發(fā)布時間：2018-04-08 22:33

  本文選題：lncRNA　切入點：大鼠肝細胞BRL-3A　出處：《河南師范大學》2017年碩士論文

【摘要】：近些年來,研究證明lncRNA能夠調(diào)控多種生理和病理進程,包括對大鼠肝再生的調(diào)控。但是lncRNA對肝再生的調(diào)控機制還不清楚。為了了解lncRNA是如何調(diào)控肝再生,本研究采用高通量測序、RT-PCR檢測大鼠2/3肝切除誘導的肝再生3個時間點肝細胞中表達有意義的lncRNA。結(jié)果表明在大鼠肝再生3個時間點肝細胞中表達變化有意義的lncRNA有770條,其中差異表達的lncRNA有28條。分析在肝再生中有意義表達的lncRNA的具體表達情況,發(fā)現(xiàn)有254條表達發(fā)生上調(diào),464條表達下調(diào),52條表達上/下調(diào)。差異表達的28條lncRNA中13條表達上調(diào),12條表達下調(diào),3條表達上/下調(diào)。為了解這些差異表達的lncRNA對肝再生的調(diào)節(jié)作用,采用生物信息學方法預測其靶基因,結(jié)果表明lncRNA的共表達(TRANS作用)基因中表達差異的465條。lncRNA的調(diào)控(CIS作用)基因差異表達的10條。為進一步篩選出待研究的lncRNA及其在肝再生中的作用,用DESeq,結(jié)合GO和KEGG信息分析基因功能發(fā)現(xiàn)基因在細胞中具有多種作用。用Ingenuity中的Ingenuity Pathway Analysis(IPA)檢測大鼠肝再生不同時間點差異表達的基因相應的蛋白質(zhì)在細胞中作用,結(jié)果顯示相關(guān)基因在細胞生長、增殖、分化、細胞通訊、信號傳導、分子運輸、細胞增殖、作為信號分子等方面具有一定的調(diào)控作用。IPA分析信號通路發(fā)現(xiàn)差異表達lncRNA的靶基因主要參與的信號通路有ILK通路、ERK/MAPK通路、SAPK/JNK Signaling通路等。本文從差異表達lncRNA中篩選其中的兩個并研究其對肝細胞增殖的調(diào)控作用。高通量測序結(jié)果表明lncRNA TCONS_00027980和TCONS_00042303在肝再生不同時間點表達水平顯著變化,RT-PCR檢測不同時間點再生肝組織中兩種lncRNA的RNA表達水平,實驗結(jié)果表明高通量測序結(jié)果是可信的。本文首先用干涉技術(shù)降低lncRNA在大鼠BRL-3A細胞中的表達水平,MTT法檢測BRL-3A細胞的活性,流式細胞術(shù)檢測BRL-3A增殖情況的變化,RT-PCR和Western-blot用來檢測與增殖相關(guān)基因的表達情況。檢測結(jié)果表明MTT法檢測表明實驗組比對照組細胞活性增加(P0.05);流式細胞術(shù)實驗檢測顯示與對照組相比,實驗組的S+G2/M期細胞數(shù)在一定程度上增加了(P0.05);而real-time PCR(RT-PCR)檢測mRNA表達水平,結(jié)果表明實驗組與對照組相比,細胞增殖相關(guān)基因MYC、CCNA2、CCND1、BCL-2表達上調(diào)(P0.05),凋亡相關(guān)基因的BAX表達下調(diào)(P0.05);蛋白質(zhì)免疫印跡(Western-blot)檢測表明,實驗組細胞增殖相關(guān)基因MYC、CCNA2、CCND1、BCL-2表達上調(diào)(P0.05),凋亡相關(guān)基因的BAX表達下調(diào)(P0.05)。lncRNA TCONS_00027980和TCONS_00042303能夠通過降低細胞增殖相關(guān)基因的表達水平抑制大鼠肝細胞BRL-3A的細胞增殖。
[Abstract]:In recent years, it has been demonstrated that lncRNA can regulate various physiological and pathological processes, including the regulation of rat liver regeneration.However, the regulatory mechanism of lncRNA on liver regeneration is unclear.In order to understand how lncRNA regulates liver regeneration, we used high-throughput sequencing RT-PCR to detect the expression of LNC in rat hepatocytes induced by 2 / 3 hepatectomy at three time points.The results showed that there were 770 differentially expressed lncRNA in rat liver cells at three time points of liver regeneration, among which 28 were differentially expressed lncRNA.By analyzing the specific expression of lncRNA in liver regeneration, it was found that there were 254 up-regulated, 464 up-regulated and 52 up-/ down-regulated expression of lncRNA.Among 28 differentially expressed lncRNA, 13 were up-regulated, 12 were down-regulated, and 3 were up-/ down-regulated.In order to understand the regulatory effect of differentially expressed lncRNA on liver regeneration, the target genes were predicted by bioinformatics.The results showed that there were 10 differentially expressed genes in the coexpression of lncRNA.In order to screen out the lncRNA and its role in liver regeneration, we used DESeq, go and KEGG information to analyze the gene function and found that the gene has many functions in the cell.Ingenuity Pathway analysis in Ingenuity was used to detect the role of proteins of differentially expressed genes at different time points in rat liver regeneration. The results showed that the related genes were involved in cell growth, proliferation, differentiation, cell communication, signal transduction and molecular transport.IPA analysis showed that the target genes involved in the differential expression of lncRNA were mainly involved in the ILK pathway, ERK / MAPK pathway, SAPKP / JNK Signaling pathway and so on.Two of them were screened from differentially expressed lncRNA and their effects on hepatocyte proliferation were studied.The results of high-throughput sequencing showed that the expression levels of lncRNA TCONS_00027980 and TCONS_00042303 were significantly changed at different time points during liver regeneration. RT-PCR was used to detect the RNA expression levels of two kinds of lncRNA in regenerated liver tissues at different time points. The results showed that the results of high-throughput sequencing were reliable.In this paper, the expression level of lncRNA in rat BRL-3A cells was reduced by interference technique. The activity of BRL-3A cells was detected by MTT assay. The changes of BRL-3A proliferation were detected by flow cytometry. RT-PCR and Western-blot were used to detect the expression of proliferation-related genes.The results showed that the cell activity of the experimental group was higher than that of the control group by MTT, the number of S G _ 2 / M phase cells of the experimental group was increased to some extent than that of the control group, and the expression of mRNA was detected by real-time PCRRT-PCR.The results showed that the expression of MYCnCCNA2CCND1 / BCL-2 was up-regulated, the expression of apoptosis-related gene BAX was down-regulated (P0.05), and the Western blot assay showed that the expression of MYC- CCNA2CCND1- BCL-2 was down-regulated by Western blot (Western blot), and the expression of apoptosis-related gene was down-regulated.In the experimental group, the expression of MYCnCCNA2CCND1 / BCL-2 gene was up-regulated, and the BAX expression of apoptosis-related gene down-regulated P0.05. lncRNA TCONS_00027980 and TCONS_00042303 could inhibit the proliferation of rat hepatocyte BRL-3A by decreasing the expression level of cell proliferation-related genes.
【學位授予單位】：河南師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575

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本文編號：1723673