本文關(guān)鍵詞： 硒 滲出性素質(zhì)雞 靜脈 內(nèi)皮細(xì)胞 mir-223 Lnc RNA-1600 Inpp4a 凋亡　出處：《東北農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：硒是動(dòng)物和人必須的微量元素之一,能夠廣泛的參與到體內(nèi)各種生理生化活動(dòng),發(fā)揮著十分重要的作用。硒缺乏以及血漿硒水平的不良狀態(tài)會(huì)引起心血管疾病。硒還可以通過對(duì)抗炎癥的特性為心血管疾病提供保護(hù)。內(nèi)皮細(xì)胞的花生四烯酸的級(jí)聯(lián)效應(yīng)也可以通過硒進(jìn)行調(diào)節(jié)。目前,硒的生物學(xué)功能受到廣泛重視,但缺硒對(duì)雞血管損傷的研究報(bào)道較少。已有研究應(yīng)用mi RNA組學(xué)和Lnc RNA組學(xué)對(duì)心血管疾病的發(fā)病機(jī)理進(jìn)行探討�；趍i RNA組學(xué)和Lnc RNA組學(xué)的特點(diǎn),組學(xué)分析已經(jīng)大大促進(jìn)研究人員對(duì)人類及動(dòng)物疾病相關(guān)機(jī)理的認(rèn)識(shí)及研究,因此將組學(xué)分析應(yīng)用到畜禽疾病的研究上就顯得更加重要。本試驗(yàn)在建立雞缺硒滲出性素質(zhì)模型的基礎(chǔ)上,對(duì)靜脈組織進(jìn)行顯微和超微的結(jié)構(gòu)觀察,試劑盒檢測(cè)氧化應(yīng)激相關(guān)因子的含量和/或酶的活性,硒蛋白的m RNA變化,熱休克基因的m RNA和蛋白變化,炎癥因子的m RNA和蛋白變化,細(xì)胞因子的mRNA變化,磷酸肌醇通路基因的m RNA和蛋白變化,線粒體凋亡通路基因的mRNA和蛋白變化,確定缺硒能引起雛雞靜脈組織發(fā)生損傷。應(yīng)用高通量測(cè)序技術(shù)對(duì)靜脈組織的mi RNA和Lnc RNA進(jìn)行測(cè)序,篩選差異表達(dá)的miRNA和Lnc RNA,應(yīng)用RT-PCR進(jìn)行組織驗(yàn)證,應(yīng)用GO和KEGG富集mi RNA和Lnc RNA指導(dǎo)的靶基因所在的通路。敲低和/或過表達(dá)特異mi RNA和Lnc RNA,驗(yàn)證其指導(dǎo)的下游通路,探討miRNA和Lnc RNA在缺硒引起的雞滲出性素質(zhì)的靜脈損傷中的重要作用。試驗(yàn)結(jié)果如下:(1)飼喂低硒日糧的雛雞靜脈組織中,病理組織學(xué)觀察到管壁增厚,內(nèi)膜層結(jié)構(gòu)部分缺失,平滑肌層纖維走向紊亂、排列不完整,平滑肌細(xì)胞結(jié)構(gòu)不規(guī)則,外膜層缺損,局部有炎性細(xì)胞和淋巴細(xì)胞浸潤。超微結(jié)構(gòu)觀察到內(nèi)皮細(xì)胞和平滑肌細(xì)胞的細(xì)胞核染色質(zhì)邊集,線粒體空泡化,細(xì)胞崩解。Tunel染色表明飼喂低硒日糧的雞靜脈組織凋亡率升高,表明缺硒能引起雞靜脈組織損傷,同時(shí)伴有細(xì)胞凋亡的發(fā)生。(2)缺硒能夠降低雞靜脈組織21種硒蛋白的mRNA表達(dá)。其中受硒缺乏影響較為明顯的硒蛋白是GPx1、SPS2、GPx2、SELS、SEPX1、GPx4、SELI、SEPW1、SELK和SEP15。揭示硒缺乏能夠降低靜脈中硒蛋白的轉(zhuǎn)錄水平。(3)硒缺乏能夠顯著升高雞靜脈組織中熱休克蛋白HSP60、HSP70以及HSP90的m RNA表達(dá)水平以及HSP60、HSP70和HSP90的蛋白水平,表明HSP60、HSP70和HSP90參與了缺硒引起的靜脈損傷。(4)硒缺乏能顯著升高炎癥因子PTGE和COX-2的m RNA和蛋白表達(dá)水平,上調(diào)IL-1β,IL-6,IL-8和IFN-γ的m RNA表達(dá)水平,下調(diào)IL-4和IL-12 mRNA的表達(dá)水平,表明了缺硒能夠引起靜脈炎癥反應(yīng)的發(fā)生。(5)缺硒引起雞靜脈組織中485個(gè)Lnc RNA表達(dá)上調(diào)(P0.05),147個(gè)Lnc RNA表達(dá)下調(diào)(P0.05)。表明缺硒能影響雞靜脈組織中Lnc RNA的表達(dá)。同時(shí)應(yīng)用RT-PCR對(duì)部分差異轉(zhuǎn)錄本進(jìn)行驗(yàn)證,證實(shí)了測(cè)序的準(zhǔn)確性。對(duì)差異表達(dá)的Lnc RNA的靶m RNA進(jìn)行GO和KEGG富集,發(fā)現(xiàn)其參與的主要通路為氧化還原通路、糖代謝通路、磷酸肌醇通路、凋亡通路。(6)缺硒引起雞靜脈組織中109個(gè)miRNA表達(dá)上調(diào),121個(gè)mi RNA下調(diào)(P0.05)。表明缺硒能影響雞靜脈組織中mi RNA的表達(dá)。應(yīng)用RT-PCR對(duì)部分差異轉(zhuǎn)錄本進(jìn)行驗(yàn)證,證實(shí)了測(cè)序的準(zhǔn)確性。對(duì)差異表達(dá)的mi RNA的靶m RNA進(jìn)行GO和KEGG富集,發(fā)現(xiàn)其指導(dǎo)的主要通路包括肌動(dòng)蛋白細(xì)胞骨架的調(diào)節(jié),磷酸肌醇通路,細(xì)胞因子受體互作等通路。將組學(xué)檢測(cè)中mi RNA和Lnc RNA的靶基因富集到的通路進(jìn)行比較,篩選出了分子功能通路、腎上腺素能信號(hào)、脂肪酸代謝、甘油磷脂代謝通路、刺猬信號(hào)通路、磷酸肌醇通路和血管平滑肌收縮通路。(7)在Lnc RNA組學(xué)結(jié)果中,篩選出23個(gè)與氧化還原有關(guān)的Lnc RNA,及其指導(dǎo)的19個(gè)靶mRNA。應(yīng)用RT-PCR對(duì)缺硒和正常雞的靜脈組織篩選出的與氧化還原有關(guān)的Lnc RNA和m RNA進(jìn)行驗(yàn)證,結(jié)果表明23個(gè)Lnc RNA和19個(gè)mRNA的表達(dá)都發(fā)生了顯著變化(P0.05)。在體外培養(yǎng)的內(nèi)皮細(xì)胞中進(jìn)行硒和過氧化氫處理,建立抗氧化和促氧化的細(xì)胞模型,結(jié)果顯示硒和過氧化氫能影響這23個(gè)Lnc RNA的表達(dá)。表明23個(gè)Lnc RNA能夠參與由硒引起的氧化還原反應(yīng)。(8)經(jīng)Lnc RNA組學(xué)和mi RNA組學(xué)聯(lián)合比較分析,應(yīng)用生物信息學(xué)的方法,預(yù)測(cè)到Lnc RNA-ALDBGALG0000001600(Lnc RNA-1600)—mir-223—Inpp4a具有靶向關(guān)系。建立mir-223的過表達(dá)模型和Lnc RNA-1600的敲低模型,在體內(nèi)、體外模型中證明其表達(dá)以負(fù)調(diào)控關(guān)系存在。通過熒光素酶報(bào)告基因試驗(yàn)確定它們的靶向關(guān)系。證實(shí)了Lnc RNA-1600能調(diào)控mir-223,再指導(dǎo)Inpp4a的轉(zhuǎn)錄。(9)應(yīng)用RT-PCR和WB檢測(cè)發(fā)現(xiàn),缺硒能下調(diào)組織中磷酸肌醇通路中Inpp4a、Impa1和Impa2,凋亡通路Bcl-2的表達(dá),上調(diào)線粒體凋亡通路中Bax、caspase3和caspase9的表達(dá)。在敲低和過表達(dá)mir-223模型和si Lnc RNA-1600模型中,應(yīng)用RT-PCR和WB檢測(cè)磷酸肌醇通路中mRNA和蛋白的變化,以及線粒體凋亡通路中Bax、Bcl-2、caspase3和caspase9的表達(dá)情況,應(yīng)用hoechst染色和FCM檢測(cè)細(xì)胞周期,發(fā)現(xiàn)過表達(dá)mir-223,敲低Lnc RNA-1600都能引起內(nèi)皮細(xì)胞凋亡的發(fā)生,表明Lnc RNA-1600—mir-223—Inpp4a通路能調(diào)控靜脈內(nèi)皮細(xì)胞的凋亡。綜上所述,缺硒可以下調(diào)雞靜脈組織中硒蛋白的轉(zhuǎn)錄水平,影響氧化還原水平、炎癥因子和細(xì)胞因子的表達(dá)水平、熱休克蛋白的表達(dá)、磷酸肌醇通路基因的表達(dá)以及凋亡基因的表達(dá),引起靜脈組織損傷。通過對(duì)mi RNA和Lnc RNA進(jìn)行組學(xué)分析,篩選出特異表達(dá)的mi RNA和Lnc RNA,并篩選出特異通路,證實(shí)Lnc RNA-1600—mir-223—Inpp4a通路誘導(dǎo)滲出性素質(zhì)雞靜脈內(nèi)皮凋亡的發(fā)生。本結(jié)果填補(bǔ)了缺硒和正常雞靜脈組織中mi RNA和Lnc RNA表達(dá)譜的空白,豐富了缺硒引起靜脈組織凋亡的病理學(xué)機(jī)制,為探討缺硒引起雞靜脈組織損傷的機(jī)制奠定理論基礎(chǔ)和試驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.31

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