NK细胞活化受体NKG2D及sMICA在晚期肺癌免疫监视中的作用

背景与目的 NK细胞活化受体NKG2D及sMICA是近来肿瘤研究领域热点之一.本研究旨在观察晚期肺癌患者外周血中NK细胞受体NKG2D及sMICA表达水平的变化,并探讨它们在晚期肺癌免疫监控中的作用及其临床意义.方法 采用流式细胞术榆测115例肺癌患者外周血NK细胞受体NKG2D、T淋巴细胞哑群及NK细胞百分比,采用酶联免疫吸附反应检测肺癌患者外周血sMICA值,并以50例健康人作为对照.结果 晚期肺癌患者外周血sMICA、CD8+T细胞、NK细胞数量较对照组明显升高,而NK细胞受体NKG2D、CD3+T细胞、CD4+>T细胞、CD4+T/CD8+T值较对照组下降.NK细胞受体NKG2D和sMICA呈负相关(r=-0.319,P<0.05).NK细胞受体NKG2D与CD4+T细胞、CD4+T/CD8+T成正相关(P0.05),与CD8+T细胞成负相关(P<0.05);sMICA与CD4+T细胞、CD4+T/CD8+T成负相关(P<0.05),与CD8+T细胞成正相关(P<0.05);它们与CD3+T、NK细胞均无相关性(P>0.05).结论 外周血sMICA上调介导NK细胞活化受体NKG2D下调机制参与了晚期肺癌以肿瘤为中心抑制免疫网络的形成,它们可作为监视晚期肺癌患者免疫状态的参考指标,也可作为评估肺癌发生、发展的参考依据.     

Role of MICA expression,anti-MICA antibodies and serum MICA during acute rejection in a rat-to-mouse cardiac transplantation model

Background and objective: Human major histocompatibility complex class I-related gene A (MICA) is reportedly associated with poor transplant outcomes and a high risk of acute and chronic rejection in solid organ transplantation. However, studies on these risks have found conflicting results. In the present study, we investigate the MICA expression and serum MICA (sMICA) as well as the MICA antibodies (anti-MICA) in serum of recipients during acute rejection (AR) in a rat-to-mouse cardiac transplantation model. Methods: Construct rat-to-mouse concordant cardiac transplantation models, histological examination of the heart in recipients during AR at 2-6 hours time point was done. We then studied the MICA gene expression of the heart in recipients during AR at 2-6 hours time point by western blot and RT-PCR assay. We latter studied the anti-MICA and sMICA levels in serum of recipients during AR at 2-6 hours time point by Flow cytometry and ELISA measurement. Results: We found that Lewis rat hearts transplanted into BALB/c mice developed typical AR in 6 days. The level of severity of xenograft rejection from 2 d to 6 d was increased in a time-dependant way. MICA protein and MICA mRNA was also increased in time-dependant way and reached the highest value at 6 h. The prevalence of anti-MICA was significantly higher among those with severe acute rejection. However, sMICA was significantly increased during AR at 2 hours, then gradually decreased, and reached the lowest value at 6 h. Conclusions: MICA expression in recipients’ heart and anti-MICA antibodies in recipients’ sera may associated with high risk of AR in rat-to-mouse transplantation. sMICA showed a negative association with acute rejection and may be a good predictor of heart transplant outcomes.     

食管癌、贲门癌患者外周血CD+8 NKT细胞活化受体NKG2D及其分泌性配体sMICA检测的临床意义

 目的 　探讨外周血CD+8 NKT细胞活化受体NKG2D及其分泌性配体sMICA检测在食管癌、贲门癌诊断及术后疗效评价中的临床意义。方法　对53例患者确诊后（29例手术患者术前14 d及术后14 d）及30名健康对照组采用流式细胞术对外周血CD+8 NKT细胞中活化受体NKG2D阳性细胞百分比测定,应用酶联免疫吸附法进行sMICA含量测定,并各自进行差异性分析,同时对两者进行依从性分析。结果　患者外周血CD+8 NKT细胞中NKG2D阳性细胞百分比为（77.632±8.972）%,明显低于对照组的（89.053±6.515）%（t＝-6.113,P＜0.05）;TNM分期Ⅱ、Ⅲ、Ⅳ期患者依次降低（F＝99.251,P＜0.01）;有区域淋巴结转移者均低于无区域淋巴结转移者（t＝-10.384,P＜0.01）;鳞状细胞癌高于腺癌（t＝9.899,P＜0.01）;术前均低于术后（t＝-4.319,P＜0.01）。患者血清sMICA的含量为（326.28±85.407）pg／ml,明显高于对照组的（210.00±22.560）pg／ml（t＝7.292,P＜0.01）;Ⅱ、Ⅲ、Ⅳ期患者依次增高（F＝63.355,P＜0.01）;有区域淋巴结转移者均高于无区域淋巴结转移者（t＝7.770,P＜0.01）;鳞状细胞癌低于腺癌（t＝-7.593,P＜0.01）;术前均高于术后（t＝7.027,P＜0.01）。血清sMICA对外周血CD+8 NKT细胞活化受体NKG2D有抑制作用（F＝142.773,P＜0.05）,决定系数R2＝0.7368。结论　外周血CD+8 NKT细胞活化受体NKG2D及其分泌性配体sMICA含量的测定有助于食管癌、贲门癌分期的临床辅助诊断,对判断其生物学行为及预后有重要临床意义,并可作为患者手术治疗效果指标。     

MICA基因第5外显子与乙肝后肝硬变的相关性研究

目的研究MICA基因多态性、sMICA分子与乙肝后肝硬化（livercirrhosis,LC）的相关性。方法采用MICA—STR微卫星基因分型技术检测101例HBsAg阳性LC患者和141例健康对照者MICA基因第5外显子的基因多态性,并进行统计学分析。结果在101例湖南汉族HBsAg阳性LC患者中检测到5种MICA基因第5外显子等位基因,频率分别为：MICA＊A4（10．9％）,MICA＊A5（37．1％）,MICA＊A5．1（34．2％）,MICA＊A6（7．4％）,MICA＊A9（10．4％）。15种基因型分别为MICA＊A4／A4,MICA＊A4／A5．MICA＊A4／A5．1．MICA＊A4／A6,MICA＊A4／A9,MICA＊A5／A5,MICA＊A5／A5．1,MICA＊A5／A6．MICA＊A5／A9,MICA＊A5．1／A5．1,MICA＊A5．1／A6,MICA＊A5．1／A9,MICA＊A6／A6,MICA＊A6／A9,MICA＊A9／A9。MICA＊A5．1基因与LC发病正相关（P=0．042）,OR值为1．398,与患者性别无关（χ^2=0．08,P=0．778）。结论MICA＊A5．1基因与乙肝后LC发病正相关,提示该基因可能是乙肝后出现LC的重要易感基因。     

IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA

NK group 2D (NKG2D)-expressing NK cells exhibit cytolytic activity against various tumors after recognition of the cellular ligand MHC class I chain-related gene A (MICA). However, release of soluble MICA (sMICA) compromises NKG2D-dependent NK-cell cytotoxicity leading to tumor escape from immunosurveillance. Although some molecular details of the NKG2D-MICA interaction have been elucidated, its impact for donor NK (dNK) cell-based therapy of solid tumors has not been studied. Within an ongoing phase I/II trial, we used allogeneic IL-2 activated dNK cells after haploidentical stem cell transplantation for immunotherapy of patients with high-risk stage IV neuroblastoma. NKG2D levels on activated dNK cells increased strongly when compared with freshly isolated dNK cells and correlated with enhanced NK-cell cytotoxicity. Most importantly, elevated sMICA levels in patients plasma correlated significantly with impaired dNK-cell-mediated cytotoxicity. This effect could be reversed by high-dose infusion of activated dNK cells, which display high levels of surface NKG2D. Our data suggest that the provided excess of NKG2D leads to clearance of sMICA and preserves cytotoxicity of dNK cells via non-occupied NKG2D. In conclusion, our results identify this tumor immune escape mechanism as a target to improve immunotherapy of neuroblastoma and presumably other tumors.     

乳腺癌血清sMICA含量检测的临床意义

目的 检测乳腺癌血清中sMICA水平,研究其单独及联合CA153检测对于乳腺癌诊断的意义.方法 ELISA法检测56例乳腺癌血清、69例乳腺良性病血清及75例健康体检者血清中sMICA水平,分析其与临床病理参数的关系.雅培免疫发光法检测其CA153浓度,并分析其与sMICA对于乳腺癌诊断的优缺点.结果 乳腺癌患者血清中sMICA及CA153浓度均显著高于乳腺良性病患者及健康体检者;其中sMICA水平与乳腺癌临床分期呈正相关,淋巴结转移者sMICA浓度显著高于未转移者,与年龄及分级无明显相关性;sMICA、CA153及两者联合检测诊断乳腺癌的敏感度分别为67.86％、71.43％和92.86％.结论 sMICA可为乳腺癌病情评价提供新的参考指标,联合CA153能提高诊断性能,降低临床漏检率.     

Soluble MICA-NKG2D interaction upregulates IFN-γ production by activated CD3CD56 NK cells: Potential impact on chronic graft versus host disease

A soluble isoform of MHC class I chain-related molecule A (soluble MICA), generated by proteolytic shedding from the membrane-bound MICA of various tumor cells, has been shown to downregulate both the expression of natural killer group 2-member D receptor and the cytotoxic function of effectors cells and was postulated as a mechanism for tumor immune evasion. Its effect on the expression of cytokines by the effector cells remained unexplored. Here we demonstrate that the sMICA molecules upregulate interferon gamma expression by interleukin-12/interleukin-18-activated CD3⁻CD56⁺ natural killer cells, witnessing the pro-inflammatory effect of soluble MICA. Overall, these data are in line with our previous observations that the raised serum levels of soluble MICA, following allogeneic hematopoietic stem cell transplantation, confer susceptibility to and the presence of pre-transplantation anti-MICA antibodies in the patient’s serum confer protection against chronic graft versus host disease.     

3-溴丙酮酸对人结肠癌SWIII-C细胞增值和凋亡的影响

目的研究恶性肿瘤患者以及不同感染性疾病患者血清中sMICA分子的含量及临床意义。方法采用ELISA法检测1041例恶性肿瘤及感染性疾病患者和141例正常对照个体血清sMICA分子含量,分析其相关性及诊断价值。结果与正常对照组相比,肝癌患者的血清sMICA含量明显升高(P<0.05)。某些细菌(肠杆菌、结核杆菌、非发酵革兰阴性菌和革兰阳性球菌)、病毒(HBV和HCV)以及梅毒螺旋体感染患者的血清sMICA含量也明显高于正常对照组(P<0.05)。结论血清中sMICA分子含量对肝癌及部分感染性疾病具有一定的辅助诊断价值。肠杆菌、结核杆菌、非发酵革兰阴性菌、革兰阳性球菌、HBV、HCV及梅毒螺旋体感染可导致血清sMICA分子增高。     

结直肠癌患者NKG2D及其配体MICA/B的表达

目的:观察结直肠癌患者NKG2D及其配体MICA/B的表达,研究自然杀伤细胞免疫状态及肿瘤逃避免疫监视的机制。方法:流式细胞术检测40例结直肠癌患者及30例健康体检者外周血自然杀伤细胞绝对计数,NKG2D及其配体MICA/B的表达。ELISA检测血清可溶性MICA的浓度。结果:结直肠癌患者自然杀伤细胞绝对计数、NKG2D、MICA/B的表达均降低,可溶性MICA浓度增高,与对照组比较差异有统计学意义。NKG2D的表达与结直肠癌组织分化程度呈负相关(P<0.01),与病理分期无关(P>0.05);血清可溶性MICA浓度与结直肠癌的病理分期呈正相关(P<0.01),与组织分化程度无关(P>0.05)。结论:结直肠癌患者血清高水平的可溶性MICA,抑制了NKG2D介导的抗肿瘤免疫,可能是肿瘤逃避免疫监视的机制之一。外周血NKG2D的表达与血清可溶性MICA浓度可以作为判断上皮类肿瘤恶性程度的指标。     

MICA在上皮性卵巢癌中的表达及意义

目的:观察膜型和可溶型MICA在上皮性卵巢癌组织中的表达,探讨上皮性卵巢癌中MICA基因表达的意义。方法:采用RT-PCR和Western Blotting方法检测28例上皮性卵巢癌组织和12例正常卵巢组织中MICA基因的表达。结果:MICA mRNA水平在卵巢癌组织中有显著表达,在卵巢癌和正常卵巢组织中的表达阳性率分别为89.3%和16.7%,差异有统计学意义(P<0.05);mMICA蛋白在上皮性卵巢癌组织中比在正常卵巢中表达降低。结论:尽管MICA mRNA的表达上调,其翻译的mMICA的表达却是降低的,可能MICA mRNA合成的蛋白大部分以sMICA的形式入血清,卵巢癌组织中mMICA表达降低可能是机体对肿瘤产生免疫逃逸的原因。