慢性HBV 感染者IFN-γ+ NK 细胞与Treg 及IL-10 的关系研究

目的:探讨慢性HBV感染者外周血自然杀伤(NK)细胞分泌的IFN-γ与CD4~+CD25~+Foxp3~+调节性T细胞(Treg)及IL-10等细胞因子含量的关系。方法:收集119例慢性HBV感染肝病患者,其中CHB患者57例,LC患者40例,HCC患者22例,另收集同期健康志愿者(HC) 27例。流式细胞仪检测外周血Treg、NK细胞及IFN-γ~+NK细胞比例,CBA细胞因子(IL-10、IFN-γ、IL-2、IL-4、TNF及IL-5)含量,全自动生化分析仪检测血清ALT、AST水平。结果:CHB、LC、HCC患者外周血Treg百分比和血清中IL-10水平均显著高于HC组, CHB、HCC患者外周血Treg绝对数值显著高于HC组(P0.05),CHB、LC及HCC患者Treg与IL-10呈正相关(P0.01,r=0.564;P0.05,r=0.426;P0.05,r=0.476)。NK细胞绝对数、IFN-γ~+ NK细胞百分比及血清中IFN-γ含量在CHB、LC、HCC组中均显著低于HC组(P0.01)。同时CHB患者组IFN-γ~+NK细胞比例与Treg细胞比例、血清IL-10水平均负相关(P0.05,r=-0.618;P0.05,r=-0.635),血清IFN-γ水平亦与Treg负相关(P0.01,r=-0.342);LC患者中,外周血中NK细胞绝对数值与Treg细胞比例负相关(P0.05,r=-0.317),IFN-γ~+NK细胞比例与血清中IL-10水平负相关(P0.05,r=-0.514);HCC患者组IFN-γ~+NK细胞比例与Treg细胞比例、血清IL-10水平均负相关(P0.01,r=-0.676;P0.01,r=-0.615)。结论:CHB患者及其进展疾病如LC和HCC外周血IFN-γ~+NK细胞比例与Treg比例、血清IL-10水平负相关,提示Treg和IL-10参与抑制IFN-γ~+ NK细胞可能是慢性HBV感染及其进展的重要因素之一,因此逆转Treg及IL-10所致的免疫耐受,将会有力提高NK细胞抗HBV慢性感染及恶性进展。     

NK细胞和NKT细胞在慢性乙型肝炎中的表型和功能特征

目的检测慢性乙型肝炎(CHB)患者外周血自然杀伤(NK)细胞、自然杀伤T(NKT)细胞的比例、NKG2D/NKG2A水平和γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)的水平。方法采集并分离CHB患者外周血单个核细胞(PBMC),流式细胞术检测NK、NKT细胞占PBMC的比例,NKG2D、NKG2A水平,经佛波酯(PMA)、布雷菲德菌素A(BFA)、离子霉素(ionomycin)处理后,流式细包术检测IFN-γ、TNF-α的水平,分析各指标与乙肝病毒载量和血清丙氨酸氨基转移酶的关系,采用独立样本t检验与Pearson相关性分析。结果与正常对照者相比,CHB患者外周血NK细胞和NKT细胞比例明显降低;NKG2A阳性的NK细胞和NKT细胞明显升高;经PMA、BFA、ionomycin刺激后,IFN-γ阳性的NK细胞和NKT细胞明显降低。结论 CHB患者NK、NKT细胞存在不同程度的减少、受体表达失调和细胞因子分泌功能下降等功能紊乱。     

复发性自然流产患者外周血自然杀伤细胞免疫表型检测

目的 检测复发性自然流产(RSA)患者外周血自然杀伤(NK)细胞的免疫表型。方法 应用流式细胞术检测27例RSA妊娠患者、41例RSA非妊娠患者、32例正常妊娠者和25例正常非妊娠者外周血CD56dimCD 16+、CD56brightCD16+/-、CD69+、HLA-DR+ NK细胞表达并进行比较。结果RSA妊娠组、RSA非妊娠组、正常妊娠组和正常非妊娠组外周血CD56dimCD16+ NK细胞占总NK细胞的比例分别为(88.69.±5.86)％、(79.25±9.31)％、(79.24±10.09)％、(75.49±11.96)％;CD56brightCD16+/- NK细胞的比例分别为(8.18±5.54)％、( 12.20±6.49)％、( 13.13±8.65)％、( 11.53±6.23)％;CD69+ NK细胞的比例分别为(3.42±2.13)％、(2.36±1.72)％、(2.68±1.81)％、(2.08±1.73)％;HLA-DR+ NK细胞的比例分别为(15.26±9.32)％、(9.96±7.18)％、( 10.57±8.05)％、(9.64±6.12)％。与RSA非妊娠组、正常妊娠组及正常非妊娠组比较,RSA妊娠组CD56dimCD16+、CD69+、HLA-DR+ NK细胞显著升高（均P＜0.05）。与RSA非妊娠组及正常妊娠组比较,RSA妊娠组CD56brightCD16+/- NK细胞明显降低（均P＜0.05）。结论 RSA妊娠患者外周血中NK细胞CD56dimCD16+、CD69+和HLA-DR+表达增高,而CD56brightCD16+/-表达下降,可能在RSA的发生发展中起着一定的作用。     

人血淋巴细胞亚群杀伤K562细胞活性的比较及其相互作用

应用单克隆抗体盤选法,从人外周血中分离出CD4、CD8、CD19(B)和CD57(NK)四种淋巴细胞亚群,发现各群细胞对K562肿瘤细胞均有杀伤活性,其中以CD57细胞的杀伤活性最强.CD4和CD57细胞混合培养后的杀伤活性超过两者之和,而CD8、CD19分别与CD57细胞混合培养后,杀伤活性与两者单独培养之和相比无显著性差异.结果表明,人外周血杀伤K562细胞活性受到多种亚群细胞的影响,因此常规用K562作靶细胞测定外周血NK活性所反映的可能并非单纯NK细胞的杀伤活性.     

结肠癌患者单核或树突状细胞MICA的表达及其与NK细胞活性的关联

目的:观察结肠癌患者单核或树突状细胞是否表达MHCⅠ类相关抗原A(MICA),并分析MICA+单核细胞的频率是否与患者体内NK细胞的活性相关联。方法:首先利用流式细胞仪检测MICA在外周血单核细胞和由单核细胞诱导而来树突状细胞(DCs)上的表达;并用IL-15和IFNα-刺激树突状细胞,观察MICA表达的变化;然后检测患者外周血NKG2D+、CD16+、CD69+NK细胞的频率及NK细胞的杀伤活性;最后分析MICA+单核细胞的频率是否与NK细胞表达NKG2D及其杀伤活性关联。结果:与健康个体相比,结肠癌患者MICA+单核细胞的频率无显著变化。未成熟DCs表达MICA,但受IL-15和IFNα-刺激后,结肠癌来源的DCs表面MICA的表达无显著上调。结肠癌患者外周血NKG2D+、CD69+NK细胞的频率显著降低,NK细胞的杀伤活性显著降低,而CD16+NK细胞的频率无明显变化。结肠癌患者MICA+单核细胞的频率与NK细胞表达NKG2D无明显关联,但与NK细胞杀伤靶细胞时CD107a的表达正相关。结论:结肠癌患者体内单核细胞表达MICA与NK细胞表达NKG2D无关,但高频率MICA阳性的单核细胞与NK细胞的抗肿瘤活性正相关。     

CD49a阳性NK细胞参与类风湿关节炎疾病进程

目的 研究外周血中CD49a阳性NK细胞的功能及其在类风湿关节炎中的作用。方法 流式细胞术检测健康志愿者和类风湿关节炎患者外周血NK细胞及其亚群包括CD56brightNK、CD56dimNK细胞群上CD49a及IFN-γ的表达;ELISA检测健康志愿者和类风湿关节炎患者血浆中IFN-γ的表达。结果 与健康志愿者相比,RA患者外周血NK细胞及其2个亚群上CD49a的表达均显著升高。在RA患者血浆、外周血NK细胞及其亚群上IFN-γ的表达显著升高;同时CD49a与IFN-γ双阳性的NK细胞比例增加,且CD49a阳性NK细胞较CD49a阴性NK细胞表达更高水平的IFN-γ。结论 CD49a阳性NK细胞参与类风湿关节炎疾病进程。     

慢性肝脏疾病中CD3^-CD161^＋NK和CD3^＋CD161^＋NKT细胞亚群的变化研究

目的探讨CD3^-CD161^＋NK、CD3^＋CD161^＋NKT细胞在慢性肝炎／肝硬化及肝细胞癌患者肝脏组织及外周血中表达及意义。方法利用流式细胞仪对31例肝细胞癌患者、59例慢性肝炎／肝硬化患者肝脏组织及外周血、15例正常肝脏组织、48例正常人外周血中的CD3^-CD161^＋NK和CD3^＋CD161^＋NKT进行定量分析。结果慢性肝炎／肝硬化组CD3^-CD161^＋NK细胞[（13．4±1．3）％]和肝细胞癌癌周组CD3^-CD161^＋NK细胞[（16．7±4．8）％]及远离肝癌组的肝脏组织CD3^-CD161^＋NK细胞[（22．0±4．4）％]与正常肝脏组织CD3^＋CD161^＋NK细胞[（35．1±7．2）％]相比,肝细胞癌癌周肝脏组织内含量最低（t值分别为2．301、2．137、2．034,P〈0．05）;外周血中肝细胞癌组CD3^-CD161^＋NK细胞f（11．6±6．3％）]、CD3^＋CD161^＋NKT细胞[（14．7±6．2）％]与慢性肝炎／肝硬化组CD3^-CD161^＋NK细胞[（10．8±1．7）％]、CD3^＋CD161^＋NKT细胞[（12．5±0．8）％]、正常对照组CD3^＋CD161^＋NK细胞[（7．5±0．8）％]、CD3^-CD161^＋NKT细胞[（13．8±1．7）％]相比肝癌组CD3^-CD161^＋NKT细胞含量最高（t值分别为2．134,2．099,P〈0．05）,肝癌组CD3^＋CD161^＋NKT细胞含量最高（t值分别为2．125,2．154,P〈0．05）。结论由于NK细胞及NKT细胞数量减少或／和活性降低,使肿瘤细胞逃逸了免疫监视,可能促进了肿瘤的发生、发展及转移。     

IFN-γ上调的单核细胞表面MHC-I类链相关分子促进NK细胞活化

目的:研究IFN-γ诱导上调表达的单核细胞MHC-I类链相关分子(MICs)分子对NK细胞的活化作用.方法:采用密度梯离离心法分离人外周血单个核细胞(PBMC),以免疫磁珠法从PBMC中特异性分选单核细胞及NK细胞,以细胞因子IFN-γ、TNF-α刺激单核细胞后,再将单核细胞与NK细胞共培养,以流式细胞术(FCM)检测NK细胞表面CD69分子及胞内IFN-γ表达,以51Cr释放试验检测NK细胞对K562细胞的杀伤效应.结果:IFN-γ上调人单核细胞表面MICs表达;IFN-γ刺激的单核细胞能促进异体NK细胞CD69及胞内IFN-γ表达,能增强NK细胞对K562细胞的杀伤效应;单核细胞的这种效应至少部分依赖于IFN-γ上调的MICs分子,因为用抗MIC抗体封闭或用细胞小室阻断细胞接触,可以明显地抑制NK细胞的活化.结论:IFN-γ上调人单核细胞表面MICs分子介导了NK细胞的活化.     

乙型肝炎病毒转基因小鼠C57-TgN(adr2.0)SMMU品系的免疫病理研究

目的: 研究乙型肝炎病毒 (HBV)转基因小鼠C57 TgN(adr2. 0型)SMMU品系的免疫病理学特征, 并与临床人慢性乙肝相比较。方法: 以 20只SPF级肝脏有明显病变的HBV转基因小鼠为研究对象, 用间接免疫荧光法通过流式细胞仪, 分别检测转基因小鼠及正常C57BL/6小鼠外周血淋巴细胞表面CD3、CD4和CD8表达的水平, 同时取其肝组织和5例本院病理科存档确诊为慢性中度乙型肝炎患者的肝组织石蜡标本, 用EnVision免疫组化染色法检查肝组织内T细胞亚群的分布。结果: 转基因小鼠外周血淋巴细胞表面CD3、CD4和CD8表达的水平低于正常对照组小鼠; 肝组织内浸润的单个核细胞多数为CD3 CD4 细胞, 未发现CD57 、CD8 细胞。慢性乙型肝炎患者的肝组织内浸润的单个核细胞主要为CD3 CD4 或CD3 CD8 细胞和少量CD57 细胞。结论: C57 TgN(adr2. 0型 )SMMU的HBV转基因小鼠外周血及肝组织中CD的表达与人慢性乙肝患者的外周血及肝组织有明显不同。     

早孕外周血及蜕膜中NK细胞表型及T淋巴细胞亚群的变化

目的: 检测孕妇外周血及蜕膜中T淋巴细胞亚群和NK细胞表型, 探讨它们与母-胎界面免疫耐受的关系.方法: 收集20例早孕同一患者的蜕膜组织及外周血, 密度梯度离心法分离出淋巴细胞, 流式细胞术(FCM)检测两组中NK细胞、 T细胞含量及其表面分子CD16、 NKG2A、 NKG2D表达水平.结果: 蜕膜自然杀伤(dNK)细胞占蜕膜淋巴细胞(57.15±4.0)%, 外周血自然杀伤(pNK)细胞占外周血淋巴细胞(11.46±1.58)%; dNK细胞表面CD16的表达明显低于pNK细胞, 二者分别(10.3±3.9)%与(95.6±2.6)%(P＜0.05); dNK细胞表面NKG2A的表达明显高于pNK细胞, 二者分别为(87.10±4.5) %与(27.5±4.2)% (P＜0.01), dNK细胞NKG2D的表达水平与外周血NK细胞相近, 分别为(88.70±4.1)%与(93.10±3.6)% (P＜0.05); 蜕膜中的CD4 T淋巴细胞的表达低于外周血中CD4 T淋巴细胞, 二者分别为(13.70±1.0)%与(15.85±2.4)% (P＜0.05), 蜕膜中CD8 T淋巴细胞表达明显低于外周血中CD8 T淋巴细胞, 二者分别为(15.23±1.5)%与(18.85±1.73)%(P＜0.01).结论: 妊娠期蜕膜中的NK细胞及T淋巴细胞可能是同维持母-胎界面的免疫耐受的重要原因.     

Human liver caudate lobe and liver segment

Recently, the caudate lobe has seemed to be the final target for aggresive cancer surgery of the liver. This lobe has five surfaces: the dorsal, left and hilar-free surfaces and the right and ventral-border planes. Surgeons have divided the caudate lobe into three parts: Spiegel’s lobe, which is called the ‘caudate lobe and papillary process’ by anatomists, the caudate process, viewed as almost the same entity by anatomists, and the paracaval portion corresponding to the dorsally located parenchyma in front of the inferior vena cava. All three parts are supplied by primary branches originating from the left and right portal veins, including the hilar bifurcation area. The hilar bifurcation branch often (50%) supplies the paracaval portion and it sometimes (29%) extends its territory to Spiegel’s lobe. It was postulated by Couinaud that the paracaval portion or the S9 is not defined by its supplying portal vein branch but by its ‘dorsal location’ in the liver. Couinaud’s caudate lobe or dorsal-liver concept caused, and still now causes, great logical confusion for surgeons. We attempt here to describe the margins of the lobe, border branches of the portal vein, the left/ right territorial border of the portal vein or Cantlie’s line and other topics closely relating to the surgery within these contexts. Finally, the caudate lobe as a liver segment will be discussed.     

生物人工肝的临床应用及其生物成分研究中的几个热点问题

生物型人工肝最早于1987年应用于临床,目前临床可应用的生物成分仍然来源于肝细胞(系).本文就临床应用现状、肝细胞来源、组织工程三个方面进行了综述.     

Phlogogenic and hemopoiesis-stimulating activities of liver and lung macrophages upon liver regeneration

The phagocytizing and colony-stimulating activities of liver macrophages are increased 36 h after partial hepatectomy. Although the blood count of phagocytizing cells is increased, the rate of the formation of zymosan-induced granulomas in the liver and lungs is decreased. Administration of zymosan after partial hepatectomy inhibits granulomonocytopoiesis in the bone marrow and reduces colony-stimulating activity of Kupffer cells and phagocytizing activity of liver and lung macrophages. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 11, pp. 494–498, November, 1996     

Assessment and improvement of liver specific function of the AMC-bioartificial liver

The variety of methods for measuring bioactive mass and functionality of bioartificial livers (BAL) is confusing and prevents accurate comparison of reported data. Here we present a comparison of different hepatocyte quantification methods and propose that estimation of cell pellet volume after centrifugation generates a reliable, useful and fast method. In addition a correlation is made between several function tests performed in 26 bioreactors to assess their predictive value. The ammonia eliminating capacity was found to be most predictive for other liver functions, except for lidocaine elimination as a measure of mixed function oxidase activity, which should therefore be determined separately. The oxygen consumption test proved to be an easy and predictive parameter as well. The first generation of our BAL system needed further development to assure optimal treatment of acute liver failure (ALF) patients. Changes in the porcine hepatocyte isolation method and bioreactor loading as well as changes in bioreactor configuration, including use of different materials, resulted in a significantly improved level and maintenance of in vitro BAL function. A fourfold increase in ammonia eliminating capacity, which is only reduced to 75% after seven days of culturing, offers promising prospects for further clinical application. Conclusion: The current second generation of our BAL and improvement of hepatocyte isolation and testing protocols have led to a significant increase in the level as well as the maintenance of hepatocyte specific function in our BAL. Finally, consensus on definition of the bioactive mass to be loaded in the bioreactor and insight in the variation and reliability of the functional and metabolic parameters enhances comparison of the different types of bioartificial livers presented in literature.     

Interconversion of liver and serum proteins in homogenates of rats' liver

Summary It was shown with the aid of proteins labeled by glycine-1-C¹⁴ in vivo that serum albumin is transformed into globulins in the homogenates of rat's liver. This takes place mostly after disintegration to amino acids of the added albumins. In these conditions hepatic globulins are transformed into albumins. There is no production of albumins from the serum -and -globulins added to the homogenates.Presented by Active Member Acad. Med. Sci. USSR, V. N. Orekhovich     

炎症应激反应与肝切除、肝损伤

炎症反应是生物体在进化过程中选择的一套复杂的防御体系,能激发适当的应答。然而手术和创伤可能诱发过度炎症级联反应,导致严重损伤甚至死亡。肝脏手术后的过度炎症反应可能更为显著和关键。该文提醒广大临床医生要高度重视研究过度炎症反应的损害与广泛肝切除的关系以及过度炎症反应对肝切除后剩余肝的损害。初期的抗炎症介质治疗研究主要集中在各种促炎因子的抗体、受体拮抗剂和竞争性受体拮抗剂上。本文介绍了美国FDA批准的第一个有关治疗严重败血症的药物(Drotrecogin Alfa),也介绍了作者在阻断肝切除后过度炎症反应、保护剩余肝细胞方面的实验研究。     

Alcohol and liver

The first part of this article is devoted to the metabolism of alcohol and the mechanisms underlying its hepatotoxicity. The second part describes the clinical features of the various patterns of alcohol-related liver disease (ARLD). The third part focuses on the characteristics, semiological value, and limitations of serum markers used in ARLD. Tests used to screen for alcohol abuse (blood alcohol, MCV, GGT, CDT, and FAEE) differ from those used to monitor alcohol withdrawal and to detect early-stage liver disease (ALT, AST, ASTm, alphaGST, and redox status).