小鼠CYP2R1基因的序列分析、真核表達及其對Hela細胞增殖作用的研究
發(fā)布時間：2018-12-24 14:00
【摘要】：維生素D需經(jīng)過兩步的酶促反應(yīng)(25-羥基化作用和1α-羥基化作用),才能產(chǎn)生有活性的1α,25(OH)2D。其中,CYP2R1是生物體內(nèi)催化維生素D的25-羥基化作用的關(guān)鍵酶,該過程是活性維生素D合成的重要環(huán)節(jié)。目前,國內(nèi)外對小鼠CYP2R1的認識還非常有限,其蛋白結(jié)構(gòu)、理化性質(zhì)和病理學(xué)功能等也尚不清楚。此外,隨著維生素D缺乏癥的發(fā)生,維生素D藥物的需求量增加,其生物學(xué)轉(zhuǎn)化也具有廣闊的應(yīng)用前景,因此,維生素D 25-羥基化酶CYP2R1的相關(guān)研究顯得尤為重要。本研究首先利用生物信息學(xué)軟件對小鼠CYP2R1序列進行分析,為小鼠CYP2R1理化性質(zhì)的研究及其定向改造提供參考;其次,克隆小鼠CYP2R1基因并在宮頸癌(Hela)細胞中進行表達,以構(gòu)建真核高表達載體,為活性維生素D的生物學(xué)轉(zhuǎn)化提供實驗基礎(chǔ);此外,通過細胞劃痕實驗、MTT檢測和qPCR檢測,初步探討小鼠CYP2R1對Hela細胞增殖的影響,為小鼠CYP2R1的相關(guān)功能研究提供了實驗基礎(chǔ)。主要結(jié)果如下:(1)多物種CYP2R1一級結(jié)構(gòu)序列的C端保守性均強于N端,以人CYP2R1序列為標(biāo)準(zhǔn),確定了小鼠CYP2R1與人CYP2R1序列中P450結(jié)構(gòu)功能域差異性位點31個,包括Cys53,Ser149,Ser164,Gln288,Tyr354等。預(yù)測了小鼠CYP2R1序列中的血紅素結(jié)合位點Arg109,Trp133,His381,Ser442,Arg446位點、底物結(jié)合區(qū)Thr344,Val375,Ile379,Met487,Thr488等位點及氧化還原配體結(jié)合位點Arg131,Arg138,Lys434,Lys435,Arg445,Arg455。以結(jié)構(gòu)明確的人CYP2R1為標(biāo)準(zhǔn),比較了11個物種CYP2R1的二級結(jié)構(gòu);并以人CYP2R1結(jié)構(gòu)為模板,對小鼠CYP2R1進行了同源建模,比較了小鼠CYP2R1與人CYP2R1三級結(jié)構(gòu)的差異。(2)利用融合有His標(biāo)簽的pcDNA3.1(+)構(gòu)建了小鼠CYP2R1真核表達載體pcDNA3.1-CYP2R1,該載體能有效提高CYP2R1的mRNA和蛋白表達水平,其中mRNA水平提高了82524.59倍。(3)小鼠CYP2R1能夠同時上調(diào)細胞周期調(diào)控因子CyclinD1、P27和P21基因的表達,其中重組質(zhì)粒轉(zhuǎn)染組的CyclinD1和P27的相對表達量相比空載質(zhì)粒轉(zhuǎn)染組具有極顯著差異(p0.01)。推測CYP2R1參與了CyclinD1和P27對細胞周期的調(diào)控,但可能形成反饋調(diào)節(jié)系統(tǒng)CyclinD1-CDK-P27,導(dǎo)致Hela細胞的增殖變化不明顯。
[Abstract]:Vitamin D needs two steps of enzymatic reaction (25-hydroxylation and 1 偽 -hydroxylation) to produce active 1 偽, 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.
【學(xué)位授予單位】：陜西理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R591.44
本文編號：2390717
[Abstract]:Vitamin D needs two steps of enzymatic reaction (25-hydroxylation and 1 偽 -hydroxylation) to produce active 1 偽, 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.
【學(xué)位授予單位】：陜西理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R591.44
【參考文獻】
相關(guān)期刊論文 前2條
1 曹雨娜;張虹;;維生素D抗腫瘤作用的研究進展[J];中國臨床藥學(xué)雜志;2014年02期
2 尤程程;王艷林;尤昌昌;黃益玲;任東明;黃利鳴;;高表達p27基因?qū)ψ訉m頸癌HeLa細胞增殖的影響[J];臨床與實驗病理學(xué)雜志;2012年06期
，本文編號：2390717
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