小鼠CYP2R1基因的序列分析、真核表达及其对Hela细胞增殖作用的研究
发布时间：2018-12-24 14:00
【摘要】：维生素D需经过两步的酶促反应(25-羟基化作用和1α-羟基化作用),才能产生有活性的1α,25(OH)2D。其中,CYP2R1是生物体内催化维生素D的25-羟基化作用的关键酶,该过程是活性维生素D合成的重要环节。目前,国内外对小鼠CYP2R1的认识还非常有限,其蛋白结构、理化性质和病理学功能等也尚不清楚。此外,随着维生素D缺乏症的发生,维生素D药物的需求量增加,其生物学转化也具有广阔的应用前景,因此,维生素D 25-羟基化酶CYP2R1的相关研究显得尤为重要。本研究首先利用生物信息学软件对小鼠CYP2R1序列进行分析,为小鼠CYP2R1理化性质的研究及其定向改造提供参考;其次,克隆小鼠CYP2R1基因并在宫颈癌(Hela)细胞中进行表达,以构建真核高表达载体,为活性维生素D的生物学转化提供实验基础;此外,通过细胞划痕实验、MTT检测和qPCR检测,初步探讨小鼠CYP2R1对Hela细胞增殖的影响,为小鼠CYP2R1的相关功能研究提供了实验基础。主要结果如下:(1)多物种CYP2R1一级结构序列的C端保守性均强于N端,以人CYP2R1序列为标准,确定了小鼠CYP2R1与人CYP2R1序列中P450结构功能域差异性位点31个,包括Cys53,Ser149,Ser164,Gln288,Tyr354等。预测了小鼠CYP2R1序列中的血红素结合位点Arg109,Trp133,His381,Ser442,Arg446位点、底物结合区Thr344,Val375,Ile379,Met487,Thr488等位点及氧化还原配体结合位点Arg131,Arg138,Lys434,Lys435,Arg445,Arg455。以结构明确的人CYP2R1为标准,比较了11个物种CYP2R1的二级结构;并以人CYP2R1结构为模板,对小鼠CYP2R1进行了同源建模,比较了小鼠CYP2R1与人CYP2R1三级结构的差异。(2)利用融合有His标签的pcDNA3.1(+)构建了小鼠CYP2R1真核表达载体pcDNA3.1-CYP2R1,该载体能有效提高CYP2R1的mRNA和蛋白表达水平,其中mRNA水平提高了82524.59倍。(3)小鼠CYP2R1能够同时上调细胞周期调控因子CyclinD1、P27和P21基因的表达,其中重组质粒转染组的CyclinD1和P27的相对表达量相比空载质粒转染组具有极显著差异(p0.01)。推测CYP2R1参与了CyclinD1和P27对细胞周期的调控,但可能形成反馈调节系统CyclinD1-CDK-P27,导致Hela细胞的增殖变化不明显。
[Abstract]:Vitamin D needs two steps of enzymatic reaction (25-hydroxylation and 1 伪 -hydroxylation) to produce active 1 伪, 25 (OH) 2D. Among them, CYP2R1 is the key enzyme to catalyze the 25-hydroxylation of vitamin D in vivo, and this process is an important step in the synthesis of active vitamin D. At present, the understanding of mouse CYP2R1 at home and abroad is very limited, and its protein structure, physicochemical properties and pathological function are not clear. In addition, with the occurrence of vitamin D deficiency and the increasing demand for vitamin D drugs, the biological transformation of vitamin D has a broad prospect. Therefore, the study of vitamin D 25-hydroxylase CYP2R1 is particularly important. In this study, the CYP2R1 sequence of mice was analyzed by bioinformatics software, which provided a reference for the study of physicochemical properties of mouse CYP2R1 and its directional modification. Secondly, mouse CYP2R1 gene was cloned and expressed in cervical cancer (Hela) cells to construct eukaryotic expression vector, which provided experimental basis for biological transformation of active vitamin D. In addition, the effects of mouse CYP2R1 on the proliferation of Hela cells were preliminarily studied by cell scratch test, MTT detection and qPCR detection, which provided the experimental basis for the study of the related function of mouse CYP2R1. The main results are as follows: (1) the C-terminal conservation of multispecies CYP2R1 primary structure sequence is stronger than that of N terminal. According to the standard of human CYP2R1 sequence, 31 P450 functional domain differences in mouse CYP2R1 and human CYP2R1 sequences were identified, including Cys53,Ser149,Ser164,Gln288,. Tyr354 et al. The heme binding site (Arg109,Trp133,His381,Ser442,Arg446), substrate binding site (Thr344,Val375,Ile379,Met487,Thr488) and redox ligand binding site (Arg131,Arg138,Lys434,Lys435,Arg445,Arg455.) in mouse CYP2R1 sequence were predicted. The secondary structures of CYP2R1 in 11 species were compared according to the standard of human CYP2R1. Using human CYP2R1 structure as template, the homologous model of mouse CYP2R1 was established, and the difference of tertiary structure between mouse CYP2R1 and human CYP2R1 was compared. (2) the mouse CYP2R1 eukaryotic expression vector pcDNA3.1-CYP2R1, was constructed by using pcDNA3.1 () with His tag. The vector could effectively increase the expression of mRNA and protein in CYP2R1, and the level of mRNA increased by 82524.59 times. (3) Mouse CYP2R1 could up-regulate the expression of CyclinD1,P27 and P21 genes at the same time. The relative expression of CyclinD1 and P27 in the recombinant plasmid transfection group was significantly higher than that in the no-load plasmid transfection group (p0.01). It is speculated that CYP2R1 is involved in the regulation of cell cycle by CyclinD1 and P27, but CyclinD1-CDK-P27, may form a feedback regulatory system, which leads to no obvious changes in the proliferation of Hela cells.
【学位授予单位】：陕西理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R591.44
本文编号：2390717
[Abstract]:Vitamin D needs two steps of enzymatic reaction (25-hydroxylation and 1 伪 -hydroxylation) to produce active 1 伪, 25 (OH) 2D. Among them, CYP2R1 is the key enzyme to catalyze the 25-hydroxylation of vitamin D in vivo, and this process is an important step in the synthesis of active vitamin D. At present, the understanding of mouse CYP2R1 at home and abroad is very limited, and its protein structure, physicochemical properties and pathological function are not clear. In addition, with the occurrence of vitamin D deficiency and the increasing demand for vitamin D drugs, the biological transformation of vitamin D has a broad prospect. Therefore, the study of vitamin D 25-hydroxylase CYP2R1 is particularly important. In this study, the CYP2R1 sequence of mice was analyzed by bioinformatics software, which provided a reference for the study of physicochemical properties of mouse CYP2R1 and its directional modification. Secondly, mouse CYP2R1 gene was cloned and expressed in cervical cancer (Hela) cells to construct eukaryotic expression vector, which provided experimental basis for biological transformation of active vitamin D. In addition, the effects of mouse CYP2R1 on the proliferation of Hela cells were preliminarily studied by cell scratch test, MTT detection and qPCR detection, which provided the experimental basis for the study of the related function of mouse CYP2R1. The main results are as follows: (1) the C-terminal conservation of multispecies CYP2R1 primary structure sequence is stronger than that of N terminal. According to the standard of human CYP2R1 sequence, 31 P450 functional domain differences in mouse CYP2R1 and human CYP2R1 sequences were identified, including Cys53,Ser149,Ser164,Gln288,. Tyr354 et al. The heme binding site (Arg109,Trp133,His381,Ser442,Arg446), substrate binding site (Thr344,Val375,Ile379,Met487,Thr488) and redox ligand binding site (Arg131,Arg138,Lys434,Lys435,Arg445,Arg455.) in mouse CYP2R1 sequence were predicted. The secondary structures of CYP2R1 in 11 species were compared according to the standard of human CYP2R1. Using human CYP2R1 structure as template, the homologous model of mouse CYP2R1 was established, and the difference of tertiary structure between mouse CYP2R1 and human CYP2R1 was compared. (2) the mouse CYP2R1 eukaryotic expression vector pcDNA3.1-CYP2R1, was constructed by using pcDNA3.1 () with His tag. The vector could effectively increase the expression of mRNA and protein in CYP2R1, and the level of mRNA increased by 82524.59 times. (3) Mouse CYP2R1 could up-regulate the expression of CyclinD1,P27 and P21 genes at the same time. The relative expression of CyclinD1 and P27 in the recombinant plasmid transfection group was significantly higher than that in the no-load plasmid transfection group (p0.01). It is speculated that CYP2R1 is involved in the regulation of cell cycle by CyclinD1 and P27, but CyclinD1-CDK-P27, may form a feedback regulatory system, which leads to no obvious changes in the proliferation of Hela cells.
【学位授予单位】：陕西理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R591.44
【参考文献】
相关期刊论文 前2条
1 曹雨娜;张虹;;维生素D抗肿瘤作用的研究进展[J];中国临床药学杂志;2014年02期
2 尤程程;王艳林;尤昌昌;黄益玲;任东明;黄利鸣;;高表达p27基因对子宫颈癌HeLa细胞增殖的影响[J];临床与实验病理学杂志;2012年06期
，本文编号：2390717
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