抑制腸道病毒71型復制的micro RNA和先導化合物的研究
發(fā)布時間：2019-03-21 17:19
【摘要】：近年來,手足口病在世界多個地區(qū),尤其是亞洲爆發(fā)并流行,且其感染和死亡率逐年增高,危害十分嚴重。腸道病毒71(Enterovirus71, EV71)是手足口病(Hand, foot, and mouth disease, HFMD)的主要病原體,以感染嬰幼兒為主,其感染常伴隨神經系統(tǒng)并發(fā)癥,嚴重可導致兒童死亡。因此,EV71的分子生物學研究,預防及治療EV71感染的藥物開發(fā),已成為當前病毒學研究領域的熱點。 miRNAs是在真核生物中發(fā)現(xiàn)的一類內源性長度約為22個核苷酸單鏈非編碼RNA,通過降解靶mRNA分子或抑制靶基因mRNA的翻譯,參與轉錄后基因表達的調控過程。已經證實miRNAs在很多生物學過程中起著重要的作用,越來越多的研究表明miRNAs還參與調控病毒在宿主細胞內的感染和復制過程,本文對miRNAs在宿主細胞內調控EV71病毒的復制的作用進行了研究。 首先構建了miRNAs靶基因篩選系統(tǒng)。我們在Luciferase雙檢測體系的pMIR載體插入待檢基因,如果插入的基因序列能被細胞內的miRNAs靶向調控,報告基因的表達將發(fā)生變化。通過實驗,我們發(fā)現(xiàn)插入EV71病毒5'-UTR基因的pMIR載體,報告基因的表達下降了2.5倍左右。進一步將5'-UTR基因打斷成260bp左右的片段,同樣進行檢測,發(fā)現(xiàn)5'-UTR-1(1-270)片段使Luciferase的表達下降了2.5倍左右,所以推測5'-UTR-1片段可能是miRNAs的作用靶標。 隨后我們利用在線分析軟件,預測可能作用于5'-UTR-1基因片段的1niRNAs,選擇其中的miR-373和miR-542-5p合成minics,檢測niRNAs對5'-UTR-1基因片段的作用,結果顯示,二者都可以下調報告基因的表達。但miRNAs分子對5'-UTR-1基因的調控作用是否可以體現(xiàn)在EV71病毒的復制過程中?為此,我們選擇了miR-373和miR-542-5p做了進一步研究。我們在RD細胞中轉染miRNAs minics,6h后感染EV71病毒。利用Western blot和real-time PCR實驗檢驗病毒的復制情況。結果顯示,miR-373和miR-542-5p可以抑制EV71病毒在RD細胞中的復制。但是miR-373和miR-542-5p的具體作用機制還有待進一步研究。 本論文的另一部分內容是進行抗EV71病毒復制的先導化合物的篩選。新藥的發(fā)現(xiàn)路徑大致為化合物庫的合成和靶的開發(fā),高通量的篩選,選出有希望的先導化合物,接下來進行先導化合物的最優(yōu)化,臨床實驗直到新藥的上市。本實驗由武漢大學提供6種化學合成的醫(yī)藥中間體,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-對羥基苯甘氨酸甲酯(Methyl D-(-)-4-hydroxy-phenylglycinate),4-氯苯甘氨酸((R)-4-Chlorophenyl glycine),3,4-二羥基苯乙酸甲酯(Methyl3,4-dihydroxyphenylacetatate),4-氯-2-異丙基-5-甲苯酚(4-Chloro-5-methyl-2-(1-methylethyl)-phenol)以及2種五肽化合物—P010157和P010158。 首先通過Western blot進行篩選,發(fā)現(xiàn)3,4-二羥基苯乙酸甲酯對EV71在RD細胞中的復制具有明顯的抑制效果。接下來利用MTT法初步檢測3,4-二羥基苯乙酸甲酯的細胞毒性,結果表明CC50為0.0726μg/μL,細胞毒性較低。Real-time PCR檢驗3,4-二羥基苯乙酸甲酯對EV71病毒復制的影響,當終濃度為0.01μg/μL時對EV71病毒復制的抑制率為76.83+2.47%。以上結果表明3,4-二羥基苯乙酸甲酯對EV71病毒在RD細胞中的復制具有抑制作用。感染EV71病毒的一日齡ICR乳鼠注射3,4-二羥基苯乙酸甲酯,生長狀態(tài)明顯好于陽性對照組,real-time PCR檢測3,4-二羥基苯乙酸甲酯對EV71病毒在乳鼠體內的復制具有很好的抑制效果。以上結果證明3,4-二羥基苯乙酸甲酯是一種具有開發(fā)潛力的抗EV71病毒復制藥物,但具體的作用機制還有待進一步研究,并且還需要根據(jù)EV71病毒的基因組特點,優(yōu)化3,4-二羥基苯乙酸甲酯的結構,合成效果更好的衍生物。 采用同樣的思路對比2種五肽化合物的實驗結果,Western blot發(fā)現(xiàn)P010157能夠抑制EV71病毒在RD細胞中的復制,CC50大約可達到155.7378μg/μL。Real-time PCR結果顯示,當P010157終濃度為0.1μg/μL時對EV71病毒復制的抑制率為91.84+2.04%。以上結果說明P010157具有很好的開發(fā)價值。 本文從miRNAs和小分子化合物兩方面研究了它們對EV71病毒在宿主細胞中復制的調控作用,有了初步的研究結果,為利用1miRNAs和小分子化合物抑制EV71病毒復制的相關研究提供了有價值的參考。
[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.
【學位授予單位】：遼寧師范大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373
本文編號：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.
【學位授予單位】：遼寧師范大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373
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