本文主要研究内容
作者马淑慧（2019）在《钟基因cBmall/cClock在单色光影响鸡松果体细胞分泌褪黑激素中的作用机制》一文中研究指出：禽类生物节律稳定性的维持依赖于下丘脑视交叉上核、松果体和视网膜等中枢起搏器的相互协作。其中,松果体呈昼低夜高的节律性分泌褪黑激素,是研究禽类生物节律系统的理想模型。禽类生物钟的维持还受到由正调控钟基因(Bmal1、Bmal2、Clock)和负调控钟基因(Cry1、Cry2、Per2、Per3)组成的负反馈环路的介导。前期研究发现单色绿光可以促进血浆褪黑激素的分泌并维持其昼夜节律性,但其调节机制还不明确。本研究分别在12 h光照:12 h黑暗的LD循环条件和持续黑暗条件(DD)下培养鸡松果体原代细胞,探究鸡松果体细胞的内源性节律振荡特性及其对光刺激的敏感性。同时在白光(400-760 nm,WL)、红光(660 nm,RL)、绿光(560 nm,GL)和蓝光(480nm,BL)四种不同光,光照制度为12L:12D的条件下培养鸡松果体原代细胞,研究单色光对鸡松果体细胞昼夜节律的影响。随后通过siRNA干扰技术和载体过表达试验进一步探究视蛋白cOpnp、正调控钟基因cBmal1和cClock在单色绿光影响鸡松果体细胞分泌褪黑激素的作用机制。研究结果如下:1.鸡松果体细胞的内源性节律振荡和光敏感性鸡松果体细胞在DD和LD条件下,正调控钟基因、负调控钟基因、cAanat和褪黑激素均呈现明显的昼夜节律表达。与DD组相比,LD降低了正调控钟基因的中值,增加了负调控钟基因的振幅,导致cAanat和褪黑激素的振幅和中值下降。LD还使cClock和cBmal2的相位提前而延迟了除cCry1外负调控钟基因的相位。以上结果表明鸡松果体细胞不仅具有内源性的节律振荡,并且还可以接受外界光刺激调控生物钟钟基因节律性变化。2.单色光对鸡松果体细胞生物钟系统昼夜节律的影响钟基因:不同光色下的鸡松果体细胞钟基因均具昼夜节律,但蓝光条件下cCry1除外。与白光相比,绿光增加了正调控钟基因的中值和振幅,而降低负调控钟基因cPer2/3的中值。相反,红光明显增加了负调控钟基因cCry1和cPer3的振幅以及cPer2/3的中值。cAanat和褪黑激素:不同光色下cAanat和褪黑激素均呈昼夜节律性。与白光相比,绿光增加了 cAanat和褪黑激素的中值和振幅,提前了两者的相位;而红光和蓝光的作用却与绿光相反。视蛋白:不同光色下鸡松果体细胞中的视蛋白cOpnp和cOpn4-1的mRNA水平变化呈现明显的昼夜节律性。与白光相比,绿光增加了 cOpnp的中值和振幅,且相位提前;而红光降低了 cOpnp的中值和振幅,相位延迟。各光组cOpn4-1的表达量与白光相比均降低。以上试验结果表明单色绿光可以增加褪黑激素的合成分泌,该过程可能与单色绿光刺激cOpnp和正调控钟基因的表达、降低负调控钟基因的表达有关。3.cOpnp在绿光影响鸡松果体细胞生物钟系统中的调控作用cOpnp干扰后,钟基因cBmal1、cCry2、cPer3、cAanat和褪黑激素的昼夜节律性消失,cBmal2和cClock的中值和振幅下降,cClock和cCry1的相位明显提前。cOpnp过表达后,钟基因cBmal1、cCry2、cAanat和褪黑激素昼夜节律消失,cPer2和cPer3的中值和振幅均增加,cCry1的相位提前。结果表明cOpnp参与绿光影响鸡松果体细胞合成和分泌褪黑激素的调控。4.cBmall在绿光影响鸡松果体细胞生物钟系统中的调控作用cBmal1的干扰使除cCry1外的所有基因和褪黑激素的昼夜节律均消失,并且导致正调控钟基因、cAanat和褪黑激素的表达水平下降。cBmal1过表达使负调控钟基因cCry1/2、cAanat和褪黑激素的昼夜表达的节律性消失,同时使所有钟基因、cAanat和褪黑激素表达水平增加。结果说明cBmal1在调控绿光影响鸡松果体细胞褪黑激素的合成和分泌中发挥重要作用。5.cClock在绿光影响鸡松果体细胞生物钟系统中的调控作用cClock干扰导致cClock、ccBmal2、cCry2和cPer3的昼夜节律性消失,但cBmal1、cCry1、cPer2、cAanat和褪黑激素昼夜节律性均未受到影响。cClock干扰后明显抑制cAanat和褪黑激素主观夜间的表达和分泌,同时降低cCry1和cPer2的中值和振幅,并且改变cCry1的相位。cClock过表达只干扰了 cCry1/2的昼夜节律性,增加了cBmal2、cPer2、cPer3以及褪黑激素的中值和振幅,导致正调控钟基因和褪黑激素的相位延迟。这表明cClock可能参与调节绿光影响鸡松果体细胞褪黑激素的合成,但不会干扰其节律性的分泌。6.cAMP信号通路在绿光影响鸡松果体细胞生物钟系统中的调控作用松果体细胞添加PKA抑制剂H89后,导致所有钟基因、cAanat和褪黑激素的表达水平均降低。而添加cAMP的激活剂FSK和cAMP的类似物Dibutyry1-cAMP后,随着正调控钟基因的表达量增加,以及负调控钟基因的表达量下降,cAanat mRNA表达量和褪黑激素的分泌水平也明显增加。以上结果表明绿光影响鸡松果体细胞钟基因的表达和褪黑激素昼夜节律分泌的调节是通过cAMP信号通路实现的。结论:鸡松果体细胞具有内源性的昼夜节律性振荡,并且可以接受外界不同波长光的刺激牵引体内生物钟系统发生改变。单色绿光通过影响cOpnp的表达量激活cAMP信号通路,增加正调控钟基因cBmall的表达,从而促进褪黑激素的节律性分泌。
Abstract
qin lei sheng wu jie lv wen ding xing de wei chi yi lai yu xia qiu nao shi jiao cha shang he 、song guo ti he shi wang mo deng zhong shu qi bo qi de xiang hu xie zuo 。ji zhong ,song guo ti cheng zhou di ye gao de jie lv xing fen bi tui hei ji su ,shi yan jiu qin lei sheng wu jie lv ji tong de li xiang mo xing 。qin lei sheng wu zhong de wei chi hai shou dao you zheng diao kong zhong ji yin (Bmal1、Bmal2、Clock)he fu diao kong zhong ji yin (Cry1、Cry2、Per2、Per3)zu cheng de fu fan kui huan lu de jie dao 。qian ji yan jiu fa xian chan se lu guang ke yi cu jin xie jiang tui hei ji su de fen bi bing wei chi ji zhou ye jie lv xing ,dan ji diao jie ji zhi hai bu ming que 。ben yan jiu fen bie zai 12 hguang zhao :12 hhei an de LDxun huan tiao jian he chi xu hei an tiao jian (DD)xia pei yang ji song guo ti yuan dai xi bao ,tan jiu ji song guo ti xi bao de nei yuan xing jie lv zhen dang te xing ji ji dui guang ci ji de min gan xing 。tong shi zai bai guang (400-760 nm,WL)、gong guang (660 nm,RL)、lu guang (560 nm,GL)he lan guang (480nm,BL)si chong bu tong guang ,guang zhao zhi du wei 12L:12Dde tiao jian xia pei yang ji song guo ti yuan dai xi bao ,yan jiu chan se guang dui ji song guo ti xi bao zhou ye jie lv de ying xiang 。sui hou tong guo siRNAgan rao ji shu he zai ti guo biao da shi yan jin yi bu tan jiu shi dan bai cOpnp、zheng diao kong zhong ji yin cBmal1he cClockzai chan se lu guang ying xiang ji song guo ti xi bao fen bi tui hei ji su de zuo yong ji zhi 。yan jiu jie guo ru xia :1.ji song guo ti xi bao de nei yuan xing jie lv zhen dang he guang min gan xing ji song guo ti xi bao zai DDhe LDtiao jian xia ,zheng diao kong zhong ji yin 、fu diao kong zhong ji yin 、cAanathe tui hei ji su jun cheng xian ming xian de zhou ye jie lv biao da 。yu DDzu xiang bi ,LDjiang di le zheng diao kong zhong ji yin de zhong zhi ,zeng jia le fu diao kong zhong ji yin de zhen fu ,dao zhi cAanathe tui hei ji su de zhen fu he zhong zhi xia jiang 。LDhai shi cClockhe cBmal2de xiang wei di qian er yan chi le chu cCry1wai fu diao kong zhong ji yin de xiang wei 。yi shang jie guo biao ming ji song guo ti xi bao bu jin ju you nei yuan xing de jie lv zhen dang ,bing ju hai ke yi jie shou wai jie guang ci ji diao kong sheng wu zhong zhong ji yin jie lv xing bian hua 。2.chan se guang dui ji song guo ti xi bao sheng wu zhong ji tong zhou ye jie lv de ying xiang zhong ji yin :bu tong guang se xia de ji song guo ti xi bao zhong ji yin jun ju zhou ye jie lv ,dan lan guang tiao jian xia cCry1chu wai 。yu bai guang xiang bi ,lu guang zeng jia le zheng diao kong zhong ji yin de zhong zhi he zhen fu ,er jiang di fu diao kong zhong ji yin cPer2/3de zhong zhi 。xiang fan ,gong guang ming xian zeng jia le fu diao kong zhong ji yin cCry1he cPer3de zhen fu yi ji cPer2/3de zhong zhi 。cAanathe tui hei ji su :bu tong guang se xia cAanathe tui hei ji su jun cheng zhou ye jie lv xing 。yu bai guang xiang bi ,lu guang zeng jia le cAanathe tui hei ji su de zhong zhi he zhen fu ,di qian le liang zhe de xiang wei ;er gong guang he lan guang de zuo yong que yu lu guang xiang fan 。shi dan bai :bu tong guang se xia ji song guo ti xi bao zhong de shi dan bai cOpnphe cOpn4-1de mRNAshui ping bian hua cheng xian ming xian de zhou ye jie lv xing 。yu bai guang xiang bi ,lu guang zeng jia le cOpnpde zhong zhi he zhen fu ,ju xiang wei di qian ;er gong guang jiang di le cOpnpde zhong zhi he zhen fu ,xiang wei yan chi 。ge guang zu cOpn4-1de biao da liang yu bai guang xiang bi jun jiang di 。yi shang shi yan jie guo biao ming chan se lu guang ke yi zeng jia tui hei ji su de ge cheng fen bi ,gai guo cheng ke neng yu chan se lu guang ci ji cOpnphe zheng diao kong zhong ji yin de biao da 、jiang di fu diao kong zhong ji yin de biao da you guan 。3.cOpnpzai lu guang ying xiang ji song guo ti xi bao sheng wu zhong ji tong zhong de diao kong zuo yong cOpnpgan rao hou ,zhong ji yin cBmal1、cCry2、cPer3、cAanathe tui hei ji su de zhou ye jie lv xing xiao shi ,cBmal2he cClockde zhong zhi he zhen fu xia jiang ,cClockhe cCry1de xiang wei ming xian di qian 。cOpnpguo biao da hou ,zhong ji yin cBmal1、cCry2、cAanathe tui hei ji su zhou ye jie lv xiao shi ,cPer2he cPer3de zhong zhi he zhen fu jun zeng jia ,cCry1de xiang wei di qian 。jie guo biao ming cOpnpcan yu lu guang ying xiang ji song guo ti xi bao ge cheng he fen bi tui hei ji su de diao kong 。4.cBmallzai lu guang ying xiang ji song guo ti xi bao sheng wu zhong ji tong zhong de diao kong zuo yong cBmal1de gan rao shi chu cCry1wai de suo you ji yin he tui hei ji su de zhou ye jie lv jun xiao shi ,bing ju dao zhi zheng diao kong zhong ji yin 、cAanathe tui hei ji su de biao da shui ping xia jiang 。cBmal1guo biao da shi fu diao kong zhong ji yin cCry1/2、cAanathe tui hei ji su de zhou ye biao da de jie lv xing xiao shi ,tong shi shi suo you zhong ji yin 、cAanathe tui hei ji su biao da shui ping zeng jia 。jie guo shui ming cBmal1zai diao kong lu guang ying xiang ji song guo ti xi bao tui hei ji su de ge cheng he fen bi zhong fa hui chong yao zuo yong 。5.cClockzai lu guang ying xiang ji song guo ti xi bao sheng wu zhong ji tong zhong de diao kong zuo yong cClockgan rao dao zhi cClock、ccBmal2、cCry2he cPer3de zhou ye jie lv xing xiao shi ,dan cBmal1、cCry1、cPer2、cAanathe tui hei ji su zhou ye jie lv xing jun wei shou dao ying xiang 。cClockgan rao hou ming xian yi zhi cAanathe tui hei ji su zhu guan ye jian de biao da he fen bi ,tong shi jiang di cCry1he cPer2de zhong zhi he zhen fu ,bing ju gai bian cCry1de xiang wei 。cClockguo biao da zhi gan rao le cCry1/2de zhou ye jie lv xing ,zeng jia le cBmal2、cPer2、cPer3yi ji tui hei ji su de zhong zhi he zhen fu ,dao zhi zheng diao kong zhong ji yin he tui hei ji su de xiang wei yan chi 。zhe biao ming cClockke neng can yu diao jie lu guang ying xiang ji song guo ti xi bao tui hei ji su de ge cheng ,dan bu hui gan rao ji jie lv xing de fen bi 。6.cAMPxin hao tong lu zai lu guang ying xiang ji song guo ti xi bao sheng wu zhong ji tong zhong de diao kong zuo yong song guo ti xi bao tian jia PKAyi zhi ji H89hou ,dao zhi suo you zhong ji yin 、cAanathe tui hei ji su de biao da shui ping jun jiang di 。er tian jia cAMPde ji huo ji FSKhe cAMPde lei shi wu Dibutyry1-cAMPhou ,sui zhao zheng diao kong zhong ji yin de biao da liang zeng jia ,yi ji fu diao kong zhong ji yin de biao da liang xia jiang ,cAanat mRNAbiao da liang he tui hei ji su de fen bi shui ping ye ming xian zeng jia 。yi shang jie guo biao ming lu guang ying xiang ji song guo ti xi bao zhong ji yin de biao da he tui hei ji su zhou ye jie lv fen bi de diao jie shi tong guo cAMPxin hao tong lu shi xian de 。jie lun :ji song guo ti xi bao ju you nei yuan xing de zhou ye jie lv xing zhen dang ,bing ju ke yi jie shou wai jie bu tong bo chang guang de ci ji qian yin ti nei sheng wu zhong ji tong fa sheng gai bian 。chan se lu guang tong guo ying xiang cOpnpde biao da liang ji huo cAMPxin hao tong lu ,zeng jia zheng diao kong zhong ji yin cBmallde biao da ,cong er cu jin tui hei ji su de jie lv xing fen bi 。
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论文作者分别是来自中国农业大学的马淑慧，发表于刊物中国农业大学2019-01-17论文，是一篇关于单色光论文,松果体细胞论文,昼夜节律论文,钟基因论文,视蛋白论文,褪黑激素论文,干扰论文,中国农业大学2019-01-17论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自中国农业大学2019-01-17论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
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