Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were ...Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.展开更多
文摘Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.
文摘目的 为优化小型猪体细胞核移植技术（SCNT）技术,探讨供体细胞不同类型、不同传代数以及蚜肠霉素（Aphidicolin,APC）处理各供体细胞对重构胚体外发育的影响.方法 小型猪输卵管上皮细胞（OEC）,耳成纤维细胞（EFC）和卵丘细胞（CC）分别经血清饥饿或血饥饿后再使用0.1μg/mlAPC处理.核移植重构胚体外培养7d,以检测其发育能力.结果 OEC经APC处理后,重构胚的卵裂率和囊胚形成均显著高于仅血清饥饿处理组（61.82％ vs 56.25％,24.55％ vs 17.86％; P＜0.05）.以EFC为供体细胞,APC组重构胚卵裂率显著高于血清饥饿组（63.36％ vs 57.01％; P＜0.05）.注入CC,APC处理能显著提高重构胚的囊胚形成率（29.63％; P＜0.05）.三种细胞经APC处理,CC作为核供体,重构胚的卵裂率和囊胚形成率最高.三种细胞中,无论哪一种作为核供体,其传代数对重构胚的发育没有影响.结论 以CC为供体细胞,血清饥饿后再使用APC处理,更适合小型猪的体细胞核移植.
