基于RNA-seq技术的乙肝相关肝硬化肝细胞转录组学研究

发布时间：2018-03-13 04:02

本文选题：肝硬化　切入点：原代肝细胞　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：背景和目的在肝脏疾病的治疗中,针对病因的抗病毒治疗及针对症状抗肝纤维化治疗均非常重要。目前,随着核苷(酸)类药物的普及以及DAA的出现,CHB和CHC的治疗已取得飞跃进展,并在很大程度上逆转了肝纤维化的进程。但对于已经形成的肝硬化的逆转和治愈仍是难于解决的问题。所以抗纤维化,特别是抗肝硬化的治疗就成了当前需要亟待解决的难题。随着新一代测序技术的出现,转录水平的研究也越来越多,但肝硬化相关的转录组学研究,多取材于肝组织,是多种细胞的混合物,并且以实验动物的研究为多。有关单一种类细胞水平的研究,特别是来自病人的细胞水平的转录组学研究非常少见。本研究拟利用病人肝细胞,采用RNA-Seq技术研究乙肝相关肝硬化发生发展过程中肝细胞的表达谱改变,筛选出肝硬化特异性的m RNA和lnc RNA,以期发现肝纤维化,肝硬化的发病机制,找到肝纤维化,肝硬化治疗相关的分子靶点。方法本研究采用改良的EGTA/胶原酶四步灌流法分离正常肝组织及硬化肝组织中的原代肝细胞。经胶原酶消化肝组织后采用密度离心及Percoll梯度密度离心法分离纯化原代肝细胞。采用台盼蓝拒染观察细胞活性,细胞计数板计数细胞数量。评价患者年龄、肝脏重量、热缺血时间(WIT)、丙氨酸氨基转移酶(alanine aminotransferase,ALT)、总胆红素(total bilrubin,TBIL)等对肝细胞活性及数量的影响。选取分离得到的5例正常肝细胞和6例乙肝相关肝硬化肝细胞。Trizol法提取细胞中的总RNA并用Agilent 2100鉴定合格后采用RNA-seq技术进行转录组测序。对得到的m RNA和lnc RNA进行转录组表达谱分析,采用火山图,聚类分析等方法,筛选出肝硬化相关的差异表达m RNA和lnc RNA。继而利用生物信息学方法对肝硬化相关的差异表达的m RNA和lnc RNA进行GO和KEGG分析,差异表达lnc RNA-m RNA互作分析;预测差异表达m RNA和lnc RNA可能参与的生物学功能。根据生物信息学结果及文献查阅找出具有研究价值的差异表达基因。结果本研究共对41例患者肝组织进行了原代肝细胞的分离,其中20块成功分离出原代肝细胞。肝硬化组12例,无肝硬化(正常组)8例。患者的平均年龄为49±13.75岁,肝组织块重量为63.9±17.42g,WIT为3.31±1.57h,ALT为142.41±217.46 U/L,TBIL为58.35±90.30umol/l。获取肝细胞的数量和活性分别为24.4±13.83×105/g,83.3%±8.44。相较于正常组,肝硬化组WIT长,TBIL水平高,且两组间有统计学差异(p≤0.05),但患者的年龄、肝脏重量、ALT水平无明显差异;肝硬化组分离获取肝细胞的数量和活性均较正常组差(p≤0.05)。当WIT≤3h,肝脏重量≤60g,TBIL≤30umol/l时,分离获取的肝细胞数量和活性不受上述因素影响,当超过上述范围后,细胞的数量及活性呈下降趋势;细胞数量和活性与患者的年龄、ALT水平无明显相关性。通过RNA-seq技术,我们发现了与肝硬化相关的190条差异表达m RNA,上调的2条,下调的188条;87条差异表达lnc RNA,上调的55条,下调的32条;72489个差异剪接基因(differentially splicing genes,DSGs)。对差异表达m RNA和lnc RNA进行GO和KEGG分类和功能富集分析,我们发现差异表达的m RNA参与肝硬化的多种生物学过程,如物质代谢、炎症反应、多种酶的活性调节等过程。病毒感染性疾病相关的通路共富集了14个差异表达m RNA,分别为FGL2、CHEK1、RANBP3L、FGB、WDR72、PTTG3P、ANKRD37、CYCSP52、IL18、C3P1、AZGP1、POLR3GL、TBPL1、novel_G001217,其中FGL2、IL18、AZGP1是已知与肝硬化明确相关的。差异表达的lnc RNA共表达m RNA显著富集的病毒感染性疾病基因为AKT1、KLHDC2、SH3BP4、RANBP1、TRAF5、WNT2B。显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与HBV致癌相关。lnc RNA-m RNA互作分析共发现6条lnc RNA和13条m RNA。XLOC_058660同时调节11个m RNA的表达,UGT1A1同时被4个lnc RNA调节。差异剪接分析发现发生2次及以上DSGs有PZP、AFM、RNF121、ATF3、DNAH14、BHLHE40、ELF3、GMDS-AS1、L3MBTL4、LOC728040、PPOX、WASH3P。结论本研究前期采用EGTA/胶原酶四步灌流法成功地从正常及硬化的肝组织中分离出数量及活性均较好的原代肝细胞。应尽量减少WIT时间,修整肝脏使之形态及重量均适宜灌注以提高细胞数量及活性。从原代肝细胞水平建立了肝硬化相关差异表达m RNA和lnc RNA谱,为筛选肝硬化相关靶基因提供了依据;预测差异表达m RNA和lnc RNA可能参与了肝硬化发生发展的多条信号通路;预测了差异表达lnc RNA可能调控的m RNA;寻找到了可能与肝硬化相关的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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.2;R512.62

【相似文献】