MEASUREMENT OF MICRONUCLEI IN LYMPHOCYTES
MEASUREMENT OF MICRONUCLEI IN LYMPHOCYTES
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DOI：
10.1016/0165-1161(85)90015-9
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发表时间：
1985-01-01
期刊：
影响因子：
--
通讯作者：
MORLEY, AA
中科院分区：
文献类型：
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作者：
FENECH, M;MORLEY, AA
微核技术被提议作为一种测量有丝分裂原刺激的人淋巴细胞染色体损伤的方法。微核需要1次细胞分裂才能表达，因此，传统的微核技术非常不精确，因为仅经历1次分裂的细胞及其中的微核不能从总淋巴细胞群中单独鉴定。为了克服这个问题，开发了两种方法来鉴定已经经历第一次有丝分裂的细胞。使用放射自显影技术，在培养48 h时用[3 H]胸苷脉冲标记淋巴细胞，使其进行有丝分裂，在72 h和84 h之间通过放射自显影进行鉴定，并对其中的微核进行评分。不可能选择本身不产生体内损伤的放射性标记浓度。该方法能定量测定体外照射淋巴细胞产生的微核。在第二种方法中，使用细胞松弛素B阻断胞质分裂。在分裂阻断的细胞中对微核进行评分。由于它们的双核外观，这些细胞易于识别，并且通过在44小时加入3.0 μ g/ml细胞松弛素B并在72小时评分，可以积累大量细胞。细胞松弛素B本身不产生微核。微核阻断法操作简单，正常人体内微核率为4.4 ±。2.6微核/500个微核阻断细胞;对于体外照射的淋巴细胞，辐射剂量与诱导的微核数之间存在线性关系。微核阻断法似乎是定量淋巴细胞微核的首选方法。
The micronucleus technique was proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require 1 cell divison to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only 1 division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, 2 methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labeled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce damage present in vivo. It was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the 2nd method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognizable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 .mu.g/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the in vivo micronucleus frequency in normal individuals was 4.4 .+-. 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.