Application of vital staining with Acridine Orange (A.O.) to investigation of embryonic growth at the stage of organogenesis

本研究采用荧光显微镜吖啶橙活体染色法观察胚胎着床后的正常和异常生长情况。注射AO的母鼠胚胎在注射后5分钟发出强烈的二次荧光，24小时仍发出微弱的二次荧光。后。但是48小时。之后，胚胎只发出自身荧光而不发出次生荧光。怀孕小鼠在妊娠第9天同时给予丝裂霉素C和咖啡因。众所周知，这种治疗导致胚胎畸形的发生率很高。然后，在连续第10天和第11天，1小时。母鼠注射AO后，取子宫角胚，荧光显微镜观察。从时间过程调查中，我们证实丝裂霉素C和咖啡因处理后的胚胎损伤组织细胞的二次荧光存在时间比对照组长，这是由于活胚胎细胞可以分泌AO，而损伤细胞不能分泌AO。胚胎全身弥漫性可见大量颗粒状细胞团，具有强烈的黄绿色荧光，在肢芽间充质组织中特异性高密度存在，未形成数字射线。综上所述，利用AO活体染色的荧光显微镜，可以通过荧光的颜色和强度来详细研究正常和异常的胚胎生长。结合全胚培养的进一步研究有望在正常和异常胚胎生长方面取得丰硕的成果。

In this study, normal and abnormal growth of post-implantation embryos was investigated by means of vital staining with acridine orange using fluorescence microscope. Embryos from mother mice injected with AO, emitted intense secondary fluorescence five minutes after injection and still did emit weak secondary one 24 hrs. after. But 48 hrs. after, embryos did illuminate only auto-fluorescence without the secondary one.Pregnant mice were administered with Mitomycin C and Caffeine simultaneously on 9th day of gestation. This treatment has been well known to cause digital malformation of the embryo at a high incidence. Then, on the successive 10th and 11th day of gestation, one hr. after AO injection into mother mice, embryos were taken out of uterine horns and investigated by fluorescence microscope.From the time course investigation, we confirmed that the secondary fluorescence existed longer in damaged tissue cells of embryos by treatment with Mitomycin C and caffeine than in control ones, as living embryonic cell can excrete AO while damaged cells can not.There were many granule-like cell masses with intense yellowish-green fluorescence diffusely on the whole body of embryo and those specifically existed with high density at the mesenchymal tissue of limb buds without formation of digital rays.From the results, above mentioned, normal and abnormal embryonic growth can be investigated in detail by color and intensity of fluorescence using fluorescence microscopy through AO vital staining.Further investigation in combination with whole embryo culture is expected to bring us a fruitful result about normal and abnormal embryonic growth.