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遗传性牙釉质发育不全相关基因Fam83h突变的鉴定及亚细胞定位的研究
The Identification of Amelogenesis Imperfecta-causing Fam83h Mutation and Study of the Subcellular Localization of Fam83h
【作者】 丁玉梅;
【导师】 张汉东;
【作者基本信息】 华中科技大学 , 口腔医学, 2009, 博士
【摘要】 第一部分遗传性牙釉质发育不全相关基因Fam83h突变的鉴定与分析[目的]对五个患有遗传性牙釉质发育不全的家族进行Fam83h测序分析,以探讨其发病是否与Fam83h突变有关。[方法]对五个家族所有成员取外周血2ml,用QIAGEN公司的QIAamp? DNABlood Maxi Kit提取基因组DNA。根据人Fam83h基因组DNA全长序列设计7对引物,首先PCR扩增出五个家族先证者的编码区Fam83h DNA,然后对所有PCR产物进行测序,对比Genebank的人Fam83h源序列,寻找Fam83h突变位点。若先证者的某Fam83h位点存在突变,再扩增该家族其他所有招募成员的Fam83h编码序列并测序,若所有患者在此位点均有相同变异,而未受影响成员在此位点无变异,则可确认该位点变异为致病突变。[结果]检测的五个AI家族中,有两个家族鉴定出两个新的致病性的Fam83h突变。家族1为西班牙人,按照标准命名法,Fam83h变异位点为:Fam83h c.1330C>T,p.Q444X,位于外显子5,突变类型为过早引入终止密码子,C末端氨基酸缺失735个;家族2为高加索人,Fam83h突变位于Fam83h c.1192C>T,p.Q398X,位于外显子5,突变类型也为过早引入终止密码子,C末端氨基酸缺失781个。结合遗传方式及表型分析,此两个家族均为常染色体显性遗传钙化不全型AI(ADHCAI)。另外三个招募的AI家族未筛选到Fam83h变异。[结论]Fam83h cDNA1330位点和1192位点C>T的突变会导致ADHCAI。第二部分Fam83h亚细胞定位的研究实验一Fam83h真核表达载体的构建[目的]构建融合绿色荧光的Fam83h真核表达载体。[方法]从日本Yokohama公司购买含有小鼠Fam83h cDNA的质粒pBluescriptⅡKS+-Fam83h cDNA,设计Fam83h引物,引入SalⅠ和BglⅡ酶切位点,PCR扩增出小鼠Fam83h cDNA全长编码区后,连入PCR2.1-TOPO T载体,转化大肠杆菌TOP10,铺板,扩增,提取PCR2.1-TOPO-Fam83h质粒,SalⅠ和BglⅡ双酶切初步鉴定正确克隆,将酶切正确的克隆测序。SalⅠ和BglⅡ双酶切测序正确的克隆从而释放出Fam83h,连入BamHⅠ和SalⅠ双酶切过的phrGFP-C绿色荧光表达载体,转化大肠杆菌TOP10,铺板,扩增,提取phrGFP-C-Fam83h质粒,SalⅠ和NdeⅠ双酶切初步鉴定正确克隆,将酶切正确的克隆再次测序确认。[结果]成功扩增出3651bp的小鼠Fam83h编码区序列,PCR2.1-TOPO-Fam83h质粒测序结果显示扩增出的Fam83h序列和genebank收录的Fam83h源序列完全一致。phrGFP-C-Fam83h质粒测序结果显示Fam83h序列被连入到phrGFP-C正确区域。[结论]成功构建phrGFP-C-Fam83h荧光真核表达载体,为实验二和实验三打下基础。实验二检测Fam83h是否为细胞内蛋白[目的]体外检测Fam83h是否为胞内蛋白或为分泌性蛋白。[方法]复苏HEK293细胞,待细胞生长状态良好时,接种入腔室载玻片进行培养,接种密度为2-6×105个细胞/cm2。24小时后,将实验一构建的phrGFP-C-Fam83h质粒,与脂质体Lipofectamine 2000按1μg:1μl的比例转染入HEK293细胞,放入培养箱继续培养,6小时后更换培养液。24-72小时后倒置显微镜下观察细胞形态及存活率,4%多聚甲醛固定细胞,分别用DiI和DAPI进行胞膜和胞核染色,封片,在荧光显微镜下观察来自Fam83h的绿色荧光是否位于细胞内并拍照。[结果]在HEK293细胞中,来自Fam83h的绿色荧光总是位于染成红色的胞膜之内,这充分表明Fam83h是一个胞内蛋白,而不是被分泌性到胞外。而且,Fam83h的绿色荧光信号很少和胞核重叠,绝大多数是位于胞核周围,特别是高尔基体常在的胞核前沿内陷处,排除拍照的角度问题,我们推测Fam83h很可能位于胞核周围的细胞器,特别是高尔基体。[结论] Fam83h是一个非分泌性蛋白,它位于细胞内,但不在胞核内。Fam83h很可能位于胞核周围的细胞器,特别是与高尔基体密切相关。实验三检测Fam83h的亚细胞定位[目的]体外探讨Fam83h亚细胞定位与高尔基体的关系。[方法]复苏HEK293细胞,待细胞生长状态良好时,接种入腔室载玻片进行培养,接种密度为2-6×105个细胞/cm2。24小时后,将实验一构建的phrGFP-C-Fam83h质粒,与脂质体Lipofectamine 2000按1μg:1μl的比例转染入HEK293细胞,放入培养箱继续培养,6小时后更换培养液。24-72小时后倒置显微镜下观察细胞形态及存活率,4%多聚甲醛固定细胞,并用DAPI进行胞核染色及BODIPY进行高尔基体染色,封片,在荧光显微镜下观察Fam83h绿色荧光信号与高尔基体的关系并拍照。[结果]在HEK293细胞中,来自Fam83h的绿色荧光总是位于高尔基体的红色荧光之内,且面积总是小于红色荧光,这充分表明Fam83h是位于高尔基体之内。并且,Fam83h绿色荧光所在位置的高尔基体红色荧光明显变淡,甚至无红色荧光,表明Fam83h蛋白可能定位于高尔基体膜表面。[结论] Fam83h蛋白在细胞内定位与高尔基体密切相关,可能位于高尔基体膜表面。
【Abstract】 PartⅠThe identification of Amelogenesis ImperfectacausingFam83h mutation[Objective] To identify whether the AI phenotypes are caused by Fam83h mutationsin five AI families by DNA sequencing.[Methods] Genomic DNA was isolated from 2 mL of peripheral whole blood of allrecruited members with QIAamp? DNA Blood Maxi Kit. 7 pairs of primers weredesigned based on human Fam83h cDNA. Full length of Fam83h encoding regions offive probands were first amplified by PCR. All the PCR products were sequenced andthen compared with the original data of Genebank to detect whether there wereFam83h mutations. If there was some Fam83h mutation in the proband, then wewould amplify Fam83h encoding regions of all the recruited members of this familyand send for sequence. If all the affected people had the same Fam83h mutation andall the unaffected people were normal in this site, we could determine that it was thisFam83h mutation that caused AI.[Results] Two new AI-causing Fam83h mutations were found in two families amongfive recruited families. The proband of family 1 was Hispanic. DNA sequencingchromatograms revealed a C to T transition in one of the two FAM83H alleles,changing codon 444 from a glutamine (Q) to a stop (X) codon (g.2452C>T;c.1330C>T; p.Q444X), the following 735 amino acids were lost. The proband of family 2 was Caucasian. DNA sequencing chromatograms showed an A to Gtransition in one of the two FAM83H alleles, changing codon 398 from a glutamine(Q) to a stop (X) codon (g.2314C>T; c.1192C>T; p.Q398X), the following 781 aminoacids were lost. No Fam83h mutation was found in the other three families[Conclusion] Mutations C>T at Fam83h cDNA 1330 and 1192 sites lead to AI. PartⅡSubcellular localization of Fam83hSectionⅠConstruction of Fam83h eukaryotic expression vector[Objective] To construct a Fam83h eukaryotic expression vector fused to greenfluorescence.[Methods] Full-length mouse Fam83h cDNA encoding region was amplified by PCRfrom pBluescriptⅡKS+-Fam83h obtained from K.K. DNAFORM (Yokohama, Jpn).SalⅠand BglⅡdigestion sites were introduced into the forward and reverse primersrespectively. The PCR product was then inserted into PCR2.1-TOPO vector andtransformed into TOP10. We plated, shaked bacterias and then extracted the PCR2.1-TOPO-Fam83h plasminds. The correct plasmids were first checked by SalⅠ/ BglⅡdouble digestion and then sended for sequencing. Fam83h was released from correctPCR2.1-TOPO-Fam83h by BglⅡ/SalⅠdouble digestion and then linked into phrGFPCvector which was digested by BamHⅠ/SalⅠ. We Transformed Phr-GFP-C-Fam83hinto TOP10 and plated, shaked bacterias and then extracted the phrGFP-C-Fam83hplasminds. The correct plasmids were first checked by SalⅠ/NdeⅠdouble digestionand then sended for sequencing again.[Results] 3651bp mouse Fam83h encoding region was amplified successfully.Sequence results of PCR2.1-TOPO-Fam83h were coincidence with the genebank data.Sequence results of phrGFP-C-Fam83h showed that Fam83h was inserted into correctregion of phrGFP-C.[Conclusion] We constructed phrGFP-C-Fam83h plasmid successfully, which laidthe first stone for SectionⅡand SectionⅢ SectionⅡDetecting if Fam83h is an intracellular protein[Objective] To determine if Fam83h is an intracellular or secreted protein in vitro.[Methods] HEK293 cells were refreshed and then seeded into chamber slides whenthey were in good condition, with seeding density 2-6×105 cells/cm2. 24 hrs later, thecorrect phrGFP-Fam83h plasmid was transfected into HEK293 cell lines by use ofLipofectamine 2000. The ratio of phrGFP-Fam83h: Lipofectamine 2000 was 1μg: 1μl.Cell culture media was changed for normal media after 6 hrs. 24-72 hrs later aftertransfection cells were fixed by 4% PFA, the cell membrane was stained into red byDiI and nucleus were stained into blue by DAPI. The green fluorescence fromFam83h was detected in fluorescence microscope and then taken pictures.[Results] We detected green fluorescence of Fam83h was always within the redmembrane in HEK293 cell lines, which suggested definitely that Fam83h should bean intracellular protein instead of being secreted extracellularly. Further more, wefound that green fluorescence of Fam83h seldom overlapped with nucleus, most ofthem located in perinuclear region, especially the front edge of membraneinvagination, where usually contains Golgi apparatus. Regard the inaccuracy causedby angle of taking pictures, we propose that Fam83h probably locates in cell organsaround nucleus, especially Golgi apparatus[Conclusion] Fam83h is an intracellular protein, but not in nucleus. The subcellularlocalization of Fam83h is probably in cell organs around nucleus, it is speculatedclosely related with Golgi complex. SectionⅢStudy of the subcellular localization of Fam83h[Objective] To determine the relation between the subcellular localization of Fam83hand Golgi complex.[Methods] HEK293 cells were refreshed and then seeded into chamber slides whenthey were in good condition, with seeding density 2-6×105 cells/cm2. 24 hrs later, thecorrect phrGFP-Fam83h plasmid was transfected into HEK293 cell lines by use ofLipofectamine 2000. The ratio of phrGFP-Fam83h: Lipofectamine 2000 was 1μg: 1μl.Cell culture media was changed for normal media after 6 hrs. 24-72 hrs later aftertransfection cells were fixed by 4% PFA, the nucleus were stained into blue by DAPIand Golgi complex was stained into red by BODIPY. The relation between greenfluorescence from Fam83h and red fluorescence from Golgi complex was detected influorescence microscope and then taken pictures.[Results] In HEK293 cells, green fluorescence signal from Fam83h was always inand smaller than the red fluorescence from Golgi complex, which suggested stronglythat Fam83h shoul be in Golgi complex. Typically the Golgi stain was weaker ornegative where the GFP localized, it seemed that Fam83h located in the membrane ofGolgi complex.[Conclusion] The subcellular localization of Fam83h is closely related with Golgicomplex, Fam83h may locate in the membrane of Golgi complex.
【Key words】 Fam83h; ADHCAI; mutation; gene sequence; T vector; phrGFP-C; transformation; intracellular protein; transfection; Golgi complex; AI;