肝癌患者红细胞补体受体I型分子及其基因多态性变化

目的探讨红细胞补体受体Ⅰ型分子（CR1）在肝癌疾病发生、发展中的意义。方法采用聚合酶链反应(PCR)和HindIII酶切技术测定红细胞CR1分子基因型,采用酶联法定量测定红细胞CR1分子的数量,以红细胞天然免疫粘附试验测定红细胞CR1分子粘附活性。结果104例肝癌患者红细胞CR1分子密度相关基因多态性分布（HH70.2%,HL24.0%,LL5.8%）与75名正常人(HH74.7%,HL21.3%,LL4.0%)比较差异无显著意义;肝癌患者红细胞CR1分子的数量（0.83±0.22）及粘附活性（47.1±6.5）均显著低于正常人组,差异有非常显著意义（1.26±0.33、62.4±7.6,P<0.01）;并且CR1分子基因多态性为高表达的肝癌患者其红细胞CR1分子数量表达与粘附活性都明显下降;伴有肝功能明显异常的肝癌患者其红细胞CR1分子数量及粘附活性显著低于早期或肝功能正常的肝癌患者。结论提示肝癌患者红细胞CR1分子数量及粘附活性的变化主要是后天因素引起,肝癌患者红细胞CR1分子数量及粘附活性的变化与病情发展及严重程度密切相关。     

慢性重型肝炎患者血清sCR1动态变化与肝脏功能损伤程度的关系研究

目的对慢性重型肝炎患者血清sCR1浓度变化进行动态研究,探讨慢性重型肝炎sCR1浓度变化与该病发生、发展及预后的关系,探索预警慢性重型肝炎发生和发展的有效检测指标。方法选择枣庄市立医院78例慢性重型肝炎、76例慢性肝炎、61例肝硬化患者分别为研究组及28例健康人为对照组,采用酶联免疫夹心法测定血清sCR1,应用统计学方法比较分析。结果测定血清sCR1平均值,正常对照组（34．3±9．1）ng／ml慢性肝炎组（72．5±15．3）ng／ml,肝硬化组（91．8±28．1）ng／ml,慢性重型肝炎组（163．2±56．5）ng／ml。慢性重型肝炎及肝硬化患者sCR1浓度≥100ng／ml90例,〈100ng／ml59例。结论慢性重型肝炎患者血清sCR1浓度的变化与肝功能损伤程度密切相关,该指标可作为慢性重型肝炎患者病情严重程度的有效检测指标。     

2型糖尿病患者CR1基因多态性与红细胞CR1数量、黏附活性相关性研究

目的研究2型糖尿病(T2DM)患者1型补体受体(CR1)基因多态性与红细胞CR1数量、黏附活性的相关性。方法应用限制性内切酶HindⅢ聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术测定132例T2DM患者和124名正常健康人群CR1基因多态性,用酶联免疫吸附试验(ELISA)测定红细胞的CR1数量,检测红细胞黏附活性,并做统计学分析。结果 T2DM患者红细胞表面的CR1数量和黏附活性均低于正常对照组(P<0.01)。CR1基因型在2组的分布频率均为HH型最高、HL型次之、LL型最低。T2DM组CR1基因等位基因L频率为14.8%,高于对照组(11.3%),但2组CR1基因型频率和等位基因频率差异均无统计学意义(P>0.05)。2组组内比较,HH型与HL型红细胞CR1数量及黏附活性差异均无统计学意义(Ρ均>0.05)。T2DM组HH型与HL型红细胞CR1数量与黏附活性均低于正常对照组(Ρ<0.05)。结论 T2DM患者与正常对照者间CR1基因型频率和等位基因频率无差异,而T2DM患者红细胞CR1数量及黏附活性减低,提示T2DM的发生与红细胞免疫功能低下有关。     

红细胞CR1密度相关基因组多态性与血细胞免疫活性相关性研究

目的　对正常人红细胞CR1密度相关基因组多态性与血细胞免疫活性相关的意义进行探讨。方法　采用CR1基因PCR PFLP方法和红细胞或淋巴细胞粘附肿瘤细胞的方法以及混合淋巴细胞培养3H TdR法测定红细胞CR1密度相关基因组多态性和血细胞的天然免疫和特异免疫活性。结果　发现红细胞CR1基因组HH型正常人 (32例 ) ,血细胞天然免疫活性都比HLA型 (1 7例 )或LL型 (1例 )天然免疫活性高。在供者中红细胞CR1基因HH型混淋刺激指数明显高于HL型或LL型。结论　红细胞CR1密度相关基因组多态性与血细胞免疫活性之间相关性在外周血干细胞移植治疗中的临床应用价值 ,值得深入研究     

急性脑梗死患者红细胞补体受体I型基因组密度多态性的变化

目的研究脑梗死患者红细胞补体受体Ⅰ型(ECR1)基因多态性分布,探讨脑梗死患者ECR1活性下降与ECR1基因缺陷的关系及脑梗死发病与ECR1基因缺陷的关系.方法采用PCR和hindⅢ酶切技术测定68例患者及30例正常对照的ECR1分子基因型,采用C3b受体花环实验测定其中30例患者和30例对照的ECR1活性,并对两个脑梗死家系进行研究分析.结果脑梗死患者组HH型比率比正常对照组明显降低(P＜0.05);40～49岁及60岁以上组患者HH型相对同年龄组正常人比率明显降低(P＜0.01);男性脑梗死患者比男性对照组HH型比率降低(P＜0.01);两个脑梗死家系的研究显示与正常对照组脑梗死组相比,HH型明显降低而HL型明显升高(P＜0.05);HH型、HL型ECR1活性明显低于对照组(P＜0.05).结论脑梗死患者组ECR1密度相关基因点突变率增高,HH型红细胞免疫功能低下,主要为后天获得性改变;而HL型为混合性红细胞免疫功能低下;年轻和老年患者的红细胞免疫功能降低与ECR1基因缺陷有关,男性患者ECR1基因突变在脑梗死发病中有重要作用.     

慢性重型肝炎血清sCR1与肝脏损伤的关系研究

目的探讨慢性重型肝炎sCR1浓度变化与该病发生、发展及预后的关系。方法采用酶联免疫夹心法测定血清sCR1,研究慢性重型肝炎血清sCR1动态变化与肝脏功能损伤的程度的关系。结果(1)慢性重型肝炎死亡组,存活组的早期、中期及肝硬化病人血清sCR1浓度均显著高于正常人,且慢性重型肝炎死亡组、存活组的早期、中期病人血清sCR1浓度升高的幅度明显大于肝硬化患者,并随CHE的降低,PT的延长及PTA的降低而逐渐升高;(2)慢性重型肝炎死亡组和存活组的早期中期无明显差异性,但两组的晚期和恢复期有明显差异性。结论慢性重型肝炎病人血清sCR1浓度的变化与肝功能损伤程度密切相关,该指标可作为慢性重型肝炎患者病情严重程度、判断病情发展及预后的重要参考指标。     

HBeAg阳性慢性乙型肝炎患者红细胞补体受体Ⅰ型分子基因点突变与HBV DNA含量的关系

目的研究免疫应答和耐受HBeAg阳性慢性乙型肝炎患者红细胞补体受体Ⅰ型分子(CR1)基因点突变与HBV DNA含量变化的关系,探讨其临床意义。方法选择肝脏功能正常、血清HBV DNA大于106IU/ml的114例HBeAg阳性慢性乙型肝炎患者作为免疫耐受组研究对象,选择初次发生转氨酶高于正常值2倍以上伴或不伴有肝细胞性黄疸的110例HBeAg阳性慢性乙型肝炎患者为免疫应答组研究对象。采用PCR和HindⅢ酶切技术对红细胞CR1分子基因多态性进行分类。用细胞核酸释放剂(CNR)裂解细胞,释放HBV DNA,对白细胞HBV DNA进行实时荧光PCR检测。结果免疫应答组HBeAg阳性慢性乙型肝炎患者的红细胞CR1基因点突变率明显低于免疫耐受组和正常人群(P<0.05)。在免疫应答组中,CR1基因发生点突变患者的血清和白细胞HBV DNA含量均明显高于未发生点突变者(P<0.05);在免疫耐受组中,红细胞CR1基因发生点突变和无点突变的患者其血清和白细胞HBV DNA差异均无统计学意义。结论检测HBeAg阳性慢性乙型肝炎患者红细胞CR1基因点突变对了解乙型肝炎患者清除HBV的能力、评估病情发展、判断预后具有一定的指导意义。     

免疫学检验与临床

,重型肝炎患者血清sCR1含量变化与红细胞粘附活性的相关性研究，C3、C4、AAG、AMG的检测对胸腔积液的诊断价值,急性脑梗死病人血清Ig与补体C3的动态变化及临床意义,华支睾吸虫病人治疗前后血清中IgG／IgG4抗体的变化,母、脐血清Ig和补体水平与胎膜早破关系的探讨,特异性体液免疫与传染性非典型肺炎患者病情的关系研究双抗体夹心酶联免疫吸附法检测IgGo的研究及应用     

HBeAg 阳性慢性乙肝患者红细胞补体受体Ⅰ型分子基因点突变的分布及意义

目的:研究免疫耐受与应答组HBeAg阳性慢性乙肝患者红细胞补体受体Ⅰ型分子 (complement receptor type 1,CR1)基因点突变分布特点及数量水平,并分析其临床意义.方法:选择114例肝脏功能正常、血清HBV DNA含量大于106 IU/mL的HBeAg阳性慢性乙肝患者作为免疫耐受组研究对象,选择110例初次转氨酶高于正常值2倍以上伴或不伴有肝细胞性黄疸的HBeAg阳性慢性乙肝患者为免疫应答组研究对象.采用PCR和Hind Ⅲ酶切技术对红细胞CR1分子基因点突变进行分类,并采用酶联法定量测定红细胞CR1分子数量水平.结果:免疫耐受组红细胞CR1基因无点突变的患者明显低于应答组(P ＜ 0.05);免疫耐受与应答组的女性患者其无点突变百分率明显高于男性患者(P ＜ 0.05);在红细胞CR1基因无点突变的患者中,耐受组患者CR1分子数量较应答组明显下降(P ＜ 0.05);发生点突变个体的CR1分子数量明显低于未发生点突变的患者(P ＜ 0.01).结论:红细胞CR1基因点突变与HBeAg阳性慢性乙肝患者的免疫反应状态有关,女性患者以无点突变类型为多.     

重型肝炎患者红细胞CR1粘附活性与肝功能损伤程度的相关性研究

目的　研究重型肝炎患者红细胞CR1粘附活性的变化 ,探讨其与肝功能损伤严重程度的相关性。方法　采用红细胞天然免疫功能实验对 38例重型肝炎、5 8例慢性病毒性肝炎、30例正常人的红细胞CR1粘附活性进行测定 ,并与肝功能指标比较。结果　重型肝炎及慢性病毒性肝炎患者红细胞CR1粘附活性均低于正常人群 (P <0 .0 1) ,但重型肝炎患者红细胞CR1粘附活性下降的程度显著重于慢性病毒性肝炎患者 (P <0 .0 1) ;在疾病恢复期红细胞CR1粘附活性则明显回升 ;重型肝炎患者红细胞CR1粘附活性的变化与CHE、PT及PTA等指标的变化密切相关。结论　重型肝炎患者红细胞CR1粘附活性的变化与肝功能损伤严重程度密切相关 ,该项可作为分析重型肝炎患者病情严重程度、判断病情发展及预后的重要参考指标。     

慢性乙型肝炎患者红细胞天然免疫黏附功能与血清sCR1变化的相关性研究

目的研究乙型肝炎病毒e抗原(HBeAg)阳性慢性乙型肝炎患者红细胞天然免疫黏附功能(RNIAF)与血清可溶性Ⅰ型补体受体(sCR1)的浓度变化,分析二者的相关性,并探讨其临床意义。方法选择48例肝功能正常、血清HBV DNA大于106 U/mL的HBeAg阳性慢性乙型肝炎患者作为肝功能正常组,48例转氨酶高于正常值2倍以上的HBeAg阳性慢性乙型肝炎患者作为肝功能异常组。检测RNIAF,同时采用酶联免疫定量检测试剂盒测定血清sCR1含量。结果肝功能异常组的HBeAg阳性慢性乙型肝炎患者RNIAF较肝功能正常组及健康对照组显著下降,而sCR1含量显著升高,差异有统计学意义(P<0.01)。动态观察持续肝功能异常的HBeAg阳性慢性乙型肝炎患者RNIAF与血清sCR1含量变化,显示二者呈明显负相关(r=-0.91)。HBeAg阳性慢性乙型肝炎患者血清sCR1含量与凝血酶原活动度也呈明显负相关(r=-0.87)。结论 HBeAg阳性慢性乙型肝炎患者RNIAF与血清sCR1的含量变化与肝脏功能损伤严重程度密切相关,可作为临床判断肝病患者病情严重程度及预后的参考指标。     

人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的评价与应用

目的对研制的人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的重复性、稳定性进行评价,并了解其实际应用效果。方法选取中、晚期肝硬化患者50例和健康体检者50例分别作为肝病组和正常对照组,以鼠抗人CD35单克隆抗体、兔抗人sCR1多克隆抗体和辣根过氧化物酶标记的山羊抗兔IgG为包被抗体、夹心抗体和检测抗体,以纯化后的重组人sCR1蛋白为标准品,建立人血清微量sCR1蛋白双抗体夹心ELISA试剂盒,进行试剂盒重复性和稳定性检验,并应用该试剂盒检测两组血清sCR1蛋白水平。结果双抗体夹心ELISA法检测人血清微量sCR1蛋白的线性范围是15.60~250.00ng/mL;血清sCR1蛋白水平对吸光度值的回归方程为Y=112.10 X2+18.21 X+1.694(r2=0.998);重复性检测中,高、低水平标准品检测值的批内相对标准偏差(RSD)分别为6.20%、7.40%,批间RSD分别为6.70%和7.90%;试剂盒稳定性检测中,RSD均不大于0.01;肝病组血清sCR1表达水平显著高于正常对照组(P0.01)。结论人血清微量sCR1蛋白双抗体夹心ELISA试剂盒线性范围广、重复性好、易于保存,适合于临床和科研检测工作。     

儿童系膜增生性肾小球肾炎红细胞免疫黏附功能的实验研究

目的从红细胞免疫角度探讨儿童系膜增生性肾小球肾炎（MsPGN）的免疫发病机制。方法检测34例MsPGN患儿的红细胞C3b受体花环率（RCR）、肿瘤红细胞花环率（TRR）作为红细胞免疫黏附功能指标;聚合酶链反应（PCR）联合Hindm酶切技术测定红细胞第1补体受体（CRl）密度相关基因多态性（HH型、HL型、LL型）。结果MsPGN患儿的RCR、TRR均低于正常儿童（P〈0．01）;MsPGN患儿红细胞CRl密度相关基因多态性分布以HH型占大多数,但百分比较对照组明显下降（P〈0．05）,未检出LL型。在相同基因人群中,MsPGN患儿TRR明显低于对照组（P〈0．01）;无论是MsPGN组还是对照组,HH型TRR均比HL型高（P〈0．01）。结论MsPGN患儿红细胞膜上CRl活性降低,识别、黏附、清除免疫复合物（Ic）的能力下降,循环中的Ic在肾小球滤过时沉积于系膜区,引起系膜细胞增生反应,MsPGN患儿红细胞免疫黏附功能显著下降在该病的发病机制中起重要作用。红细胞CRl密度相关基因型别与红细胞免疫黏附功能有关,MsPGN患儿红细胞免疫功能下降可能为继发性下降。     

Membrane-bound C4b interacts endogenously with complement receptor CR1 of human red cells

Activation of the classical complement pathway on the membrane of autologous cells results in the deposition of C4b on their surface and in the assembly of the C3 convertase C4b2a, one of the amplifying enzymes of the cascade. Here we study the sequence of events leading to irreversible inactivation of the potentially harmful C4b bound to human red cells. We show that deposited C4b interacts endogenously with complement receptor type 1 (CR1) present on the membrane of the same red cell. Complexes containing CR1 and C4b are found in extracts of membranes of C4b-bearing red cells after treatment of the intact cells with a bifunctional crosslinking reagent. The amount of complexed CR1 increases with the number of deposited C4b molecules. Only small amounts of free CR1 are observed on red cells bearing as few as 1,900 molecules of C4b, suggesting that the binding avidity between C4b and endogenous CR1 is high. In agreement with this observation, we find that the deposited C4b inhibits the exogenous cofactor activity of the red cell CR1 for the factor I-mediated cleavage of target-bound clustered C3b. The C4b bound to the human red cells is cleaved by the serum enzyme C3b/C4b inactivator (factor I) and a large fragment (C4c) is released in the incubation medium. The cleavage is totally inhibited by mAbs against CR1, showing that the complement receptor is an essential cofactor for the activity of I. When the number of bound C4b per red cell is relatively small (less than 1,000 molecules) the substrate for the enzymatic activity of factor I is mostly or exclusively the C4b bound endogenously to CR1. Indeed, the kinetics or the extent of cleavage of C4b are not affected by greatly augmenting the concentration of exogenous CR1 or of C4b-bearing red cells in the incubation mixture, thereby increasing the frequency of collisions between CR1 on the surface of one cell with C4b deposited on the membrane of a different cell. On the basis of the present and prior observations, we speculate that both DAF and CR1 act endogenously to inactivate the function of autologous red cell-bound C4b and prevent the progression of the cascade. DAF binding prevents the formation of the C3 convertase, C4b2a. The cleavage and irreversible inactivation of C4b only occurs after the concerted activities of endogenous CR1 and serum factor I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complement receptor 1/CD35 is a receptor for mannan-binding lectin

Mannan-binding lectin (MBL), a member of the collectin family, is known to have opsonic function, although identification of its cellular receptor has been elusive. Complement C1q, which is homologous to MBL, binds to complement receptor 1 (CR1/CD35), and thus we investigated whether CR1 also functions as the MBL receptor. Radioiodinated MBL bound to recombinant soluble CR1 (sCR1) that had been immobilized on plastic with an apparent equilibrium dissociation constant of 5 nM. N-acetyl-d-glucosamine did not inhibit sCR1-MBL binding, indicating that the carbohydrate binding site of MBL is not involved in binding CR1. C1q inhibited MBL binding to immobilized sCR1, suggesting that MBL and C1q might bind to the same or adjacent sites on CR1. MBL binding to polymorphonuclear leukocytes (PMNs) was associated positively with changes in CR1 expression induced by phorbol myristate acetate. Finally, CR1 mediated the adhesion of human erythrocytes to immobilized MBL and functioned as a phagocytic receptor on PMNs for MBL-immunoglobulin G opsonized bacteria. Thus, MBL binds to both recombinant sCR1 and cellular CR1, which supports the role of CR1 as a cellular receptor for the collectin MBL.     

Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes

Complement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1). By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 +/- 1.15 micrograms/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoaffinity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-CR1 and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney. uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CR1- coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.