【作者】 朱怀虚；

【导师】 邬玲仟； 潘乾； 龙志高；

【作者基本信息】 中南大学 ， 遗传学， 2008， 硕士

【摘要】 背景:间隙连接蛋白(Connexins)是一类可以在细胞的膜上组装成为间隙连接通道(Gap Junctions)以介导相邻细胞间小分子物质(＜1000Da)的重要蛋白家族。通过这些物质和信息的交换,使得细胞间对外界刺激作出协调一致的反应。Connexin31(CX31)是间隙连接蛋白家族的一员,其突变可导致显、隐性红皮肤角化病(dominant and/recessive EKV),显、隐性神经性耳聋(dominantand/recessive hearing impairment),或者显性神经性耳聋-周围神经病(dominanthearing impairment combined with peripheral neuropathy)等。CX31的基因敲除小鼠中没有发现耳聋或皮肤病的表型,以人CX31蛋白为启动子的转基因小鼠研究发现其F137L突变体小鼠表皮可出现部分角质化。CX31对于细胞稳态维持、细胞增殖、分化具有重要作用。但对CX31通道组装、通道通透性、通道功能调节和细胞内运输等CX31分子特征和细胞行为的认识还很粗略。目的:增强CX31在细胞中的表达水平,尤其是突变体在某一特定组织中的表达水平,将有可能使动物模型出现相应的表型。本研究用Keratin14蛋白启动子构建CX31野生型和F137L突变体的表达载体,并研究该载体的皮肤组织表达能力和表达特异性。得到一种可望用于转基因动物模型的皮肤组织特异性表达的CX31载体。方法:我们使用PCR技术,将CX31产物连接到,含有Keratin14启动子、兔globin和Keratin14的3’非翻译区的载体中。然后将该载体用Lipifectamine LTX转染Hacat细胞,应用免疫沉淀和免疫荧光技术对CX31的表达进行检测。为了研究其组织表达的特异性,我们还用磷酸钙法转染了HEK 293T细胞,并用免疫荧光技术对CX31的表达情况进行了检测。结果:通过酶切及测序证实我们得到了CX31野生型和F137L突变体载体;Hacat细胞的免疫沉淀和免疫荧光检测到了CX31的表达,且野生型CX31可以形成间隙连接斑(gap junction plaques),F137L突变的蛋白则不能;对HEK293T细胞的免疫荧光检测没有发现以Keratin14启动子的载体的CX31的表达,而转染了pCDNA3.1A(-)-CX31-myc的HEK 293T细胞阳性对照中则能够通过免疫荧光检测到蛋白表达和形成的间隙连接斑。结论:本研究构建的CX31组织特异性表达载体能有效表达,在线性化去除其部分骨架后可望用于转基因动物模型的构建。更多还原

【Abstract】 BackgroundConnexins is a protein family that can form a Gap Junction structure on the membrane and allows the exchange of small molecules(＜1kD) between cells.The cells then synchronize their response of foreign stimulus.Connexin31(CX31)is one member of the Connexin family which its mutations are found to cause dominant or recessive EKV,dominant or recessive hearing impairment and dominant hearing impairment combined with peripheral neuropathy.There is no hearing loss or skin disease phenotype found in CX31 knockout mouse and a research which created a transgenic mouse in using of human CX31 promoter found partially keratinise of the epidermis.CX31 plays very important role in cell stability,proliferation and differentiation.But we still know little of CX31 character and activity in Gap Junction assembling,permeability, function regulation and intra-cellular trafficking.Research goalsTo enhance the expression level of CX31,especially some mutation’s expression in specific tissue may cause the phenotype in animal models.We employed a Keratin 14 promoter and made an expression construct of CX31 wt and F137L mutant,then studied its expression ability and specificity in skin sourced cell line.We obtain a skin tissue specifically expressed construct which also can be used to create a transgenic animal model.MethodsWe use PCR to amplify CX31 wt and F137L mutant and ligate it into a vector contains Keratin14 promoter,rabbit globin and Keratin 14 3’ Untanslated region elements.Thereafter,tansfect the constructs in to Hacat cell use Lipofectamine LTX and detect the CX31 with immunoprecipitation and immunostaining.We also transfect the HEK 293T cell by calcium phosphate method and detect its expression level by immunostaining in order to evaluate the ability of tissue specific expression.Results:The constructs are established by enzyme digestion and sequencing. The expression in Hacat cell is detected by immunoprecipitation and immunostaining,which the CX31 wild type protein can form the Gap Junction plaques while the F137L mutant can not.We do not detect the CX31 expression in HEK 293T cells by using Keratinl4 promoter,but the positive control which was transfected with pCDNA3.1A(-)-CX31-myc has the expression and Gap Junction plaques.Conclusions The two constructs made in this research can successfully and specifically express in skin tissue sourced cell line.They also are available in the creation of transgenic animal model after linearization to remove their vector backbones.更多还原