IMMUNOHISTOCHEMICAL DETECTION OF DNA REPLICATING CELLS IN THE DEVELOPING NERVOUS SYSTEM: USE OF BROMODEOXYURIDINE AND ITS MONOCLONAL ANTIBODY TO RAT FETUSES
IMMUNOHISTOCHEMICAL DETECTION OF DNA REPLICATING CELLS IN THE DEVELOPING NERVOUS SYSTEM: USE OF BROMODEOXYURIDINE AND ITS MONOCLONAL ANTIBODY TO RAT FETUSES
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发育中神经系统DNA复制细胞的免疫组织化学检测：溴脱氧尿嘧啶及其大鼠胎儿单克隆抗体的应用
DOI：
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发表时间：
1987
期刊：
影响因子：
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通讯作者：
F. Ikuta
中科院分区：
文献类型：
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作者：
Y. Yoshida;M. Yamada;K. Wakabayashi;F. Ikuta
本文观察了溴脱氧尿苷（BrdU）对大鼠胎脑成神经细胞形成期DNA合成细胞的影响。静脉注射BrdU后27 min，用抗BrdU单克隆抗体，在多聚甲醛固定的石蜡包埋组织切片中，用抗生物素蛋白-生物素复合物过氧化物酶法清楚地检测到细胞核中的BrdU。免疫组化所需BrdU剂量为20 mg/kg体重。用这种方法，显示了基质细胞核的升降运动和神经母细胞在迁移带和皮质板中的迁移曲线。在神经母细胞形成过程中的大脑新皮层的基质细胞的标记指数基本上等于或甚至高于以前报道的氚化胸苷放射自显影的值。BrdU在胚胎阶段也可在3月龄大鼠中检测到。在胚胎第16天接受20-60 mg/kg BrdU的大鼠在行为和形态上没有发现明显异常。结果表明，BrdU免疫组织化学方法是一个有用的工具，在发育中的神经系统在体内的细胞动力学研究。自1982年Gratzner（9）使用溴脱氧尿苷（BrdU）（一种胸苷类似物）的单克隆抗体建立DNA合成细胞的免疫组织化学检测方法以来，已报道了几项关于体外或人类和实验动物成年期各种组织中肿瘤细胞细胞动力学的分析研究（20，22，23）。这种方法在显示体内DNA合成细胞方面比胸苷放射自显影法具有更大的优点。该方法耗时少得多，在使用放射性同位素时不需要预防措施（11，25），并且其反应产物在高分辨率下清晰可见（37）。虽然这种方法似乎非常有用的细胞动力学研究在不同领域的发展中的动物，很少有关于BrdU的免疫组织化学适用性的动物在胎儿发育过程中的信息。在1982年之前，已经指出掺入核DNA中的BrdU在体外细胞中引起细胞毒性（16），或在胚胎中引起致畸性（27-29）。这些工作试图评估卤代核苷BrdU作为哺乳动物胚胎的致畸剂（5，28，34），并估计其作为肿瘤组织X射线照射中的放射增敏化学品（18，30，36）。因此，i i } i
The effects of bromodeoxyuridine (BrdU), an analogue of thymidine, on the DNA synthesizing cells of the rat fetal brain were examined at the stage of neuroblast formation. BrdU in the cell nuclei was clearly detected by avidin-biotin complex peroxidase method using an anti-BrdU monoclonal antibody in paraformaldehydefixed, paraffin-embedded tissue sections 27 min after the intravenous administration ofBrdU. The sufficient dose ofBrdU required for immunohistochemistry was 20 mg/kg of body weight. By this method, the elevator movement of matrix cell nuclei and the migration profiles of the neuroblasts in the migrating zone and cortical plate were demonstrated. The labeling indices of the matrix cells in the cerebral neopallium during the neuroblast formation gave essentially equal or even higher values than those previously reported by tritiated thymidine autoradiography. BrdU incorporated at the embryonic stage was also detectable in 3-month-old rats. Rats that received 20-60 mg/kg BrdU on the embryonic day 16 revealed no significant abnormalities in behaviors and morphology. The results suggest that the BrdU immunohistochemical method is a useful tool in cell kinetic studies of the developing nervous system in vivo. Since the establishment of immunohistochemical detection method of DNA synthesizing cells using a monoclonal antibody to bromodeoxyuridine (BrdU), an analogue of thymidine, by Gratzner (9) in 1982, several analytic studies on cell kinetics of neoplastic cells in vitro or in various tissues of humans and experimental animals in adulthood have been reported (20, 22, 23). This method is of greater advantage than thymidine autoradiography in visualizing the DNA-synthesizing cells in viva. The method is much less timeconsuming, does not require precaution in working with radioisotopes (ll, 25), and its reaction products are visualized clearly "at a high resolution (37). Although this method seems very useful in diverse fields of cell kinetic study in developing animals, little information is available concerning the immunohistochemical applicability of BrdU to animals during the fetal development. Before 1982, it has been pointed out that BrdU incorporated into the nuclear DNA causes cytotoxicity in cells in vitr0i(16), or teratogenicity in embryos (27-29). These works attempted to evaluate the halogenated nucleoside, BrdU, as a teratogen to mammalian embryos (5, 28,34) and to estimate it as a radiosensitizing chemical in X-ray irradiation to neoplastic tissues (18, 30, 36). Accordingly, i i } i