LncRNA-ALDBGALG0000001600-mir-223-Inpp4a通路在渗出性素质鸡静脉内皮细胞凋亡中作用

发布时间：2018-02-02 20:36

本文关键词： 硒渗出性素质鸡静脉内皮细胞 mir-223 Lnc RNA-1600 Inpp4a 凋亡　出处：《东北农业大学》2017年博士论文　论文类型：学位论文

【摘要】：硒是动物和人必须的微量元素之一,能够广泛的参与到体内各种生理生化活动,发挥着十分重要的作用。硒缺乏以及血浆硒水平的不良状态会引起心血管疾病。硒还可以通过对抗炎症的特性为心血管疾病提供保护。内皮细胞的花生四烯酸的级联效应也可以通过硒进行调节。目前,硒的生物学功能受到广泛重视,但缺硒对鸡血管损伤的研究报道较少。已有研究应用mi RNA组学和Lnc RNA组学对心血管疾病的发病机理进行探讨。基于mi RNA组学和Lnc RNA组学的特点,组学分析已经大大促进研究人员对人类及动物疾病相关机理的认识及研究,因此将组学分析应用到畜禽疾病的研究上就显得更加重要。本试验在建立鸡缺硒渗出性素质模型的基础上,对静脉组织进行显微和超微的结构观察,试剂盒检测氧化应激相关因子的含量和/或酶的活性,硒蛋白的m RNA变化,热休克基因的m RNA和蛋白变化,炎症因子的m RNA和蛋白变化,细胞因子的mRNA变化,磷酸肌醇通路基因的m RNA和蛋白变化,线粒体凋亡通路基因的mRNA和蛋白变化,确定缺硒能引起雏鸡静脉组织发生损伤。应用高通量测序技术对静脉组织的mi RNA和Lnc RNA进行测序,筛选差异表达的miRNA和Lnc RNA,应用RT-PCR进行组织验证,应用GO和KEGG富集mi RNA和Lnc RNA指导的靶基因所在的通路。敲低和/或过表达特异mi RNA和Lnc RNA,验证其指导的下游通路,探讨miRNA和Lnc RNA在缺硒引起的鸡渗出性素质的静脉损伤中的重要作用。试验结果如下:(1)饲喂低硒日粮的雏鸡静脉组织中,病理组织学观察到管壁增厚,内膜层结构部分缺失,平滑肌层纤维走向紊乱、排列不完整,平滑肌细胞结构不规则,外膜层缺损,局部有炎性细胞和淋巴细胞浸润。超微结构观察到内皮细胞和平滑肌细胞的细胞核染色质边集,线粒体空泡化,细胞崩解。Tunel染色表明饲喂低硒日粮的鸡静脉组织凋亡率升高,表明缺硒能引起鸡静脉组织损伤,同时伴有细胞凋亡的发生。(2)缺硒能够降低鸡静脉组织21种硒蛋白的mRNA表达。其中受硒缺乏影响较为明显的硒蛋白是GPx1、SPS2、GPx2、SELS、SEPX1、GPx4、SELI、SEPW1、SELK和SEP15。揭示硒缺乏能够降低静脉中硒蛋白的转录水平。(3)硒缺乏能够显著升高鸡静脉组织中热休克蛋白HSP60、HSP70以及HSP90的m RNA表达水平以及HSP60、HSP70和HSP90的蛋白水平,表明HSP60、HSP70和HSP90参与了缺硒引起的静脉损伤。(4)硒缺乏能显著升高炎症因子PTGE和COX-2的m RNA和蛋白表达水平,上调IL-1β,IL-6,IL-8和IFN-γ的m RNA表达水平,下调IL-4和IL-12 mRNA的表达水平,表明了缺硒能够引起静脉炎症反应的发生。(5)缺硒引起鸡静脉组织中485个Lnc RNA表达上调(P0.05),147个Lnc RNA表达下调(P0.05)。表明缺硒能影响鸡静脉组织中Lnc RNA的表达。同时应用RT-PCR对部分差异转录本进行验证,证实了测序的准确性。对差异表达的Lnc RNA的靶m RNA进行GO和KEGG富集,发现其参与的主要通路为氧化还原通路、糖代谢通路、磷酸肌醇通路、凋亡通路。(6)缺硒引起鸡静脉组织中109个miRNA表达上调,121个mi RNA下调(P0.05)。表明缺硒能影响鸡静脉组织中mi RNA的表达。应用RT-PCR对部分差异转录本进行验证,证实了测序的准确性。对差异表达的mi RNA的靶m RNA进行GO和KEGG富集,发现其指导的主要通路包括肌动蛋白细胞骨架的调节,磷酸肌醇通路,细胞因子受体互作等通路。将组学检测中mi RNA和Lnc RNA的靶基因富集到的通路进行比较,筛选出了分子功能通路、肾上腺素能信号、脂肪酸代谢、甘油磷脂代谢通路、刺猬信号通路、磷酸肌醇通路和血管平滑肌收缩通路。(7)在Lnc RNA组学结果中,筛选出23个与氧化还原有关的Lnc RNA,及其指导的19个靶mRNA。应用RT-PCR对缺硒和正常鸡的静脉组织筛选出的与氧化还原有关的Lnc RNA和m RNA进行验证,结果表明23个Lnc RNA和19个mRNA的表达都发生了显著变化(P0.05)。在体外培养的内皮细胞中进行硒和过氧化氢处理,建立抗氧化和促氧化的细胞模型,结果显示硒和过氧化氢能影响这23个Lnc RNA的表达。表明23个Lnc RNA能够参与由硒引起的氧化还原反应。(8)经Lnc RNA组学和mi RNA组学联合比较分析,应用生物信息学的方法,预测到Lnc RNA-ALDBGALG0000001600(Lnc RNA-1600)—mir-223—Inpp4a具有靶向关系。建立mir-223的过表达模型和Lnc RNA-1600的敲低模型,在体内、体外模型中证明其表达以负调控关系存在。通过荧光素酶报告基因试验确定它们的靶向关系。证实了Lnc RNA-1600能调控mir-223,再指导Inpp4a的转录。(9)应用RT-PCR和WB检测发现,缺硒能下调组织中磷酸肌醇通路中Inpp4a、Impa1和Impa2,凋亡通路Bcl-2的表达,上调线粒体凋亡通路中Bax、caspase3和caspase9的表达。在敲低和过表达mir-223模型和si Lnc RNA-1600模型中,应用RT-PCR和WB检测磷酸肌醇通路中mRNA和蛋白的变化,以及线粒体凋亡通路中Bax、Bcl-2、caspase3和caspase9的表达情况,应用hoechst染色和FCM检测细胞周期,发现过表达mir-223,敲低Lnc RNA-1600都能引起内皮细胞凋亡的发生,表明Lnc RNA-1600—mir-223—Inpp4a通路能调控静脉内皮细胞的凋亡。综上所述,缺硒可以下调鸡静脉组织中硒蛋白的转录水平,影响氧化还原水平、炎症因子和细胞因子的表达水平、热休克蛋白的表达、磷酸肌醇通路基因的表达以及凋亡基因的表达,引起静脉组织损伤。通过对mi RNA和Lnc RNA进行组学分析,筛选出特异表达的mi RNA和Lnc RNA,并筛选出特异通路,证实Lnc RNA-1600—mir-223—Inpp4a通路诱导渗出性素质鸡静脉内皮凋亡的发生。本结果填补了缺硒和正常鸡静脉组织中mi RNA和Lnc RNA表达谱的空白,丰富了缺硒引起静脉组织凋亡的病理学机制,为探讨缺硒引起鸡静脉组织损伤的机制奠定理论基础和试验依据。
[Abstract]:Selenium is one of the trace elements in animal and human must, can be widely involved in various physiological and biochemical activities, plays a very important role. Selenium deficiency and abnormal state of plasma selenium levels can cause cardiovascular disease. Selenium can fight inflammation disease characteristics to provide protection for cardiovascular endothelial cells. The cascade effect of peanut four acid also can be adjusted by selenium. At present, the biological function of selenium has been paid attention to, but the lack of selenium research reports on chicken vascular injury is less. The existing research application of MI RNA were studied on the pathogenesis of cardiovascular disease and Lnc RNA were discussed. The characteristics of MI RNA and Lnc RNA group group based on proteomic analysis has been greatly promote the understanding and study of human and animal disease related mechanism, so the group analysis applied to the study of animal disease becomes more serious To the test on the base of the chicken. Selenium deficiency exudative diathesis model, observe the structure and micro vein tissue, the content of oxidative stress detection kit related factors and / or enzyme activity, m RNA change of selenoproteins, changes of M RNA and heat shock protein genes, RNA and M change protein mRNA changes of inflammatory factors, cytokines, changes of M RNA and protein phosphoinositide pathway genes, mRNA and protein changes of mitochondrial apoptotic pathway genes, determine the selenium deficiency can cause injury of tissues of chickens. Intravenous application of high-throughput sequencing technology on MI RNA and Lnc vein RNA were sequenced to screen differentially expressed miRNA and Lnc RNA, using RT-PCR to organize validation pathway target genes using GO and KEGG mi RNA and Lnc RNA enrichment guide is located. Knockdown and / or overexpression of specific mi RNA and Lnc RNA, to verify the pathways downstream of its guidance, To explore the important role of miRNA and Lnc RNA in venous injury caused by selenium deficiency in chicken exudative diathesis. The results are as follows: (1) chicks fed low selenium diet vein in histopathology observed wall thickening and intimal layer structure excalation, smooth muscle fibers arranged to disorder, incomplete. Smooth muscle cells of irregular structure, outer layer defect, local inflammatory cells and lymphocytes. By observation of ultrastructure of endothelial cell and smooth muscle cell nuclear chromatin margination, mitochondrial vacuolization, cell apoptosis of chicken tissue disintegration vein.Tunel staining show fed low selenium diet increased rate, showed that selenium deficiency can cause tissue chicken vein injury, accompanied by apoptosis. (2) the lack of selenium can reduce the expression of 21 kinds of chicken tissue vein mRNA. By which the selenoprotein selenium deficiency obviously affect selenoprotein is GPx1, SPS2, GPx2, SELS SEPX1, GPx4, SELI, SEPW1, SELK, and SEP15. revealed the lack of selenium can reduce the transcription level of selenoprotein. The vein (3) can significantly increase the heat shock protein HSP60 in chicken tissue vein selenium deficiency, HSP70 m and RNA HSP90 expression level and protein level of HSP60, HSP70, and HSP90 showed that HSP60, HSP70 and HSP90 is involved in the vein injury caused by selenium deficiency. (4) m RNA protein and selenium deficiency can increase the inflammatory cytokines PTGE and COX-2 expression, up regulation of IL-1 IL-6 m RNA IL-8, beta, and gamma IFN- expression levels, the expression of IL-12 and down-regulation of IL-4 water level mRNA, indicated that selenium deficiency can cause vein the occurrence of inflammation. (5) selenium deficiency induced by 485 Lnc RNA expression of chicken vein (P0.05), 147 Lnc (P0.05). RNA expression showed that selenium deficiency can affect the expression of Lnc RNA in chicken vein tissue. At the same time on the part of the application to verify the differential transcription of RT-PCR, confirmed The sequencing of the m RNA on the target accuracy. Lnc RNA differential expression of GO and KEGG enrichment, found that the main pathway participates in the redox pathway, glucose metabolism pathway, phosphoinositide pathway, apoptosis pathway. (6) selenium deficiency induced by 109 miRNA expression of chicken vein, 121 mi RNA down (P0.05). The results indicated that selenium deficiency can affect the expression of MI RNA in chicken vein tissue. The application of RT-PCR on the part of the differential transcription is verified, confirmed the accuracy of sequencing. The target m RNA on the MI RNA expression of GO and KEGG enrichment, found mainly in the guidance of regulatory pathways including the actin cytoskeleton, the phosphoinositide pathway, cytokine receptor interaction pathway. The pathway group target gene RNA and Lnc MI in the detection of RNA enrichment to compare selected molecular pathways, adrenergic signaling, fatty acid metabolism, glycerophospholipid metabolism pathway, thorn The hedgehog signaling pathway, phosphoinositide pathway and vascular smooth muscle contraction pathway. (7) in the Lnc RNA group results, selected 23 Lnc RNA related to the redox of vein tissue selenium deficiency and normal chicken and guide the 19 target mRNA. application selected by RT-PCR and redox related Lnc RNA m and RNA were verified, the results showed that the expression of 23 Lnc RNA and 19 mRNA were significantly changed (P0.05). Selenium and hydrogen peroxide in cultured endothelial cells, cell model of antioxidant and oxidant, hydrogen peroxide results showed that selenium and influence the expression of the 23 Lnc RNA that 23 Lnc RNA could be involved in the oxidation caused by selenium reduction reaction. (8) by Lnc RNA and MI RNA group were combined with comparative analysis, based on bioinformatics analysis and prediction to Lnc RNA-ALDBGALG0000001600 (Lnc RNA-1600) - mir-223 - Inpp4a Targeted. Establish a knockdown expression model, Lnc model and RNA-1600 mir-223 in vivo in negative regulation of the relationship between the expression of that in vitro model. By luciferase reporter gene assay to determine their target relationship. Confirmed that Lnc RNA-1600 can regulate the transcription of mir-223, and then guide the Inpp4a (9) found. The application of RT-PCR and WB detection, selenium deficiency can decrease the tissue of the phosphoinositide pathway in Inpp4a, Impa1 and Impa2, the expression of Bcl-2 in the apoptosis pathway, apoptosis pathway. Mitochondria Bax, expression of Caspase3 and caspase9. In the knockdown and overexpression of mir-223 Lnc model and Si RNA-1600 model, using RT-PCR and WB to detect the changes of mRNA and protein phosphoinositide pathway, and mitochondrial apoptosis pathway of Bax, Bcl-2, expression of Caspase3 and caspase9, using Hoechst staining and FCM detection of cell cycle, mir-223 overexpression, knockdown of Lnc RNA-1600 Can cause endothelial cell apoptosis, Lnc showed that RNA-1600 - mir-223 - Inpp4a pathway can regulate apoptosis of venous endothelial cells. In summary, selenium deficiency can be down regulated transcription level in chicken selenoprotein vein, the influence of the redox level, the expression levels of inflammatory factors and cytokines, the expression of heat shock protein, expression of inositol phosphate the apoptosis pathway genes and genes, caused by venous tissue damage. Through the MI RNA and Lnc RNA genomics analysis, screened the specific expression of MI RNA and Lnc RNA, and screened the specific pathway, and confirmed that the Lnc RNA-1600 - mir-223 - Inpp4a pathway induced apoptosis of exudative diathesis of chicken vein endothelial occurred. The results fill the spectrum of MI RNA and Lnc RNA blank expression of tissue selenium deficiency and normal chicken vein, rich selenium deficiency caused by pathological vein tissue apoptosis mechanism of selenium deficiency induced static chicken The mechanism of injury of pulse tissue is the theoretical basis and experimental basis.

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.31

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