Application of a New 5′-Nase Monoclonal Antibody Specific for Lymphatic Endothelial Cells
Application of a New 5′-Nase Monoclonal Antibody Specific for Lymphatic Endothelial Cells
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新型淋巴内皮细胞特异性5′-Nase单克隆抗体的应用
DOI：
10.1097/01.lab.0000095685.57920.2e
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发表时间：
2003
影响因子：
5
通讯作者：
S. Kato
中科院分区：
文献类型：
--
作者：
R. Ji;P. Qu;S. Kato
5 '-核苷酸酶（5'-Nase）组织化学染色已被证明是区分初始内皮细胞与毛细血管的有效方法，这是基于其对淋巴管内皮的活性远高于对血管内皮的活性（Kato等人，1996）。然而，在不同器官和组织中发现淋巴管内皮细胞的酶活性存在相当大的差异，例如，在发育中的胃壁中淋巴管内皮表达降低，在妊娠猴中子宫内膜过度表达（Ji，1998; Ji和Kato，2003）。当考虑5 '-Nase的抗原性而不是其活性时，特异于淋巴管内皮的5'-Nase mAb，而不是腺苷5 '-单磷酸，可以作为细胞选择和体外培养的有用标记物。在这些情况下，酶组织化学不能检测到低或过表达的5 '-Nase活性。这种缺陷可以通过免疫组织化学用有效的5 '-Nase-mAb来弥补，以确定淋巴管内皮细胞而不是其他实质细胞特异性抗原的存在和位点。这一贡献的目的是产生一个敏感和特异性的标记物，用于识别淋巴管内皮细胞。将来自响尾蛇毒液的5 ′-Nase（EC 3.1.3.5，Sigma，St. Louis，密苏里州）直接注射到雄性BALB/c小鼠（5 - 8周龄）的脾脏中用于第一次免疫。在加强注射后制备脾细胞，并与X63-Ag 8.653骨髓瘤细胞系融合（科勒和米尔斯坦，1975）。在杂交瘤选择之后，最初通过ELISA和通过间接链霉亲和素-生物素过氧化物酶复合物方法在冷冻切片上进行来自生长细胞的上清液的筛选。在BALB/c胸腺细胞作为饲养细胞的情况下，通过有限稀释（每孔0.25个细胞）从阳性杂交瘤威尔斯孔中选择一个克隆JC 815。用雌性BALB/c小鼠制备腹水瘤液，用蛋白A-琼脂糖柱层析纯化。对于免疫印迹，用SDS-PAGE处理在裂解缓冲液中均质化的5 '-Nase和培养的大鼠胸导管内皮细胞（ThDEC）的蛋白样品。将分离的蛋白质在200 mA下转移到硝酸纤维素膜上3小时。用JC 815（IgG和腹水以1：800稀释）孵育修剪的片，然后与山羊抗鼠免疫球蛋白和链霉亲和素-生物素-过氧化物酶溶液反应。用水冲洗以停止片材的0.03%3，3 ′-二氨基联苯胺四盐酸盐（DAB）显影。对于免疫组织化学，将培养的ThDEC和来自小鼠和大鼠组织的5至7 μ m厚的冷冻切片与以1：200至1：400稀释的JC 815和生物素化的缀合的兔抗小鼠IgG孵育。DAB可视化通过2%OsO4固定进行部分处理，用于透射电子显微镜（TEM）以鉴定JC 815的反应性。将着床后染色超声切片与1：400稀释的JC 815孵育，然后与结合5 nm金颗粒的山羊抗鼠IgG（BBInternational，卡迪夫，英国）孵育。将阴性对照与含1%正常山羊血清的0.1 M PBS（而非JC 815）一起孵育。用5 '-Nase组织化学染色处理阳性对照，以证明淋巴管内皮细胞的酶活性。与5 '-Nase染色对照相比，JC 815免疫反应性在小鼠和大鼠的胃、横膈、皮肤、胸导管、舌、胰腺和肝脏的淋巴管上明显表达。在舌中，JC 815强烈染色上皮下淋巴管内皮，并且与5 '-Nase染色模式相似（图1，A和B）。在肝脏中，表达JC 815的淋巴管具有薄的内皮壁和不规则的轮廓，位于小叶间结缔组织中（图1C）。杂交瘤诱导的胰腺肿瘤中的淋巴管显示出不均匀的JC 815免疫反应性，在其管腔中存在许多转移性杂交瘤细胞（图1D）。在ThDEC的汇合单层中，分裂和多核培养的细胞的特征在于均匀的鹅卵石外观，显示出各种反应性DOI：10.1097/01.LAB.0000095685.57920.2E
5'-nucleotidase (5'-Nase) histochemical staining has proved to be an effective method in differentiating initial lymphatics from blood capillaries, based on its much higher activity on lymphatic than on blood vascular endothelium (Kato et al, 1996). A considerable variability in the enzyme activity of lymphatic endothelial cells, however, was revealed in different organs and tissues, eg, a reduced expression for lymphatic endothelium in the developing gastric wall and an overexpression for uterine lymphatics in pregnant monkeys (Ji, 1998; Ji and Kato, 2003). When 5'-Nase antigenity rather than its activity is considered, 5'-Nase mAb specific for lymphatic endothelium, instead of adenosine 5'monophosphate, can serve immunohistochemically as a useful marker for cell selection and in vitro cultivation. Under these circumstances, the enzyme histochemistry cannot detect underor overexpressed 5'-Nase activity. This deficiency can be made up by immunohistochemistry with an effective 5'-Nase-mAb to determine the existence and site of the antigen specific for lymphatic endothelial cells rather than other parenchymal cells. The aim of this contribution is to produce a sensitive and specific marker for the identification of lymphatic endothelial cells. 5'-Nase from Crotalus atrox venom (EC 3.1.3.5, Sigma, St. Louis, Missouri) was directly injected into the spleen of male BALB/c mice (5 to 8 weeks old) for the first immunization. The spleen cells were prepared after the booster injection and fused with X63-Ag8.653 myeloma cell line (Kohler and Milstein, 1975). Following hybridoma selection, screening of the supernatants from growing cells was initially performed by ELISA and on cryosections by the indirect streptavidin-biotinperoxidase complex method. One of the clones, JC815, was selected from positive hybridoma wells by limiting dilution (0.25 cells per well) in the presence of BALB/c thymocytes used as feeder cells. Ascites tumor fluid was produced by using female BALB/c mice and purified by the protein A–Sepharose chromatography. For immunoblotting protein samples of 5'-Nase and cultured rat thoracic duct endothelial cells (ThDECs) homogenized in lysis buffer were processed with SDS-PAGE. The separated proteins were transferred onto a nitrocellulose membrane at 200 mA for 3 hours. The trimmed sheets were incubated with JC815 (IgG and ascites diluted in 1:800) and then reacted with goat antimouse immunoglobulins and streptavidin-biotin-peroxidase solution. A water rinse was used to stop 0.03% 3,3'diaminobenzidine tetrahydrochloride (DAB) development of the sheets. For immunohistochemistry, cultured ThDECs and 5to 7m thick cryosections from mouse and rat tissues were incubated with JC815 diluted in 1:200 to 1:400 and with biotinylated conjugated rabbit antimouse IgG. DAB visualization was partly processed by 2% OsO4 fixation for transmission electron microscopy (TEM) to identify JC815 reactivity. Postembedding staining ultrasections were incubated with JC815 diluted in 1:400 and then with goat antimouse IgG bound to 5-nm gold particles (BBInternational, Cardiff, United Kingdom). Negative controls were incubated with 1% normal goat serum in 0.1 M PBS rather than JC815. Positive controls were treated with 5'-Nase histochemical staining to demonstrate the enzyme activity for lymphatic endothelial cells. JC815 immunoreactivity was distinctly expressed on the lymphatic vessels of the stomach, diaphragm, skin, thoracic duct, tongue, pancreas, and liver from mice and rats, in comparison with 5'-Nase staining controls. In the tongue, JC815 strongly stained the subepithelial lymphatic endothelium and was similar to the 5'-Nase staining pattern (Fig. 1, A and B). In the liver, JC815expressing lymphatic vessels with thin endothelial walls and irregular contours were located in the interlobular connective tissue (Fig. 1C). Lymphatic vessels in hybridoma-induced pancreatic tumors showed uneven JC815 immunoreactivity, in whose lumen there were numerous metastatic hybridoma cells (Fig. 1D). In the confluent monolayer of ThDECs, the dividing and multinuclear cultured cells were characterized by a uniform cobblestone appearance, showing various reactive DOI: 10.1097/01.LAB.0000095685.57920.2E