裸鼹鼠耐低氧关键基因的筛选及相关基因调控机制的研究

发布时间：2018-05-18 22:16

本文选题：裸鼹鼠 + 耐低氧　；参考：《第二军医大学》2016年硕士论文

【摘要】：研究背景及目的:裸鼹鼠长期生活于地底下的洞穴环境中,其周围环境氧气浓度极低,只有(10%~15%),其承受低氧环境的能力是生活于地上的哺乳类所不能比拟的。这表明,缺氧造成的损伤或病理改变在这种异常耐受低氧环境的啮齿类中较少或者被彻底清除。然而,对于这个物种耐受低氧环境的机制我们还不甚了解。在局部缺血性心脏病导致机体损伤的过程中,缺氧因素扮演着极其重要的角色。而且,在其他一些致死性损伤疾病的发展过程中往往继发缺氧。最新数据显示,局部缺血性心脏病在全球范围内特别是中等收入国家发病率呈逐年上升的态势,其是导致全球性疾病负担的主要原因。对裸鼹鼠独特的耐低氧机制进行研究,将有利于人类缺血性疾病的防治及为制定低氧环境医学保护措施提供新思路。另外,实体肿瘤的基本特征之一是局部的低氧微环境。目前已有的数据证实,局部的低氧微环境不仅是诱导肿瘤恶性转化的始动因素,而且其还可以诱导肿瘤细胞对放射化疗的抵抗。因此,实体肿瘤局部低氧微环境被认为与多种恶性肿瘤的治疗后复发、治疗效果差和不良预后密切相关。因此,阐明裸鼹鼠耐低氧的分子机制,将为恶性肿瘤的治疗提供新线索。研究方法:运用低氧舱控制氧气浓度,急性低氧环境(5%O2)处理成年(8月龄)裸鼹鼠1h、4h、8h、12h后,提取裸鼹鼠肌肉组织总RNA,运用转录组测序方法检测低氧处理后基因表达量变化,研究低氧环境对裸鼹鼠基因表达谱的影响。针对基因表达水平结果,筛选低氧处理前后差异表达的基因,并采用hierarchical聚类法对差异表达的基因进行聚类,对各组进行GO和KEGG pathway分析。研究差异表达基因GO功能分类及KEGG信号通路的富集情况。针对KEGG信号通路的富集情况,选取一个显著差异的信号通路(MAPK信号通路)里面的差异表达基因STMN1、JIP1/MAPK8IP1、JNK3/MAPK10,并根据GO功能分类,采用real-time PCR、Western Blotting验证差异基因的表达。进一步针对差异表达基因STMN1、JIP1/MAPK8IP1、JNK3/MAPK10,通过si RNA干扰技术沉默肌肉成纤维细胞中STMN1、JIP1/MAPK8IP1、JNK3/MAPK10的表达。运用流式细胞术检测细胞周期和凋亡情况,CCK8法检测细胞的增殖活性,研究STMN1、JIP1/MAPK8IP1、JNK3/MAPK10沉默对细胞增殖、凋亡和细胞周期的影响。结果:差异表达基因的筛选与对照组相比,低氧处理1h、4h、8h、12h后裸鼹鼠肌肉组织中基因表达水平发生上调的总数分别为243个、304个、717个、527个,基因表达量发生下调的基因总数分别为344个、332个、487个、250个。在任意情况下与对照组相比较,表达水平发生上调的基因总数为1450个,发生下调的基因总数为971个。采用hierarchical聚类法把2337个差异表达的基因分成6个基因功能簇(cluster),显著地聚集到4个实验处理组中。Cluster1、Cluster2、Cluster3、Cluster4、Cluster5、Cluster6中含有的差异基因数目分别为566个、566个、227个、674个、108个、196个。cluster1中的基因在低氧1h组显著聚类,cluster2中的基因在低氧4h组显著聚类,cluster4、cluster5中的基因在低氧8h组显著聚类,而cluster3、cluster6中的基因在低氧12h组显著聚类。差异表达基因GO功能富集参照经典的基因功能分类体系㧟GO功能分类体系(Gene ontology),把各个cluster中的基因进行功能的聚集后发现,cluster1、cluster2、cluster3、cluster4、cluster5、cluster6中显著性富集的GO terms分别有1682个、1300个、1384个、2185个、637个、592个。其中cluster1、cluster2、cluster3、cluster4、cluster5、cluster6中差异最显著的GO terms分别为解剖结构形态(anatomical structure morphogenesis)、单一生物过程(single-organism process)、组织细胞成分(cellular component organization)、单一生物过程(single-organism process)、离子转运(ion transport)、细胞-细胞信号(cell-cell signaling)。差异表达基因KEGG信号通路富集参考KEGG数据库初步判断各个cluster中差异基因显著性富集信号通路的情况。cluster1、cluster2、cluster3、cluster4、cluster5、cluster6中差异表达基因最显著性富集的信号通路分别为焦点粘连(Focal adhesion)、扩张型心肌病(Dilated cardiomyopathy)、昼夜节律-哺乳动物(Circadian rhythm-mammal)、MAPK信号通路(MAPK signaling pathway)、甘氨酸,丝氨酸和苏氨酸代谢(Glycine,serine and threonine metabolism)、II型糖尿病(Type II diabetes mellitus)。差异表达基因的KEGG信号通路富集性分析结果表明,调控细胞粘连,细胞增殖、分化、凋亡以及氨基酸代谢的信号路径可能在裸鼹鼠的低氧应答反应中扮演着重要角色。体外实验验证差异表达基因(STMN1、JIP1/MAPK8IP1、JINK3/MAPK10)的表达低氧培养条件处理24h后,与对照组相比,裸鼹鼠肌肉成纤维细胞中STMN1、JIP1/MAPK8IP1、JNK3/MAPK10的表达水平均表现为显著上调。STMN1、JIP1/MAPK8IP1、JNK3/MAPK10表达的沉默显著地抑制了裸鼹鼠肌肉成纤维细胞的增殖活性,诱导细胞凋亡。并且,沉默STMN1的表达阻滞裸鼹鼠肌肉成纤维细胞在G0/G1及S期,沉默JIP1/MAPK8IP1、JNK3/MAPK10的表达阻断裸鼹鼠肌肉成纤维细胞于S期。结论:运用转录组测序的方法高通量筛选出裸鼹鼠肌肉组织在低氧处理后2337个差异表达的基因,并对这些差异表达的基因进行了GO功能分类和KEGG信号通路的富集。体外实验验证了其中三个差异表达基因(STMN1、JIP1/MAPK8IP1、JNK3/MAPK10)的表达,从而提示其可能在调控裸鼹鼠耐受低氧环境中发挥重要作用。
[Abstract]:Research background and purpose: the naked mole rats have long lived in the cave environment under the ground where the ambient oxygen concentration is very low, only (10%~15%), and its ability to withstand the low oxygen environment is incomparable to the mammals living on the ground. This indicates that the damage or disease caused by hypoxia is in the rodent with the abnormally tolerable hypoxia environment. It is less or completely eliminated. However, we do not know much about the mechanism of tolerance to hypoxia in this species. Hypoxia plays an important role in the process of local ischemic heart disease causing damage to the body. Moreover, there are often secondary hypoxia in the development of some other fatal injury diseases. The incidence of local ischemic heart disease in the world, especially in middle-income countries, is increasing year by year, which is the main cause of the global disease burden. The study of the unique hypoxia tolerance mechanism for naked mole rats will be beneficial to the prevention and control of human ischemic diseases and to provide new measures for the protection of the hypoxic environment. In addition, one of the basic features of solid tumors is the local hypoxic microenvironment. Current data show that local hypoxia environment is not only a starting factor in inducing malignant transformation of tumor, but also can induce tumor cells to resist radiation chemotherapy. Therefore, local hypoxia microenvironment is considered to be associated with a variety of evil. Therefore, the molecular mechanism of hypoxia tolerance in nude mole rats will provide new clues for the treatment of malignant tumors. Study methods: using hypoxic capsule to control oxygen concentration, acute hypoxia environment (5%O2) treatment of nude mole rats 1H, 4h, 8h, 12h, to extract nude mole rat muscles The change of gene expression after hypoxia treatment was detected by the method of transcription group sequencing, and the influence of hypoxia environment on the gene expression profiles of naked mole rats was studied. According to the results of gene expression, the differentially expressed genes were screened before and after hypoxia treatment, and the differentially expressed genes were clustered by hierarchical clustering method, and GO was used in each group for GO. And KEGG pathway analysis. Study the GO functional classification of differentially expressed genes and the enrichment of KEGG signaling pathways. In view of the enrichment of KEGG signaling pathways, the differential expression of gene STMN1, JIP1/MAPK8IP1, and JNK3/ MAPK10 in a significant differential signal pathway (MAPK signaling pathway) was selected and classified according to GO function. N Blotting verifies the expression of differential genes. Further targeting differential expression genes STMN1, JIP1/MAPK8IP1, JNK3/MAPK10, Si RNA interference technique is used to silence the expression of STMN1, JIP1/MAPK8IP1, JNK3/MAPK10 in muscle fibroblasts. Flow cytometry is used to detect cell cycle and withering, CCK8 method to detect cell proliferation activity, and to study STMN1 The effect of JIP1/MAPK8IP1, JNK3/MAPK10 silence on cell proliferation, apoptosis and cell cycle. Results: the screening of differentially expressed genes was 243, 304, 717, 527, and the gene expression decreased in total gene expression after 1h, 4h, 8h and 12h. There were 344, 332, 487 and 250 respectively. In any case, the total number of genes up regulated by the control group was 1450, and the total number of down regulated genes was 971. 2337 differentially expressed genes were divided into 6 gene power energy clusters (cluster) by hierarchical clustering method, which significantly aggregated to 4 experimental groups. The number of differentially expressed genes in.Cluster1, Cluster2, Cluster3, Cluster4, Cluster5, and Cluster6 were 566, 566, 227, 674, 108, and 196.Cluster1 in the hypoxia 1H group, and the genes in cluster2 were clustered significantly in the hypoxia 4H group, cluster4, and the genes in the low oxygen group were clustering significantly. The genes in the cluster6 group were clustered in the hypoxia 12h group. The differential expression gene GO function enrichment referred to the classical functional classification system of the gene, and the GO functional classification system (Gene ontology), after the aggregation of the genes in each cluster, the significant enrichment of cluster1, cluster2, cluster3, cluster4, cluster5, and cluster5. There are 1682, 1300, 1384, 2185, 637, 592, of which the most distinct GO terms in cluster1, cluster2, cluster3, cluster4, cluster5, cluster6 is the anatomical structure (anatomical structure morphogenesis), and the single biological process (single-organism) On), single biological process (single-organism process), ion transport (ion transport), cell cell signal (cell-cell signaling). Differential expression gene KEGG signaling pathway is enriched by reference KEGG database to determine the status of the difference gene significant enrichment signal pathway in each cluster.Cluster1. The most significant enrichment of differential expression genes in luster6 is focal adhesion (Focal adhesion), dilated cardiomyopathy (Dilated cardiomyopathy), circadian rhythm mammal (Circadian rhythm-mammal), MAPK signaling pathway (MAPK signaling pathway), glycine, serine and threonine metabolism. E metabolism), type II diabetes mellitus (Type II diabetes mellitus). The analysis of the concentration of KEGG signaling pathway of differentially expressed genes shows that the signaling pathways regulating cell adhesion, cell proliferation, differentiation, apoptosis and amino acid metabolism may play an important role in the hypoxia response response in the nude mole rats. In vitro experiments verify the differential expression basis. Compared with the control group, the expression level of STMN1, JIP1/MAPK8IP1, JNK3/MAPK10 in the muscle fibroblasts of the naked mole rat showed a significant increase in.STMN1, JIP1/MAPK8IP1 and JNK3/MAPK10 apparent silence significantly inhibited the muscle fibroblasts of the naked mole rats, compared with the control group because of the low oxygen culture conditions (STMN1, JIP1/MAPK8IP1, JINK3/MAPK10). Proliferation activity and induction of apoptosis, and the expression of silent STMN1 blocking the muscle fibroblasts of nude mole rat at G0/G1 and S phase, silent JIP1/MAPK8IP1, JNK3/MAPK10 expression blocking the muscle fibroblasts of naked mole rats at S stage. Conclusion: the 2337 difference of the muscle tissue of naked mole rat was selected by high throughput screening method by the method of transcriptional sequencing. These differentially expressed genes were expressed by the GO function classification and the enrichment of KEGG signaling pathways. In vitro, three differentially expressed genes (STMN1, JIP1/MAPK8IP1, JNK3/MAPK10) were expressed in vitro, suggesting that it may play an important role in regulating the tolerance to hypoxia in naked mole rats.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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