本文主要研究内容
作者尹翠翠（2019）在《Kirrel1蛋白对骨骼肌成肌细胞分化及融合的影响》一文中研究指出：研究背景细胞-细胞融合对于多细胞生物的发育至关重要。骨骼肌是由许多肌纤维组成的独特组织,每个肌纤维都是由成百上千个成肌细胞相互融合的产物。成肌细胞融合不仅对胚胎发育过程中骨骼肌形成至关重要,而且对成人肌卫星细胞介导的肌肉再生也至关重要。为了发生成肌细胞融合,两个融合伴侣必须相互识别粘附其质膜,打开融合孔以允许细胞中的物质相互交换,并最终合并成一个细胞。在果蝇胚胎中,有两种不同类型的成肌细胞,肌肉创始细胞(founder cells,FCs)和融合感受态成肌细胞(fusion competent myoblasts,FCMs),FCs和FCMs膜表面表达的不同蛋白之间相互作用,促成它们相互识别和粘附。在FCs中,两个旁系同源物Dumbfounded(Duf)亦被称为Kin-of-Irre(Kirre)和Roughest(Rst)亦被称为Irre C,在成肌细胞融合过程中具有多种功能。在FCMs中,Sticks and stones(Sns)作为主要胞粘附分子(cell adhesion molecules,CAMs),其功能部分由其旁系同源物Hibris(Hbs)补偿。类似胚胎肌肉的发育,多核果蝇成年肌肉由表达Duf-Rst的创始细胞吸引周围表达Sns-Hbs的FCMs相互融合形成。果蝇中Rst和Kirre的哺乳动物同源物是Kirrel基因家族,包括Kirrel1、Kirrel2和Kirrel3,Kirrel1有Kirrel A和Kirrel B两种基因亚型。果蝇中Sns的哺乳动物的同源物是Nephrin,并且我们的前期实验已经证实了Nephrin在斑马鱼成肌细胞融合过程中很重要。在果蝇和斑马鱼中骨骼肌的成肌细胞融合过程已被大家熟知,但是对哺乳动物成肌细胞融合还知之甚少。故本课题旨在研究Kirrel1蛋白对小鼠骨骼肌成肌细胞分化融合的影响,以期找到证据支持Kirrel家族成员在调节小鼠骨骼肌成肌细胞融合过程起作用。研究目的1.研究Kirrel A和Kirrel B在小鼠骨骼肌成肌细胞C2C12分化融合过程中及小鼠肌肉损伤修复重建过程中m RNA的表达变化;2.探究敲低Kirrel1、Kirrel A和Kirrel B对小鼠骨骼肌成肌细胞C2C12分化融合的影响。研究方法通过半定量RT-PCR和RT-q PCR检测Kirrel A和Kirrel B在小鼠骨骼肌成肌细胞C2C12分化融合过程中及在小鼠肌肉损伤重建过程中m RNA的表达变化;通过Western Blot验证Kirrel A-、Kirrel B-、Kirrel1-sh RNA在C2C12细胞中敲低Kirrel A和Kirrel B效果;在C2C12细胞系中转染Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNA质粒,通过药筛建立稳转细胞系;通过半定量RT-PCR和RT-q PCR验证稳转细胞系的建立。免疫荧光检测Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNA细胞系在分化融合过程中分化第二天(Day2,D2)和分化第五天(Day5,D5)myosin的表达,计算融合指数;Western Blot检测Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNA细胞系在分化融合过程中D0~D5 myosin和myogenin蛋白表达量。(myosin主要在肌管中表达,反映肌管形成的多少;myogenin是促分化因子,启动细胞分化;融合指数是指在同一显微视野中,肌管中细胞核数和总细胞核数的比值。)结果1.基于半定量RT-PCR和RT-q PCR结果显示:Kirrel A和Kirrel B在C2C12细胞分化融合过程中及在小鼠肌肉损伤重建过程中m RNA的表达量明显增加,并且RT-q PCR结果显示与D0相比这种增加具有统计学意义(p<0.05,n=3)。2.转染Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNA表达质粒至C2C12细胞系中。Myosin免疫荧光结果显示:在C2C12细胞分化D2、D5和对照组相比,Kirrel A-、Kirrel B-、Kirrel1-sh RNA的融合指数均比对照组低且有统计学意义(p<0.05,n=6)。3.转染Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNA表达质粒至C2C12细胞系中。Myosin Western Blot结果显示:在C2C12细胞分化D0~D5 Kirrel A-、Kirrel B-、Kirrel1-sh RNA myosin蛋白的表达量均比对照组低且有统计学意义(p<0.05,n=3);myogenin蛋白的表达量和对照组相比表达量有差异但没有统计学意义(p>0.05,n=3)。结论1.Kirrel A和Kirrel B参与成肌细胞分化融合及肌肉损伤重建过程。2.敲低Kirrel1、Kirrel A、Kirrel B能够抑制C2C12细胞融合。
Abstract
yan jiu bei jing xi bao -xi bao rong ge dui yu duo xi bao sheng wu de fa yo zhi guan chong yao 。gu ge ji shi you hu duo ji qian wei zu cheng de du te zu zhi ,mei ge ji qian wei dou shi you cheng bai shang qian ge cheng ji xi bao xiang hu rong ge de chan wu 。cheng ji xi bao rong ge bu jin dui pei tai fa yo guo cheng zhong gu ge ji xing cheng zhi guan chong yao ,er ju dui cheng ren ji wei xing xi bao jie dao de ji rou zai sheng ye zhi guan chong yao 。wei le fa sheng cheng ji xi bao rong ge ,liang ge rong ge ban lv bi xu xiang hu shi bie nian fu ji zhi mo ,da kai rong ge kong yi yun hu xi bao zhong de wu zhi xiang hu jiao huan ,bing zui zhong ge bing cheng yi ge xi bao 。zai guo ying pei tai zhong ,you liang chong bu tong lei xing de cheng ji xi bao ,ji rou chuang shi xi bao (founder cells,FCs)he rong ge gan shou tai cheng ji xi bao (fusion competent myoblasts,FCMs),FCshe FCMsmo biao mian biao da de bu tong dan bai zhi jian xiang hu zuo yong ,cu cheng ta men xiang hu shi bie he nian fu 。zai FCszhong ,liang ge pang ji tong yuan wu Dumbfounded(Duf)yi bei chen wei Kin-of-Irre(Kirre)he Roughest(Rst)yi bei chen wei Irre C,zai cheng ji xi bao rong ge guo cheng zhong ju you duo chong gong neng 。zai FCMszhong ,Sticks and stones(Sns)zuo wei zhu yao bao nian fu fen zi (cell adhesion molecules,CAMs),ji gong neng bu fen you ji pang ji tong yuan wu Hibris(Hbs)bu chang 。lei shi pei tai ji rou de fa yo ,duo he guo ying cheng nian ji rou you biao da Duf-Rstde chuang shi xi bao xi yin zhou wei biao da Sns-Hbsde FCMsxiang hu rong ge xing cheng 。guo ying zhong Rsthe Kirrede bu ru dong wu tong yuan wu shi Kirrelji yin jia zu ,bao gua Kirrel1、Kirrel2he Kirrel3,Kirrel1you Kirrel Ahe Kirrel Bliang chong ji yin ya xing 。guo ying zhong Snsde bu ru dong wu de tong yuan wu shi Nephrin,bing ju wo men de qian ji shi yan yi jing zheng shi le Nephrinzai ban ma yu cheng ji xi bao rong ge guo cheng zhong hen chong yao 。zai guo ying he ban ma yu zhong gu ge ji de cheng ji xi bao rong ge guo cheng yi bei da jia shou zhi ,dan shi dui bu ru dong wu cheng ji xi bao rong ge hai zhi zhi shen shao 。gu ben ke ti zhi zai yan jiu Kirrel1dan bai dui xiao shu gu ge ji cheng ji xi bao fen hua rong ge de ying xiang ,yi ji zhao dao zheng ju zhi chi Kirreljia zu cheng yuan zai diao jie xiao shu gu ge ji cheng ji xi bao rong ge guo cheng qi zuo yong 。yan jiu mu de 1.yan jiu Kirrel Ahe Kirrel Bzai xiao shu gu ge ji cheng ji xi bao C2C12fen hua rong ge guo cheng zhong ji xiao shu ji rou sun shang xiu fu chong jian guo cheng zhong m RNAde biao da bian hua ;2.tan jiu qiao di Kirrel1、Kirrel Ahe Kirrel Bdui xiao shu gu ge ji cheng ji xi bao C2C12fen hua rong ge de ying xiang 。yan jiu fang fa tong guo ban ding liang RT-PCRhe RT-q PCRjian ce Kirrel Ahe Kirrel Bzai xiao shu gu ge ji cheng ji xi bao C2C12fen hua rong ge guo cheng zhong ji zai xiao shu ji rou sun shang chong jian guo cheng zhong m RNAde biao da bian hua ;tong guo Western Blotyan zheng Kirrel A-、Kirrel B-、Kirrel1-sh RNAzai C2C12xi bao zhong qiao di Kirrel Ahe Kirrel Bxiao guo ;zai C2C12xi bao ji zhong zhuai ran Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNAzhi li ,tong guo yao shai jian li wen zhuai xi bao ji ;tong guo ban ding liang RT-PCRhe RT-q PCRyan zheng wen zhuai xi bao ji de jian li 。mian yi ying guang jian ce Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNAxi bao ji zai fen hua rong ge guo cheng zhong fen hua di er tian (Day2,D2)he fen hua di wu tian (Day5,D5)myosinde biao da ,ji suan rong ge zhi shu ;Western Blotjian ce Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNAxi bao ji zai fen hua rong ge guo cheng zhong D0~D5 myosinhe myogenindan bai biao da liang 。(myosinzhu yao zai ji guan zhong biao da ,fan ying ji guan xing cheng de duo shao ;myogeninshi cu fen hua yin zi ,qi dong xi bao fen hua ;rong ge zhi shu shi zhi zai tong yi xian wei shi ye zhong ,ji guan zhong xi bao he shu he zong xi bao he shu de bi zhi 。)jie guo 1.ji yu ban ding liang RT-PCRhe RT-q PCRjie guo xian shi :Kirrel Ahe Kirrel Bzai C2C12xi bao fen hua rong ge guo cheng zhong ji zai xiao shu ji rou sun shang chong jian guo cheng zhong m RNAde biao da liang ming xian zeng jia ,bing ju RT-q PCRjie guo xian shi yu D0xiang bi zhe chong zeng jia ju you tong ji xue yi yi (p<0.05,n=3)。2.zhuai ran Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNAbiao da zhi li zhi C2C12xi bao ji zhong 。Myosinmian yi ying guang jie guo xian shi :zai C2C12xi bao fen hua D2、D5he dui zhao zu xiang bi ,Kirrel A-、Kirrel B-、Kirrel1-sh RNAde rong ge zhi shu jun bi dui zhao zu di ju you tong ji xue yi yi (p<0.05,n=6)。3.zhuai ran Control-、Kirrel A-、Kirrel B-、Kirrel1-sh RNAbiao da zhi li zhi C2C12xi bao ji zhong 。Myosin Western Blotjie guo xian shi :zai C2C12xi bao fen hua D0~D5 Kirrel A-、Kirrel B-、Kirrel1-sh RNA myosindan bai de biao da liang jun bi dui zhao zu di ju you tong ji xue yi yi (p<0.05,n=3);myogenindan bai de biao da liang he dui zhao zu xiang bi biao da liang you cha yi dan mei you tong ji xue yi yi (p>0.05,n=3)。jie lun 1.Kirrel Ahe Kirrel Bcan yu cheng ji xi bao fen hua rong ge ji ji rou sun shang chong jian guo cheng 。2.qiao di Kirrel1、Kirrel A、Kirrel Bneng gou yi zhi C2C12xi bao rong ge 。
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