智力低下相关蛋白FXR1P与CMAS相互作用生物学效应的研究

发布时间：2018-03-18 08:15

本文选题：FXR1P　切入点：CMAS　出处：《南华大学》2012年硕士论文　论文类型：学位论文

【摘要】：脆性X综合征（fragile X syndrome，FXS）是常见的遗传性智力低下疾病之一，99%的患者是由FMR1（智力低下基因1）的5’端非编码区的CGG三核苷酸重复序列异常扩增或其上游的CpG岛过度甲基化引起的FMR1的表达产物FMRP（fragile X mental retardation protein，智力低下蛋白）表达下降或缺失所致。定位于3q28的智力低下相关基因FXR1与FMR1属同一基因家族，编码的蛋白质都具有与蛋白质和RNA结合特性。本课题在本实验室前期工作已证明FXR1P与CMAS相互作用的基础上，进一步研究FXR1P与CMAS相互作用后的生物学效应。目的：研究FXR1P与CMAS相互作用后的生物学效应，为阐明FXR1的基因功能提供实验依据。方法：1. PCR扩增FXR1基因，将其连接到pcDNA3.1(-)载体上，构建pcDNA3.1(-)/FXR1重组载体，并进行酶切鉴定和测序分析。2.通过Lipofectamine2000转染pcDNA3.1(-)/FXR1重组载体至大鼠肾上腺嗜铬细胞瘤PC12细胞和大鼠血管平滑肌细胞VSMC细胞，并通过western blot实验检测转染效率。3. pcDNA3.1(-)/FXR1重组载体转染PC12细胞和VSMC细胞后，观察细胞形态的变化，使用大鼠抗神经节苷脂抗体（GM1）ELISA检测试剂盒检测FXR1过表达引起的神经节苷脂浓度的变化，超微量ATP酶测试盒检测Na+-K+~ATP酶、Ca2+-Mg2+~ATP酶活力的变化，流式细胞术（FCM）检测细胞凋亡情况。结果：1.酶切鉴定和测序分析显示pcDNA3.1(-)/FXR1重组载体构建成功。2. pcDNA3.1(-)/FXR1重组载体成功转染至PC12细胞和VSMC细胞，使FXR1在PC12细胞和VSMC细胞中过表达。3. FXR1的过表达不影响PC12细胞的形态，而FXR1过表达的VSMC细胞呈不规则形；FXR1过表达能显著提高PC12细胞GM1的浓度，而不影响VSMC细胞GM1的浓度；并显著提高PC12细胞Na+-K+~ATP酶和Ca2+-Mg2+~ATP酶的活性，而不影响VSMC细胞Na+-K+~ATP酶和Ca2+-Mg2+~ATP酶的活性；可以显著降低PC12细胞的凋亡率，而不影响VSMC细胞的凋亡率。结论： 1. FXR1基因的过表达可能通过提高PC12细胞中与其相互作用的CMAS的活性，进而提高细胞中GM1的浓度，，对PC12细胞的生长产生保护作用。 2. FXR1P调节GM1的浓度具有组织特异性，PC12细胞是其效应细胞之一，在VSMC细胞中未呈现这种调节作用。
[Abstract]:Fragile X syndrome FXS is one of the most common inherited mental retardation. 99% of the patients were amplified by abnormal CGG trinucleotide repeats in the 5 '-terminal noncoding region of FMR1 or its upstream CpG island hypermethylmethyl. The expression of FMRP(fragile X mental retardation protein (FMRP(fragile X mental retardation protein), the expression product of FMR1, was decreased or deleted. FXR1, a gene associated with mental retardation located at 3q28, belonged to the same gene family as FMR1. The encoded proteins have the properties of binding to proteins and RNA. Based on the previous work in our laboratory, we have proved the interaction between FXR1P and CMAS, and further study the biological effects of FXR1P / CMAS interaction. Aim: to study the biological effects of FXR1P and CMAS interaction in order to elucidate the gene function of FXR1. Methods the FXR1 gene was amplified by PCR and ligated into pcDNA3.1 ~ (-1) vector to construct the recombinant pcDNA3.1(-)/FXR1 vector. The recombinant pcDNA3.1(-)/FXR1 vector was transfected into rat adrenal pheochromocytoma PC12 cells and rat vascular smooth muscle cells VSMC cells by Lipofectamine2000. The transfection efficiency was detected by western blot assay. After transfection of pcDNA3.1(-)/FXR1 recombinant vector into PC12 cells and VSMC cells, the morphological changes of the cells were observed. The changes of ganglioside concentration induced by over-expression of FXR1 were detected by rat anti-ganglioside antibody GM1 Elisa kit, the activity of Na -K ATPase Ca 2 mg 2 + ATPase was detected by ultramicro ATP enzyme test box, and apoptosis was detected by flow cytometry (FCM). Results 1. Restriction endonuclease analysis and sequencing analysis showed that pcDNA3.1(-)/FXR1 recombinant vector was successfully constructed. 2.The recombinant vector of pcDNA3.1(-)/FXR1 was successfully transfected into PC12 cells and VSMC cells. The overexpression of FXR1 in PC12 cells and VSMC cells did not affect the morphology of PC12 cells. The overexpression of FXR1 in VSMC cells with irregular FXR1 significantly increased the concentration of GM1 in PC12 cells, but did not affect the concentration of GM1 in VSMC cells, and significantly increased the activities of Na -K ATPase and Ca2 mg 2 + ATPase in PC12 cells. The activity of Na K ATPase and Ca2 mg 2 + ATPase in VSMC cells was not affected, but the apoptosis rate of PC12 cells was significantly decreased, but the apoptosis rate of VSMC cells was not affected. Conclusion:. 1. Overexpression of FXR1 gene may have protective effect on the growth of PC12 cells by increasing the activity of CMAS interacting with it and increasing the concentration of GM1 in PC12 cells. 2. The effect of FXR1P on the concentration of GM1 was found to be one of the effector cells in VSMC cells, but not in VSMC cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.94

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