low T细胞与正常对照组比较没有差异,但CD4+CD25+、CD4+ CD25high、CD4+ CD25high CD127lwo T细胞与正常对照组比较差异具有显著性;初诊组CD4+ CD25+T细胞的比例与化疗后组相比没有差异,但其CD4+ CD25high 、CD4+ CD25+ CD127low、CD4+ CD25high CD127low T细胞的比例均低于化疗后组,差异具有统计学意义.2)37例化疗后MM患者:稳定期与活动期的CD4+ CD25+、CD4+CD25+CD127low T细胞相比没有差异,但稳定期的CD4+ CD25high、CD4+ CD25high CD127low T细胞明显低于活动期.结论 CD4+ CD25+ Tregs在MM患者外周血中比例明显升高,且化疗可能影响其在外周血的比例;活动期MM患者CD4+ CD25+ Tregs的比例明显高于稳定期.这些提示CD4+ CD25+ Tregs可能是MM免疫抑制的一个重要原因.     

免疫荧光双标记监测白血病微小残留病临床研究

目的 :观察 Td T及系列相关抗原双标记在急性白血病细胞中的表达及其在预测白血病复发过程中的作用。方法 :采用免疫荧光双标记技术 ,配合流式细胞术检测急性白血病细胞的 Td T及系列相关抗原双标记及急性白血病完全缓解后免疫双标记抗原变化。结果 :64例急性白血病中免疫双标记表达 36例 ( 56.2 5% ) ,其中 32例 AL L全部表达免疫双标记 ,32例 AML 仅有 4例表达( 12 .5% ) ,在 12例获 CR的表达免疫双标记的急性白血病中 ,4例免疫双标记细胞占单个核细胞0 .0 1%～ 0 .0 8% ,并有长期缓解 ,而 8例免疫双标记细胞占单个核细胞 1.5%～ 95.38%的急性白血病 ,均于近期骨髓复发。结论 :免疫荧光双标记技术在部分急性白血病的微小残留监测中具有一定的临床意义。     

B细胞活化因子在部分肾移植受者外周血淋巴细胞异常高表达及可能的生物学意义

背景:B细胞活化因子(B cell activating factor belonging to TNF family,BAFF),经与其受体结合,对B细胞分化、成熟、生存和抗体分泌发挥重要作用,在T细胞应答过程中亦可能起着重要作用.BAFF信号是否在同种肾移植排斥反应过程中起作用值得探讨.目的:分析肾移植受者外周血淋巴细胞上BAFF的表达情况及其可能的生物学作用.设计、时间及地点:病例观察,于2006-06/2007-03在苏州大学附属第三医院泌尿外科进行.对象:86例肾移植随访患者,男60例,女26例,年龄12～62岁.患者均为首次肾移植,血肌酐值在65～267 μmol/L.方法:取患者外周血,以EDTA-Na2抗凝.收集部分患者的移植肾活检标本.主要观察指标:分析外周血单个核细胞上BAFF+、BAFF-R+、CD4+、CD8+、CD4+CD25+CD127/-low、CD134+、CD4+CD134+和CD19+ BAFF-R+的表达率,计算CD4/CD8比值;对活检组织进行病理分析和免疫组织化学分析.结果:肾移植受者外周血单个核细胞上BAFF的表达率在0.18%～76.97%之间.将15%设为临界值,所有数据分成≥15%组和＜15%组进行统计分析.在≥15%组,BAFF表达率为36.91%,与CD4/CD8比值、CD4+CD25+ CD127-/low T细胞之间不相关.然而,BAFF+细胞数与CD134+细胞和CD4+CD134+细胞存在显著相关性(分别为P＜0.01和P＜0.05).而在＜15%组,各指标之间没有显著相关性.病理诊断证实慢性排斥的移植肾组织,BAFF表达于肾小管上皮细胞胞浆/胞膜.结论:BAFF在肾移植受者外周血单个核细胞的异常高表达可能与同种肾移植排斥反应相关.     

CD_(34)~+急性早幼粒细胞白血病的临床特点及预后

急性早幼粒细胞白血病(APL)的主要特点为染色体t(15;17)(q22;q12)易位形成PML/RARot融合基因,早期常伴有严重的凝血功能障碍而危及生命.FAB分型将APL分为2个亚型:典型粗颗粒型M3和细颗粒型M3v.APL免疫表型特点为CD_(13)~+,CD_(33)~+,CD_9~+和CD_(14)~-.目前以全反式维甲酸(ATRA)联合蒽环类为基础的化疗,使初治APL患者完全缓解(CR)率达90%～95%,早期死亡率明显下降,CR后巩固强化及维持治疗使得APL复发率明显下降,生存期明显延长[1].尽管大多数APL经过上述治疗取得了非常满意的疗效,仍有少部分患者表现为极差的临床过程.     

活化性杀伤细胞免疫球蛋白样受体阳性的NK细胞对靶细胞的杀伤作用

目的 研究活化性杀伤细胞免疫球蛋白样受体(KIR)KIR2DS1阳性的自然杀伤(NK)细胞对急性髓系白血病(AML)靶细胞的杀伤作用;并探讨NK细胞KIR及人类白细胞抗原Cw位点(HLA-Cw)与靶细胞HLA-Cw错配对靶细胞的杀伤机制.方法 获取健康供者的外周血,经Dnyal磁珠负选高纯度NK细胞;取初治确诊的AML患者新鲜骨髓液,分离后的白血病细胞作为靶细胞.用抗CD158a和CD158b单克隆抗体封闭NK细胞抑制性KIR(如:KIR2DL1、KIR2DL2和KIR2DL3),并用噻唑蓝(MTT)比色法分别检测抑制性KIR封闭前和封闭后NK细胞对靶细胞的杀伤作用.采用聚合酶链反应和顺序特异性引物(PCR-SSP)基因分型技术分别检测NK细胞及靶细胞的KIR基因和HLA-Cw;根据HLA-Cw将NK细胞和靶细胞分别分为C1组(表达HLA-Cw 01、03、07、08、12、14、16分子)、C2组(表达HLA-Cw02、04、05、06、15、17、18分子)和C1/C2组(既表达C1组的等位基因又表达C2组的等位基因).结果 分选后的NK细胞经流式细胞仪检测,其纯度为(90.8±6.08)%;NK细胞抑制性KIR封闭后与封闭前比较,其对靶细胞的杀伤作用明显增强(t=-3.00,P=0.005);KIR2DS1阳性的NK细胞C1组对靶细胞C2组的杀伤作用高于靶细胞C1及C1/C2组,杀伤率分别为(57.370±1.400)%、(44.190±4.666)%和(36.770±6.560)%(F=11.87,P=0.021);NK细胞抑制性KIR封闭后,其对各组靶细胞的杀伤率更高(F=18.72,P=0.009).结论 NK细胞抑制性KIR封闭后其活性增强,KIR2DS1阳性的NK细胞对靶细胞的杀伤率高于KIR2DS1阴性的NK细胞;NK细胞KIR及HLA-Cw与靶细胞HLA-Cw错配,能够介导KIR2DS1阳性的NK细胞C1组抗原的异源反应性,实现"丢失自我"的识别作用,从而使NK细胞对靶细胞杀伤率增强.     

浅谈抗生素的合理使用

抗生素如今已被广泛应用于临床,治疗各种感染性疾病和预防某些传染病.抗生素的合理使用对细菌感染的治疗起到了举足轻重的作用.不合理使用抗生素则会使细菌耐药性增强,在医院的环境内引起交叉感染,甚至发生耐药菌的爆发流行,并向全社会传播,给广大患者及家庭、医院乃至社会造成不良的影响及后果.因此,合理使用抗生素已是一项非常重要的问题,应引起广大医务工作者的高度重视.本文就抗生素的合理使用谈谈笔者的看法.     

γ射线对白血病细胞B7分子、HLA抗原及抗原递呈能力的调节

协同刺激分子B7-1(CD80)、B7-2(CD86)及人类白细胞抗原(HLA)的缺乏是许多肿瘤逃逸免疫作用的重要机制之一.有效抗原递呈是诱导抗原特异免疫应答的始动环节及先决条件.在抗原递呈细胞(APC)向T细胞递呈抗原及激活T细胞的过程中,组织相容性复合物(MHC)分子及CD80、CD86、细胞内黏附分子-1(ICAM-1)、血管细胞黏附分子(VCAM-1)等协同刺激分子起着关键作用[1].白血病细胞表面共刺激分子、HLA分子的调节研究对了解白血病逃避免疫作用和设计白血病免疫治疗方案有重要的意义.