本文選題：肝硬化　切入點(diǎn)：原代肝細(xì)胞　出處：《吉林大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景和目的在肝臟疾病的治療中,針對(duì)病因的抗病毒治療及針對(duì)癥狀抗肝纖維化治療均非常重要。目前,隨著核苷(酸)類(lèi)藥物的普及以及DAA的出現(xiàn),CHB和CHC的治療已取得飛躍進(jìn)展,并在很大程度上逆轉(zhuǎn)了肝纖維化的進(jìn)程。但對(duì)于已經(jīng)形成的肝硬化的逆轉(zhuǎn)和治愈仍是難于解決的問(wèn)題。所以抗纖維化,特別是抗肝硬化的治療就成了當(dāng)前需要亟待解決的難題。隨著新一代測(cè)序技術(shù)的出現(xiàn),轉(zhuǎn)錄水平的研究也越來(lái)越多,但肝硬化相關(guān)的轉(zhuǎn)錄組學(xué)研究,多取材于肝組織,是多種細(xì)胞的混合物,并且以實(shí)驗(yàn)動(dòng)物的研究為多。有關(guān)單一種類(lèi)細(xì)胞水平的研究,特別是來(lái)自病人的細(xì)胞水平的轉(zhuǎn)錄組學(xué)研究非常少見(jiàn)。本研究擬利用病人肝細(xì)胞,采用RNA-Seq技術(shù)研究乙肝相關(guān)肝硬化發(fā)生發(fā)展過(guò)程中肝細(xì)胞的表達(dá)譜改變,篩選出肝硬化特異性的m RNA和lnc RNA,以期發(fā)現(xiàn)肝纖維化,肝硬化的發(fā)病機(jī)制,找到肝纖維化,肝硬化治療相關(guān)的分子靶點(diǎn)。方法本研究采用改良的EGTA/膠原酶四步灌流法分離正常肝組織及硬化肝組織中的原代肝細(xì)胞。經(jīng)膠原酶消化肝組織后采用密度離心及Percoll梯度密度離心法分離純化原代肝細(xì)胞。采用臺(tái)盼藍(lán)拒染觀察細(xì)胞活性,細(xì)胞計(jì)數(shù)板計(jì)數(shù)細(xì)胞數(shù)量。評(píng)價(jià)患者年齡、肝臟重量、熱缺血時(shí)間(WIT)、丙氨酸氨基轉(zhuǎn)移酶(alanine aminotransferase,ALT)、總膽紅素(total bilrubin,TBIL)等對(duì)肝細(xì)胞活性及數(shù)量的影響。選取分離得到的5例正常肝細(xì)胞和6例乙肝相關(guān)肝硬化肝細(xì)胞。Trizol法提取細(xì)胞中的總RNA并用Agilent 2100鑒定合格后采用RNA-seq技術(shù)進(jìn)行轉(zhuǎn)錄組測(cè)序。對(duì)得到的m RNA和lnc RNA進(jìn)行轉(zhuǎn)錄組表達(dá)譜分析,采用火山圖,聚類(lèi)分析等方法,篩選出肝硬化相關(guān)的差異表達(dá)m RNA和lnc RNA。繼而利用生物信息學(xué)方法對(duì)肝硬化相關(guān)的差異表達(dá)的m RNA和lnc RNA進(jìn)行GO和KEGG分析,差異表達(dá)lnc RNA-m RNA互作分析;預(yù)測(cè)差異表達(dá)m RNA和lnc RNA可能參與的生物學(xué)功能。根據(jù)生物信息學(xué)結(jié)果及文獻(xiàn)查閱找出具有研究?jī)r(jià)值的差異表達(dá)基因。結(jié)果本研究共對(duì)41例患者肝組織進(jìn)行了原代肝細(xì)胞的分離,其中20塊成功分離出原代肝細(xì)胞。肝硬化組12例,無(wú)肝硬化(正常組)8例。患者的平均年齡為49±13.75歲,肝組織塊重量為63.9±17.42g,WIT為3.31±1.57h,ALT為142.41±217.46 U/L,TBIL為58.35±90.30umol/l。獲取肝細(xì)胞的數(shù)量和活性分別為24.4±13.83×105/g,83.3%±8.44。相較于正常組,肝硬化組WIT長(zhǎng),TBIL水平高,且兩組間有統(tǒng)計(jì)學(xué)差異(p≤0.05),但患者的年齡、肝臟重量、ALT水平無(wú)明顯差異;肝硬化組分離獲取肝細(xì)胞的數(shù)量和活性均較正常組差(p≤0.05)。當(dāng)WIT≤3h,肝臟重量≤60g,TBIL≤30umol/l時(shí),分離獲取的肝細(xì)胞數(shù)量和活性不受上述因素影響,當(dāng)超過(guò)上述范圍后,細(xì)胞的數(shù)量及活性呈下降趨勢(shì);細(xì)胞數(shù)量和活性與患者的年齡、ALT水平無(wú)明顯相關(guān)性。通過(guò)RNA-seq技術(shù),我們發(fā)現(xiàn)了與肝硬化相關(guān)的190條差異表達(dá)m RNA,上調(diào)的2條,下調(diào)的188條;87條差異表達(dá)lnc RNA,上調(diào)的55條,下調(diào)的32條;72489個(gè)差異剪接基因(differentially splicing genes,DSGs)。對(duì)差異表達(dá)m RNA和lnc RNA進(jìn)行GO和KEGG分類(lèi)和功能富集分析,我們發(fā)現(xiàn)差異表達(dá)的m RNA參與肝硬化的多種生物學(xué)過(guò)程,如物質(zhì)代謝、炎癥反應(yīng)、多種酶的活性調(diào)節(jié)等過(guò)程。病毒感染性疾病相關(guān)的通路共富集了14個(gè)差異表達(dá)m RNA,分別為FGL2、CHEK1、RANBP3L、FGB、WDR72、PTTG3P、ANKRD37、CYCSP52、IL18、C3P1、AZGP1、POLR3GL、TBPL1、novel_G001217,其中FGL2、IL18、AZGP1是已知與肝硬化明確相關(guān)的。差異表達(dá)的lnc RNA共表達(dá)m RNA顯著富集的病毒感染性疾病基因?yàn)锳KT1、KLHDC2、SH3BP4、RANBP1、TRAF5、WNT2B。顯著富集的通路包括酒精中毒和病毒致癌,病毒致癌通路中的SRC與HBV致癌相關(guān)。lnc RNA-m RNA互作分析共發(fā)現(xiàn)6條lnc RNA和13條m RNA。XLOC_058660同時(shí)調(diào)節(jié)11個(gè)m RNA的表達(dá),UGT1A1同時(shí)被4個(gè)lnc RNA調(diào)節(jié)。差異剪接分析發(fā)現(xiàn)發(fā)生2次及以上DSGs有PZP、AFM、RNF121、ATF3、DNAH14、BHLHE40、ELF3、GMDS-AS1、L3MBTL4、LOC728040、PPOX、WASH3P。結(jié)論本研究前期采用EGTA/膠原酶四步灌流法成功地從正常及硬化的肝組織中分離出數(shù)量及活性均較好的原代肝細(xì)胞。應(yīng)盡量減少WIT時(shí)間,修整肝臟使之形態(tài)及重量均適宜灌注以提高細(xì)胞數(shù)量及活性。從原代肝細(xì)胞水平建立了肝硬化相關(guān)差異表達(dá)m RNA和lnc RNA譜,為篩選肝硬化相關(guān)靶基因提供了依據(jù);預(yù)測(cè)差異表達(dá)m RNA和lnc RNA可能參與了肝硬化發(fā)生發(fā)展的多條信號(hào)通路;預(yù)測(cè)了差異表達(dá)lnc RNA可能調(diào)控的m RNA;尋找到了可能與肝硬化相關(guān)的DSGs。
[Abstract]:Background and purpose in the treatment of liver disease, and antiviral therapy for the cause and symptoms in treatment of hepatic fibrosis are very important. At present, with nucleoside (acid) drugs as well as the popularity of DAA, the treatment of CHB and CHC has been made progress, and to a great extent reversed the process of liver fibrosis but the formation of liver cirrhosis and reversal treatment are still difficult to solve problems. So the anti fibrosis, especially the treatment of anti liver cirrhosis has become the problem urgently to be solved. With the emergence of a new generation of sequencing technology, research on the transcriptional level is also increasing, but the relevant study on the transcriptome of cirrhosis, multiple based on the liver tissue, is a mixture of cells, and to study the experimental animal for research. The single cell type level, especially the transcriptome research from patients at the cellular level Very rare. This study intends to use the liver cells, liver cells expression profiles of utilizing RNA-Seq technology to study the occurrence and development of HBV related liver cirrhosis, liver cirrhosis screening specific m RNA and LNC RNA, in order to find the pathogenesis of hepatic fibrosis, cirrhosis, hepatic fibrosis found, therapeutic molecular targets associated with cirrhosis. Methods this study used a modified four step collagenase EGTA/ perfusion in primary hepatocytes in liver tissue and normal liver tissue hardening flow method separation. The liver tissue after collagenase digestion by density centrifugation and Percoll density gradient centrifugation separation and purification of primary liver cells by trypan blue stain to observe cell activity, cell number counting plate count cells. Evaluation of patient age, liver weight, warm ischemia time (WIT), alanine aminotransferase (alanine, aminotransferase, ALT), total bilirubin (total bilrubin, TBIL) on liver cells Effect of cell activity and quantity. 5 cases of normal liver cells were isolated and 6 cases of hepatitis B related cirrhosis of liver cell.Trizol method for extracting total RNA cells and 2100 Agilent after the identification of qualified RNA-seq technology is adopted to get the transcriptome sequencing. M RNA and LNC RNA transcriptome expression profile analysis, the volcano chart, cluster analysis, screening out the differential expression of liver cirrhosis related differential expression of M RNA and LNC RNA. by using bioinformatics methods for liver cirrhosis related M RNA and LNC RNA were analyzed by GO and KEGG, expression analysis of interaction between LNC RNA-m RNA and LNC RNA m; the biological function of RNA may be involved in the expression of prediction difference. According to the results of bioinformatics literature and identify differentially expressed genes with research value. Results a total of 41 cases of patients with liver tissue were isolated primary hepatocytes, of which 20 blocks Function of isolated primary hepatocytes. Hepatic cirrhosis group of 12 patients without cirrhosis (normal group) 8 cases. The average age of the patients was 49 + 13.75 years, liver weight was 63.9 + 17.42g, WIT = 3.31 + 1.57h, ALT = 142.41 + 217.46 U/L, TBIL 58.35 + 90.30umol/l. to obtain the number and activity of liver cells were 24.4 + 13.83 * 105/g, 83.3% + 8.44. compared to the normal group, liver cirrhosis group WIT, TBIL level is high, and the significant difference between the two groups (P = 0.05), but the patients' age, liver weight, no significant differences in the level of ALT; the number and activity of liver cells isolated from liver cirrhosis group the difference compared with the normal group (P < 0.05). When the WIT is less than or equal to 3h, the liver weight less than or equal to 60g, TBIL or 30umol/l, the number and activity of liver cells obtained from the above factors, when the above range, the amount and activity of cells decreased; the cell number and activity of patients age, ALT level Obvious correlation. By using RNA-seq technology, we found 190 differentially expressed m RNA associated with liver cirrhosis, up 2, down 188; LNC RNA 87 differentially expressed, up 55, down 32; 72489 differentially spliced genes (differentially splicing, genes, DSGs). The expression of M RNA LNC and RNA were GO and KEGG classification and functional enrichment analysis for differences, we found that the expression of M RNA in liver cirrhosis in various biological processes, such as metabolism, inflammatory reaction, enzymatic activity and regulation and so on. Viral infection related pathways were enriched 14 differentially expressed m RNA and FGL2 respectively. CHEK1, RANBP3L, FGB, WDR72, PTTG3P, ANKRD37, CYCSP52, IL18, C3P1, AZGP1, POLR3GL, TBPL1, novel_G001217, FGL2, IL18, AZGP1, and liver cirrhosis is known clearly related to the significant enrichment of M. RNA virus co expressing LNC RNA differential expression Disease gene AKT1, KLHDC2, SH3BP4, RANBP1, TRAF5, WNT2B. pathway significantly enriched including alcoholism and viral carcinogenesis, cancer and virus SRC in HBV pathway.Lnc RNA-m RNA oncogene related interaction analysis were found in expression of 6 LNC RNA and 13 m RNA.XLOC_058660 and 11 m RNA regulation, UGT1A1 by 4 LNC RNA regulation. Alternative splicing analysis showed that 2 times and above DSGs PZP, AFM, RNF121, ATF3, DNAH14, BHLHE40, ELF3, GMDS-AS1, L3MBTL4, LOC728040, PPOX, WASH3P. early conclusion of this study using EGTA/ four step collagenase perfusion method successfully from normal liver tissues and hardening of the separation the number and activity of good primary hepatocytes. Try to reduce the WIT time, morphology and weight which are suitable for dressing liver perfusion in order to increase the number and activity of cells from primary liver cells. The level of the differential expression of M and LNC related cirrhosis RNA RN A spectrum provides a basis for screening related target genes in liver cirrhosis. Prediction of differential expression of M RNA and LNC RNA may be involved in multiple signaling pathways of liver cirrhosis, predict m RNA with differential expression of LNC RNA, and find possible DSGs. related to cirrhosis.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R575.2;R512.62

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