抑制肠道病毒71型复制的micro RNA和先导化合物的研究
发布时间：2019-03-21 17:19
【摘要】：近年来,手足口病在世界多个地区,尤其是亚洲爆发并流行,且其感染和死亡率逐年增高,危害十分严重。肠道病毒71(Enterovirus71, EV71)是手足口病(Hand, foot, and mouth disease, HFMD)的主要病原体,以感染婴幼儿为主,其感染常伴随神经系统并发症,严重可导致儿童死亡。因此,EV71的分子生物学研究,预防及治疗EV71感染的药物开发,已成为当前病毒学研究领域的热点。 miRNAs是在真核生物中发现的一类内源性长度约为22个核苷酸单链非编码RNA,通过降解靶mRNA分子或抑制靶基因mRNA的翻译,参与转录后基因表达的调控过程。已经证实miRNAs在很多生物学过程中起着重要的作用,越来越多的研究表明miRNAs还参与调控病毒在宿主细胞内的感染和复制过程,本文对miRNAs在宿主细胞内调控EV71病毒的复制的作用进行了研究。 首先构建了miRNAs靶基因筛选系统。我们在Luciferase双检测体系的pMIR载体插入待检基因,如果插入的基因序列能被细胞内的miRNAs靶向调控,报告基因的表达将发生变化。通过实验,我们发现插入EV71病毒5'-UTR基因的pMIR载体,报告基因的表达下降了2.5倍左右。进一步将5'-UTR基因打断成260bp左右的片段,同样进行检测,发现5'-UTR-1(1-270)片段使Luciferase的表达下降了2.5倍左右,所以推测5'-UTR-1片段可能是miRNAs的作用靶标。 随后我们利用在线分析软件,预测可能作用于5'-UTR-1基因片段的1niRNAs,选择其中的miR-373和miR-542-5p合成minics,检测niRNAs对5'-UTR-1基因片段的作用,结果显示,二者都可以下调报告基因的表达。但miRNAs分子对5'-UTR-1基因的调控作用是否可以体现在EV71病毒的复制过程中?为此,我们选择了miR-373和miR-542-5p做了进一步研究。我们在RD细胞中转染miRNAs minics,6h后感染EV71病毒。利用Western blot和real-time PCR实验检验病毒的复制情况。结果显示,miR-373和miR-542-5p可以抑制EV71病毒在RD细胞中的复制。但是miR-373和miR-542-5p的具体作用机制还有待进一步研究。 本论文的另一部分内容是进行抗EV71病毒复制的先导化合物的筛选。新药的发现路径大致为化合物库的合成和靶的开发,高通量的筛选,选出有希望的先导化合物,接下来进行先导化合物的最优化,临床实验直到新药的上市。本实验由武汉大学提供6种化学合成的医药中间体,3,4-二甲氧基苯甲酸甲酯(Methyl3,4-dimethoxybenzoate),3,4-二甲氧基苯乙酸甲酯(Methyl3,4-dimethoxyphenylacetate),D-对羟基苯甘氨酸甲酯(Methyl D-(-)-4-hydroxy-phenylglycinate),4-氯苯甘氨酸((R)-4-Chlorophenyl glycine),3,4-二羟基苯乙酸甲酯(Methyl3,4-dihydroxyphenylacetatate),4-氯-2-异丙基-5-甲苯酚(4-Chloro-5-methyl-2-(1-methylethyl)-phenol)以及2种五肽化合物—P010157和P010158。 首先通过Western blot进行筛选,发现3,4-二羟基苯乙酸甲酯对EV71在RD细胞中的复制具有明显的抑制效果。接下来利用MTT法初步检测3,4-二羟基苯乙酸甲酯的细胞毒性,结果表明CC50为0.0726μg/μL,细胞毒性较低。Real-time PCR检验3,4-二羟基苯乙酸甲酯对EV71病毒复制的影响,当终浓度为0.01μg/μL时对EV71病毒复制的抑制率为76.83+2.47%。以上结果表明3,4-二羟基苯乙酸甲酯对EV71病毒在RD细胞中的复制具有抑制作用。感染EV71病毒的一日龄ICR乳鼠注射3,4-二羟基苯乙酸甲酯,生长状态明显好于阳性对照组,real-time PCR检测3,4-二羟基苯乙酸甲酯对EV71病毒在乳鼠体内的复制具有很好的抑制效果。以上结果证明3,4-二羟基苯乙酸甲酯是一种具有开发潜力的抗EV71病毒复制药物,但具体的作用机制还有待进一步研究,并且还需要根据EV71病毒的基因组特点,优化3,4-二羟基苯乙酸甲酯的结构,合成效果更好的衍生物。 采用同样的思路对比2种五肽化合物的实验结果,Western blot发现P010157能够抑制EV71病毒在RD细胞中的复制,CC50大约可达到155.7378μg/μL。Real-time PCR结果显示,当P010157终浓度为0.1μg/μL时对EV71病毒复制的抑制率为91.84+2.04%。以上结果说明P010157具有很好的开发价值。 本文从miRNAs和小分子化合物两方面研究了它们对EV71病毒在宿主细胞中复制的调控作用,有了初步的研究结果,为利用1miRNAs和小分子化合物抑制EV71病毒复制的相关研究提供了有价值的参考。
[Abstract]:In recent years, hand-foot-mouth disease is in many parts of the world, especially in Asia, and its infection and mortality are increasing year by year, and the harm is very serious. Enterovirus 71 (EV71) is the main pathogen of hand-foot-mouth disease (HFMD), which is the main pathogen of hand-foot-mouth disease (HFMD). Therefore, the molecular biological study of EV71, the prevention and treatment of the drug development of the EV71 infection, has become a hot spot in the current virology research field. MiRNAs are a class of non-coding RNAs with an intrinsic length of about 22 nucleotides found in eukaryotes, and are involved in the regulation of post-transcriptional gene expression by degrading the target mRNA molecule or inhibiting the translation of the target gene mRNA. It has been shown that miRNAs play an important role in many biological processes, and more and more studies have shown that miRNAs are also involved in the process of controlling the infection and replication of the virus in the host cell. The effect of miRNAs on the replication of the EV71 virus in the host cell has been studied in this paper. Research. First, the miRNAs target gene screen was constructed. The gene is inserted into the pMIR vector of the Lucifasse double-detection system, and if the inserted gene sequence can be targeted and controlled by miRNAs in the cell, the expression of the reporter gene will be generated. The results showed that the expression of the reporter gene decreased by 2.5 in the pMIR vector of the 5 '-UTR gene inserted into the EV71 virus. The 5 '-UTR-1 (1-270) fragment was further detected to reduce the expression of Lucifasse by about 2.5 times, so it was suggested that the 5'-UTR-1 fragment could be miRNAs. The target is used. Then we use the online analysis software to predict the 1 niRNAs that may act on the 5 '-UTR-1 gene fragment, select the miR-373 and miR-542-5p synthesis mintics, to detect the effect of niRNAs on the 5'-UTR-1 gene fragment, and the results show that both can downregulate the report. The effect of miRNAs on the 5 '-UTR-1 gene can be reflected in the EV71 virus. During the replication process, we selected miR-373 and miR-542-5p for this purpose Further study. We transfect miRNAs minics in RD cells for 6 h post-infection E V71 virus. The virus was tested by Western blot and real-time PCR. The results show that miR-373 and miR-542-5p can inhibit EV71 virus from being fine in RD Replication in the cell. However, the specific mechanism of action of miR-373 and miR-542-5p is yet to be Further study. Another part of this paper is the first step in the replication of the anti-EV71 virus screening of the guide compound. The discovery path of the new drug is generally the synthesis of the compound library and the development of the target, high-throughput screening, the selection of the desired pilot compound, the subsequent optimization of the pilot compound, the clinical experiment, Up to the marketing of new drugs,6 chemical intermediates, 3,4-dimethxybenzoate, 3,4-dimethoxyphenylacetate, D-p-hydroxyphenylglycine ((R) -4-chlorophenyl, D-p-hydroxyphenylglycine ((R) -4-chlorophenyl), and 4-chlorophenylglycine ((R) -4-chlorophenyl) are provided by the Wuhan University. lycine, 3,4-dihydroxyphenylacetate,4-chloro-2-isopropyl-5-methylphenol (4-chloroo-5-methyl-2-(1-methylethyl)-phrenol) and two pentapeptide compounds, pP010157 and P010158. The screening was carried out by Western blot and the replication of the 3,4-dihydroxyphenylacetate to the EV71 in the RD cells was found. The cytotoxicity of 3,4-dihydroxyphenylacetic acid methyl ester was detected by MTT method. The results showed that the CC50 was 0.0726. The effect of 3,4-dihydroxyphenylacetate on the replication of EV71 virus was tested by Real-time PCR, and the inhibition rate for EV71 virus replication was 76 when the final concentration was 0.01. m u.g/. .83 + 2.47%. The above results indicate that the 3,4-dihydroxyphenylacetic acid methyl ester is used for EV71 virus in RD cells The replication of EV71 virus was significantly better than that of the positive control group. The real-time PCR was used to detect the replication of the 3,4-dihydroxyphenylacetic acid methyl ester to the EV71 virus in the rat. The above results show that the 3,4-dihydroxyphenylacetic acid methyl ester is an anti-EV71 virus replication drug with the development potential, but the specific mechanism of action is to be further studied, and it is also necessary to optimize the 3,4-dihydroxyphenylacetate according to the genomic characteristics of the EV71 virus. the structure and the combination of the phenylacetic acid methyl ester The results showed that P010157 could inhibit the replication of EV71 virus in RD cells. The results showed that when the final concentration of P010157 was 0.1. m u.g/. 91.84 + 2.04%. The above results indicate P0101 57. The effect of their replication on the host cells of the EV71 virus was studied from the aspects of miRNAs and small molecular compounds. The results of the preliminary study were as follows:1 miRNAs and small molecular compounds were used to inhibit the replication of the EV71 virus.
【学位授予单位】：辽宁师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
本文编号：2445147
[Abstract]:In recent years, hand-foot-mouth disease is in many parts of the world, especially in Asia, and its infection and mortality are increasing year by year, and the harm is very serious. Enterovirus 71 (EV71) is the main pathogen of hand-foot-mouth disease (HFMD), which is the main pathogen of hand-foot-mouth disease (HFMD). Therefore, the molecular biological study of EV71, the prevention and treatment of the drug development of the EV71 infection, has become a hot spot in the current virology research field. MiRNAs are a class of non-coding RNAs with an intrinsic length of about 22 nucleotides found in eukaryotes, and are involved in the regulation of post-transcriptional gene expression by degrading the target mRNA molecule or inhibiting the translation of the target gene mRNA. It has been shown that miRNAs play an important role in many biological processes, and more and more studies have shown that miRNAs are also involved in the process of controlling the infection and replication of the virus in the host cell. The effect of miRNAs on the replication of the EV71 virus in the host cell has been studied in this paper. Research. First, the miRNAs target gene screen was constructed. The gene is inserted into the pMIR vector of the Lucifasse double-detection system, and if the inserted gene sequence can be targeted and controlled by miRNAs in the cell, the expression of the reporter gene will be generated. The results showed that the expression of the reporter gene decreased by 2.5 in the pMIR vector of the 5 '-UTR gene inserted into the EV71 virus. The 5 '-UTR-1 (1-270) fragment was further detected to reduce the expression of Lucifasse by about 2.5 times, so it was suggested that the 5'-UTR-1 fragment could be miRNAs. The target is used. Then we use the online analysis software to predict the 1 niRNAs that may act on the 5 '-UTR-1 gene fragment, select the miR-373 and miR-542-5p synthesis mintics, to detect the effect of niRNAs on the 5'-UTR-1 gene fragment, and the results show that both can downregulate the report. The effect of miRNAs on the 5 '-UTR-1 gene can be reflected in the EV71 virus. During the replication process, we selected miR-373 and miR-542-5p for this purpose Further study. We transfect miRNAs minics in RD cells for 6 h post-infection E V71 virus. The virus was tested by Western blot and real-time PCR. The results show that miR-373 and miR-542-5p can inhibit EV71 virus from being fine in RD Replication in the cell. However, the specific mechanism of action of miR-373 and miR-542-5p is yet to be Further study. Another part of this paper is the first step in the replication of the anti-EV71 virus screening of the guide compound. The discovery path of the new drug is generally the synthesis of the compound library and the development of the target, high-throughput screening, the selection of the desired pilot compound, the subsequent optimization of the pilot compound, the clinical experiment, Up to the marketing of new drugs,6 chemical intermediates, 3,4-dimethxybenzoate, 3,4-dimethoxyphenylacetate, D-p-hydroxyphenylglycine ((R) -4-chlorophenyl, D-p-hydroxyphenylglycine ((R) -4-chlorophenyl), and 4-chlorophenylglycine ((R) -4-chlorophenyl) are provided by the Wuhan University. lycine, 3,4-dihydroxyphenylacetate,4-chloro-2-isopropyl-5-methylphenol (4-chloroo-5-methyl-2-(1-methylethyl)-phrenol) and two pentapeptide compounds, pP010157 and P010158. The screening was carried out by Western blot and the replication of the 3,4-dihydroxyphenylacetate to the EV71 in the RD cells was found. The cytotoxicity of 3,4-dihydroxyphenylacetic acid methyl ester was detected by MTT method. The results showed that the CC50 was 0.0726. The effect of 3,4-dihydroxyphenylacetate on the replication of EV71 virus was tested by Real-time PCR, and the inhibition rate for EV71 virus replication was 76 when the final concentration was 0.01. m u.g/. .83 + 2.47%. The above results indicate that the 3,4-dihydroxyphenylacetic acid methyl ester is used for EV71 virus in RD cells The replication of EV71 virus was significantly better than that of the positive control group. The real-time PCR was used to detect the replication of the 3,4-dihydroxyphenylacetic acid methyl ester to the EV71 virus in the rat. The above results show that the 3,4-dihydroxyphenylacetic acid methyl ester is an anti-EV71 virus replication drug with the development potential, but the specific mechanism of action is to be further studied, and it is also necessary to optimize the 3,4-dihydroxyphenylacetate according to the genomic characteristics of the EV71 virus. the structure and the combination of the phenylacetic acid methyl ester The results showed that P010157 could inhibit the replication of EV71 virus in RD cells. The results showed that when the final concentration of P010157 was 0.1. m u.g/. 91.84 + 2.04%. The above results indicate P0101 57. The effect of their replication on the host cells of the EV71 virus was studied from the aspects of miRNAs and small molecular compounds. The results of the preliminary study were as follows:1 miRNAs and small molecular compounds were used to inhibit the replication of the EV71 virus.
【学位授予单位】：辽宁师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
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