抗人脑胶质瘤单克隆抗体SZ-39特异性及其识别抗原的初步鉴定

以人脑恶性胶质瘤细胞系 SHG-44为抗原免疫BALB/c 小鼠,通过杂交瘤技术获得了一株稳定地分泌抗胶质瘤细胞单克隆抗体的杂交瘤细胞株 SZ-39。经 ELISA、间接免疫荧光法和 ABC 免疫组化法分别检测了 SZ-39与各种肿瘤和正常细胞,脑瘤和正常人体组织的反应性。结果显示 SZ-39与10种被检测恶性胶质瘤细胞系中9种发生强结合反应,而与血液细胞等7种正常细胞及脑组织等12种人体组织无交叉反应。ABC 免疫组化检查了50例颅内肿瘤,结果提示 SZ-39与84.7%星形细胞瘤发生反应,尤以恶性星形细胞瘤反应最强。McAbSZ-39在移植性胶质瘤裸小鼠体内显示出良好的特异性。经免疫转移电泳法证实该抗体识别抗原为胶质瘤细胞膜上 Mw180000糖蛋白。     

人脑胶质瘤免疫抑制因子研究

实验表明人脑胶质瘤原代培养细胞及４个人脑恶性胶质瘤体外细胞系细胞培养上清液（Ｓｕ－ｐｅｒｎａｔａｎｔ，ＳＮ）显著抑制植物血凝素ＰＨＡ－Ｐ刺激的正常人或胶质瘤患者自体和异体外周血淋巴细胞增殖。我们发现用抗转化生长因子－β_２（ＴＧＦ－β_２）单克隆抗体可显著降低胶质瘤ＳＮ的免疫抑制活性。４个人脑胶质瘤体外细胞系应用抗ＴＧＦ－β_２单抗免疫组化染色呈阳性反应，３例正常脑组织呈阴性反应。Ｎｏｒｔｈｅｒｎ杂交实验表明上述４个人脑胶质瘤体外细胞系均表达６ｋｂ的ＴＧＦ－β_２ｍＲＮＡ，而４例人胎脑则无表达。这些结果提示人脑胶质瘤细胞可以自体分泌免疫抑制因子抑制患者的免疫功能，而这抑制因子的主要成份是ＴＧＦ－β_２。     

抗人脑恶性胶质瘤单克隆抗体的制备及若干方法的改进

用胶质瘤细胞系BT325和人脑胶质瘤组织各免疫Balb/c小鼠,取脾细胞和骨髓瘤细胞P_3×63-Ag_8653融合,经检测筛选,共取得15株分泌抗人脑胶质瘤的单克隆抗体杂交瘤细胞系,对其中E_4-A_6、E_4-B_6、3B_2-A_1三株进行生物学特性分析,证明对人脑恶性胶质瘤有一定特异性,对正常脑组织、正常细胞系无交叉反应。本文着重介绍了用半固体琼脂糖培养和免疫吸附检测法直接制备单克隆抗体的优点以及间接免疫荧光和改良的ELISA检测抗体的方法,应用这些方法使我们的实验取得了稳定而满意的结果。     

抗人脑胶质瘤单克隆抗体SZ39单链抗体的构建及初步表达

目的：研究人脑胶质瘤基因工程单抗。方法：采用基因重组技术分别将抗人脑胶质瘤单抗ＳＺ３９重链和轻链可变区基因片段连接，构建表达载体，并在大肠杆菌中表达。结果：表达的ＳｃＦｖ抗体为可溶性，ＥＬＩＳＡ和ＷｅｓｔｅｒｎＢｌｏｔ检测证实能特异性地与人脑胶质瘤细胞系ＳＨＧ－４４表面抗原结合，其表达量为２２０μｇ／Ｌ。结论：成功地构建了抗人脑胶质瘤单抗ＳＺ３９ＳｃＦｖ表达质粒，并得到了与胶质瘤细胞表面抗原相结合的单链抗体。     

抗人脑胶质瘤单克隆抗体SZ—39轻链可变区基因的克隆与序列分析

从抗人脑胶质瘤单克隆抗体杂交瘤细胞中抽提总ＲＮＡ，并以Ｏｌｉｇｏ柱纯化ｍＲＮＡ，经逆转录－聚合酶链反应直接扩增出３１８ｂｐ的轻链可变区ｃＤＮＡ片断，经核苷酸序列分析研究，证实已获得了ＳＺ－３９单抗轻链可变区基因。这为今后进一步制备基因工程抗体打下基础。     

胶质瘤基因工程单链抗体的制备及其抗瘤效应的初步研究

目的：构建并高效表达抗胶质瘤单链抗体（ScFv)，为基因工程双功能抗体及免疫毒素的制备奠定基础。方法：以抗胶质瘤杂交瘤细胞重、轻链可变区基因构建ScFv基因，并克隆入pET－20b表达载体，在大肠 杆菌BL21 DE3中诱导表达，以Western印迹及酶联免疫吸附测定（ELISA）检测表达产物。结果：测序证实基因构建正确，表达产物相对分子质量（Mr）为28000，表达量占菌体总蛋白的15．0％，且保留了胶质瘤相关抗原的特异结合活性。结论：初步得到有活性的胶质瘤ScFv，为脑肿瘤的分子导向诊治提供了新的选择。     

白细胞介素-6在人脑胶质细胞瘤的表达

白细胞介素-6(IL-6)是一种具有多种功能的生物活性多肽,其功能之一是作为自分泌生长因子参与某些肿瘤的发生发展过程。已证实在实验条件下人脑胶质瘤细胞株可自发分泌IL-6,但关于IL-6在人脑胶质瘤中表达情况的研究尚不多见,而且结论也不一致。本试验采用逆转录-聚合酶链反应(RT-PCR)半定量法检测IL-6在人脑胶质瘤中的表达情况,以探讨IL-6与人脑胶质瘤的关系。1资料1.1标本:采用中国医科大学第二临床学院神经外科2002~2004年间胶质瘤患者术中新鲜标本62例。全部病例均经病理证实,按WHO分类法(2000年)包括胶质母细胞瘤18例,间变性星形…     

抗人脑胶质瘤单克隆抗体SZ－39轻链可变区基因的克隆与序列分析

从抗人脑胶质瘤单克隆抗体杂交瘤细胞中抽提总ＲＮＡ，并以Ｏｌｉｇｏ（ｄＴ）柱纯化ｍＲＮＡ，经逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）直接扩增出３１８ｂｐ的轻链可变区（ＶＬ）ｃＤＮＡ片断，经核苷酸序列分析研究，证实已获得了ＳＺ－３９单抗轻链可变区基因。这为今后进一步制备基因工程抗体打下基础。     

抗猪囊尾蚴抗原McAb建立和临床应用的初步观察

本文就我所1987～1988年建立的抗猪囊尾蚴单克隆抗体(McAb),及其应用于脑囊虫病诊断的初步观察作一简要报道: 实验和结果 一、McAb的建立及鉴定:以猪囊尾蚴囊液抗原免疫BALB/C小白鼠。取免疫的小鼠脾细胞与SP2/O骨髓瘤细胞进行融合杂交。经选择培养,测定上清抗体,克隆化,获一株特异的抗猪囊尾蚴抗原的McAb杂交瘤细胞(4G_2)。将4G_2株杂交瘤细胞进行冰冻保种,并接种小鼠腹腔诱生小鼠腹水McAb。经鉴定     

人体脑瘤的免疫学鉴别诊断

1967年发现甲_2-糖蛋白(α_2-glycoprotein)抗原,对人体中枢神经系统的胶质细胞似有特异性。此抗原在胎儿脑胶质细胞的髓鞘形成期,即胚胎第14～18周之间,开始出现。通过治疗性子宫切开术可获得此时期的胎脑,亦可用成人脑以亲和层析法(Affinity Chromatography)来制备此抗原。将此抗原注射于兔子,便可产生其抗体。37例人体脑瘤手术时切除的肿块,一部分作病     

人脑胶质瘤干／祖细胞系SU-2放射耐受及其相关机制的初步研究

目的研究胶质瘤干细胞在胶质瘤耐受放射中的作用,为克服恶性胶质瘤放射耐受寻找新的干预靶点。方法干细胞培养条件下培养自建人脑胶质瘤干/祖细胞系SU-2,以及人脑胶质瘤细胞系U251和SHG-44,观察不同剂量直线加速器照射前后细胞形态变化、Hoechst 33342-细胞和CD133 细胞比例、肿瘤细胞存活率和裸小鼠致瘤率等项指标,并以实时荧光定量PCR方法检测人脑胶质瘤细胞系U251和人脑胶质瘤干/祖细胞系SU-2照射前后MGMT基因表达水平。结果当照射剂量为1～15Gy时,人脑胶质瘤干/祖细胞系SU-2中的Hoechst 33342-细胞和CD133 细胞比例明显增加,高达18.73%和13.70%,细胞存活率升高、致瘤率(8/8)增加、侵袭性增强,MGMT基因表达水平轻度升高。结论经一定剂量的X线照射后,胶质瘤干细胞因具有强于其他肿瘤细胞的放射耐受性而出现选择性存活且细胞比例升高,其生物学特性如细胞存活率、体内致瘤率增加,侵袭性增强。可能与胶质瘤干细胞DNA损伤修复能力提高有关,确切的分子学机制值得进一步深入研究。     

Isolation and characterization of a monoclonal antibody specific for fibrinogen and fibrin of human origin

A hybridoma cell line secreting a monoclonal antibody directed against human fibrinogen has been isolated. The antigenic determinant recognized is present on both the fibrinogen and fibrin molecules but is apparently absent from the D and E fragments and from fibrinopeptides A and B. The antibody seems to recognize a conformational structure present in native fibrinogen and in which several or possibly all the fibrinogen polypeptide chains may participate.     

Monoclonal antibodies against human astrocytomas and their reactivity pattern

The establishment of hybridomas after fusion of X63-Ag8.653 mouse myeloma cells and splenocytes from BALB/c mice hyperimmunized against human astrocytomas is presented. The animals were primed with 5 × 10⁶ chemically modified uncultured or cultured glioma cells. Six weeks after the last immunization step an intrasplenal booster injection was administrated and 3 days later the spleen cells were prepared for fusion experiments. According to the specificity analysis of the generated antibodies 7 hybridoma products (MUC 7-22, MUC 8-22, MUC 10-22, MUC 11-22, MUC 14-22, MUC 15-22 and MUC 2-63) react with gliomas, neuroblastomas and melanomas as well as with embryonic and fetal cells but do not recognize non-neurogenic tumors. The selected monoclonal antibodies (McAbs) of IgG₁ and IgG_2a isotypes are not extensively characterized but these antibodies have been demonstrated to be reactive with a panel of glioma cell lines with varying patterns of antigen distribution. Using the McAbs described above and a series of cryosections of glioma biopsies and paraffin sections of the same material as well as glioma cultures established from these, variable antigenic profiles among glioma cell populations could be demonstrated. From these results it is evident that there is not only a distinct degree of antigenic heterogeneity among and within brain tumors, but also that the pattern of antigenic expression can change continuously. Some of the glioma associated antigens recognized by the selected antibodies persist after fixation with methanol/acetone and Karnovsky's fixative and probably are oncoembryonic/oncofetal antigen(s). The data suggest that the use of McAbs recognizing tumor associated oncofetal antigens in immunohistochemistry facilitates objective typing of intracranial malignancies and precise analysis of fine needle brain/tumor biopsies in a sensitive and reproducible manner.     

A new antigen common to the rat nervous and immune systems: I. Detection with a hybridoma

An antibody secreting cell line has been obtained using the technology developed by Milstein, Kohler, and colleagues by fusion between ×63 myeloma cells and spleen cells from a mouse previously immunized with PC12 cultured rat pheochromocytoma cells. This antibody bound to particulate protein from adult rat brain and to a lesser extent spinal cord and retina but not adrenal. Lower levels of binding were observed also with spleen, bone marrow, and peritoneal exudate cells. Cells or particulate protein from seven nonneural, nonimmune tissues showed essentially no specific binding. Analysis of adherent and nonadherent peritoneal exudate cells indicated specific antibody binding to both populations. The specific antibody bound was greater in the non-adherent fraction. The antigen has been provisionally named G5 after the antibody secreting clone. Like the Thy 1 antigen of rodents, it is expressed by subpopulation of cells from the nervous and immune systems. However the antigen could not be detected on the PC1 2 cell line used for immunization suggesting that Balb/C mice spontaneously produce antibody to this rat differentiation antigen.     

Overexpression of the 18A2/mts1 gene and down–regulation of the TIMP–2 gene in invasive human glioma cell lines in vitro

Invasion of the reconstituted extracellular matrix composite, Matrigel, by eight human glioma–derived cell lines and human fetal brain cells was assessed in vitro using 8 um polycarbonate filters in a modified Boyden migration chamber. With the exception of one low grade glioma derived cell line, all lines studied proved to be invasive while normal fetal brain cells failed to invade. This invasive potential was independent of the histological grade of the tumour from which the cell lines originated. In addition, the expression of the metastasis–associated gene 18A2lmts1 as well as the tissue inhibitor of metalloproteinases–2 (TIMP–2) was analysed in each of the glioma–derived cell lines. The 18A2/mtsl was expressed in all the cells studied with the exception of fetal brain cells and the low grade non–invasive glioma derived IPRK–7 cell line. The 18A2/mtsl related genes coding for the S100 subfamily of calcium binding proteins were found to be differentially and overexpressed in invasive cell lines. TIMP–2 was expressed only in noninvasive cell lines. These results suggest that the 18A2/ mtsl and TIMP–2 genes could play an important role in the invasive behaviour of human glioma cells in vitro. .     

Interleukin-2 expression and glioma cell proliferation following Vaceinia vector gene transfection in vivo

BACKGROUND: The effectiveness of gene therapy is closely related to the efficiency of vector transfection and expression.OBJECTIVE: This study was designed to transfect a human brain glioma cell line with recombinant Vaccinia virus expressing the interleukin-2 (rVV-IL-2) gene, and to observe IL-2 expression and glioma cell proliferation potential after transfection. DESIGN: Experimental observation. SETTING: Department of Neurosurgery, Shenyang Military Area Command of Chinese PLA. MATERIALS: The rVV-IL-2 vectors were obtained through homologous recombination and screening in the Second Military Medical University of Chinese PLA. The human brain glioma cell line and IL-2-dependent cells were produced by the Second Military Medical University of Chinese PLA. Human IL-2 was produced by Genzyme Corporation. MAIN OUTCOME MEASURES: IL-2 expression at different time points after transfection of human brain glioma cells with varying MOI of Vaccinia viral vectors; in vitro proliferation capacity of human brain glioma cells among the 4 groups. RESULTS: IL-2 expression was detectable 4 hours after Vaccinia viral vector transfection and reached 300 kU/L by 8 hours. There was no significant difference in the proliferating rate of human brain glioma cells among the 4 groups (P > 0.05).CONCLUSION: Vaccinia viral vectors can transfect human brain glioma cells in vitro and express high levels of IL-2. Vaccinia virus and high IL-2 expression do not influence the proliferation rate of human brain glioma cells in vitro.     

The molecular structure of microtubule-associated protein 1A (MAP1A) in vivo and in vitro. An immunoelectron microscopy and quick-freeze, deep-etch study

We studied the distribution of microtubule-associated protein 1A (MAP1A) in Purkinje cell dendrites by means of electronmicroscopic immunocytochemistry, using a monoclonal antibody (McAb) against MAP1A; this was combined with the observation of the 3-dimensional cytoskeletal ultrastructure in dendrites via the quick-freeze, deep-etch technique (QF-DE). We prepared a McAb against rat brain MAP1. This McAb recognized MAP1A on a nitrocellulose filter through use of the immunoblotting method, and stained immunofluorescently Purkinje cell perikarya, dendrites, and axons. Using the McAb, we labeled rat cerebellum extracted with Triton X-100 and simultaneously fixed with aldehyde, followed by gold-labeled rabbit anti-mouse IgG. Gold particles were attached to the filamentous, fuzzy materials, mostly those connected to microtubules (MTs), but were hardly localized on those attached to neurofilaments (NFs). The 3-dimensional cytoskeletal ultrastructure of fresh Purkinje cell dendrites was revealed by QF-DE. In Purkinje cell dendrites, MT was a predominant cytoskeletal element, whereas only a few NFs were found. Fine, elaborate cross-bridges filled up the interstices among MTs, and between MTs and other cellular components. Cross-bridges linking MTs to one another were composed mainly of a fine filamentous structure, frequently branching and anastomosing at several sites, and appeared somewhat granular. We ensured that the cross-bridges observed in saponin-extracted tissues were not a result of artifactual condensations or precipitations of soluble proteins during deep etching. The molecular structure of MAP1A was further investigated by the rotary shadowing technique. The affinity-purified MAP1A was a long, thin, filamentous, and very flexible molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hax-1对胶质瘤细胞增殖、迁移和侵袭的作用

目的 探讨造血干细胞特异性相关结合蛋白-1（Hax-1）对胶质瘤细胞增殖、迁移和侵袭的影响。方法 选择2018年9月至2019年6月手术切除并得到术后病理证实的胶质瘤组织35例和颅脑损伤内减压术切除的正常脑组织35例,采用qRT-PCR检测Hax-1 mRNA水平;同时检测胶质瘤细胞系（U87、A172、T98及U343）和人星形胶质细胞（NHAs）Hax-1 mRAN水平。使用Lipofectamine 2000法将Hax-1 siRNAs和阴性对照siRNA转染U87细胞构建Hax-1低表达细胞株,将Hax-1高表达质粒和PCDNA3.1空载质粒转染U343细胞构建Hax-1过表达细胞株,使用蛋白印迹法验证转染效果,采用CCK-8法检测细胞增殖,采用Transwell小室实验检测细胞迁移和侵袭,免疫印迹法检测NF-κB信号通路相关蛋白表达水平（NF-κB、CCND1、C-myc、MMP-2、MMP-9）。结果 胶质瘤组织Hax-1 mRNA水平明显高于正常脑组织（P<0.05）;胶质瘤细胞系（U87、A172、T98及U343）Hax-1 mRNA水平明显高于人星形胶质细胞（P<0.05）,其中U87细胞Hax-1 mRNA水平最高,U343细胞最低。U343细胞转染Hax-1过表达质粒后,Hax-1蛋白水平显著增高（P<0.05）,细胞增殖、侵袭、迁移能力均明显增强（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显增高（P<0.05）。U87细胞转染Hax-1 siRNA后Hax-1蛋白水平显著降低（P<0.05）,细胞增殖、侵袭、迁移能力均明显降低（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显降低（P<0.05）。结论 胶质瘤组织Hax-1呈高表达,明显促进肿瘤细胞增殖、侵袭和迁移,其机制可能与激活NF-κB信号通路有关。     

下调RNF138基因对胶质瘤细胞株U251增殖、凋亡及细胞周期影响的研究

目的探讨RNA干扰下调胶质瘤细胞株高表达基因RNF138对胶质瘤细胞株U251体外增殖、凋亡以及细胞周期的影响。方法构建下调RNF138基因的siRNA,通过慢病毒转染导入胶质瘤细胞株U251,设立阴性干扰对照组及空白对照组,荧光显微镜观察转染效率;实时荧光定量PCR检测敲减效率;运用Cellomics仪器连续检测U251体外增殖情况;流式细胞仪检测U251凋亡及细胞周期变化情况。结果慢病毒载体高效、稳定转染U251细胞,靶向RNF138的siRNA有效抑制RNF138基因表达,使RNF138mRNA表达减少60%,U251体外增殖明显减缓,凋亡明显增加,细胞阻滞在G2/M期。结论 RNA干扰抑制RNF138基因表达可以明显抑制胶质瘤细胞株U251体外增殖,促进其凋亡,影响细胞周期,阻滞细胞分裂。