人外周血淋巴细胞和粒细胞褪黑素受体亚型mt1 的检测

目的检测人外周血淋巴细胞和粒细胞的褪黑素受体(MR)亚型mt1和其细胞内定位.方法分离出健康成年人外周血淋巴细胞和粒细胞,应用RT-PCR技术分别检测其亚型mt1的mRNA,阳性产物用自动测序仪测序;同时将所分离的淋巴细胞和粒细胞制成细胞涂片,应用免疫组化染色检测mt1在淋巴细胞和粒细胞上的分布.结果 RT-PCR产物电泳显示人外周血淋巴细胞和粒细胞均存在mt1阳性条带,片段长度约370 bp,测序结果显示扩增产物与GenBank的人mt1受体亚型的基因序列相吻合;免疫组化结果显示人外周血淋巴细胞和粒细胞均存在mt1受体蛋白,分布于细胞膜、细胞浆和细胞核,以细胞膜和细胞浆为主.结论人外周血淋巴细胞和粒细胞中均存在MR的亚型mt1,外周血淋巴细胞和粒细胞为褪黑素作用的外周靶器官,提示褪黑素在生理及病理情况下可能对免疫系统有调节作用.     

Graves病及正常甲状腺组织褪黑素受体亚型表达差异的研究

用RT-PCR半定量分析结果显示,Graves病甲状腺组织及甲状腺腺瘤瘤旁正常甲状腺组织(各5例)均存在褪黑素受体亚型mt1mRNA的表达,未检测到MT2亚型。手术时经药物控制甲状腺功能已正常的Graves病与甲状腺机能仍亢进的Graves病甲状腺组织mt1表达量无差异(P>0.05);Graves病甲状腺组织mt1表达量较正常甲状腺组织明显增加(P<0.01)。提示褪黑素的免疫调节作用可能通过mt1介导,Graves病发病可能与mt1亚型激活有关。     

甲状腺滤泡细胞癌组织褪黑素受体表达的研究

目的研究成人甲状腺滤泡细胞癌及癌旁正常甲状腺组织褪黑素受体(MR)表达的改变及意义。方法选取第二军医大学长征医院2004年2月至2005年6月手术的成人甲状腺滤泡细胞癌组织及癌旁正常组织,用放射配体结合法检测肿瘤组织MR表达,用逆转录-聚合酶链反应半定量分析MR亚型mRNA表达的改变。结果甲状腺滤泡细胞癌组织125I褪黑素(Mel)特异结合Scatchard分析结果为最大结合容量(Bmax)(0.63±0.07)fmol/mg,平衡解离常数(Kd)值为(45.3±9.3)pmol/L;正常甲状腺组织Bmax(0.58±0.04)fmol/mg,Kd值为(43.5±8.2)pmol/L。甲状腺滤泡细胞癌及瘤旁正常甲状腺组织均有mt1、MT2受体表达;甲状腺滤泡细胞癌MT2受体亚型表达量比正常甲状腺组织显著增加(P<0.05)。结论褪黑素受体亚型MT2与甲状腺滤泡细胞癌的发生发展有一定关联。     

桥本甲状腺炎及正常甲状腺组织褪黑素受体亚型基因表达差异的研究

目的　研究成人桥本甲状腺炎及甲状腺腺瘤旁正常甲状腺组织褪黑素受体亚型基因表达的差异及意义。方法　取成人桥本甲状腺炎患者手术切除的甲状腺组织及甲状腺腺瘤瘤旁1. 0cm外正常甲状腺组织,抽提总RNA,合成mt1、MT2 受体引物,用逆转录多聚酶链反应半定量分析褪黑素受体亚型mRNA表达的改变。结果　成人桥本甲状腺炎、甲状腺腺瘤瘤旁正常甲状腺均存在mt1 受体表达,未检测到MT2 受体亚型。桥本甲状腺炎甲状腺组织mt1 受体表达量较正常甲状腺组织明显减少,统计学分析有显著性差异(P<0. 05)。结论　桥本甲状腺炎发病可能与mt1亚型受体抑制有关。褪黑素免疫调节作用可能通过mt1 受体介导。     

蜱中人粒细胞埃立克体DNA的PCR检测及序列分析

目的了解吉林地区蜱种感染人粒细胞埃立克体的情况。方法采用16S rRNA基因特异引物进行半套式PCR,检测吉林省部分林区蜱中人粒细胞埃立克体DNA,并对扩增产物进行克隆和测序,与已知序列进行同源性比较。结果从采集于吉林地区的全沟硬蜱中检出人粒细胞埃立克体的特异性DNA片段,阳性率为1.98%。扩增产物经克隆、测序发现吉林地区人粒细胞埃立克体扩增片段与美国人粒细胞埃立克体分离株(GenBank注册号为U02521)16SrRNA基因序列相对应片段相差2个核苷酸,同源性为99.7%。结论吉林地区的全沟硬蜱携带人粒细胞埃立克体,提示吉林地区可能存在人粒细胞埃立克体病的自然疫源地。     

系统性红斑狼疮患者自体外周血造血干细胞移植前后粒细胞形态变化

目的观察25例系统性红斑狼疮患者自体外周血造血干细胞移植前后的粒细胞形态变化。方法采用环磷酰胺和重组人粒细胞集落刺激因子（rhGM-CSF）动员后获得的外周血造血干细胞,经马利兰、环磷酰胺、抗人淋巴细胞球蛋白、甲基强的松龙预处理后,回输自体外周血干细胞,用rhGM-CSF刺激恢复造血,注射rhGM-CSF前后采集静脉血制备血涂片,pH 6.4~6.8条件下瑞特-姬姆萨混合染色后在油镜下观察中性粒细胞形态,同时进行中性粒细胞碱性磷酸酶（NAP）染色,pH 5.4条件下姬氏染色观察中毒颗粒。结果应用rhGM-CSF治疗前1 d中性粒细胞形态无明显异常,中毒颗粒、NAP积分值均在正常范围;应用rhGM-CSF治疗后中毒颗粒、NAP积分明显增高,96%的中性粒细胞胞体偏大,胞浆内见＂中毒＂性颗粒、空泡数量增加。结论系统性红斑狼疮患者应用rhGM-CSF可引起外周血中性粒细胞出现明显的形态学改变。     

粒细胞颗粒的核糖核酸酶—金探针标记研究

应用核糖核酸酶－金探针对胃癌、胃炎患者的胃粘膜活检组织中的炎性细胞以及正常人和粒细胞白血病患者之外周血白细胞的核糖核酸（ＲＮＡ）进行了超微结构水平上的定位。电镜下发现，胃粘膜组织中单核细胞、淋巴细胞的胞浆嗜天青颗粒不被ＲＮａｓｅ－Ｇ标记，但中性、嗜酸性及嗜碱性粒细胞的各种胞浆颗粒上均明确标记有ＲＮａｓｅ－Ｇ探针。正常人外周血白细胞的ＲＮａｓｅ－Ｇ标记结果与以上相同。研究结果表明，各种粒细胞颗粒内均     

粒细胞颗粒的核糖核酸酶－金探针标记研究

应用核糖核酸酶－金（ＲＮａｓｅ－Ｇ）探针对胃癌、胃炎患者的胃粘膜活检组织中的炎性细胞以及正常人和粒细胞性白血病患者之外周血白细胞的核糖核酸（ＲＮＡ）进行了超微结构水平上的定位。电镜下发现，胃粘膜组织中单核细胞、淋巴细胞的胞浆嗜天青颗粒不被ＲＮａｓｅ－Ｇ标记，但中性、嗜酸性及嗜碱性粒细胞的各种胞浆颗粒上均明确标记有ＲＮａｓｅ－Ｇ探针。正常人外周血白细胞的ＲＮａｓｅ－Ｇ标记结果与以上相同。研究结果表明，各种粒细胞颗粒内均含有ＲＮＡ成份。这一发现的重要意义在于它对粒细胞颗粒成份认识的传统观念提出了修正。此外，我们尚观察到粒细胞性白血病患者外周血白血病细胞的胞浆颗粒不被或甚少被ＲＮａｓｅ－Ｇ标记，与正常人明显不同，因此，ＲＮａｓｅ－Ｇ技术还具有重要的临床实用开发潜能。     

病毒性心肌炎患者心肌细胞线粒体DNA缺失的定量分析

为探讨病毒性心肌炎 (VMC)患者心肌细胞线粒体 DNA(mt NDA)缺失突变情况及意义 ,用定量 PCR法检测 2 0例 VMC患者心肌细胞及其外周血淋巴细胞 mt DNA4 977碱基对 (mt DNA4 977)和 mt DNA74 36 碱基对 (mt D-NA74 36 )缺失率。取 10例健康意外死亡者心肌和 2 0例献血员外周血淋巴细胞作正常对照。结果显示 ,正常对照者和 VMC患者心肌细胞均存在 m t DNA4 977及 mt DNA74 36缺失 ,合计缺失率分别为 0 .176 %、0 .384 % ,二者差异显著 ,P<0 .0 5 ;VMC患者外周淋巴细胞 mt DNA缺失程度与心肌细胞呈一致性改变 ,且有良好的相关性 (r=0 .92 0 ,P<0 .0 0 1)。提示 mt DNA缺失可能是 VMC发病过程中重要的心肌损伤机制 ;外周淋巴细胞在研究心肌细胞 mt DNA缺失中的作用值得进一步探讨     

山东省1例人粒细胞无形体病调查

目的结合1例可疑人粒细胞无形体病病例个案调查,探讨PCR技术在现场流行病学中的应用。方法对1例可疑人粒细胞无形体病病例进行流行病个案调查,采用套式-聚合酶链反应技术检测病例血液中人粒细胞无形体16SrRNA基因。结果DNA检测为阳性,测序分析显示与浙江、吉林野鼠检出的无形体相应序列的同源性在99％以上。结论山东省存在人粒细胞无形体感染病例,进一步开展自然疫源地调查工作十分必要。     

Melatonin receptor subtype MT2 (Mel 1b) and not mt1 (Mel 1a) is associated with melatonin-induced enhancement of cell-mediated and humoral immunity

Individuals of many vertebrate species undergo seasonal changes in immune function in addition to marked seasonal changes in reproductive, metabolic, and other physiological processes. Despite growing evidence that photoperiod mediates seasonal changes in immunity, little is known regarding the neuroendocrine mechanisms underlying these changes. Enhanced immune function in short days is correlated with increased duration of nightly melatonin secretion, and recent studies indicate that melatonin can act directly on immune cells to enhance immune function. It remains unknown, however, which melatonin receptor subtype mediates immune enhancement by melatonin. The present study examined the contribution of specific melatonin receptor subtypes, mt1 (Mel 1a) and MT2 (Mel 1b), in mediating melatonin-induced enhancement of cell-mediated and humoral immune function in mice. Melatonin enhanced both splenocyte proliferation and anti-keyhole limpet hemocyanin (KLH) IgG concentrations in both wild-type (WT) and mice lacking a functional gene for melatonin receptor mt1 (mt1 -/-), suggesting that the mt1 receptor does not mediate these responses. In addition, luzindole, an MT2 receptor antagonist, attenuated melatonin-induced enhancement of splenocyte proliferation in both WT and mt1 -/- mice. Taken together, these results suggest that receptor subtype mt1 is not necessary for mediating melatonin-induced enhancement of immune function and provide the first evidence for a specific melatonin receptor subtype, MT2, that may be involved in melatonin-induced immune enhancement.     

Human cytotoxic T-lymphocyte recognition of an HLA-A3 gene product expressed on murine L cells: the only human gene product required on the target cells for lysis is the class I heavy chain.

To dissect the molecular basis for T-cell recognition of class I major histocompatibility complex antigens, we have examined the ability of human cytotoxic T lymphocytes (CTL) to recognize murine L cells transformed with a human class I gene. Three transformed L-cell lines were generated that expressed the human HLA-A3 gene from donor E1 at levels comparable to those of the endogenous L-cell H-2Kk molecules. CTL were generated in secondary and tertiary mixed lymphocyte culture against the HLA-A3 subtype of donor E1 by culturing irradiated E1 peripheral blood lymphocytes with the peripheral blood lymphocytes of responder donor M3 (M3 shares all defined class I antigens with E1 but expresses a different HLA-A3 subtype). Each of the HLA-A3-transformed L cells was lysed by M3 anti-E1 CTL in a short-term 51Cr release assay and this recognition was blocked by a monoclonal anti-HLA-A3 antibody. Antibodies specific for the human T8 and LFA-1 molecules on the CTL effectors (but absent from the transformed targets) also blocked lysis of each of the HLA-A3 transformed L-cell targets. Antibodies to other T-cell molecules failed to block lysis. The present results demonstrate that human CTL can recognize human class I molecules on targets that do not express any other human gene product and further suggest that effector T-cell molecules T8 and LFA-1 are functionally involved in this recognition process.     

Mitochondrial functionality and mitochondrial DNA content in lymphocytes of vertically infected human immunodeficiency virus-positive children with highly active antiretroviral therapy-related lipodystrophy.

Mitochondria functionality and apoptosis were studied in peripheral blood lymphocytes (PBL) of human immunodeficiency virus type 1-infected children, with or without lipodystrophy (LD), who were receiving highly active antiretroviral therapy (HAART) and in PBL of healthy control subjects (HCs). By flow cytometry, mitochondrial (mt) membrane potential, mt mass, intra-mt cardiolipin distribution, and early and late apoptosis in fresh PBL or in PBL cultured with different stimuli were assessed. mtDNA content was evaluated in fresh PBL by an original double-competitive quantitative polymerase chain reaction method, which enabled direct quantification of the number of mtDNA copies present in human lymphocytes. PBL from LD-positive and LD-negative children and from HCs were similar in mt functionality and in their tendency to undergo apoptosis. mtDNA content was also similar in PBL of LD-positive children and HCs, suggesting that normal mt functionality and normal tendency to undergo apoptosis are present in PBL of children with HAART-associated LD.     

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.     

mt1 Melatonin receptor in the primate adrenal gland: inhibition of adrenocorticotropin-stimulated cortisol production by melatonin

The pineal hormone melatonin participates in circadian, seasonal, and reproductive physiology. The presence of melatonin binding sites in human brain and peripheral tissues is well documented. However, in the mammalian adrenal gland, low-affinity melatonin binding sites have been detected only in the rat by some but not all authors. Conflicting evidence for a regulatory role of melatonin on adrenal cortisol production, prompted us to investigate this possibility in a New World primate, the capuchin monkey. Expression of melatonin receptors in the adrenal cortex was demonstrated through pharmacological characterization and autoradiographic localization of 2-[125I]iodomelatonin binding sites (dissociation constant = 96.9 +/- 15 pM; maximal binding capacity = 3.8 +/- 0.4 fmol/mg protein). The mt1 identity of these receptors was established by cDNA sequencing. Melatonin treatment of dispersed cells and explants from adrenal gland did not affect basal cortisol production. However, cortisol production stimulated by 100 nM ACTH was significantly inhibited by low melatonin concentrations (0.1-100 nM); this inhibitory effect was reversed by the mt1/MT2 melatonin antagonist luzindole. Melatonin also inhibited dibutyril-cAMP-stimulated cortisol production, suggesting that melatonin acts through a cAMP-independent signaling pathway. The present data demonstrate that the primate adrenal gland cortex expresses functional mt1 melatonin receptors and shows that melatonin inhibits ACTH-stimulated cortisol production.     

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines

The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.     

Immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells

Abstract: This paper reviews the evidence that supports the hypothesis of the existence of specific binding sites for melatonin on immune cells. These binding sites have been described in human blood lymphocytes and granulocytes, and thymus, spleen, and bursa of Fabricius from different rodents and birds. The dissociation constant values of these binding sites are in the 0.1 -1 nM range, suggesting that melatonin may play a physiological role in lymphocyte regulation. Moreover, melatonin binding sites appear to be modulated by guanine nucleotides. Therefore, in addition to other mechanisms described for the regulation of immune function by melatonin, a direct mechanism of regulation can be involved via binding of melatonin by immunocompetent cells.     

Expression of CD61 (beta3 integrin subunit) on canine cells

A monoclonal antibody (JM2E5) specific for the integrin beta3 chain, or CD61 or GPIIIa subunit, has been employed to determine the expression of the canine homologue CD41/CD61 or CD51/CD61 complex on different canine cells in peripheral blood lymphocytes, monocytes, granulocytes, platelets, erythrocytes, lymph-node cells, spleen cells and breast tumour cells). The canine homologue CD41/CD61 or CD51/61 was present on peripheral blood lymphocytes, monocytes, granulocytes, breast tumour cells and spleen cells as well as on platelets and it was absent from erythrocytes and lymph-node cells. An antigen with components of molecular masses of 25/100/120 kDa (under reducing conditions) was immunoprecipitated from canine peripheral lymphocytes and platelets, but not from granulocytes or monocytes. Expression on canine lymphocytes of the canine homologue of the human beta3 integrin chain was unexpected, based on the expression pattern of this molecule in human tissue.