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延边大学医学学报　２０２２年３月　第４５卷　第１期与疾病报告２０２０概要［Ｊ］．中国循环杂志，２０２１，３６（６）：５２１－５４５．［２］　ＭＥＤＩＮＡ－ＬＥＹＴＥ　ＤＪ，ＺＥＰＥＤＡ－ＧＡＲＣíＡ　Ｏ，ＤＯＭíＮＧ－ＵＥＺＰéＲＥＺ　Ｍ，ｅｔ　ａｌ．．Ｅｎｄｏｔｈｅｌｉａｌ　ｄｙｓｆｕｎｃｔｉｏｎ，ｉｎｆｌａｍ－ｍａｔｉｏｎ　ａｎｄ　ｃｏｒｏｎａｒｙ　ａｒｔｅｒｙ　ｄｉｓｅａｓｅ：ｐｏｔｅｎｔｉａｌ　ｂｉｏｍａｒｋ－ｅｒｓ　ａｎｄ　ｐｒｏｍｉｓｉｎｇ　ｔｈｅｒａｐｅｕｔｉｃａｌ　ａｐｐｒｏａｃｈｅｓ［Ｊ］．Ｉｎｔ　ＪＭｏｌ　Ｓｃｉ，２０２１，２２（８）：３８５０．［３］　ＨＵＡＮＧ　Ｃ，ＨＵＡＮＧ　ＷＨ，ＷＡＮＧ　Ｒ，ｅｔ　ａｌ．．Ｕｌｉｎａｓｔａｔｉｎｉｎｈｉｂｉｔｓ　ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ，ｉｎｖａｓｉｏｎ　ａｎｄ　ｐｈｅｎｏｔｙｐｉｃｓｗｉｔｃｈｉｎｇ　ｏｆ　ＰＤＧＦ－ＢＢ－ｉｎｄｕｃｅｄ　ＶＳＭＣｓ　ｖｉａ　Ａｋｔ／ｅＮＯＳ／ＮＯ／ｃＧＭＰ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ［Ｊ］．Ｄｒｕｇ　Ｄｅｓ　Ｄｅｖｅｌ　Ｔｈｅｒ，２０２０，１４：５５０５－５５１４．［４］　赵高峰．延边地区朝鲜族ＳＭＡＲＣＡ４基因ＳＮＰｒｓ１１２２６０８与冠心病相关性研究［Ｄ］．延吉：延边大学，２０２０．［５］　ＩＭ　ＰＫ，ＭＩＬＬＷＯＯＤ　ＩＹ，ＫＡＲＴＳＯＮＡＫＩ　Ｃ，ｅｔ　ａｌ．．Ａｌ－ｃｏｈｏｌ　ｄｒｉｎｋｉｎｇ　ａｎｄ　ｒｉｓｋｓ　ｏｆ　ｔｏｔａｌ　ａｎｄ　ｓｉｔｅ－ｓｐｅｃｉｆｉｃ　ｃａｎｃ－ｅｒｓ　ｉｎ　Ｃｈｉｎａ：Ａ　１０－ｙｅａｒ　ｐｒｏｓｐｅｃｔｉｖｅ　ｓｔｕｄｙ　ｏｆ　０．５ｍｉｌｌｉｏｎａｄｕｌｔｓ［Ｊ］．Ｉｎｔ　Ｊ　Ｃａｎｃｅｒ，２０２１，１４９（３）：５２２－５３４．［６］　ＲＯＳＣＨ　ＰＪ．Ｃｏｕｌｄ　ｔｈｅ　ｂｅｎｅｆｉｔｓ　ｏｆ　ｄｒｉｎｋｉｎｇ　ａｌｃｏｈｏｌ　ｏｕｔ－ｗｅｉｇｈ　ａｌｌ　ｉｔｓ　ｒｉｓｋｓ［Ｊ］．Ｓｔｒｅｓｓ　Ｈｅａｌｔｈ，２０１３．［７］　ＷＡＬＬＥＲＡＴＨ　Ｔ，ＰＯＬＥＯ　Ｄ，ＬＩ　Ｈ，ｅｔ　ａｌ．．Ｒｅｄ　ｗｉｎｅ　ｉｎ－ｃｒｅａｓｅｓ　ｔｈｅ　ｅｘｐｒｅｓｓｉｏｎ　ｏｆ　ｈｕｍａｎ　ｅｎｄｏｔｈｅｌｉａｌ　ｎｉｔｒｉｃ　ｏｘｉｄｅｓｙｎｔｈａｓｅ：ａ　ｍｅｃｈａｎｉｓｍ　ｔｈａｔ　ｍａｙ　ｃｏｎｔｒｉｂｕｔｅ　ｔｏ　ｉｔｓ　ｂｅｎｅ－ｆｉｃｉａｌ　ｃａｒｄｉｏｖａｓｃｕｌａｒ　ｅｆｆｅｃｔｓ［Ｊ］．ＪＡＣＣ，２００３，４１（３）：４７１－４７８．［８］　ＺＨＡＯ　Ｆ，ＪＩ　Ｚ，ＣＨＩ　Ｊ，ｅｔ　ａｌ．．Ｅｆｆｅｃｔｓ　ｏｆ　Ｃｈｉｎｅｓｅ　ｙｅｌｌｏｗｗｉｎｅ　ｏｎ　ｎｉｔｒｉｃ　ｏｘｉｄｅ　ｓｙｎｔｈａｓｅ　ａｎｄ　ｉｎｔｅｒｃｅｌｌｕｌａｒ　ａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅ－１ｅｘｐｒｅｓｓｉｏｎｓ　ｉｎ　ｒａｔ　ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｃｅｌｌｓ［Ｊ］．Ａｃｔａ　Ｃａｒｄｉｏｌｏｇｉｃａ，２０１６，７１（１）：２７－３４．［９］　金春花，刘文博，关宏锏，等．一氧化氮合酶各种亚型的信号通路对心力衰竭的作用机制与治疗的研究进展［Ｊ］．中国循环杂志，２０１７，３２（８）：８３０－８３２．［１０］ＷＡＧＮＥＲ　ＭＣ，ＹＥＬＩＧＡＲ　ＳＭ，ＬＯＵ　ＡＢ，ｅｔ　ａｌ．．ＰＰＡＲγｌｉｇａｎｄｓ　ｒｅｇｕｌａｔｅ　ＮＡＤＰＨ　ｏｘｉｄａｓｅ，ｅＮＯＳ，ａｎｄ　ｂａｒｒｉｅｒｆｕｎｃｔｉｏｎ　ｉｎ　ｔｈｅ　ｌｕｎｇ　ｆｏｌｌｏｗｉｎｇ　ｃｈｒｏｎｉｃ　ａｌｃｏｈｏｌ　ｉｎｇｅｓｔｉｏｎ［Ｊ］．Ａｌｃｏｈｏｌ　Ｃｌｉｎ　Ｅｘｐ　Ｒｅｓ，２０１２，３６（２）：１９７－２０６．［１１］ＴＩＲＡＰＥＬＬＩ　ＣＲ，ＬＥＯＮＥ　ＡＦ，ＹＯＧＩ　Ａ，ｅｔ　ａｌ．．Ｅｔｈａｎｏｌｃｏｎｓｕｍｐｔｉｏｎ　ｉｎｃｒｅａｓｅｓ　ｂｌｏｏｄ　ｐｒｅｓｓｕｒｅ　ａｎｄ　ａｌｔｅｒｓ　ｔｈｅ　ｒｅ－ｓｐｏｎｓｉｖｅｎｅｓｓ　ｏｆ　ｔｈｅ　ｍｅｓｅｎｔｅｒｉｃ　ｖａｓｃｕｌａｔｕｒｅ　ｉｎ　ｒａｔｓ［Ｊ］．Ｊ　Ｐｈａｒｍ　Ｐｈａｒｍａｃｏｌ，２００８，６０（３）：３３１－３４１．［１２］ＣＨＥＮ　ＳＳ，ＷＡＮＧ　ＺＹ，ＺＨＯＵ　Ｈ，ｅｔ　ａｌ．．Ｉｃａｒｉｉｎ　ｒｅ－ｄｕｃｅｓ　ｈｉｇｈ　ｇｌｕｃｏｓｅ－ｉｎｄｕｃｅｄ　ｅｎｄｏｔｈｅｌｉａｌ　ｐｒｏｇｅｎｉｔｏｒ　ｃｅｌｌｄｙｓｆｕｎｃｔｉｏｎ　ｖｉａ　ｉｎｈｉｂｉｔｉｎｇ　ｔｈｅ　ｐ３８／ＣＲＥＢ　ｐａｔｈｗａｙ　ａｎｄａｃｔｉｖａｔｉｎｇ　ｔｈｅ　Ａｋｔ／ｅＮＯＳ／ＮＯ　ｐａｔｈｗａｙ［Ｊ］．Ｅｘｐ　ＴｈｅｒＭｅｄ，２０１９，１８（６）：４７７４－４７８０．■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■［基金项目］　国家自然科学基金项目（编号：８１５６０１７９）．［收稿日期］　２０２２－０１－０３［作者简介］　黄银珠（１９９５—），女，硕士，研究方向为口腔颌面外科．［通信作者］　金成日，Ｅｍａｉｌ：ｊｃｒ１１０５＠１６３．ｃｏｍ．初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路影响口腔鳞状细胞癌增殖研究黄银珠，李京旭，金成日（延边大学附属医院口腔科，吉林延吉１３３０００）［摘要］　［目的］探讨初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路对口腔鳞状细胞癌增殖能力产生的影响．［方法］选择人舌鳞癌Ｔｃａ８１１３细胞株作为研究对象，分为ＫＩＦ３Ａ－ｓｉＲＮＡ转染组和对照组．采用蛋白印迹实验和实时荧光定量实验检测各组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１蛋白及ｍＲＮＡ的相对表达水平；利用ＭＴＴ细胞增殖实验观察Ｔｃａ８１１３细胞增殖能力的变化．［结果］蛋白印迹实验结果显示，与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１蛋白表达水平均明显下调（Ｐ＜０．０５）；实时荧光定量实验结果显示，与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１ｍＲＮＡ表达水平均明显下调（Ｐ＜０．０１）；ＭＴＴ细胞增殖实验结果显示，细胞培养２４ｈ时两组细胞增殖间差异无统计学意义（Ｐ＞０．０５），细胞培养４８、７２ｈ时ＫＩＦ３Ａ－ｓｉＲＮＡ转染组增殖率较对照组明显降低（Ｐ＜０．０１）．［结论］沉默初级纤毛相关基因ＫＩＦ３Ａ可能通过调控Ｈｅｄｇｅｈｏｇ信号通路抑制口腔鳞癌的增殖能力．［关键词］　口腔鳞状细胞癌；初级纤毛；ＫＩＦ３Ａ；Ｈｅｄｇｅｈｏｇ信号通路ＤＯＩ：１０．１６０６８／ｊ．１０００－１８２４．２０２２．０１．００２［中图分类号］　Ｒ　７３９．８ ［文献标志码］　Ａ ［文章编号］　１０００－１８２４（２０２２）０１－０００７－０４·７·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ　Ｍａｒ．２０２２　Ｖｏｌ．４５　Ｎｏ．１Ｅｆｆｅｃｔｓ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｏｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｙ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙＨＵＡＮＧ　Ｙｉｎｚｈｕ，ＬＩ　Ｊｉｎｇｘｕ，ＪＩＮ　Ｃｈｅｎｇｒｉ（Ｄｅｐａｒｔｍｅｎｔ　ｏｆ　Ｓｔｏｍａｔｏｌｏｇｙ，Ａｆｆｉｌｉａｔｅｄ　Ｈｏｓｐｉｔａｌ　ｏｆ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ，Ｙａｎｊｉ　１３３０００，Ｊｉｌｉｎ，Ｃｈｉｎａ）ＡＢＳＴＲＡＣＴ：ＯＢＪＥＣＴＩＶＥＴｏ　ｅｘｐｌｏｒｅ　ｔｈｅ　ｅｆｆｅｃｔ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｏｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓｃｅｌｌ　ｃａｒｃｉｎｏｍａ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｔｈｒｏｕｇｈ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ．ＭＥＴＨＯＤＳ　Ｈｕｍａｎ　ｔｏｎｇｕｅ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ　Ｔｃａ８１１３ｃｅｌｌ　ｌｉｎｅ　ｗａｓ　ｓｅｌｅｃｔｅｄ　ａｎｄ　ｄｉｖｉｄｅｄ　ｉｎｔｏ　ＫＩＦ３Ａ－ｓｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ａｎｄ　ｃｏｎｔｒｏｌｇｒｏｕｐ．Ｗｅｓｔｅｒｎ　ｂｌｏｔ　ａｎｄ　ｒｅａｌ－ｔｉｍｅ　ｑＰＣＲ　ｗｅｒｅ　ｕｓｅｄ　ｔｏ　ｄｅｔｅｃｔ　ｔｈｅ　ｒｅｌａｔｉｖｅ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｐｒｏｔｅｉｎｓ　ａｎｄ　ｍＲＮＡ　ｉｎ　ｅａｃｈ　ｇｒｏｕｐ．ＭＴＴ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ａｓｓａｙ　ｗａｓ　ｕｓｅｄ　ｔｏ　ｏｂｓｅｒｖｅ　ｔｈｅｐｒｏｌｉｆｅｒａｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ．ＲＥＳＵＬＴＳ　Ｗｅｓｔｅｒｎ　ｂｌｏｔ　ｒｅｓｕｌｔｓ　ｓｈｏｗｅｄ　ｔｈａｔ　ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｔｈｅ　ｃｏｎｔｒｏｌ　ｇｒｏｕｐ，ｔｈｅ　ｐｒｏｔｅｉｎ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈ　ａｎｄ　Ｇｌｉ１ｉｎ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙ　ｄｏｗｎ－ｒｅｇｕｌａｔｅｄ（Ｐ＜０．０５）．Ｒｅａｌ－ｔｉｍｅ　ｑＰＣＲ　ｒｅｓｕｌｔｓ　ｓｈｏｗｅｄ　ｔｈａｔ　ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｔｈｅ　ｃｏｎｔｒｏｌｇｒｏｕｐ，ｔｈｅ　ｍＲＮＡ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈ　ａｎｄ　Ｇｌｉ１ｉｎ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙ　ｄｏｗｎ－ｒｅｇｕｌａｔｅｄ（Ｐ＜０．０１）．ＭＴＴ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ａｓｓａｙ　ｓｈｏｗｅｄ　ｔｈａｔ　ｔｈｅｒｅ　ｗａｓ　ｎｏ　ｓｉｇｎｉｆｉｃａｎｔｄｉｆｆｅｒｅｎｃｅ　ｉｎ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｅｔｗｅｅｎ　ｔｈｅ　ｔｗｏ　ｇｒｏｕｐｓ　ａｆｔｅｒ　２４ｈｃｅｌｌ　ｃｕｌｔｕｒｅ（Ｐ＞０．０５）．Ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎｒａｔｅ　ｏｆ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗａｓ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ｌｏｗｅｒ　ｔｈａｎ　ｔｈａｔ　ｏｆ　ｔｈｅ　ｃｏｎｔｒｏｌ　ｇｒｏｕｐ　ａｆｔｅｒ　ｃｅｌｌｃｕｌｔｕｒｅ　ｆｏｒ　４８ａｎｄ　７２ｈ（Ｐ＜０．０１）．ＣＯＮＣＬＵＳＩＯＮＳｉｌｅｎｃｉｎｇ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｍａｙｉｎｈｉｂｉｔ　ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｏｆ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ　ｂｙ　ｒｅｇｕｌａｔｉｎｇ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ．Ｋｅｙｗｏｒｄｓ：ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ；ｐｒｉｍａｒｙ　ｃｉｌｉａ；ＫＩＦ３Ａ；Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ 口腔癌人数约占全世界罹患恶性肿瘤人数的３％，其中鳞状细胞癌比例超过９０％．２００５年，ＷＨＯ将口腔鳞状细胞癌（ＯＳＣＣ）定义为一种具有不同分化程度和侵袭性的肿瘤，早期可出现广泛的淋巴结转移，好发于４０～７０岁的烟酒爱好者［１］．口腔癌患者５年生存率约为５０％［２］，患者术后预后差和５年生存率低的主要原因为患者未及时就诊、淋巴结转移及复发［３］．控制ＯＳＣＣ的侵袭转移能力对预后至关重要，认为可偿试从分子水平出发找出更好的治疗方法［４］．初生纤毛属于一种细胞表面的细胞器，在脊椎动物发育和人类遗传疾病中发挥着重要作用．纤毛对发育信号的响应是必需的，而越来越多的证据表明初级纤毛是专属于Ｈｅｄｇｅｈｏｇ信号传导的［５］，而Ｈｅｄｇｅｈｏｇ信号传导对肿瘤的发生发展起着重要作用．研究［６］结果显示，多种肿瘤组织中均存在Ｈｅｄｇｅｈｏｇ信号的异常激活，如肺小细胞癌、胰腺癌、前列腺癌、乳腺癌、恶性胶质瘤、髓母细胞瘤及多发性骨髓瘤等．ＫＩＦ３Ａ属于初级纤毛内运输系统，具有维持和发挥初级纤毛正常功能的作用［７］．本研究探讨了初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路对ＯＳＣＣ增殖能力产生的影响．１　材料与方法１．１　细胞　人舌鳞癌细胞株Ｔｃａ８１１３购自美国菌种保藏中心（ＡＴＣＣ），置于延边大学附属医院中心实验室封存．１．２细胞培养与转染　取Ｔｃａ８１１３细胞置于温度３７℃、二氧化碳体积分数５％、饱和湿度９５％的恒温孵育箱中，用含１０％（质量分数）ＦＢＳ、青链霉素的ＤＭＥＭ高糖型培养基传代培养．在转染前１ｄ，将Ｔｃａ８１１３细胞分为对照组和ＫＩＦ３Ａ－ｓｉＲＮＡ转染组后接种至６孔板中，待生长至８０％时进行细胞转染．ＫＩＦ３Ａ－ｓｉＲＮＡ转染组转染ＫＩＦ３Ａ－ｓｉＲＮＡ，对照组未进行转染．转染操作按照ＬｉｐｏｆｅｃｔａｍｉｎｅＴＭ２０００Ｒｅ－ａｇｅｎ转染说明书进行．ＫＩＦ３Ａ－ｓｉＲＮＡ序列（５′－３′）：ＵＡＡ　ＧＧＡ　ＡＵＧ　ＣＵＧ　ＡＡＧ　ＡＡＧ　ＡＣＧ　ＡＧＵ　Ｃ．１．３　蛋白印迹实验　转染２４ｈ后分别收集两组细胞，并加入ＲＩＰＡ与ＰＭＳＦ混合（１００:１）冰上裂解细胞，提取细胞总蛋白，采用ＢＣＡ试剂盒测定总蛋白水平．取各组标本行８％（质量分数）十二烷基硫酸钠聚丙烯酰胺凝胶（ＳＤＳ－ＰＡＧＥ）电泳，转膜，室温下用５％（质量分数）脱脂奶粉封闭２ｈ．ＴＢＳＴ洗涤３·８·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■延边大学医学学报　２０２２年３月　第４５卷　第１期次，每次１０ｍｉｎ，加入兔抗人Ｋｉｆ３ａ一抗（１:１　０００）、兔抗人Ｓｈｈ一抗（１:２　０００）、兔抗人Ｇｌｉ１（１:１　０００）及兔抗人ＧＡＰＤＨ（１:１　０００）后置于湿盒中，于４℃冰箱中孵育过夜；次日ＴＢＳＴ洗涤３次，每次１０ｍｉｎ，分别加入山羊抗兔ＩｇＧ二抗（１:１０　０００）后置于湿盒中室温孵育２ｈ；ＴＢＳＴ洗涤３次，每次１０ｍｉｎ，加入增强型化学发光剂ＥＣＬ显影．以ＧＡＰＤＨ为内参，采用Ｉｍａｇｉｎｅ　Ｊ软件分析蛋白条带灰度值．１．４　实时荧光定量实验　转染２４ｈ后分别收集两组细胞，采用ＴＲＮｚｏｌ法提取细胞中的总ＲＮＡ，反转录为ｃＤＮＡ，按照ＲＴ－ＰＣＲ试剂盒说明书以ｃＤＮＡ为模板进行扩增．测定循环阈值（Ｃｔ），采用２－△△Ｃｔ法进行统计．实验重复３次．引物序列，ＫＩＦ３Ａ－正向：５′－ＡＧＡ　ＧＣＧ　ＴＣＡ　ＡＣＧ　ＡＧＧ　ＴＧＴ　ＴＴ－３′；ＫＩＦ３Ａ－反向：５′－ＴＡＴ　ＴＧＡ　ＴＣＧ　ＧＣＡ　ＴＣＴ　ＴＧＧ　ＣＣＣ－３′；Ｓｈｈ－正向：５′－ＣＴＣ　ＧＣＴ　ＧＣＴ　ＧＧＴ　ＡＴＧ　ＣＴＣ　Ｇ－３′；Ｓｈｈ－反向：５′－ＡＴＣ　ＧＣＴ　ＣＧＧ　ＡＧＴ　ＴＴＣ　ＴＧＧＡＧＡ－３′；Ｇｌｉ１－正向：５′－ＡＧＣ　ＧＴＧ　ＡＧＣ　ＣＴＧ　ＡＡＴＣＴＧ　ＴＧ－３′；Ｇｌｉ１－反向：５′－ＣＡＧ　ＣＡＴ　ＧＴＡ　ＣＴＧＧＧＣ　ＴＴＴ　ＧＡＡ－３′；ＧＡＰＤＨ－正向：５′－ＧＧＡ　ＧＣＧＡＧＡ　ＴＣＣ　ＣＴＣ　ＣＡＡ　ＡＡＴ－３′；ＧＡＰＤＨ－反向：５′－ＧＧＣ　ＴＧＴ　ＴＧＴＣＡＴＡＣＴ　ＴＣＴＣＡＴＧＧ－３′．１．５　ＭＴＴ细胞增殖实验　转染２４ｈ后分别收集两组细胞，以５　０００个／孔接种于９６孔板中，每组设置５个复孔．培养２４、４８、７２ｈ后弃去旧培养液，每孔加入１００μＬ　ＭＴＴ（５　ｇ／Ｌ），于３７℃、５％（体积分数）二氧化碳、９５％饱和湿度的恒温孵育箱中培养４　ｈ，弃去上清液终止反应，每孔加入１００μＬ　ＤＭＳＯ，置于振荡器３　ｍｉｎ溶解结晶后在酶标仪波长４９０　ｎｍ处测定各孔的光密度（ＯＤ）值，计算相对细胞增殖率．相对细胞增殖率（％）＝（实验组ＯＤ值／对照组ＯＤ值）×１００％．１．６　统计学分析　采用Ｇｒａｐｈｐａｄ　Ｐｒｉｓｍ　７．０软件进行数据分析．计量数据以均数±标准偏差（ ｘ±ｓ）表示，进行两个样本均数的ｔ检验，以Ｐ＜０．０５为差异有统计学意义．２　结果２．１　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞Ｈｅｄｇｅｈｏｇ信号通路中Ｓｈｈ、Ｇｌｉ１蛋白表达水平的影响　与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组２４ｈ时ＫＩＦ３Ａ、Ｓｈｈ及Ｇｌｉ１蛋白表达水平均明显降低，差异均具有统计学意义（Ｐ＜０．０５），见Ｆｉｇｕｒｅ　１．ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊Ｐ＜０．０５．Ｆｉｇｕｒｅ　１　Ｔｈｅ　ｐｒｏｔｅｉｎ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｄｅｃｒｅａｓｅｄ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ２．２　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞中Ｈｅｄｇｅｈｏｇ信号通路Ｓｈｈ、Ｇｌｉ１ｍＲＮＡ表达水平的影响　与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染２４ｈ组ＫＩＦ３Ａ、Ｓｈｈ及Ｇｌｉ１ｍＲＮＡ表达水平均显著降低，差异均具有统计学意义（Ｐ＜０．０１），见Ｆｉｇｕｒｅ　２．ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊＊Ｐ＜０．０１．Ｆｉｇｕｒｅ　２　Ｔｈｅ　ｍＲＮＡ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｄｅｃｒｅａｓｅｄ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ２．３　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞增殖能力的影响　ＭＴＴ检测结果显示，细胞培养２４ｈ时ＫＩＦ３Ａ－ｓｉＲＮＡ转染组与对照组细胞增殖间差异无统计学意义（Ｐ＞０．０５）；细胞培养４８、７２ｈ时，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组与对照组比较增殖显著受到抑制（Ｐ＜０．０１），见Ｆｉｇｕｒｅ　３．·９·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ　Ｍａｒ．２０２２　Ｖｏｌ．４５　Ｎｏ．１ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊＊Ｐ＜０．０１．Ｆｉｇｕｒｅ　３　Ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｒａｔｅ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ３　讨论初级纤毛为多数分化型细胞表面上的感觉附属物，具有化学感觉和机械感觉功能，在细胞周期控制中具有重要的作用．以往的研究认为初级纤毛在快速增殖的细胞上是找不到的，如癌症细胞，但ＫＯＷＡＬ等［８］通过一种初级纤毛标志物Ａｒｌ１３ｂ在ＨｅＬａ和ＭＧ６３细胞中发现了初级纤毛．未在ＯＳＣＣ中查看是否存在初级纤毛是本研究的不足之处．ＫＩＦ３Ａ属于初级纤毛内运输系统，在维持和发挥初级纤毛功能中起重要作用．ＨＯＡＮＧ－ＭＩＮＨ等［９］的研究结果显示，沉默ＫＩＦ３Ａ基因表达后可降低胶质母细胞瘤中初级纤毛的数量，最终促进肿瘤的发生．玄云泽等［１０］首次在ＯＳＣＣ中发现有初级纤毛，后来有学者亦在口腔白斑与ＯＳＣＣ组织中发现了初级纤毛，ＥＧＦＲ－Ａｕｒｏｒａ　Ａ异常信号被激活后，口腔黏膜癌变过程中原发性纤毛逐渐减少［１１］．本研究结果显示，利用ｓｉＲＮＡ技术沉默ＫＩＦ３Ａ基因后ｍＲＮＡ和蛋白表达水平明显降低，提示初级纤毛的检出率降低．Ｈｅｄｇｅｈｏｇ信号通路对胚胎发育及肿瘤的发生发展具有重要意义．研究［１２－１３］结果显示，Ｈｅｄｇｅｈｏｇ信号通路的异常激活与皮肤、乳腺、肺及消化道等多种恶性肿瘤的发生、增殖密切相关．程志芬等［１２］利用Ｈｅｄｇｅｈｏｇ信号通路特异性抑制剂环巴胺处理Ｔｃａ８１１３细胞株后，Ｓｍｏｏｔｈｅｎｅｄ的ｍＲＮＡ和蛋白表达明显减少，且细胞增殖受到抑制．ＫＵＲＯＤＡ等［１４］的研究结果显示，给予Ｈｅｄｇｅｈｏｇ信号通路抑制剂可抑制肿瘤前沿血管内皮细胞的增殖、迁移，进而抑制肿瘤的增殖和迁移．在小细胞肺癌中，沉默ＫＩＦ３Ａ后初级纤毛明显减少，且Ｓｈｈ信号通路介导的Ｇｌｉ１ｍＲＮＡ表达水平明显降低［１５］，此结果与本研究结果相符．研究［１６］结果显示，Ｈｅｄｇｅｈｏｇ信号通路转录因子Ｇｌｉ１的激活是恶性肿瘤的一个不良预后因素，利用环巴胺阻断Ｈｅｄｇｅｈｏｇ信号通路可抑制Ｇｌｉ１激活，并增强头颈部鳞癌对放疗的敏感性，提示Ｈｅｄｇｅｈｏｇ信号通路影响头颈部鳞癌的放疗预后．总之，本研究揭示了初级纤毛相关基因ＫＩＦ３Ａ在人舌鳞癌Ｔｃａ８１１３细胞Ｈｅｄｇｅｈｏｇ信号通路中的作用，初步解释了沉默ＫＩＦ３Ａ后可能通过抑制Ｈｅｄｇｅｈｏｇ信号通路影响ＯＳＣＣ细胞的增殖能力，为ＯＳＣＣ患者的靶向治疗提供了理论依据．［参　考　文　献］［１］　ＤＩ　ＰＡＲＤＯ　ＢＪ，ＢＲＯＮＳＯＮ　ＮＷ，ＤＩＧＧＳ　ＢＳ，ｅｔ　ａｌ．．Ｔｈｅｇｌｏｂａｌ　ｂｕｒｄｅｎ　ｏｆ　ｅｓｏｐｈａｇｅａｌ　ｃａｎｃｅｒ：ａ　ｄｉｓａｂｉｌｉｔｙ－ａｄｊｕｓｔｅｄｌｉｆｅ－ｙｅａｒ　ａｐｐｒｏａｃｈ［Ｊ］．Ｗｏｒｌｄ　Ｊ　Ｓｕｒｇ，２０１６，４０（２）：３９５－４０１．［２］　ＢＲＯＣＫＬＥＨＵＲＳＴ　ＰＲ，ＢＡＫＥＲ　ＳＲ，ＳＰＥＩＧＨＴ　ＰＭ．Ｏｒａｌｃａｎｃｅｒ　ｓｃｒｅｅｎｉｎｇ：ｗｈａｔ　ｈａｖｅ　ｗｅ　ｌｅａｒｎｔ　ａｎｄ　ｗｈａｔ　ｉｓ　ｔｈｅｒｅｓｔｉｌｌ　ｔｏ　ａｃｈｉｅｖｅ［Ｊ］．Ｆｕｔｕｒｅ　Ｏｎｃｏｌ，２０１０，６（２）：２９９－３０４．［３］　ＢＡＶＬＥ　ＲＭ，ＶＥＮＵＧＯＰＡＬ　Ｒ，ＫＯＮＤＡ　Ｐ，ｅｔ　ａｌ．．Ｍｏ－ｌｅｃｕｌａｒ　ｃｌａｓｓｉｆｉｃａｔｉｏｎ　ｏｆ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ［Ｊ］．ＪＣＤＲ，２０１６，１０（９）：Ｚ１８－Ｚ２１．［４］　陈新，徐文华，周健，等．口腔鳞状细胞癌现状［Ｊ］．口腔医学，２０１７，３７（５）：４６２－４６５．［５］　ＧＯＥＴＺ　ＳＣ，ＡＮＤＥＲＳＯＮ　ＫＶ．Ｔｈｅ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ：ａ　ｓｉｇｎａｌｌｉｎｇ　ｃｅｎｔｒｅ　ｄｕｒｉｎｇ　ｖｅｒｔｅｂｒａｔｅ　ｄｅｖｅｌｏｐｍｅｎｔ［Ｊ］．Ｎａｔ　Ｒｅｖ　Ｇｅｎｅ，２０１０，１１（５）：３３１－３４４．［６］　那玉岩，刘万林．纤毛相关疾病：细胞学机制及转化应用进展［Ｊ］．中国组织工程研究，２０１６，２０（２４）：３６４２－３６４８．［７］　ＮＩＥＷＩＡＤＯＭＳＫＩ　Ｐ，ＮＩＥＤＺＩółＫＡ　ＳＭ，ＭＡＲＫＩＥＷＩＣＺŁ，ｅｔ　ａｌ．．Ｇｌｉ　ｐｒｏｔｅｉｎｓ：ｒｅｇｕｌａｔｉｏｎ　ｉｎ　ｄｅｖｅｌｏｐｍｅｎｔ　ａｎｄｃａｎｃｅｒ［Ｊ］．Ｃｅｌｌｓ，２０１９，８（２）：１４７．［８］　ＫＯＷＡＬ　ＴＪ，ＦＡＬＫ　ＭＭ．Ｐｒｉｍａｒｙ　ｃｉｌｉａ　ｆｏｕｎｄ　ｏｎ　ＨｅＬａａｎｄ　ｏｔｈｅｒ　ｃａｎｃｅｒ　ｃｅｌｌｓ［Ｊ］．Ｃｅｌｌ　Ｂｉｏｌ　Ｉｎｔ，２０１５，３９（１１）：１３４１－１３４７．［９］　ＨＯＡＮＧ－ＭＩＮＨ　ＬＢ，ＤＥＬＥＹＲＯＬＬＥ　ＬＰ，ＳＩＥＢＺＥＨ－ＮＲＵＢＬ　Ｄ，ｅｔ　ａｌ．．Ｄｉｓｒｕｐｔｉｏｎ　ｏｆ　ＫＩＦ３Ａｉｎ　ｐａｔｉｅｎｔ－ｄｅ－ｒｉｖｅｄ　ｇｌｉｏｂｌａｓｔｏｍａ　ｃｅｌｌｓ：ｅｆｆｅｃｔｓ　ｏｎ　ｃｉｌｉｏｇｅｎｅｓｉｓ，ｈｅｄｇｅ－ｈｏｇ　ｓｅｎｓｉｔｉｖｉｔｙ，ａｎｄ　ｔｕｍｏｒｉｇｅｎｅｓｉｓ［Ｊ］．Ｏｎｃｏｔａｒｇｅｔ，２０１６，７（６）：７０２９－７０４３．［１０］玄云泽，李书进，金成日，等．初级纤毛相关基因ＫＩＦ３Ａ通过调控ＥＭＴ过程在口腔鳞状细胞癌中的作用机制研究［Ｊ］．重庆医学，２０２０，４９（１）：７－１２．［１１］ＹＩＮ　Ｆ，ＣＨＥＮ　Ｑ，ＳＨＩ　Ｙ，ｅｔ　ａｌ．．Ａｃｔｉｖａｔｉｏｎ　ｏｆ　ＥＧＦＲ－Ａｕ－ｒｏｒａ　Ａ　ｉｎｄｕｃｅｓ　ｌｏｓｓ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉａ　ｉｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ［Ｊ］．Ｏｒａｌ　Ｄｉｓ，２０２２，２８（３）：６２１－６３０．［１２］程志芬，崔演，玄延花．Ｐｔｃｈ和Ｓｍｏ在舌鳞状细胞癌Ｔｃａ８１１３细胞中的表达及其意义［Ｊ］．口腔医学研究，２０１５，３１（５）：４３３－４３６．［１３］ＫＯＮＳＴＡＮＴＩＮＯＵ　Ｄ，ＢＥＲＴＡＵＸ－ＳＫＥＩＲＩＫ　Ｎ，ＺＡＶＲＯＳＹ．Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｉｎ　ｔｈｅ　ｓｔｏｍａｃｈ［Ｊ］．Ｃｕｒｒ　ＯｐｉｎＰｈａｒｍａｃｏｌ，２０１６，３１：７６－８２．［１４］ＫＵＲＯＤＡ　Ｈ，ＫＵＲＩＯ　Ｎ，ＳＨＩＭＯ　Ｔ，ｅｔ　ａｌ．．Ｏｒａｌ　ｓｑｕａｍｏｕｓｃｅｌｌ　ｃａｒｃｉｎｏｍａ－ｄｅｒｉｖｅｄ　ｓｏｎｉｃ　ｈｅｄｇｅｈｏｇ　ｐｒｏｍｏｔｅｓ　ａｎｇｉｏｇｅｎｅ－ｓｉｓ［Ｊ］．Ａｎｔｉｃａｎｃｅｒ　Ｒｅｓ，２０１７，３７（１２）：６７３１－６７３７．［１５］ＣＯＣＨＲＡＮＥ　ＣＲ，ＶＡＧＨＪＩＡＮＩ　Ｖ，ＳＺＣＺＥＰＮＹ　Ａ，ｅｔ　ａｌ．．Ｔｒｐ５３ａｎｄ　Ｒｂ１ｒｅｇｕｌａｔｅ　ａｕｔｏｐｈａｇｙ　ａｎｄ　ｌｉｇａｎｄ－ｄｅｐｅｎｄｅｎｔＨｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ［Ｊ］．Ｊ　Ｃｌｉｎ　Ｉｎｖｅｓｔ，２０２０，１３０（８）：４００６－４０１８．［１６］ＧＡＮ　ＧＮ，ＥＡＧＬＥＳ　Ｊ，ＫＥＹＳＡＲ　ＳＢ，ｅｔ　ａｌ．．Ｈｅｄｇｅｈｏｇｓｉｇｎａｌｉｎｇ　ｄｒｉｖｅｓ　ｒａｄｉｏｒｅｓｉｓｔａｎｃｅ　ａｎｄ　ｓｔｒｏｍａ－ｄｒｉｖｅｎｔｕｍｏｒ　ｒｅｐｏｐｕｌａｔｉｏｎ　ｉｎ　ｈｅａｄ　ａｎｄ　ｎｅｃｋ　ｓｑｕａｍｏｕｓ　ｃａｎｃｅｒｓ［Ｊ］．Ｃａｎｃｅｒ　Ｒｅｓ，２０１４，７４（２３）：７０２４－７０３６．■·０１·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■。

第47卷第6期2021年11月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.47No.6Nov.2021DOI：10.13481/j.1671‑587X.20210606棕榈酸对骨骼肌细胞脂质沉积的诱导作用何勇,洪莉,黄国涛,赵芷涵（武汉大学人民医院妇产科，湖北武汉430060）［摘要］目的目的：探讨棕榈酸（PA）对骨骼肌细胞脂质沉积的作用及其最佳作用浓度和时间。

方法：在C2C12细胞生长至70%~80%时分为对照组和不同浓度（50、75、100和125μmol·L－1）PA组。

不同浓度PA组细胞采用含不同浓度PA的生长培养基培养，对照组细胞不给予PA（0μmol·L－1PA）。

CCK-8法检测各组细胞增殖活性，定量检测细胞中甘油三酯（TG）水平，油红O染色观察细胞中脂滴形成情况，2%马血清诱导细胞分化并评价各组C2C12细胞分化状态。

结果结果：与对照组比较，50、75和100μmol·L－1PA组C2C12细胞增殖活性差异无统计学意义（P>0.05），125μmol·L－1PA组C2C12细胞增殖活性明显降低（P<0.01），50、75和100μmol·L－1PA组细胞中TG水平升高（P<0.01），100μmol·L－1PA组C2C12细胞中TG水平升高最明显（P<0.01）；与培养24h比较，100μmol·L－1 PA培养48h后，C2C12细胞中TG水平升高（P<0.01）。

分化培养后，PA组和对照组C2C12细胞中均有较多肌管形成。

结论结论：100μmol·L－1PA作用48h可有效促进C2C12细胞脂质沉积。

［关键词］盆底功能障碍；肥胖；高脂；棕榈酸；脂质沉积［中图分类号］R711.4［文献标志码］AEffect of palmitic acid on induction of lipid deposition inskeletal muscle cellsHE Yong,HONG Li,HUANG Guotao,ZHAO Zhihan（Department of Obstetrics and Gynecology，People’s Hospital，Wuhan University，Wuhan430060，China）ABSTRACT Objective：To explore the effect of palmitic acid（PA）on the lipid deposition in the skeletal muscle cells，and to discuss the optimum action concentration and time.Methods：The C2C12cells were divided into control group and different concentrations（50，75，100，and125μmol·L－1）of PA groups when the C2C12cells grew to70%－80%.The C2C12cells in different concentrations of PA groups were given growth media containing different concentrations of PA，while the C2C12cells in control group were not treated with PA（0μmol·L－1PA）.CCK-8method was used to detect the cell proliferation activities of cells in various groups，the triglyceride（TG）levels in the C2C12cells in various groups were detected，Oil Red O staining was used to observe the formation of lipid droplets in the C2C12cells in various groups，and2% horse serum was used to induce the cell differentiation and evaluate the differentiation of C2C12cells in various groups.Results：Compared with control group，there were no significant differences in the proliferation activities of the C2C12cells in50，75，and100μmol·L－1PA groups（P>0.05），the [文章编号]1671‑587X(2021)06‑1380‑06[收稿日期]2021‑02‑03[基金项目]国家自然科学基金面上项目（81971364，81771562）[作者简介]何勇（1994－），男，湖北省恩施州人，在读硕士研究生，主要从事女性盆底功能障碍性疾病和妇科肿瘤方面的研究。

DOI: 10.1126/science.1094786, 441 (2004);304Science et al.Mitchell S. Abrahamsen,Cryptosporidium parvum Complete Genome Sequence of the Apicomplexan, (this information is current as of October 7, 2009 ):The following resources related to this article are available online at/cgi/content/full/304/5669/441version of this article at:including high-resolution figures, can be found in the online Updated information and services,/cgi/content/full/1094786/DC1 can be found at:Supporting Online Material/cgi/content/full/304/5669/441#otherarticles , 9 of which can be accessed for free: cites 25 articles This article 239 article(s) on the ISI Web of Science. cited by This article has been /cgi/content/full/304/5669/441#otherarticles 53 articles hosted by HighWire Press; see: cited by This article has been/cgi/collection/genetics Genetics: subject collections This article appears in the following/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS.is a Science 2004 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. 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of1mm3)T1-weightedmagnetization-prepared rapid gradient-echo pulse se-quence.The fMRI data were preprocessed and analyzedby statistical parametric mappingwith SPM99software(http://www.fi/spm99).25.S.E.Petersen et al.,Nature331,585(1988).26.B.T.Gold,R.L.Buckner,Neuron35,803(2002).27.E.Halgren et al.,J.Psychophysiol.88,1(1994).28.E.Halgren et al.,Neuroimage17,1101(2002).29.M.K.Tanenhaus et al.,Science268,1632(1995).30.J.J.A.van Berkum et al.,J.Cognit.Neurosci.11,657(1999).31.P.A.M.Seuren,Discourse Semantics(Basil Blackwell,Oxford,1985).32.We thank P.Indefrey,P.Fries,P.A.M.Seuren,and M.van Turennout for helpful discussions.Supported bythe Netherlands Organization for Scientific Research,grant no.400-56-384(P.H.).Supporting Online Material/cgi/content/full/1095455/DC1Materials and MethodsFig.S1References and Notes8January2004;accepted9March2004Published online18March2004;10.1126/science.1095455Include this information when citingthis paper.Complete Genome Sequence ofthe Apicomplexan,Cryptosporidium parvumMitchell S.Abrahamsen,1,2*†Thomas J.Templeton,3†Shinichiro Enomoto,1Juan E.Abrahante,1Guan Zhu,4 Cheryl ncto,1Mingqi Deng,1Chang Liu,1‡Giovanni Widmer,5Saul Tzipori,5GregoryA.Buck,6Ping Xu,6 Alan T.Bankier,7Paul H.Dear,7Bernard A.Konfortov,7 Helen F.Spriggs,7Lakshminarayan Iyer,8Vivek Anantharaman,8L.Aravind,8Vivek Kapur2,9The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals,and causes an unrelenting infection in immuno-compromised individuals such as AIDS patients.We report the complete ge-nome sequence of C.parvum,type II isolate.Genome analysis identifies ex-tremely streamlined metabolic pathways and a reliance on the host for nu-trients.In contrast to Plasmodium and Toxoplasma,the parasite lacks an api-coplast and its genome,and possesses a degenerate mitochondrion that has lost its genome.Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected.Elu-cidation of the core metabolism,including enzymes with high similarities to bacterial and plant counterparts,opens new avenues for drug development.Cryptosporidium parvum is a globally impor-tant intracellular pathogen of humans and animals.The duration of infection and patho-genesis of cryptosporidiosis depends on host immune status,ranging from a severe but self-limiting diarrhea in immunocompetent individuals to a life-threatening,prolonged infection in immunocompromised patients.Asubstantial degree of morbidity and mortalityis associated with infections in AIDS pa-tients.Despite intensive efforts over the past20years,there is currently no effective ther-apy for treating or preventing C.parvuminfection in humans.Cryptosporidium belongs to the phylumApicomplexa,whose members share a com-mon apical secretory apparatus mediating lo-comotion and tissue or cellular invasion.Many apicomplexans are of medical or vet-erinary importance,including Plasmodium,Babesia,Toxoplasma,Neosprora,Sarcocys-tis,Cyclospora,and Eimeria.The life cycle ofC.parvum is similar to that of other cyst-forming apicomplexans(e.g.,Eimeria and Tox-oplasma),resulting in the formation of oocysts1Department of Veterinary and Biomedical Science,College of Veterinary Medicine,2Biomedical Genom-ics Center,University of Minnesota,St.Paul,MN55108,USA.3Department of Microbiology and Immu-nology,Weill Medical College and Program in Immu-nology,Weill Graduate School of Medical Sciences ofCornell University,New York,NY10021,USA.4De-partment of Veterinary Pathobiology,College of Vet-erinary Medicine,Texas A&M University,College Sta-tion,TX77843,USA.5Division of Infectious Diseases,Tufts University School of Veterinary Medicine,NorthGrafton,MA01536,USA.6Center for the Study ofBiological Complexity and Department of Microbiol-ogy and Immunology,Virginia Commonwealth Uni-versity,Richmond,VA23198,USA.7MRC Laboratoryof Molecular Biology,Hills Road,Cambridge CB22QH,UK.8National Center for Biotechnology Infor-mation,National Library of Medicine,National Insti-tutes of Health,Bethesda,MD20894,USA.9Depart-ment of Microbiology,University of Minnesota,Min-neapolis,MN55455,USA.*To whom correspondence should be addressed.E-mail:abe@†These authors contributed equally to this work.‡Present address:Bioinformatics Division,Genetic Re-search,GlaxoSmithKline Pharmaceuticals,5MooreDrive,Research Triangle Park,NC27009,USA.R E P O R T S SCIENCE VOL30416APRIL2004441o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mthat are shed in the feces of infected hosts.C.parvum oocysts are highly resistant to environ-mental stresses,including chlorine treatment of community water supplies;hence,the parasite is an important water-and food-borne pathogen (1).The obligate intracellular nature of the par-asite ’s life cycle and the inability to culture the parasite continuously in vitro greatly impair researchers ’ability to obtain purified samples of the different developmental stages.The par-asite cannot be genetically manipulated,and transformation methodologies are currently un-available.To begin to address these limitations,we have obtained the complete C.parvum ge-nome sequence and its predicted protein com-plement.(This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accession AAEE00000000.The version described in this paper is the first version,AAEE01000000.)The random shotgun approach was used to obtain the complete DNA sequence (2)of the Iowa “type II ”isolate of C.parvum .This isolate readily transmits disease among numerous mammals,including humans.The resulting ge-nome sequence has roughly 13ϫgenome cov-erage containing five gaps and 9.1Mb of totalDNA sequence within eight chromosomes.The C.parvum genome is thus quite compact rela-tive to the 23-Mb,14-chromosome genome of Plasmodium falciparum (3);this size difference is predominantly the result of shorter intergenic regions,fewer introns,and a smaller number of genes (Table 1).Comparison of the assembled sequence of chromosome VI to that of the recently published sequence of chromosome VI (4)revealed that our assembly contains an ad-ditional 160kb of sequence and a single gap versus two,with the common sequences dis-playing a 99.993%sequence identity (2).The relative paucity of introns greatly simplified gene predictions and facilitated an-notation (2)of predicted open reading frames (ORFs).These analyses provided an estimate of 3807protein-encoding genes for the C.parvum genome,far fewer than the estimated 5300genes predicted for the Plasmodium genome (3).This difference is primarily due to the absence of an apicoplast and mitochondrial genome,as well as the pres-ence of fewer genes encoding metabolic functions and variant surface proteins,such as the P.falciparum var and rifin molecules (Table 2).An analysis of the encoded pro-tein sequences with the program SEG (5)shows that these protein-encoding genes are not enriched in low-complexity se-quences (34%)to the extent observed in the proteins from Plasmodium (70%).Our sequence analysis indicates that Cryptosporidium ,unlike Plasmodium and Toxoplasma ,lacks both mitochondrion and apicoplast genomes.The overall complete-ness of the genome sequence,together with the fact that similar DNA extraction proce-dures used to isolate total genomic DNA from C.parvum efficiently yielded mito-chondrion and apicoplast genomes from Ei-meria sp.and Toxoplasma (6,7),indicates that the absence of organellar genomes was unlikely to have been the result of method-ological error.These conclusions are con-sistent with the absence of nuclear genes for the DNA replication and translation machinery characteristic of mitochondria and apicoplasts,and with the lack of mito-chondrial or apicoplast targeting signals for tRNA synthetases.A number of putative mitochondrial pro-teins were identified,including components of a mitochondrial protein import apparatus,chaperones,uncoupling proteins,and solute translocators (table S1).However,the ge-nome does not encode any Krebs cycle en-zymes,nor the components constituting the mitochondrial complexes I to IV;this finding indicates that the parasite does not rely on complete oxidation and respiratory chains for synthesizing adenosine triphosphate (ATP).Similar to Plasmodium ,no orthologs for the ␥,␦,or εsubunits or the c subunit of the F 0proton channel were detected (whereas all subunits were found for a V-type ATPase).Cryptosporidium ,like Eimeria (8)and Plas-modium ,possesses a pyridine nucleotide tran-shydrogenase integral membrane protein that may couple reduced nicotinamide adenine dinucleotide (NADH)and reduced nico-tinamide adenine dinucleotide phosphate (NADPH)redox to proton translocation across the inner mitochondrial membrane.Unlike Plasmodium ,the parasite has two copies of the pyridine nucleotide transhydrogenase gene.Also present is a likely mitochondrial membrane –associated,cyanide-resistant alter-native oxidase (AOX )that catalyzes the reduction of molecular oxygen by ubiquinol to produce H 2O,but not superoxide or H 2O 2.Several genes were identified as involved in biogenesis of iron-sulfur [Fe-S]complexes with potential mitochondrial targeting signals (e.g.,nifS,nifU,frataxin,and ferredoxin),supporting the presence of a limited electron flux in the mitochondrial remnant (table S2).Our sequence analysis confirms the absence of a plastid genome (7)and,additionally,the loss of plastid-associated metabolic pathways including the type II fatty acid synthases (FASs)and isoprenoid synthetic enzymes thatTable 1.General features of the C.parvum genome and comparison with other single-celled eukaryotes.Values are derived from respective genome project summaries (3,26–28).ND,not determined.FeatureC.parvum P.falciparum S.pombe S.cerevisiae E.cuniculiSize (Mbp)9.122.912.512.5 2.5(G ϩC)content (%)3019.43638.347No.of genes 38075268492957701997Mean gene length (bp)excluding introns 1795228314261424ND Gene density (bp per gene)23824338252820881256Percent coding75.352.657.570.590Genes with introns (%)553.9435ND Intergenic regions (G ϩC)content %23.913.632.435.145Mean length (bp)5661694952515129RNAsNo.of tRNA genes 454317429944No.of 5S rRNA genes 6330100–2003No.of 5.8S ,18S ,and 28S rRNA units 57200–400100–20022Table parison between predicted C.parvum and P.falciparum proteins.FeatureC.parvum P.falciparum *Common †Total predicted proteins380752681883Mitochondrial targeted/encoded 17(0.45%)246(4.7%)15Apicoplast targeted/encoded 0581(11.0%)0var/rif/stevor ‡0236(4.5%)0Annotated as protease §50(1.3%)31(0.59%)27Annotated as transporter ࿣69(1.8%)34(0.65%)34Assigned EC function ¶167(4.4%)389(7.4%)113Hypothetical proteins925(24.3%)3208(60.9%)126*Values indicated for P.falciparum are as reported (3)with the exception of those for proteins annotated as protease or transporter.†TBLASTN hits (e Ͻ–5)between C.parvum and P.falciparum .‡As reported in (3).§Pre-dicted proteins annotated as “protease or peptidase”for C.parvum (CryptoGenome database,)and P.falciparum (PlasmoDB database,).࿣Predicted proteins annotated as “trans-porter,permease of P-type ATPase”for C.parvum (CryptoGenome)and P.falciparum (PlasmoDB).¶Bidirectional BLAST hit (e Ͻ–15)to orthologs with assigned Enzyme Commission (EC)numbers.Does not include EC assignment numbers for protein kinases or protein phosphatases (due to inconsistent annotation across genomes),or DNA polymerases or RNA polymerases,as a result of issues related to subunit inclusion.(For consistency,46proteins were excluded from the reported P.falciparum values.)R E P O R T S16APRIL 2004VOL 304SCIENCE 442 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mare otherwise localized to the plastid in other apicomplexans.C.parvum fatty acid biosynthe-sis appears to be cytoplasmic,conducted by a large(8252amino acids)modular type I FAS (9)and possibly by another large enzyme that is related to the multidomain bacterial polyketide synthase(10).Comprehensive screening of the C.parvum genome sequence also did not detect orthologs of Plasmodium nuclear-encoded genes that contain apicoplast-targeting and transit sequences(11).C.parvum metabolism is greatly stream-lined relative to that of Plasmodium,and in certain ways it is reminiscent of that of another obligate eukaryotic parasite,the microsporidian Encephalitozoon.The degeneration of the mi-tochondrion and associated metabolic capabili-ties suggests that the parasite largely relies on glycolysis for energy production.The parasite is capable of uptake and catabolism of mono-sugars(e.g.,glucose and fructose)as well as synthesis,storage,and catabolism of polysac-charides such as trehalose and amylopectin. Like many anaerobic organisms,it economizes ATP through the use of pyrophosphate-dependent phosphofructokinases.The conver-sion of pyruvate to acetyl–coenzyme A(CoA) is catalyzed by an atypical pyruvate-NADPH oxidoreductase(Cp PNO)that contains an N-terminal pyruvate–ferredoxin oxidoreductase (PFO)domain fused with a C-terminal NADPH–cytochrome P450reductase domain (CPR).Such a PFO-CPR fusion has previously been observed only in the euglenozoan protist Euglena gracilis(12).Acetyl-CoA can be con-verted to malonyl-CoA,an important precursor for fatty acid and polyketide biosynthesis.Gly-colysis leads to several possible organic end products,including lactate,acetate,and ethanol. The production of acetate from acetyl-CoA may be economically beneficial to the parasite via coupling with ATP production.Ethanol is potentially produced via two in-dependent pathways:(i)from the combination of pyruvate decarboxylase and alcohol dehy-drogenase,or(ii)from acetyl-CoA by means of a bifunctional dehydrogenase(adhE)with ac-etaldehyde and alcohol dehydrogenase activi-ties;adhE first converts acetyl-CoA to acetal-dehyde and then reduces the latter to ethanol. AdhE predominantly occurs in bacteria but has recently been identified in several protozoans, including vertebrate gut parasites such as Enta-moeba and Giardia(13,14).Adjacent to the adhE gene resides a second gene encoding only the AdhE C-terminal Fe-dependent alcohol de-hydrogenase domain.This gene product may form a multisubunit complex with AdhE,or it may function as an alternative alcohol dehydro-genase that is specific to certain growth condi-tions.C.parvum has a glycerol3-phosphate dehydrogenase similar to those of plants,fungi, and the kinetoplastid Trypanosoma,but(unlike trypanosomes)the parasite lacks an ortholog of glycerol kinase and thus this pathway does not yield glycerol production.In addition to themodular fatty acid synthase(Cp FAS1)andpolyketide synthase homolog(Cp PKS1), C.parvum possesses several fatty acyl–CoA syn-thases and a fatty acyl elongase that may partici-pate in fatty acid metabolism.Further,enzymesfor the metabolism of complex lipids(e.g.,glyc-erolipid and inositol phosphate)were identified inthe genome.Fatty acids are apparently not anenergy source,because enzymes of the fatty acidoxidative pathway are absent,with the exceptionof a3-hydroxyacyl-CoA dehydrogenase.C.parvum purine metabolism is greatlysimplified,retaining only an adenosine ki-nase and enzymes catalyzing conversionsof adenosine5Ј-monophosphate(AMP)toinosine,xanthosine,and guanosine5Ј-monophosphates(IMP,XMP,and GMP).Among these enzymes,IMP dehydrogenase(IMPDH)is phylogenetically related toε-proteobacterial IMPDH and is strikinglydifferent from its counterparts in both thehost and other apicomplexans(15).In con-trast to other apicomplexans such as Toxo-plasma gondii and P.falciparum,no geneencoding hypoxanthine-xanthineguaninephosphoribosyltransferase(HXGPRT)is de-tected,in contrast to a previous report on theactivity of this enzyme in C.parvum sporo-zoites(16).The absence of HXGPRT sug-gests that the parasite may rely solely on asingle enzyme system including IMPDH toproduce GMP from AMP.In contrast to otherapicomplexans,the parasite appears to relyon adenosine for purine salvage,a modelsupported by the identification of an adeno-sine transporter.Unlike other apicomplexansand many parasitic protists that can synthe-size pyrimidines de novo,C.parvum relies onpyrimidine salvage and retains the ability forinterconversions among uridine and cytidine5Ј-monophosphates(UMP and CMP),theirdeoxy forms(dUMP and dCMP),and dAMP,as well as their corresponding di-and triphos-phonucleotides.The parasite has also largelyshed the ability to synthesize amino acids denovo,although it retains the ability to convertselect amino acids,and instead appears torely on amino acid uptake from the host bymeans of a set of at least11amino acidtransporters(table S2).Most of the Cryptosporidium core pro-cesses involved in DNA replication,repair,transcription,and translation conform to thebasic eukaryotic blueprint(2).The transcrip-tional apparatus resembles Plasmodium interms of basal transcription machinery.How-ever,a striking numerical difference is seenin the complements of two RNA bindingdomains,Sm and RRM,between P.falcipa-rum(17and71domains,respectively)and C.parvum(9and51domains).This reductionresults in part from the loss of conservedproteins belonging to the spliceosomal ma-chinery,including all genes encoding Smdomain proteins belonging to the U6spliceo-somal particle,which suggests that this par-ticle activity is degenerate or entirely lost.This reduction in spliceosomal machinery isconsistent with the reduced number of pre-dicted introns in Cryptosporidium(5%)rela-tive to Plasmodium(Ͼ50%).In addition,keycomponents of the small RNA–mediatedposttranscriptional gene silencing system aremissing,such as the RNA-dependent RNApolymerase,Argonaute,and Dicer orthologs;hence,RNA interference–related technolo-gies are unlikely to be of much value intargeted disruption of genes in C.parvum.Cryptosporidium invasion of columnarbrush border epithelial cells has been de-scribed as“intracellular,but extracytoplas-mic,”as the parasite resides on the surface ofthe intestinal epithelium but lies underneaththe host cell membrane.This niche may al-low the parasite to evade immune surveil-lance but take advantage of solute transportacross the host microvillus membrane or theextensively convoluted parasitophorous vac-uole.Indeed,Cryptosporidium has numerousgenes(table S2)encoding families of putativesugar transporters(up to9genes)and aminoacid transporters(11genes).This is in starkcontrast to Plasmodium,which has fewersugar transporters and only one putative ami-no acid transporter(GenBank identificationnumber23612372).As a first step toward identification ofmulti–drug-resistant pumps,the genome se-quence was analyzed for all occurrences ofgenes encoding multitransmembrane proteins.Notable are a set of four paralogous proteinsthat belong to the sbmA family(table S2)thatare involved in the transport of peptide antibi-otics in bacteria.A putative ortholog of thePlasmodium chloroquine resistance–linkedgene Pf CRT(17)was also identified,althoughthe parasite does not possess a food vacuole likethe one seen in Plasmodium.Unlike Plasmodium,C.parvum does notpossess extensive subtelomeric clusters of anti-genically variant proteins(exemplified by thelarge families of var and rif/stevor genes)thatare involved in immune evasion.In contrast,more than20genes were identified that encodemucin-like proteins(18,19)having hallmarksof extensive Thr or Ser stretches suggestive ofglycosylation and signal peptide sequences sug-gesting secretion(table S2).One notable exam-ple is an11,700–amino acid protein with anuninterrupted stretch of308Thr residues(cgd3_720).Although large families of secretedproteins analogous to the Plasmodium multi-gene families were not found,several smallermultigene clusters were observed that encodepredicted secreted proteins,with no detectablesimilarity to proteins from other organisms(Fig.1,A and B).Within this group,at leastfour distinct families appear to have emergedthrough gene expansions specific to the Cryp-R E P O R T S SCIENCE VOL30416APRIL2004443o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mtosporidium clade.These families —SKSR,MEDLE,WYLE,FGLN,and GGC —were named after well-conserved sequence motifs (table S2).Reverse transcription polymerase chain reaction (RT-PCR)expression analysis (20)of one cluster,a locus of seven adjacent CpLSP genes (Fig.1B),shows coexpression during the course of in vitro development (Fig.1C).An additional eight genes were identified that encode proteins having a periodic cysteine structure similar to the Cryptosporidium oocyst wall protein;these eight genes are similarly expressed during the onset of oocyst formation and likely participate in the formation of the coccidian rigid oocyst wall in both Cryptospo-ridium and Toxoplasma (21).Whereas the extracellular proteins described above are of apparent apicomplexan or lineage-specific in-vention,Cryptosporidium possesses many genesencodingsecretedproteinshavinglineage-specific multidomain architectures composed of animal-and bacterial-like extracellular adhe-sive domains (fig.S1).Lineage-specific expansions were ob-served for several proteases (table S2),in-cluding an aspartyl protease (six genes),a subtilisin-like protease,a cryptopain-like cys-teine protease (five genes),and a Plas-modium falcilysin-like (insulin degrading enzyme –like)protease (19genes).Nine of the Cryptosporidium falcilysin genes lack the Zn-chelating “HXXEH ”active site motif and are likely to be catalytically inactive copies that may have been reused for specific protein-protein interactions on the cell sur-face.In contrast to the Plasmodium falcilysin,the Cryptosporidium genes possess signal peptide sequences and are likely trafficked to a secretory pathway.The expansion of this family suggests either that the proteins have distinct cleavage specificities or that their diversity may be related to evasion of a host immune response.Completion of the C.parvum genome se-quence has highlighted the lack of conven-tional drug targets currently pursued for the control and treatment of other parasitic protists.On the basis of molecular and bio-chemical studies and drug screening of other apicomplexans,several putative Cryptospo-ridium metabolic pathways or enzymes have been erroneously proposed to be potential drug targets (22),including the apicoplast and its associated metabolic pathways,the shikimate pathway,the mannitol cycle,the electron transport chain,and HXGPRT.Nonetheless,complete genome sequence analysis identifies a number of classic and novel molecular candidates for drug explora-tion,including numerous plant-like and bacterial-like enzymes (tables S3and S4).Although the C.parvum genome lacks HXGPRT,a potent drug target in other api-complexans,it has only the single pathway dependent on IMPDH to convert AMP to GMP.The bacterial-type IMPDH may be a promising target because it differs substan-tially from that of eukaryotic enzymes (15).Because of the lack of de novo biosynthetic capacity for purines,pyrimidines,and amino acids,C.parvum relies solely on scavenge from the host via a series of transporters,which may be exploited for chemotherapy.C.parvum possesses a bacterial-type thymidine kinase,and the role of this enzyme in pyrim-idine metabolism and its drug target candida-cy should be pursued.The presence of an alternative oxidase,likely targeted to the remnant mitochondrion,gives promise to the study of salicylhydroxamic acid (SHAM),as-cofuranone,and their analogs as inhibitors of energy metabolism in the parasite (23).Cryptosporidium possesses at least 15“plant-like ”enzymes that are either absent in or highly divergent from those typically found in mammals (table S3).Within the glycolytic pathway,the plant-like PPi-PFK has been shown to be a potential target in other parasites including T.gondii ,and PEPCL and PGI ap-pear to be plant-type enzymes in C.parvum .Another example is a trehalose-6-phosphate synthase/phosphatase catalyzing trehalose bio-synthesis from glucose-6-phosphate and uridine diphosphate –glucose.Trehalose may serve as a sugar storage source or may function as an antidesiccant,antioxidant,or protein stability agent in oocysts,playing a role similar to that of mannitol in Eimeria oocysts (24).Orthologs of putative Eimeria mannitol synthesis enzymes were not found.However,two oxidoreductases (table S2)were identified in C.parvum ,one of which belongs to the same families as the plant mannose dehydrogenases (25)and the other to the plant cinnamyl alcohol dehydrogenases.In principle,these enzymes could synthesize protective polyol compounds,and the former enzyme could use host-derived mannose to syn-thesize mannitol.References and Notes1.D.G.Korich et al .,Appl.Environ.Microbiol.56,1423(1990).2.See supportingdata on Science Online.3.M.J.Gardner et al .,Nature 419,498(2002).4.A.T.Bankier et al .,Genome Res.13,1787(2003).5.J.C.Wootton,Comput.Chem.18,269(1994).Fig.1.(A )Schematic showing the chromosomal locations of clusters of potentially secreted proteins.Numbers of adjacent genes are indicated in paren-theses.Arrows indicate direc-tion of clusters containinguni-directional genes (encoded on the same strand);squares indi-cate clusters containingg enes encoded on both strands.Non-paralogous genes are indicated by solid gray squares or direc-tional triangles;SKSR (green triangles),FGLN (red trian-gles),and MEDLE (blue trian-gles)indicate three C.parvum –specific families of paralogous genes predominantly located at telomeres.Insl (yellow tri-angles)indicates an insulinase/falcilysin-like paralogous gene family.Cp LSP (white square)indicates the location of a clus-ter of adjacent large secreted proteins (table S2)that are cotranscriptionally regulated.Identified anchored telomeric repeat sequences are indicated by circles.(B )Schematic show-inga select locus containinga cluster of coexpressed large secreted proteins (Cp LSP).Genes and intergenic regions (regions between identified genes)are drawn to scale at the nucleotide level.The length of the intergenic re-gions is indicated above or be-low the locus.(C )Relative ex-pression levels of CpLSP (red lines)and,as a control,C.parvum Hedgehog-type HINT domain gene (blue line)duringin vitro development,as determined by semiquantitative RT-PCR usingg ene-specific primers correspondingto the seven adjacent g enes within the CpLSP locus as shown in (B).Expression levels from three independent time-course experiments are represented as the ratio of the expression of each gene to that of C.parvum 18S rRNA present in each of the infected samples (20).R E P O R T S16APRIL 2004VOL 304SCIENCE 444 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

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HOLZFORSCHUNG HOLZFORSCHUNG0018-3830F ORESTRY HUMAN-COMPUTER INTERACTION HUM-COMPUT INTERACT0737-0024C OMPUTER SC HUMAN-COMPUTER INTERACTION HUM-COMPUT INTERACT0737-0024C OMPUTER SC HYDROMETALLURGY HYDROMETALLURGY0304-386X M ETALLURGY IEEE COMMUNICATIONS MAGAZINE IEEE COMMUN MAG0163-6804T ELECOMMUNIEEE COMMUNICATIONS MAGAZINE IEEE COMMUN MAG0163-6804E NGINEERING IEEE Computational Intelligence Magazine IEEE COMPUT INTELL M1556-603X C OMPUTER SC IEEE CONTROL SYSTEMS MAGAZINE IEEE CONTR SYST MAG1066-033X A UTOMATION IEEE ELECTRON DEVICE LETTERS IEEE ELECTR DEVICE L0741-3106E NGINEERING IEEE Journal of Photovoltaics IEEE J PHOTOVOLT2156-3381M ATERIALS S IEEE Journal of Photovoltaics IEEE J PHOTOVOLT2156-3381E NERGY & FU IEEE Journal of Photovoltaics IEEE J PHOTOVOLT2156-3381P HYSICS, APPIEEE J SEL AREA COMM0733-8716T ELECOMMUN IEEE JOURNAL ON SELECTED AREAS IN COIEEE J SEL AREA COMM0733-8716E NGINEERING IEEE JOURNAL ON SELECTED AREAS IN COIEEE JOURNAL OF SELECTED TOPICS IN QUIEEE J SEL 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刊名全称BRAINJOURNAL OF HEAD TRAUMA REHABILITATIONLANCET NEUROLOGYNEUROLOGYNEUROREHABILITATION AND NEURAL REPAIRPain PhysicianSTROKEAMERICAN JOURNAL OF NEURORADIOLOGYBRAIN PATHOLOGY 病理Brain StimulationCurrent Alzheimer Research老年痴呆CURRENT OPINION IN NEUROLOGYEUROPEAN JOURNAL OF PAINJOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY JOURNAL OF NEUROTRAUMAJOURNAL OF PAINNEURO-ONCOLOGY 神经肿瘤 **NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY NEUROSCIENTIST **PAINBMC Neurology 英国神经病学CEREBROVASCULAR DISEASES 脑血管CLINICAL JOURNAL OF PAINCurrent Neurology and Neuroscience Reports CURRENT NEUROVASCULAR RESEARCH 神经与血管EUROPEAN JOURNAL OF NEUROLOGY 欧洲神经病学HEADACHEJOURNAL OF NEUROLOGY 神经病学JOURNAL OF NEURO-ONCOLOGY 神经肿瘤JOURNAL OF NEUROSURGERY Neurodegenerative Diseases神经退行性病变NEUROGENETICS 神经遗传学NEUROLOGIC CLINICSNEURORADIOLOGY 神经放射学Neurosurgical FocusNEUROSURGERYSPINAL CORD 脊髓SPINE 脊柱Spine Journal 脊骨ACTA NEUROCHIRURGICA 欧亚神经外科学院Brain Tumor PathologyBRITISH JOURNAL OF NEUROSURGERY 英国神经外科CANADIAN JOURNAL OF NEUROLOGICAL SCIENCESCentral European NeurosurgeryCLINICAL NEUROLOGY AND NEUROSURGERYCLINICAL NEUROPATHOLOGYCurrent Pain and Headache ReportsCurrent Treatment Options in NeurologyDevelopmental NeurorehabilitationEUROPEAN JOURNAL OF PAEDIATRIC NEUROLOGY 小儿EUROPEAN NEUROLOGYEUROPEAN SPINE JOURNALJOURNAL OF CHILD NEUROLOGYJOURNAL OF CLINICAL AND EXPERIMENTAL NEUROPSYCHOLOGY Journal of Clinical NeurologyJOURNAL OF CLINICAL NEUROSCIENCEJOURNAL OF GERIATRIC PSYCHIATRY AND NEUROLOGY JOURNAL OF HEADACHE AND PAINJOURNAL OF NERVOUS AND MENTAL DISEASEJournal of Neurodevelopmental DisordersJOURNAL OF NEUROIMAGINGJOURNAL OF THE NEUROLOGICAL SCIENCESJOURNAL OF NEURO-OPHTHALMOLOGYJOURNAL OF NEUROPSYCHIATRY AND CLINICAL NEUROSCIENCES JOURNAL OF NEURORADIOLOGYJOURNAL OF NEUROSURGERY-SPINEJOURNAL OF SPINAL CORD MEDICINEJOURNAL OF SPINAL DISORDERS & TECHNIQUES NEUROCASENeurocritical CareNEUROLOGICAL RESEARCHNEUROLOGICAL SCIENCESNEUROLOGISTNEUROMODULATIONNEUROPATHOLOGYNEUROPHYSIOLOGIE CLINIQUE-CLINICAL NEUROPHYSIOLOGY NEUROSURGERY CLINICS OF NORTH AMERICANEUROSURGICAL REVIEWPain Research & ManagementPEDIATRIC NEUROLOGYPSYCHIATRY AND CLINICAL NEUROSCIENCESREVISTA DE NEUROLOGIASCHMERZSEIZURE-EUROPEAN JOURNAL OF EPILEPSYSEMINARS IN NEUROLOGYSeminars in Pediatric NeurologySURGICAL NEUROLOGYHUMAN BRAIN MAPPINGNEUROIMAGEAMERICAN JOURNAL OF NEURORADIOLOGYNEURORADIOLOGYPSYCHIATRY RESEARCH-NEUROIMAGINGSTEREOTACTIC AND FUNCTIONAL NEUROSURGERYCLINICAL EEG AND NEUROSCIENCEJOURNAL OF NEUROIMAGINGJournal of NeuroInterventional SurgeryJOURNAL OF NEURORADIOLOGYNEUROIMAGING CLINICS OF NORTH AMERICABrain Structure & FunctionFRONTIERS IN NEUROENDOCRINOLOGYHUMAN BRAIN MAPPINGJOURNAL OF COMPARATIVE NEUROLOGYJARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY MOLECULAR PSYCHIATRYNATURE NEUROSCIENCENATURE REVIEWS NEUROSCIENCENEUROIMAGENEURONNEUROPSYCHOPHARMACOLOGYPROGRESS IN NEUROBIOLOGYTRENDS IN COGNITIVE SCIENCESTRENDS IN NEUROSCIENCESAUDIOLOGY AND NEURO-OTOLOGYBIPOLAR DISORDERSBRAIN BEHAVIOR AND IMMUNITYBRAIN PATHOLOGYBRAIN RESEARCH REVIEWSBrain StimulationCEPHALALGIACurrent Alzheimer ResearchCURRENT OPINION IN NEUROBIOLOGYCURRENT OPINION IN NEUROLOGYEUROPEAN JOURNAL OF PAINHEARING RESEARCHHIPPOCAMPUSINTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY JOURNAL OF ALZHEIMERS DISEASEJOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM JOURNAL OF COGNITIVE NEUROSCIENCEJOURNAL OF COMPARATIVE PHYSIOLOGY A-NEUROETHOLOGY Journal of Neural EngineeringJOURNAL OF NEUROCHEMISTRYJournal of NeuroinflammationJOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY JOURNAL OF NEUROSCIENCEJOURNAL OF NEUROTRAUMAJOURNAL OF PAINJOURNAL OF PINEAL RESEARCH 松果体NEURAL COMPUTATIONNEUROBIOLOGY OF AGINGNEUROBIOLOGY OF DISEASENEUROINFORMATICSNEUROPATHOLOGY AND APPLIED NEUROBIOLOGY NEUROSCIENCE AND BIOBEHAVIORAL REVIEWS NEUROSCIENTISTNeurotherapeuticsPAINPSYCHONEUROENDOCRINOLOGYPSYCHOPHARMACOLOGYSLEEPSLEEP MEDICINE REVIEWSSocial Cognitive and Affective NeuroscienceASN NeuroBEHAVIOURAL BRAIN RESEARCHBMC NEUROSCIENCEBRAIN INJURYBRAIN AND LANGUAGECEREBELLUMCLINICAL NEUROPHYSIOLOGYCNS & Neurological Disorders-Drug TargetsCNS Neuroscience & TherapeuticsCOGNITIVE AFFECTIVE & BEHAVIORAL NEUROSCIENCECurrent Neurology and Neuroscience ReportsCURRENT NEUROVASCULAR RESEARCHDevelopmental Disabilities Research Reviews DEVELOPMENTAL NEUROSCIENCEEUROPEAN JOURNAL OF NEUROLOGYEUROPEAN JOURNAL OF NEUROSCIENCEEUROPEAN NEUROPSYCHOPHARMACOLOGYEXPERIMENTAL NEUROLOGYFrontiers in Cellular NeuroscienceFrontiers in Neural CircuitsGENES BRAIN AND BEHAVIORINTERNATIONAL REVIEW OF NEUROBIOLOGYJOURNAL OF ELECTROMYOGRAPHY AND KINESIOLOGYJOURNAL OF THE INTERNATIONAL NEUROPSYCHOLOGICAL SOCIETY JOURNAL OF NEURAL TRANSMISSIONJOURNAL OF NEUROENDOCRINOLOGYJournal of Neuroimmune PharmacologyJOURNAL OF NEUROIMMUNOLOGYJOURNAL OF NEUROPHYSIOLOGYJOURNAL OF NEUROSCIENCE RESEARCHJOURNAL OF THE PERIPHERAL NERVOUS SYSTEMJOURNAL OF PSYCHOPHARMACOLOGYJOURNAL OF SLEEP RESEARCHLEARNING & MEMORYMOLECULAR AND CELLULAR NEUROSCIENCEMolecular PainMUSCLE & NERVENETWORK-COMPUTATION IN NEURAL SYSTEMSNeural DevelopmentNEURAL NETWORKSNEUROBIOLOGY OF LEARNING AND MEMORYNEUROCHEMISTRY INTERNATIONALNeurodegenerative DiseasesNEUROENDOCRINOLOGYNEUROGASTROENTEROLOGY AND MOTILITYNEUROLOGIC CLINICSNEUROMOLECULAR MEDICINENEUROMUSCULAR DISORDERSNEUROPHARMACOLOGYNEUROPSYCHOLOGY REVIEWNEUROPSYCHOLOGIANEUROPSYCHOLOGYNEUROSCIENCENEUROSIGNALSNEUROTOXICOLOGY AND TERATOLOGYNEUROTOXICOLOGYPROGRESS IN BRAIN RESEARCHPROGRESS IN NEURO-PSYCHOPHARMACOLOGY & BIOLOGICAL PSYCHIATRY PSYCHOPHYSIOLOGYPurinergic SignallingRESTORATIVE NEUROLOGY AND NEUROSCIENCESocial NeuroscienceSTEREOTACTIC AND FUNCTIONAL NEUROSURGERYSTRESS-THE INTERNATIONAL JOURNAL ON THE BIOLOGY OF STRESS ACTA NEUROBIOLOGIAE EXPERIMENTALISACUPUNCTURE & ELECTRO-THERAPEUTICS RESEARCHAUTONOMIC NEUROSCIENCE-BASIC & CLINICALBehavioral and Brain FunctionsBEHAVIORAL NEUROSCIENCEBEHAVIOURAL PHARMACOLOGYBRAIN BEHAVIOR AND EVOLUTIONBRAIN AND COGNITIONBRAIN RESEARCHBRAIN RESEARCH BULLETINBRAIN TOPOGRAPHYCELLULAR AND MOLECULAR NEUROBIOLOGYCHEMICAL SENSESChemosensory PerceptionCLINICAL AUTONOMIC RESEARCHCLINICAL EEG AND NEUROSCIENCECognitive NeurodynamicsCognitive Systems ResearchCurrent NeuropharmacologyDevelopmental NeurobiologyEUROPEAN NEUROLOGYEXPERIMENTAL BRAIN RESEARCHFOLIA NEUROPATHOLOGICAFrontiers in Computational NeuroscienceFrontiers in Human NeuroscienceFUNCTIONAL NEUROLOGYGAIT & POSTUREHUMAN MOVEMENT SCIENCEINTERNATIONAL JOURNAL OF DEVELOPMENTAL NEUROSCIENCE INTERNATIONAL JOURNAL OF PSYCHOPHYSIOLOGY JOURNAL OF CHEMICAL NEUROANATOMYJOURNAL OF CLINICAL NEUROPHYSIOLOGYJOURNAL OF CLINICAL NEUROSCIENCEJOURNAL OF COMPUTATIONAL NEUROSCIENCEJOURNAL OF HEADACHE AND PAINJournal of Integrative NeuroscienceJOURNAL OF MOLECULAR NEUROSCIENCEJOURNAL OF MOTOR BEHAVIORJournal of Neurodevelopmental DisordersJOURNAL OF NEUROGENETICSJOURNAL OF THE NEUROLOGICAL SCIENCESJOURNAL OF NEUROLINGUISTICSJOURNAL OF NEUROPSYCHIATRY AND CLINICAL NEUROSCIENCES JOURNAL OF NEUROSCIENCE METHODSJOURNAL OF NEUROVIROLOGYJOURNAL OF PHYSIOLOGY-PARISMETABOLIC BRAIN DISEASEMOTOR CONTROLNEUROCHEMICAL RESEARCHNEUROENDOCRINOLOGY LETTERSNEUROIMAGING CLINICS OF NORTH AMERICA NEUROIMMUNOMODULATIONNEUROLOGICAL RESEARCHNEUROLOGICAL SCIENCESNeuron Glia BiologyNEUROPATHOLOGYNEUROPEPTIDESNEUROPHYSIOLOGIE CLINIQUE-CLINICAL NEUROPHYSIOLOGY NEUROPSYCHOBIOLOGYNEUROPSYCHOLOGICAL REHABILITATIONNEUROREPORTNEUROSCIENCE LETTERSNEUROSCIENCE RESEARCHNEUROTOXICITY RESEARCHNUTRITIONAL NEUROSCIENCEPHARMACOLOGY BIOCHEMISTRY AND BEHAVIORPSYCHIATRY AND CLINICAL NEUROSCIENCESPSYCHIATRIC GENETICSREVIEWS IN THE NEUROSCIENCESSEIZURE-EUROPEAN JOURNAL OF EPILEPSY SOMATOSENSORY AND MOTOR RESEARCHISSN名称小区大区0006-8950临床神经学11 Journal Of Head Trauma Rehabilitation0885-9701临床神经学131474-4422临床神经学110028-3878临床神经学11 Neurorehabilitation And Neural Repair1545-9683临床神经学121533-3159临床神经学110039-2499临床神经学110195-6108临床神经学231015-6305临床神经学221935-861X临床神经学221567-2050临床神经学22 current opinion in neurology1350-7540临床神经学22european journal of pain1090-3801临床神经学22 AL NEUROLOGY0022-3069临床神经学220897-7151临床神经学221526-5900临床神经学22 neuro-oncology 神经肿瘤 **1522-8517临床神经学220305-1846临床神经学22 Neuroscientist **1073-8584临床神经学220304-3959临床神经学221471-2377临床神经学331015-9770临床神经学330749-8047临床神经学331528-4042临床神经学331567-2026临床神经学331351-5101临床神经学33 Headache0017-8748临床神经学330340-5354临床神经学330167-594X临床神经学330022-3085临床神经学331660-2854临床神经学33 neurogenetics 神经遗传学1364-6745临床神经学33neurologic clinics0733-8619临床神经学33neuroradiology 神经放射学0028-3940临床神经学331092-0684临床神经学33 neurosurgery0148-396X临床神经学331362-4393临床神经学340362-2436临床神经学331529-9430临床神经学330001-6268临床神经学441433-7398临床神经学440268-8697临床神经学440317-1671临床神经学440044-4251临床神经学44 Clinical Neurology And Neurosurgery0303-8467临床神经学440722-5091临床神经学441531-3433临床神经学441092-8480临床神经学441751-8423临床神经学441090-3798临床神经学440014-3022临床神经学440940-6719临床神经学430883-0738临床神经学44 ROPSYCHOLOGY1380-3395临床神经学441738-6586临床神经学44 Journal Of Clinical Neuroscience0967-5868临床神经学440891-9887临床神经学441129-2369临床神经学440022-3018临床神经学441866-1947临床神经学441051-2284临床神经学440022-510X临床神经学431070-8022临床神经学44 NEUROSCIENCES0895-0172临床神经学430150-9861临床神经学441547-5654临床神经学441079-0268临床神经学441536-0652临床神经学44 Neurocase1355-4794临床神经学441541-6933临床神经学440161-6412临床神经学441590-1874临床神经学441074-7931临床神经学441094-7159临床神经学440919-6544临床神经学44 PHYSIOLOGY0987-7053临床神经学441042-3680临床神经学44 Neurosurgical Review0344-5607临床神经学441203-6765临床神经学44 Pediatric Neurology0887-8994临床神经学441323-1316临床神经学440210-0010临床神经学440932-433X临床神经学441059-1311临床神经学440271-8235临床神经学431071-9091临床神经学440090-3019临床神经学441065-9471神经成像121053-8119神经成像120195-6108神经成像230028-3940神经成像330925-4927神经成像331011-6125神经成像341550-0594神经成像441051-2284神经成像441759-8478神经成像440150-9861神经成像441052-5149神经成像441863-2653神经科学120091-3022神经科学111065-9471神经科学120021-9967神经科学12 ARCH IN OTOLARYNGOLOGY1525-3961神经科学131359-4184神经科学111097-6256神经科学111471-0048神经科学111053-8119神经科学120896-6273神经科学110166-2236神经科学111420-3030神经科学231398-5647神经科学220889-1591神经科学221015-6305神经科学220165-0173神经科学211935-861X神经科学220333-1024神经科学221567-2050神经科学220959-4388神经科学211350-7540神经科学221090-3801神经科学220378-5955神经科学231050-9631神经科学22 MACOLOGY1461-1457神经科学221387-2877神经科学220271-678X神经科学220898-929X神经科学22 LOGY SENSORY NEURAL AND BEHAVIORAL PHYSIOLO0340-7594神经科学241741-2560神经科学220022-3042神经科学221742-2094神经科学22 AL NEUROLOGY0022-3069神经科学22 Journal Of Neuroscience0270-6474神经科学210897-7151神经科学221526-5900神经科学220742-3098神经科学220899-7667神经科学220197-4580神经科学220969-9961神经科学22 neuroinformatics1539-2791神经科学230305-1846神经科学220149-7634神经科学21 Neuroscientist1073-8584神经科学220033-3158神经科学220161-8105神经科学221087-0792神经科学221749-5016神经科学221759-0914神经科学320166-4328神经科学331471-2202神经科学330269-9052神经科学340093-934X神经科学331473-4222神经科学32 Clinical Neurophysiology1388-2457神经科学331871-5273神经科学321755-5930神经科学331530-7026神经科学321528-4042神经科学331567-2026神经科学331940-5510神经科学330378-5866神经科学331351-5101神经科学330953-816X神经科学320924-977X神经科学320014-4886神经科学321662-5102神经科学321662-5110神经科学321601-1848神经科学320074-7742神经科学331050-6411神经科学34 LOGICAL SOCIETY1355-6177神经科学330300-9564神经科学330953-8194神经科学321557-1890神经科学330165-5728神经科学330022-3077神经科学330360-4012神经科学331072-0502神经科学321044-7431神经科学321744-8069神经科学320148-639X神经科学330954-898X神经科学331749-8104神经科学330893-6080神经科学321074-7427神经科学320197-0186神经科学321660-2854神经科学330028-3835神经科学331350-1925神经科学32 Neurologic Clinics0733-8619神经科学331535-1084神经科学330960-8966神经科学330028-3908神经科学321040-7308神经科学320028-3932神经科学320894-4105神经科学330306-4522神经科学331424-862X神经科学320892-0362神经科学330161-813X神经科学330079-6123神经科学33 IOLOGICAL PSYCHIATRY0278-5846神经科学330048-5772神经科学321573-9538神经科学330922-6028神经科学331747-0919神经科学321011-6125神经科学34 BIOLOGY OF STRESS1025-3890神经科学330065-1400神经科学440360-1293神经科学441566-0702神经科学440006-8977神经科学430278-2626神经科学430006-8993神经科学430361-9230神经科学430896-0267神经科学430272-4340神经科学430379-864X神经科学431936-5802神经科学430959-9851神经科学441550-0594神经科学441871-4080神经科学441389-0417神经科学441570-159X神经科学441932-8451神经科学430014-3022神经科学440014-4819神经科学431641-4640神经科学441662-5188神经科学431662-5161神经科学440393-5264神经科学440966-6362神经科学430167-9457神经科学44 EUROSCIENCE0736-5748神经科学440167-8760神经科学430891-0618神经科学430736-0258神经科学440967-5868神经科学440929-5313神经科学431129-2369神经科学440219-6352神经科学440895-8696神经科学430022-2895神经科学441866-1947神经科学440167-7063神经科学440911-6044神经科学44 NEUROSCIENCES0895-0172神经科学430165-0270神经科学431355-0284神经科学430928-4257神经科学430885-7490神经科学441087-1640神经科学440364-3190神经科学430172-780X神经科学441052-5149神经科学441021-7401神经科学440161-6412神经科学441590-1874神经科学441740-925X神经科学440919-6544神经科学440143-4179神经科学43 PHYSIOLOGY0987-7053神经科学440302-282X神经科学430960-2011神经科学440959-4965神经科学440304-3940神经科学440168-0102神经科学431029-8428神经科学431028-415X神经科学440091-3057神经科学431323-1316神经科学440955-8829神经科学430334-1763神经科学431059-1311神经科学440899-0220神经科学4410年因子-2010年平均影响备注9.2329.4422.779 2.58521.65918.0188.0177.7443.7724.2637.7937.7935.7566.4323.464 3.1684.7415.4074.964 3.9824.953 4.6855.021 5.3343.819 3.7444.190 4.6313.426 3.7364.851 4.0055.483 5.1563.625 3.5934.5775.5175.355 5.5852.797 2.5632.9873.1883.114 3.0032.697 2.4673.047 3.2833.765 3.0022.642 2.8363.853 3.0972.929 2.6682.739 2.4863.791 3.4253.488 3.3252.533 2.5342.870 2.5052.348 2.3483.298 3.1862.510 2.6423.024 2.9631.329 1.4781.1290.8780.9660.9141.179 1.1530.7160.6362009更名，原名为ZENTRALBLATT FUR NEUROCHIRURGIE1.636 1.4211.067 1.1011.450 1.4501.810 1.8101.384 1.3841.994 1.8071.760 1.7171.9942.1151.668 1.5641.805 1.9581.0970.7711.165 1.1752.131 1.9472.015 2.0091.796 1.7921.269 1.2691.287 1.6062.167 2.2831.059 1.1351.9812.2261.203 1.2621.594 1.5371.442 1.3881.333 1.3011.0690.9862.353 2.0611.621 1.5111.220 1.2581.8812.0581.605 1.731 1.693 1.3411.477 1.4562.259 1.961 1.515 1.515 1.513 1.505 1.559 1.426 1.218 1.178 1.170 1.2031.6492.0202.000 2.209 1.862 1.862 1.260 1.399 5.107 5.586 5.937 5.7903.464 3.168 2.870 2.505 2.064 2.712 1.882 1.727 1.325 1.199 1.287 1.606 1.069 1.069 1.203 1.262 1.420 1.4974.982 3.938 12.75011.1635.107 5.586 3.774 3.745 3.038 2.634 15.47014.352 14.19114.233 29.51027.311 5.937 5.790 14.02713.8199.9669.412 9.68610.777 13.32012.977 2.228 2.162 5.221 4.8943.9564.6424.7415.407 8.8427.489 4.964 3.982 4.265 3.805 4.953 4.6856.8917.401 5.021 5.334 3.819 3.744 2.428 2.313 4.609 4.584 4.699 4.650 4.261 4.3984.5225.2405.357 5.202 2.000 1.955 2.628 3.0354.337 4.2795.785 4.555 4.190 4.631 7.2717.3003.426 3.7364.851 4.0055.855 5.373 2.290 2.2816.634 6.177 5.121 4.830 3.027 2.990 3.625 3.593 9.0158.203 4.577 5.5175.355 5.585 5.168 4.383 3.817 3.8655.486 5.1216.338 6.149 4.482 4.343 3.833 3.833 3.393 3.261 3.091 2.895 1.750 1.466 3.162 3.021 3.288 3.4722.786 2.9603.618 3.962 3.492 3.091 3.512 3.5652.697 2.4673.047 3.283 3.436 2.7182.707 2.7303.765 3.0023.658 3.4874.201 3.849 4.436 4.108 3.588 3.5883.960 3.9604.061 3.915 2.183 2.892 2.372 2.084 2.910 2.767 2.597 2.457 4.650 3.867 3.528 3.1012.901 2.9673.114 3.4152.9583.0103.032 3.1523.801 3.8373.361 3.3724.607 4.2713.861 3.7884.148 4.013 2.302 2.394 0.957 1.275 3.395 3.411 1.972 2.169 3.701 3.646 3.601 3.457 3.791 3.425 3.272 3.086 3.349 3.466 2.533 2.534 4.657 3.373 2.764 2.891 4.677 3.990 4.231 4.270 3.949 4.123 3.176 3.109 3.215 3.354 3.967 3.997 2.622 2.8792.921 2.7493.134 2.9592.877 2.7793.263 3.5022.975 2.9233.349 3.014 2.823 3.4491.882 1.7272.553 2.903 1.533 1.320 1.0000.6471.671 1.8722.305 2.3212.481 2.650 2.530 2.6511.9682.4722.838 2.609 2.623 2.5272.498 2.3213.288 2.182 2.423 2.360 2.327 2.800 1.091 1.046 1.333 1.376 1.325 1.199 1.625 1.9441.000 1.0512.783 1.888 2.855 2.6401.760 1.7172.296 2.2491.222 1.1532.586 2.586 1.940 1.7441.481 1.2112.313 2.544 1.967 1.9471.938 1.9442.378 2.562 2.121 2.151 1.154 1.4551.165 1.1752.325 2.432 2.015 2.0091.216 1.2552.922 2.568 1.650 1.428 1.269 1.2691.950 1.0152.167 2.2831.742 1.6161.9812.2262.100 2.1622.243 2.1463.030 2.401 2.343 2.0421.204 1.4112.608 2.530 1.621 1.3421.420 1.4972.642 1.963 1.621 1.5111.220 1.2582.611 1.829 1.605 1.731 1.917 2.1301.693 1.3412.567 2.155 1.731 1.7591.822 1.8442.055 2.0602.096 2.2383.015 2.7611.301 1.1792.624 2.7811.559 1.4262.061 2.361 2.047 2.612 1.649 2.020 1.115 1.011。