Raji细胞免疫酶抑制法检测血清抗HLA—DR抗体

本研究利用Ｒａｊｉ细胞表面具有表达组织相容性抗原ＤＲ（ＨＬＡ－ＤＲ）特点，以及被检血清中抗ＨＬＡ－ＤＲ抗体与抗人ＨＬＡ－ＤＲ单克隆抗体（ＭｃＡｂ）竞争结合Ｒａｊｉ细胞表面ＨＬＡ－ＤＲ抗原的特征，建立检测血清抗ＨＬＡ－ＤＲ抗体的Ｒａｊｉ细胞免疫酶抑制法。对７２例系统性红斑狼疮（ＳＬＥ）患者和１１３例健康者血清检测结果表明：ＳＬＥ患者和健康者血清中抗ＨＬＡ－ＤＲ抗体阳性率分别为３９．０％和１．８％。此     

抗组织因子途径抑制物单克隆抗体的制备及鉴定

目的：制备抗组织因子途径抑制物（ＴＦＰＩ）单克隆抗体。方法：用微量ＴＦＰＩ脾内包埋免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与ＳＰ２／Ｏ细胞融合，建立了２株稳定分泌抗ＴＦＰＩ单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株，分别命名为４Ｆ４和４Ｆ８。对其中的一株４Ｆ８进行研究，其杂交瘤细胞染色体众数为９３，分泌的抗体为ＩｇＧ１。以简易正辛酸法从腹水纯化ＭｃＡｂ４Ｆ８，ＳＤＳ－ＰＡＧＥ检测其纯度，Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析显示其可特异性地识别分子量为３４８００的抗原分子。对纯化的抗体以改良过碘酸钠氧化法用辣根过氧化物酶标记后，采用双夹心ＥＬＩＳＡ法检测正常人血浆ＴＦＰＩ含量。结果：稀释的凝血酶原时间（ＰＴ）测定，ＭｃＡｂ４Ｆ８可缩短其凝固时间，且呈量效关系，ＭｃＡｂ４Ｆ８还可使乏因子Ⅸ血浆稀释的ＰＴ明显缩短。正常人血浆ＴＦＰＩ含量为１０３．２±１１．５μｇ／Ｌ，结果与美国Ｄｉａｇｎｏｓｔｉｃａ公司的ＴＦＰＩＥＬＩＳＡ试剂盒所测结果（９８．４±１０．３μｇ／Ｌ）相近（ｒ＝０．９２）。结论：ＭｃＡｂ４Ｆ８有潜在的药用价值，并可望为我国ＴＦＰＩ的研究提供精确的检测手段。     

ELISA间接法测定AchRab对重症肌无力的诊断价值

本文对４０例健康正常人及８０便重症肌无力（ＭＧ）患者进行了酶联免疫吸附试验（ＥＬＩＳＡ）间接法血清乙酰胆碱受体抗体（ＡｃｈＲａｂ）检测，结果：正常对照组ＡｃｈＲａｂ阳性率为０％，ＭＧ患者组ＡｃｈＲａｂ阳性率为６２．５％，其中全身型为６９．５７％。ＥＬＩＳＡ间接法简便、结果可靠、特异性好／敏感性较高，对检测人员无危害，易于临床推广应用。     

抗T淋巴细胞单克隆抗体对重型再生障碍性贫血造血祖细胞的影响

报告抗Ｔ淋巴细胞单克隆抗体（ＭｃＡｂ－Ｔ）对重型再生障碍性贫血（ＳＡＡ）造血祖细胞的影响。发现经ＭｃＡｂ－Ｔ体外清除（ＳＡＡ）骨髓中的ＣＤ＿３或ＣＤ＿８细胞后，１９例ＳＡＡ中有１０例患者的粒－单核系祖细胞集落及红系祖细胞集落产量明显增多（Ｐ＜０．０１）。提示部分ＳＡＡ的发病与Ｔ淋巴细胞的免疫抑制有关。在体内应用ＭｃＡｂ－Ｔ（抗ＣＤ＿３和抗ＣＤ＿８）治疗后，该１０例患者临床疗效显著，其中基本治愈、缓解共６例，明显进步４例。结果表明，ＭｃＡｂ－Ｔ能特异性地抑制相应的Ｔ淋巴细胞，解除Ｔ淋巴细胞对造血功能的直接或间接抑制作用，从而使ＳＡＡ患者的造血功能得以恢复。     

Graves‘病人TSH抗独特型抗体和TRAb相关性研究

根据自身免疫性甲状腺疾病（Ａｕｔｏｍｉｍｍｕｎｏ ｔｈｙｒｏｉｄ ｄｉｓｅａｓｅ，ＡＩＴＤ）发病机制新的独特型－抗独特型免疫网络学说，用ＴＳＨ独特型抗体（ＴＳＨＡｂ１）检测ＴＳＨ抗独特抗体（ＴＳＨＡｂ２），用＾１２５Ｉ－ｈＴ－ＳＨＡｂ１＾１２５Ｉ－１３Ｂ４（ｈＴＳＨ单抗）检测ＴＲＡｂ阳性和阴性的Ｇｒａｖｅｓ‘病人，依正常混合血清为质控标本，依ＴＡｂ阴性组结合率ｘ＋２ｓ为标准，大于此值为ＴＳＨＡｂ２     

McAb79—HSA—MTX在小鼠体内稳定性及生物学分布研究

本文用马来酰亚胺基类活泼酯法交联ＭｃＡｂ７９－ＨＳＡ－ＭＴＸ，并从尾静脉注入小鼠体内，在不同时间眼球后取血，用ＳＤＳ－ＰＡＧＥ放射自显影对其稳定性加以研究，结果表明：该偶联物在入血半小时即有７６％裂解，但在２４小时内无明显增加。本文还以ＭｃＡｂ７９－ＭＴＸ为对照，对ＭｃＡｂ７９－ＨＳＡ－ＭＴＸ在人Ｎａｌｍ－移植瘤裸鼠体内生物学分布进行了检测。结果发现：ＭｃＡｂ７９－ＭＴＸ更易在肿瘤部位浓集，并且肝     

血浆交换疗法在神经科的应用和有关实验检查

应用ＰＥ疗法治疗１５例（１００次）神经科危重病人（ＭＧ危象１０例，ＧＢＳ３例，ＡＤＥＭ２例），其中１３例（８７％）获得快速而显著的效果。３０次ＰＥ前后ＡｃｈＲａｂ的变化和１８次ＰＥ前后ＮＫ细胞的变化均有显著性差异，为ＰＥ疗法提供了理论和实验依据。尽管ＰＥ可以引起一些并发症，但仍然是一种安全的治疗方法。     

重症肌无力的诊断和治疗——神经系统疾病(7)(续前)

１　定义 重症肌无力是重点累及神经肌肉接头（ＮＭＪ）处突触后膜（ＰＳＭ）上的乙酰胆碱受体（ＡｃｈＲ），主要由乙酰胆碱受体抗体（ＡｃｈＲＡｂ）介导、细胞免疫依赖性、补体参与的自身免疫性受体病 （ＡＩＲＤ）。２　发病机制２．１　由乙酰胆碱受体抗体介导２．１．１乙酸胆碱受体抗体与重症肌无力相关　①重症肌无力病人的ＡｃｈＲＡｂ滴度高，而正常人滴度正常；②同一重症肌无力病人肌无力重时ＡｃｈＲＡｂ滴度高，轻时低。重症肌无力病人血浆交换前如肌无力病情重，则抗体滴度高，血浆交换后肌无力病情减轻，则抗体滴度低。２．…     

抗卵巢抗体的检测及其抗生育作用的研究

用ＴｒｉｔｏｎＸ－１００自人卵巢组织匀浆中提取得卵巢抗原（ＯＡｇ），建立了检测抗卵巢抗体（ＡｏＡｂ）的ＥＬＩＳＡ法。发现卵巢功能早衰、不孕症、流产患者ＡｏＡｂ水平与检出率均显著高于正常生育妇女。与ＡｏＡｂ阴性患者比较，ＡｏＡｂ阳性患者基础体温呈双相型者较少，呈黄体功能不足型者较多；Ｂ超监测发现排卵者减少，不排卵者增多；雌二醇（Ｅ２）、孕酮（Ｐ）水平显著降低。经ＡｏＡｂ阳性血清的γ球蛋白作用后，体外培养的卵巢组织细胞分泌ＩＬ－１β、ＴＮＦ－α、ｓＩＬ－２Ｒ显著增多，而分泌Ｅ２与Ｐ显著减少。研究结果提示，ＡｏＡｂ具有潜在的抗生育作用。     

SLE患者血清抗亲环素A自身抗体及其特异免疫复合物的测定与意义

用离子交换色谱和凝胶过滤技术从小牛胸腺中提取、纯化亲环素 Ａ（ＣｙＰ Ａ）。 ＳＤＳ－ＰＡＧＥ分析为单一条带，分子量为 １７．８ＫＤ，与 ＣｙＰ Ａ标准品一致。用此 ＣｙＰ Ａ建立了检测抗 ＣｙＰＡ自身抗体（ＡＣＡ）的ＥＬＩＳＡ法。在免疫豚鼠获得抗血清基础上，又建工了ＥＬＩＳＡ法检测ＣｙＰＡ特异免疫复合物（ＣｙＰ Ａ－ＩＣ）。正常对照（ｎ＝１００） ＡＣＡ和 ＣｙＰ Ａ－ＩＣ阳性率均仅 １．０％． ＳＬＥ患者（ｎ＝１０１） ＡＣＡ阳性率为 ５０．５％， ＣｙＰ Ａ－ＩＣ为 ７４．２％。结果提示，血清 ＡＣＡ和 ＣｙＰ Ａ－ＩＣ测定对ＳＬＥ的诊断有较高的敏感性和特异性。     

甲状腺激素对大鼠额叶乙酰胆碱的影响

目的研究成年期甲状腺功能减退症大鼠额叶内乙酰胆碱变化及甲状腺素替代治疗后的情况。方法 26只成年期雄性SD大鼠随机分为三组,用丙基硫氧嘧啶(PTU)建立成年期大鼠甲减模型,采用放射免疫法测定健康对照组、甲减组、甲状腺素替代治疗组(T4-6)大鼠的血清甲状腺激素水平;碱性羟胺比色法测定脑组织乙酰胆碱含量;用单因素方差分析比较三组之间各项指标差异。结果与健康对照组相比,甲减组大鼠血清T3、T4水平显著减低、TSH水平增加(P<0.05);替代治疗后,血清T3、T4、TSH恢复至正常水平;甲减组大鼠额叶内乙酰胆碱含量降低(P<0.05),替代治疗后乙酰胆碱含量与对照组相比差异无统计学意义(P>0.05)。结论成年期甲减大鼠额叶内乙酰胆碱含量减少,甲状腺素替代治疗后乙酰胆碱含量恢复正常。     

重症肌无力患者白细胞介素6水平的测定

目的　探讨白细胞介素 6 (IL 6 )在重症肌无力 (MG)发病机制中的作用。方法　采用双抗体夹心ELISA法对 18例MG患者用糖皮质激素 (GC)治疗前、治疗 3个月后和 2 0例正常对照血清IL 6、乙酰胆碱受体抗体 (AchRab)水平进行检测。结果　MG患者组血清IL 6水平显著高于对照组 (P <0 .0 1) ,MG患者组血清IL 6水平在用GC治疗 3个月后显著降低 (P <0 .0 1) ,其血清IL 6与血清AchRab水平呈正相关 (r =0 .6 89,P <0 .0 1)。结论　IL 6与MG发病密切相关 ,IL 6参与了MG的免疫病理过程 ;检测血清IL 6水平对MG临床有重要价值 ;GC可抑制IL 6合成及Ach Rab的产生。     

某些有机磷杀虫剂影响乙酰胆碱M2受体磷酸化的实验研究

目的研究有机磷类杀虫剂(OPs)对G蛋白结合受体激酶-2(GRK-2)介导的毒蕈碱乙酰胆碱m2受体(mAChR2)磷酸化的影响,进一步揭示OPs可能存在的其他作用途径。方法将纯化的大鼠脑mAChR2,GRK-2,同位素磷32标记的三磷酸腺苷([γ-P32]ATP)与不同浓度的对氧磷(PO)、氧毒死蜱(CPO)和毒死蜱(CPF)共同保温,聚丙烯酰胺凝胶电泳,凝胶片干燥后放射性自显影检测mAChR2磷酸化结果。将干燥凝胶片中放射性标记的磷酸化mAChR2蛋白带剪下,同位素液体闪烁计数器计数。结果CPO抑制大鼠脑mAChR2的磷酸化,其IC50为70?mol/L;CPF也抑制mAChR2的磷酸化,而PO与对硫磷(PT)不抑制mAChR2的磷酸化。CPO和CPF并不抑制同样由GRK2介导的β2肾上腺素受体(β2-AR)的磷酸化。结论CPO和CPF选择性地抑制GRK-2介导的mAChR2磷酸化,而PO和PT无此效应。说明某些有机磷类杀虫剂可能存在其它作用靶分子。     

Regulation of brain acetylcholine concentration by muscarinic receptors

The cholinergic agonists oxotremorine, oxotremorine-1, oxotremorine-3, arecoline and BM 123 (N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone) were used to investigate the role of muscarinic receptors in the regulation of acetylcholine (Ach) concentration in the whole mouse brain. Intravenous oxotremorine, oxotremorine-1, oxotremorine-3 and arecoline dose-dependently decreased ex vivo binding of [3H]oxotremorine-M and correspondingly increased brain Ach concentration. The correlation coefficient between the ED50's of these two parameters was 0.90. BM 123 induced percentage of reduction in muscarinic receptors correlated with percentage of decrease in response of oxotremorine, for increasing brain Ach concentration. These results indicate that the muscarinic receptor system involved in the regulation of brain Ach levels may lack spare receptors.     

Effect of acetylcholine on mitogen response of peripheral lymphocytes isolated from rats exposed to chronic stress

The purpose of this study was to clarify the effects of acetylcholine (Ach) on lymphocyte function in rats under chronic stress. The authors isolated peripheral lymphocytes from rats 5 weeks after stress treatment and then measured interleukin-2 (IL-2) production after stimulation with concanavalin A or phytohemagglutinin-L. Although mitogen-induced IL-2 production of the stress group was lower than that of the control group, the addition of Ach significantly increased mitogen-induced IL-2 production in both groups. This effect of Ach was inhibited by atropine in the control group only. The changes (increasing rates) in mitogen-induced IL-2 production from basal condition showed a negative correlation with serum corticosterone concentrations. The authors observed no correlation between the effects of Ach (changes in mitogen-induced IL-2 production with Ach compared to those without Ach) and serum corticosterone concentration. These findings suggest that stimulation of the parasympathetic nervous system improves lymphocyte function during chronic stress.     

睡眠剥夺对大鼠学习能力和海马乙酰胆碱含量的影响

目的研究不同持续时间睡眠剥夺(SD)对大鼠学习能力以及海马乙酰胆碱(Ach)含量的影响。方法采用小平台水环境法建立睡眠剥夺模型,以正常组作为对照,使用Y型迷宫测试不同持续时间睡眠剥夺对大鼠学习能力的影响,采用碱性羟胺比色法测定海马乙酰胆碱含量变化。结果与正常对照组相比,睡眠剥夺4 d、6 d组学习成绩明显降低,海马乙酰胆碱含量明显减少,睡眠剥夺2 d组学习成绩明显提高,海马乙酰胆碱含量无显著差异。结论睡眠剥夺导致的大鼠学习能力下降可能与其海马乙酰胆碱含量减少有关。     

蛋白A免疫吸附治疗重症肌无力的临床观察

目的探讨蛋白A免疫吸附治疗重症肌无力（MG）的临床疗效。方法采用病例对照方法,将24例MG患者非随机分为2组。治疗组12例,对照组12例,治疗组在传统治疗方法的基础上,给予蛋白A免疫吸附治疗,每周3次,共4周。对照组不进行蛋白A吸附治疗。治疗前和治疗后抽取清晨空腹血,测IgG、IgA、IgM、补体C3、C4和采用酶联免疫吸附法测乙酰胆碱受体抗剂（AChR-Ab）,并根据疗效标准临床相对评分法进行评分。结果治疗组总有效率为91.67%（11/12）,对照组总有效率为75%（9/12）,差异有统计学意义（P〈0.05）。治疗组免疫球蛋白IgG、IgA、IgM和AchR-Ab下降程度较对照组明显降低（P〈0.01）。结论蛋白A免疫吸附能有效降低MG血液中AchR-Ab和免疫球蛋白水平,比单纯药物治疗疗效好,是治疗MG的有效方法。     

Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [3H]acetylcholine release from rabbit retina labeled in vitro with [3H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [3H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [3H]acetylcholine with the following order of potency: apomorphine greater than or equal to SKF(R)82526 greater than SKF 85174 greater than SKF(R)38393 greater than or equal to pergolide greater than or equal to dopamine (EC50 = 4.5 microM) greater than SKF(S)82526 greater than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of [3H]acetylcholine: SCH 23390 (IC50 = 1 nM) greater than (+)-butaclamol greater than or equal to cis-flupenthixol greater than fluphenazine greater than perphenazine greater than trans-flupenthixol greater than R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating [3H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [3H]SCH 23390, or as determined by adenylate cyclase activity. [3H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [3H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [3H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [3H]SCH 23390 binding sites (r = 0.755, P greater than .05, n = 8). The potencies of antagonists to inhibit dopamine-evoked [3H]acetylcholine release were correlated with their potencies to inhibit the dopamine-stimulated adenylate cyclase (r = 0.759, P less than .05, n = 5) and with their affinities at [3H]SCH 23390 binding sites (r = 0.998, P less than .01, n = 7). We conclude that in rabbit retina dopamine evokes calcium-dependent [3H]acetylcholine release through activation of a site with the pharmacological characteristics of a D-1 dopamine receptor.     

In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes

To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated.     

Immunoenzymometric assay and radioimmunoassay measure different populations of antibody against human acetylcholine receptor

We used a "sandwich"-type immunoenzymometric assay (IEMA) and a radioimmunoassay (RIA) to measure antibody against the human nicotinic acetylcholine receptor in serum from individuals with myasthenia gravis, with markedly different results for certain specimens, as measured by the two techniques. In some cases, antibody concentrations were high by RIA but low by IEMA; in others, the reverse was found. Such differences persisted through 30 months after thymectomy. An investigation of potential causes of this disparity suggests that high IEMA measurements reflect specific anti-receptor antibody and are not artifactual. The IEMA is recommended as an adjunct to the RIA because some patients with myasthenia gravis who have low concentrations of anti-receptor antibodies as measured by RIA have significantly above-normal concentrations of anti-receptor antibodies as measured by IEMA.