光物理因子对中华大蟾蜍早期胚胎发育影响的初步研究
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摘要
激光和紫外线是非电离辐射光物理因子,为了研究其对两栖类早期胚胎发育的影响,本论文以中华大蟾蜍为主要实验材料,分别用YAG倍频激光器和302 nm中波紫外线(UVB)照射不同发育时期的中华大蟾蜍胚胎,在发育形态、蛋白质谱和核酸水平检测了照射前后的变化。1)用UVB分别以不同强度和不同时间照射中华大蟾蜍二细胞期胚胎,观察记录胚胎的发育过程、统计死亡率与照射时间和照射强度的关系。结果表明,30μW/cm~2强度的UVB分别照射二细胞期胚胎20s、60s和80μW/cm~2强度分别照射20s、60s、120s后,对中华大蟾蜍早期胚胎发育有促进作用,使发育过程中的死亡率降低,畸胚出现的类型较少且畸变程度低;80μW/cm~2强度的UVB照射二细胞期胚胎600s,100μW/cm~2强度的UVB照射20s、60s、600s和120μW/cm~2强度的UVB照射20s,导致胚胎发育过程中死亡率明显升高,畸胚数量多且畸变程度高。由此结果可见,UVB照射剂量和时间不同,对中华大蟾蜍早期胚胎发育的影响有明显差异。2)用YAG倍频激光器100μJ/cm~2、300μJ/cm~2和600μJ/cm~2分别照射中华大蟾蜍原肠胚,提取各组样品蛋白质组进行双向电泳,凝胶成像后用PDQuest软件分析。结果表明:激光照射对原肠胚蛋白质点的个数和分布都有显著影响,影响程度与照射激光的密度有关。有些点只在照射组中出现,有些则只在对照组中出现,另外一些蛋白质点的表达量在照射前后发生了明显的变化。3)为了在分子水平上检测激光照射对生物组织或细胞基因组损伤的程度,结合单细胞凝胶电泳技术建立了一种快速、定量检测小鼠淋巴细胞受激光照射后DNA损伤程度的方法。使用能量密度为600μJ/cm~2的YAG激光照射小鼠脾淋巴细胞15、25、35个脉冲后,采用单细胞凝胶电泳法检测实验组和对照组细胞DNA损伤程度,发现DNA损伤程度与激光照射脉冲数成正比。用强度为30μW/cm~2、60μW/cm~2、90μW/cm~2、120μW/cm~2、150μW/cm~2的UVB分别照射小鼠脾淋巴细胞30min,采用单细胞凝胶电泳法检测UVB照射对细胞DNA的损伤,结果表明30μW/cm~2组DNA损伤程度最高。随着照射强度的加大,60μW/cm~2、90μW/cm~2、120μW/cm~2组损伤程度有所降低,而150μW/cm~2组又出现了高DNA损伤。4)分别提取经不同强度照射UVB的四细胞期胚胎、原肠胚和神经胚的基因组DNA,然后进行琼脂糖凝胶电泳。发现各组中随着照射强度的增加DNA条带弥散的程度也加大,这可能与UVB照射胚胎生成了光产物,从而使DNA链断裂有关。5)提取未经照射的受精卵、四细胞期胚胎、囊胚、神经胚、原肠胚、肌肉感应期胚胎和经80μW/cm~2的UVB照射的四细胞期胚胎、原肠胚、神经胚的总RNA,进行甲酰胺化RNA变性琼脂糖电泳。发现与对照组相比,照射后的胚胎RNA条带均出现了不同程度的弥散。本论文所涉及的激光、UVB等环境物理因子对两栖类胚胎影响的研究,可为环境生物学研究辐射对生物体的影响提供借鉴。
Laser and ultraviolet belong to nonionization radiation and have many biological effects.The main biological effects of laser are photochemical interaction,thermal interaction and electromagnetic effect.Ultraviolet(UV)may induce structure of DNA,produce cyclobutane pyrimidine dimmer and pyrimidine(6-4)pyrimidone photoproducts and result in immunosuppression so much as to cancer.In order to examine the effects of irradiation of laser and UV on amphibian's embryos,Bufo bufo gargarizans's embryos were explored to YAG double-frequency laser microbeams and lamps of UVB at various strength and time.Compared to the control groups,the embryos which were irradiated by laser or Ultraviolet B(UVB)had many changes in morphology,nucleic acid and proteins.When Bufo bufo gargarizans's two-cell stage embryos were irradiated by UVB at various wave strength and time,many kinds of abnormal embryos could be observed in treatment groups.The results indicated that after exposed at low strength and short time,such as 30μW/cm~2 by 20s,60s and 80μW/cm~2 by 20s,60s,120s respectively,embryos' development process was promoted and death rate and degree of deformities were reduced than control and other groups. On the contrary,after irradiated by high wave strength and long time,such as 80μW/cm~2 by 600s and 100μW/cm~2 by 20s,60s,600s and 120μW/cm~2 by 20s respectively,embryos' death rate was higher than control and other groups.The results suggested that UVB had two sides of effect on Bufo bufo gargarizans's embryos,one is biostimulating and the other is killing and wounding.After irradiated by UVB with same time but various wave strength,the four-cell stage embryos,gastrulae and neurula were fetched and the results of DNA and RNA gel electrophoresis showed that the higher wave strength and the more dispersion.A new method,single-cell gel electrophoresis(comet assay),was used to determined damage grade of DNA after mouse's lymphocytes were irradiated by laser.The results indicated that high intensity laser led to severe damage of DNA.Changes of gastrula protein's expression patterns between control groups and other groups which were irradiated by YAG laser were examined by two-dimensional electrophoresis.The map of proteins showed that protein expression was influenced by laser irradiation and the total number of protein spots was related to the intensity of the laser beam.There were some proteins obviously changed in laser treatment groups compared to the control and some spots were visible in the treatment groups but not observed in the control. In further reserch,it is necessary to proceed to point out which genes and proteins change after irradiation with laser and UVB.This basic reaserch could offered reference to environmental biology.
Laser and ultraviolet belong to nonionization radiation and have many biological effects.The main biological effects of laser are photochemical interaction,thermal interaction and electromagnetic effect.Ultraviolet(UV)may induce structure of DNA,produce cyclobutane pyrimidine dimmer and pyrimidine(6-4)pyrimidone photoproducts and result in immunosuppression so much as to cancer.In order to examine the effects of irradiation of laser and UV on amphibian's embryos,Bufo bufo gargarizans's embryos were explored to YAG double-frequency laser microbeams and lamps of UVB at various strength and time.Compared to the control groups,the embryos which were irradiated by laser or Ultraviolet B(UVB)had many changes in morphology,nucleic acid and proteins.When Bufo bufo gargarizans's two-cell stage embryos were irradiated by UVB at various wave strength and time,many kinds of abnormal embryos could be observed in treatment groups.The results indicated that after exposed at low strength and short time,such as 30μW/cm~2 by 20s,60s and 80μW/cm~2 by 20s,60s,120s respectively,embryos' development process was promoted and death rate and degree of deformities were reduced than control and other groups. On the contrary,after irradiated by high wave strength and long time,such as 80μW/cm~2 by 600s and 100μW/cm~2 by 20s,60s,600s and 120μW/cm~2 by 20s respectively,embryos' death rate was higher than control and other groups.The results suggested that UVB had two sides of effect on Bufo bufo gargarizans's embryos,one is biostimulating and the other is killing and wounding.After irradiated by UVB with same time but various wave strength,the four-cell stage embryos,gastrulae and neurula were fetched and the results of DNA and RNA gel electrophoresis showed that the higher wave strength and the more dispersion.A new method,single-cell gel electrophoresis(comet assay),was used to determined damage grade of DNA after mouse's lymphocytes were irradiated by laser.The results indicated that high intensity laser led to severe damage of DNA.Changes of gastrula protein's expression patterns between control groups and other groups which were irradiated by YAG laser were examined by two-dimensional electrophoresis.The map of proteins showed that protein expression was influenced by laser irradiation and the total number of protein spots was related to the intensity of the laser beam.There were some proteins obviously changed in laser treatment groups compared to the control and some spots were visible in the treatment groups but not observed in the control. In further reserch,it is necessary to proceed to point out which genes and proteins change after irradiation with laser and UVB.This basic reaserch could offered reference to environmental biology.
引文
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