本文選題：lncRNA　切入點(diǎn)：大鼠肝細(xì)胞BRL-3A　出處：《河南師范大學(xué)》2017年碩士論文

【摘要】：近些年來(lái),研究證明lncRNA能夠調(diào)控多種生理和病理進(jìn)程,包括對(duì)大鼠肝再生的調(diào)控。但是lncRNA對(duì)肝再生的調(diào)控機(jī)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(cè)序、RT-PCR檢測(cè)大鼠2/3肝切除誘導(dǎo)的肝再生3個(gè)時(shí)間點(diǎn)肝細(xì)胞中表達(dá)有意義的lncRNA。結(jié)果表明在大鼠肝再生3個(gè)時(shí)間點(diǎn)肝細(xì)胞中表達(dá)變化有意義的lncRNA有770條,其中差異表達(dá)的lncRNA有28條。分析在肝再生中有意義表達(dá)的lncRNA的具體表達(dá)情況,發(fā)現(xiàn)有254條表達(dá)發(fā)生上調(diào),464條表達(dá)下調(diào),52條表達(dá)上/下調(diào)。差異表達(dá)的28條lncRNA中13條表達(dá)上調(diào),12條表達(dá)下調(diào),3條表達(dá)上/下調(diào)。為了解這些差異表達(dá)的lncRNA對(duì)肝再生的調(diào)節(jié)作用,采用生物信息學(xué)方法預(yù)測(cè)其靶基因,結(jié)果表明lncRNA的共表達(dá)(TRANS作用)基因中表達(dá)差異的465條。lncRNA的調(diào)控(CIS作用)基因差異表達(dá)的10條。為進(jìn)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(xì)胞中具有多種作用。用Ingenuity中的Ingenuity Pathway Analysis(IPA)檢測(cè)大鼠肝再生不同時(shí)間點(diǎn)差異表達(dá)的基因相應(yīng)的蛋白質(zhì)在細(xì)胞中作用,結(jié)果顯示相關(guān)基因在細(xì)胞生長(zhǎng)、增殖、分化、細(xì)胞通訊、信號(hào)傳導(dǎo)、分子運(yùn)輸、細(xì)胞增殖、作為信號(hào)分子等方面具有一定的調(diào)控作用。IPA分析信號(hào)通路發(fā)現(xiàn)差異表達(dá)lncRNA的靶基因主要參與的信號(hào)通路有ILK通路、ERK/MAPK通路、SAPK/JNK Signaling通路等。本文從差異表達(dá)lncRNA中篩選其中的兩個(gè)并研究其對(duì)肝細(xì)胞增殖的調(diào)控作用。高通量測(cè)序結(jié)果表明lncRNA TCONS_00027980和TCONS_00042303在肝再生不同時(shí)間點(diǎn)表達(dá)水平顯著變化,RT-PCR檢測(cè)不同時(shí)間點(diǎn)再生肝組織中兩種lncRNA的RNA表達(dá)水平,實(shí)驗(yàn)結(jié)果表明高通量測(cè)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(xì)胞中的表達(dá)水平,MTT法檢測(cè)BRL-3A細(xì)胞的活性,流式細(xì)胞術(shù)檢測(cè)BRL-3A增殖情況的變化,RT-PCR和Western-blot用來(lái)檢測(cè)與增殖相關(guān)基因的表達(dá)情況。檢測(cè)結(jié)果表明MTT法檢測(cè)表明實(shí)驗(yàn)組比對(duì)照組細(xì)胞活性增加(P0.05);流式細(xì)胞術(shù)實(shí)驗(yàn)檢測(cè)顯示與對(duì)照組相比,實(shí)驗(yàn)組的S+G2/M期細(xì)胞數(shù)在一定程度上增加了(P0.05);而real-time PCR(RT-PCR)檢測(cè)mRNA表達(dá)水平,結(jié)果表明實(shí)驗(yàn)組與對(duì)照組相比,細(xì)胞增殖相關(guān)基因MYC、CCNA2、CCND1、BCL-2表達(dá)上調(diào)(P0.05),凋亡相關(guān)基因的BAX表達(dá)下調(diào)(P0.05);蛋白質(zhì)免疫印跡(Western-blot)檢測(cè)表明,實(shí)驗(yàn)組細(xì)胞增殖相關(guān)基因MYC、CCNA2、CCND1、BCL-2表達(dá)上調(diào)(P0.05),凋亡相關(guān)基因的BAX表達(dá)下調(diào)(P0.05)。lncRNA TCONS_00027980和TCONS_00042303能夠通過(guò)降低細(xì)胞增殖相關(guān)基因的表達(dá)水平抑制大鼠肝細(xì)胞BRL-3A的細(xì)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(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R575

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