表达人膜辅助因子蛋白基因的猪内皮细胞可抑制人血 …

目的 研究转染人膜辅助因子蛋白基因的猪内皮细胞抗补体的细胞毒作用。方法 从人胎盘组织提取总ＲＮＡ,用ＲＴ－ＰＣＲ技术扩增得到人膜辅助因子蛋白基因全长ｃＤＮＡ,将其克隆到带有巨细胞病毒（ＣＭＶ）ＩＥ启动子的ｐＣＩ－ｎｅｏ哺乳动物表达载体和以ｐＣＩ－ｎｅｏ为基础构建成的带有人ＥＦ－１α启动子的ｐＥＦ－ｎｅｏ哺乳动物表达载体上,得到质粒ｐＣＩ－ｎｅｏ为基础构建成的带有人ＥＦ－１α启动子的ｐＥＦ－ｎｅｏ哺     

携带共扩增基因的CHO细胞表达载体的构建

构建携带共扩增基因的ＣＨＯ细胞表达载体。方法以质粒载体ｐＣＩ－ｎｅｏ为骨架,应用小鼠二氢叶酸还原酶基因的ｃＤＮＡ,构建了哺乳动物细胞表达载体ｐＣｄｈｆｒ１。把绿色荧光蛋白基因亚克隆到ｐＣｄｈｆｒ１的多克隆位点,构建了表达质粒ｐＦＰ。     

干扰素对人胃癌细胞IAP—2表达的调节

应用基因重组干扰素－α和－γ体外诱导三侏人胃癌细胞系ＳＧＣ７９０１，ＭＧＣ８０３和ＭＫＮ４５，ＡＢＣ－ＣＥＬＩＳＡ法测定诱导组和对照细胞表面免疫抑制酸性蛋白Ⅱ型的表达量。在基础培养条件下，三株细胞表达ＩＡＰ－２均呈低水平。低浓度（＜１０００ｕ／ｍｌ）ＩＦＮ－α或－γ反使其ＩＡＰ－２表达量显著减少。这些结果提示：（１）ＩＦＮ－α和－γ可能对胃癌细胞ＩＡＰ－２表达呈双向调节作用；（２）癌细胞ＩＡＰ－２     

人朊蛋白基因外显子Ⅰ及其上游序列具有启动子样活性

目的 鉴定朊蛋白（ＰｒＰ）基因转录启动子位置。方法 利用聚合酶链反应（ＰＣＲ）扩增人ＰｒＰ基因外显子Ⅰ及其上游序列，序列鉴定后插入ＣＡＴ报道质粒ｐＢＬ－ＣＡＴ６，分别转染ＨｅＬａ、ＣＯＳ７和Ｓｈ－ｓｙ５ｙ细胞系，检测ＣＡＴ表达值；提取三种细胞蛋白，以条带移动实验检测细胞转录激活因子ＳＰ１含量。结果 人ＰｒＰ基因外显子Ｉ及其上游序列为ＧＣ富含，带有多个ＳＰ１可能性综合位点，但无明显ＴＡＴＡ盒；瞬时转染结果显示这段序列可诱导２～３倍的ＣＡＴ表达增强；定量移动条带实验证明ＨｅＬａ细胞中含有较高浓度的ＳＰ１，而ＣＯＳ７和Ｓｈ－ｓｙ５ｙ细胞中ＳＰＩ含量极低。结论 人ＰｒＰ基因外显子Ｉ及其上游序列具有启动子样功能，为弱的非ＴＡＴＡ盒启动子；不同组织来源的细胞中ＳＰ１含量不同，在神经细胞系Ｓｈ－ｓｙ５ｙ中人ＰｒＰ基因外显子Ｉ     

抗氧化剂PDTC抑制氧化的低密度脂蛋白诱导的人肾小球系膜细胞NF_(-k)B活化

目的 研究氧化的低密度脂蛋白（Ｏｘ－ＬＤＬ）对体外培养的人肾小球系膜细胞（ＨＭＣ）核因子－ＫＢ（ＮＦ－ＫＢ）活化的影响，以及抗氧化剂毗咯二硫氨基甲酸酯（Ｐ ＤＴＣ）对ＮＦ－ＫＢ活化的抑制作用，探讨ＯＸ－ＬＤＬ介导肾损害的基因调控机制及抗氧化剂ＰＤＴＣ防治脂质肾损害的可能性。方法 将Ｏｘ－ＬＤＬ或ＰＤＴＣ与ＨＭＣ共培养后，提取细胞核蛋白进行凝胶迁移率变动分析（ＥＭＳＡ）检测ＮＦ－ＫＢ的活化，用细胞ＥＬＩＳＡ法检测细胞内ＩＫＢα蛋白含量的变化，反映ＩＫＢα的降解及免疫组化染色检测细胞内的Ｐ６５向核转位。结果 正常对照组未见ＮＦ－ＫＢ活化，当用不同浓度（１０、２５、５０及１００ｍｇ／Ｌ）的Ｏｘ－ＬＤＬ，刺激肾小球系膜细胞 ｌｈ后，均可引起细胞 ＮＦ－ＫＢ活化及 ＩＫＢα降解。与对只组相卜较差异显著（ｐ＜０．０５），以５０ｍｇ／Ｌ 的ＯＸ－ＬＤＬ刺激ＨＭＣ１ｈ，ＮＦ－ＫＢ活化及 ＩＫＢα降解最明显。ＮＦ－ＫＢ活化的同时，伴有Ｐ６５由胞浆向胞核的转位。１００μｍｏｌ／Ｌ ＰＤＴＣ，能明显抑制 ＮＦ－ＫＢ的活化、ＩＫＢα降解（ｐ＜０．０１）及 Ｐ６５的核转位。结论Ｏｘ－ＬＤＬ。能诱导ＨＭＣ的ＩＫＢαａ降解、Ｐ６５的核转位，最终使Ｎ     

TNF-α、IL-1β 、LPS对人多核白细胞、脐静脉内皮细胞血小板内皮细胞粘附分子-1表达的影响

目的：初探炎性刺激对人多核白细胞（ＰＭＮ）和脐静脉内皮细胞（ＨＵＶＥＣ）的血小板内皮细胞粘附分子－１（ＰＥＣＡＭ－１）表达的影响和ＰＥＣＡＭ－１的可能作用。方法：使用流式细胞仪和免疫组化法,观察脂多糖（ＬＰＳ）、白介素（ＩＬ）－１β和肿瘤坏死因子（ＴＮＦ）－ａ对ＰＭＮ和ＨＵＶＥＣ ＰＥＣＡＭ－１表达的变化。结果：ＬＰＳ、１Ｌ－１β和ＴＮＦ－ａ可引起ＰＭＮ表达ＰＥＣＡＭ－１蛋白减少,而对ＨＵＶＥＣ　ＰＥＣＡＭ－１的表达无显著影响,但组化显著在ＨＵ－ＶＥＣ连接部的ＰＥＣＡＭ－１染色变淡。结论：上述炎性刺激物不仅可引起ＰＭＮ表达ＰＥＣＡＭ－１减少、激活ＰＭＮ,而且可改变内皮细胞ＰＥＣＡＭ－１的分布,因此ＰＥＣＡＭ－１在炎症中可能起重要作用。     

T细胞产生的一种新型细胞因子：人IL—17

从ＣＤ４＾＋Ｔ细胞文库中克隆出人ＩＬ－１７（ｈＩＬ－１７）。外周血ＣＤ４＾＋Ｔ细胞在刺激状况下表达高水平的ｈＩＬ－１７。ｈＩＬ－１７基因由哺乳动物表达载体ｐＤＣ４０９携带转染ＣＤ１／ＥＢＮＡ细胞后，可表达为糖基化及非糖基化两种形式。ｈＩＬ－１７Ｆｃ融合蛋白及转染ｈＩＬ－１７的细胞培养上清可诱导产生ＩＬ－６和ＩＬ－８，并且还可促进人或纤维细胞表面细胞间粘附分子－１（ＩＣＡＭ－１）的表达。     

PAI—1在TGF—β1基因和反义TGF—β1基因转染人肝癌？…

目的：通过对人肝癌细胞株（ＳＭＭＣ－７７２１）转染ＴＧＦ－β１基因及反义ＴＧＦ－β１基因,观察该细胞ＰＡＩ－１表达的变化。方法：采用电穿孔法进行基因转染,并用Ｗｅｓｔｅｒｎｂｌｏｔ、Ｎｏｒｔｈｅｒｎｂｌｏｔ法另以鉴定后,分别应用Ｗｅｓｔｅｒｎｂｌｏｔ及Ｎｏｒｔｈｅｒｎｂｌｏｔ观察该细胞表达ＰＡＩ－１ｍＲＮＡ的变化。结果：过表达的ＴＧＦ－β１细胞克隆ＰＡＩ－１ｍＲＮＡ表达均高于对照组,而低表达ＴＧＦ     

ER阳性和ER阴性人乳腺癌细胞株p53、mdm-2及p21~(WAF1)蛋白表达的研究

目的研究ＥＲ阳性和ＥＲ阴性人乳腺癌细胞株ｐ５３、ｍｄｍ－２和ｐ２１ＷＡＦ１蛋白的表达及其与细胞生物学特性的关系。方法应用细胞培养、基因转染和免疫组化染色ＬＳＡＢ法等技术，检测ＥＲ阳性、表达野生型ｐ５３（ｗｔｐ５３）蛋白的ＭＣＦ－７细胞和ＥＲ阴性、表达突变型ｐ５３（ｍｔｐ５３）的ＭＤＡ－ＭＢ－２３１细胞以及ＥＲ转染阳性ＭＤＡ－ＭＢ－２３１细胞中ｐ５３、ｍｄｍ－２和ｐ２１ＷＡＦ１蛋白的表达水平，比较其与细胞生物学特性的关系。结果（１）ＭＣＦ－７细胞和ＭＤＡ－ＭＢ－２３１细胞ｐ５３蛋白的性质和分布明显不同，前者ｐ２１ＷＡＦ１和ｍｄｍ－２蛋白的表达水平明显高于后者（Ｐ＜０．０５），且前者的生物学特性较后者为好。（２）ＥＲ质粒转染ＭＤＡ－ＭＢ－２３１细胞后，其ｐ５３蛋白的表达水平降低（Ｐ＜０．０５），而ｍｄｍ－２蛋白的表达水平增加（Ｐ＜０．０５），生物学特性得以改善。结论乳腺癌细胞ＥＲ状态与ｐ５３和ｍｄｍ－２蛋白的表达水平以及生物学特性有关。     

人补体膜辅助调节蛋白MCP的基因克隆及共真核表达的研究

目的 克隆获得编码人补体膜辅助调节蛋白（ＭＣＰ）的ｃＤＮＡ，并对其在真核细胞的表达及功能进行研究。方法 应用ＲＴ－ＰＣＲ 方法，从Ｕ９３７细胞总ＲＮＡ中扩增编码人ＭＣＰ分子的ｃＤＮＡ片段，快速克隆于ｐＧＥＭ－Ｔ Ｅａｓｙ载体，测定共序列。将该片段重组于ｐＬＸＳＮ载体，电穿孔转染ＮＩＨ３Ｔ３细胞，经ＦＡＣＳ检测筛选表达ＭＣＰ的阳性细胞克隆，用补体容破试验鉴定其抑制人补体溶破的功能。结果 ＲＴ－ＰＣＲ     

Cleavage of human IgM with human lysosomal elastase

Human lysosomal elastase, a serine proteinase stored in the azurophil granules of polymorphonuclear leucocytes, cleaves human monoclonal IgM producing two fragments and dialyzable peptides. An F(ab)₂μ-like fragment, called IgMe in this report, retains some reactivity with an anti-Fcμ-antiserum and is antigenically deficient with respect to both the subunit (IgMs) produced by reduction and alkylation of IgM and the similar fragment (IgMp) produced by papain digestion. The other fragment is very similar to Fabμ generated by papain digestion, as indicated by immunochemical identity and a similar molecular weight.     

Uptake of human eosinophil peroxidase by human neutrophils.

A cytochemical analysis was carried out for study of the interaction between human eosinophil peroxidase (EPO) and human neutrophils. To this end, neutrophils with a genetic deficiency of myeloperoxidase (MPO) were used to avoid the otherwise inevitable interference of the high endogenous MPO activity of normal neutrophils. The data show that human neutrophils incubated with EPO (1 GU/ml) rapidly bind the enzyme all over the cell surface and internalize it in small vesicles. Part of bound EPO concentrates in a limited area on the cell surface and is then internalized by means of coarse tubular channels. Fusion of the small vesicles to each other or possibly with the tubular channels gives rise ultimately to EPO-containing multivesicular bodies, which, after 30 minutes of incubation, are the only peroxidase-positive structures in the cytoplasm. Under identical experimental conditions, no binding of human MPO to the neutrophils was detected. At concentrations 10 times as high as those used for EPO, a minority of neutrophils bound MPO, but the binding pattern remained diffuse on the plasma membrane and the internalization was negligible. It seems, therefore, that the EPO trapping system of human neutrophils exhibits specificity at least among leukocyte peroxidases. Furthermore, it operates at much lower concentrations of EPO than those reported for EPO uptake by mast cells and basophils. The uptake of EPO by neutrophils may serve to sequester a potentially toxic agent, thus limiting damage to the tissue in eosinophil-rich inflammatory lesions.     

Blastogenic response of human lymphocytes to human cytomegalovirus.

A method was developed for measuring the blastogenic response of human lymphocytes to human cytomegalovirus (CMV). Viral and control antigens were prepared by extracting disrupted infected and uninfected cell cultures with an alkaline buffer. Lymphocytes from ten donors with complement-fixing (CF) antibody exhibited a blastogenic response, whereas cells from ten seronegative donors did not. A relationship between the stimulation index (SI) and the results of neutralization (NT), indirect haemagglutination (IHA) or CF tests was not observed. The maximum blastogenic response occurred after 5 to 7 days of incubation and was usually greater when the cultures were supplemented with homologous plasma instead of sera. The presence of CMV antibody in the supplementary sera did not appear to affect the reactivity of the lymphocytes.     

Effect of recombinant human interferon γ against human cytomegalovirus

Summary Escherichia coli-derived human interferon- (rIFN-) inhibited the replication of human cytomegalovirus (HCMV) synergistically when combined with IFN-. The induction of HCMV DNA polymerase was inhibited in rIFN--treated cells. It is suggested that the induction of 2–5 A synthetase does not play an important role in the anti-HCMV actions of IFNs.With 2 Figures     

The binding of human IgG subclasses to human monocytes

The direct binding of human IgG subclasses to human monocytes has been measured by autoradiography using radiolabeled myeloma proteins. Only IgGl and IgG3 were found to bind strongly to the monocyte surface. This binding could be inhibited both by fresh human serum and by soluble immune complexes.     

Persistence of human parvovirus B19 in human tissues

Human parvovirus B19 infection causes various clinical symptoms, such as rash, arthropathy, anemias and fetal death, but it can also remain asymptomatic. The arthropathies and anemias can become chronic for several years, not infrequently resembling autoimmune syndromes. B19 replicates only in red blood cell precursors of bone marrow or fetal liver, resulting in high-titred short-lived viremia, but viral DNA is detectable also in cells of several other types. Recently B19 DNA has been found, by very sensitive amplification tests, in certain tissues not only of symptomatic but also of healthy individuals for several years or decades after B19 infection. The mere presence of B19 DNA in these tissues of a symptomatic patient (e.g. joints in chronic arthritis or skin in dermatomyositis) thereby does not prove that the present disease is caused by B19. The diagnosis has to be verified by other innovative means. How and why viral DNA persists in the tissues of healthy individuals is under investigation.     

Development of human monoclonal antibodies against human cytomegalovirus.

Human monoclonal antibodies (HMAbs) against human cytomegalovirus (HCMV) have been developed by fusion of human spleen cells and human lymphoblastoid cell lines (NP101 and NP197). The cell line NP101 had great advantages in its high fusion frequency and the stability of the resultant hybridomas. The specificity of HMAbs was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Two of the six HMAbs obtained, which were IgG3 subclass, neutralized viral infectivity in the absence of complement. The neutralizing activity of one of these two HMAbs was enhanced in the presence of human complement, whereas the other was not. Another IgG1 subclass HMAb neutralized viral infection only in the presence of complement. The remaining three HMAbs showed no neutralizing activity. Those HMAbs may provide an important approach to studying human immune responses to HCMV. HMAbs having neutralizing activity may prove to be useful for passive immunotherapy of HCMV diseases.     

Detection of human cytomegalovirus DNA in human tonsillar lymphocytes

The human cytomegalovirus (HCMV) was first isolated in cell cultures from the oropharynx, which is thought to be a site of primary infection. Although HCMV can be recovered from the oropharynx during reactivation phases, its exact site of latency is not known. In the present study we demonstrated evidence suggesting the presence of latent HCMV in this anatomic region--in the palatine tonsils. Samples from 30 tonsils obtained by tonsillectomy were screened for the presence of HCMV. Out of the 30 tonsil donors, 23 were seropositive for HCMV. Three methods were used in attempts to demonstrate HCMV's presence in the tonsils: (1) viral isolation attempts on various cell cultures, (2) immunohistochemical staining--immunoperoxidase method--designed to detect viral antigens, and (3) DNA dot hybridization with a HCMV-DNA probe designed to detect viral DNA. Neither infectious HCMV nor other viruses were isolated in cell cultures. No viral antigens were detected by immunoperoxidase staining in the tonsillar tissue. Four out of the 30 tonsils studied were found to contain viral DNA. In one case in which the tonsillar mononuclear (MN) fraction was separated from the polymorphonuclear (PMN) fraction, only the first fraction contained the viral DNA.     

Immortalization of human keratinocytes with human papilloma virus DNA

Human keratinocytes, derived from the cervix or foreskin, can be immortalized with the HPV-16 or HPV-18 E6 and E7 genes. Two methods of introducing the viral oncogenes into keratinocytes i.e. calcium phosphate transfection and retroviral transduction, are described below, both of which have been optimized for human keratinocytes. While the calcium phosphate transfection method can be used in a normal tissue culture facility, transduction with a retroviral vector containing oncogenes, requires a containment facility and appropriate laboratory practice.