Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts
Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts
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DOI：
10.1152/ajpheart.1997.272.1.h220
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发表时间：
1997-01-01
影响因子：
4.8
通讯作者：
Field, LJ
中科院分区：
文献类型：
--
作者：
Soonpaa, MH;Field, LJ
在正常和损伤的成年小鼠心脏上检测了心肌细胞的DNA合成。在初步研究中，通过[H-3]胸腺嘧啶核苷掺入监测DNA合成，然后对分散的细胞制剂进行放射自显影分析。当对来自正常成人心脏的20,000个心室肌细胞进行检查时，没有鉴定出合成细胞。建立了一种高通量的方法来建立成年小鼠心脏的实际标记指数。该方法利用转基因小鼠的[H-3]胸腺嘧啶核苷掺入，转基因小鼠仅在心肌细胞中表达核定位的β-半乳糖苷酶(β-Gal)报告基因。心肌细胞DNA合成在组织切片的放射自显影中表现为β-半乳糖活性和银粒的共存。对来自正常成年转基因小鼠的180,000个心室肌细胞核的检测表明，只有一个合成细胞核，最大标记指数为0.0005%。接下来，对局部烧灼左室壁损伤的心脏检测心肌细胞DNA合成。当对受损心脏外周带的36,000个心肌细胞核进行检查时，只鉴定出三个合成细胞核。当检查坏死区远端的18万个核时，没有发现额外的合成核。这些数据证实，成年小鼠心脏中的心肌细胞DNA合成极其罕见，并为转基因动物的分析提供了基线数据。
Cardiomyocyte DNA synthesis was examined in normal and injured adult mouse hearts. In preliminary studies DNA synthesis was monitored by [H-3]thymidine incorporation, followed by autoradiographic analysis of dispersed cell preparations. No synthetic cells mere identified when 20,000 ventricular cardiomyocytes from normal adult hearts were examined. A high throughput assay was developed to establish the actual labeling index for the adult mouse heart. The assay utilized [H-3]thymidine incorporation in transgenic mice which expressed a nuclear-localized beta-galactosidase (beta-Gal) reporter gene exclusively in cardiac myocytes. Cardiomyocyte DNA synthesis was evidenced by colocalization of beta-Gal activity and silver grains in autoradiograms of histological sections. Examination of 180,000 ventricular cardiomyocyte nuclei from normal adult transgenic mice identified a single synthetic nucleus, suggesting a maximum labeling index of 0.0005%. Cardiomyocyte DNA synthesis was next examined in hearts injured by focal cauterization of the left ventricular fi ee wall. Only three synthetic nuclei were identified when 36,000 cardiomyocyte nuclei in the perine-crotic zone of the injured heart were examined. No additional synthetic nuclei were identified when 180,000 nuclei in regions distal to the necrotic zone were examined. These data confirm that cardiomyocyte DNA synthesis in the adult mouse heart is extremely rare and provide baseline data for analyses in genetically modified animals.