基于RNA-Seq技术的雏鸵鸟胸腺应答硼刺激分子机制研究

发布时间：2018-05-11 06:14

  本文选题：雏鸵鸟 + 硼　； 参考：《华中农业大学》2016年博士论文

【摘要】：非洲鸵鸟具有很大的经济价值和科学价值。鸵鸟育雏期长达3个月,人工养殖的鸵鸟容易受到应激、感染和疾病的影响,使得育雏期鸵鸟的发病率和死亡率高达10%-50%。此外,鸵鸟是快速生长的鸟类,育雏期鸵鸟个体发育差别较大,免疫机能的强弱直接反应了育雏期鸵鸟生长发育状况。胸腺是机体内重要的中枢免疫器官和淋巴器官,也是机体抵抗外界抗原刺激,参与机体T淋巴细胞分化的重要免疫器官。硼是动物体必需的微量元素,具有多种生物学功能。硼在免疫系统中发挥重要的调节作用,适量的硼有利于免疫器官的生长发育。本试验选取1日龄的雏鸵鸟作为研究对象,饮水中添加梯度剂量的硼酸,饲喂至90日龄,宰杀取胸腺。研究硼对鸵鸟中枢免疫器官胸腺的调节作用,阐释硼参与鸵鸟机体免疫应答的作用机制。1.硼对雏鸵鸟胸腺生长发育相关基因的影响Foxn1参与胸腺上皮细胞的分化发育过程,并发挥关键调控作用,BMP2和BMP4参与调控胸腺T细胞的发育,Foxn1,BMP2和BMP4基因与胸腺内环境稳态密切相关。研究不同水平的硼对鸵鸟胸腺细胞凋亡的影响及对鸵鸟胸腺发育相关基因Foxn1,BMP2和BMP4的表达调控,为揭示硼对鸵鸟胸腺发育影响的相关机制提供参考。HE染色技术观察硼对雏鸵鸟胸腺组织学结构的影响,RACE PCR克隆鸵鸟Foxn1基因的序列,免疫组织化学技术和western blot技术检测Foxn1蛋白在鸵鸟胸腺内的定位表达。免疫荧光技术检测BMP2和BMP4蛋白在鸵鸟胸腺内的表达。荧光定量PCR技术检测Foxn1基因,BMP2基因和BMP4基因的mRNA表达水平。TUNEL技术检测鸵鸟胸腺内细胞凋亡情况,免疫组织化学检测活化体caspase-3在鸵鸟胸腺内的定位表达。实验结果如下:(1)HE染色观察硼对雏鸵鸟胸腺组织学结构的影响,高剂量硼组(B320组和B640组)胸腺的形态结构不完整,皮髓交界分界不清楚,胸腺皮质内出现“星空状”外观,胸腺细胞急剧减少和空竭,胸腺髓质内淋巴细胞增多,胞核固缩深染,B640组胸腺胸腺结构破坏最严重,胸腺上皮细胞空泡化,髓质部最明显。低剂量硼组(B40和B80)胸腺形态结构与对照组基本一致。(2)TUNEL技术检测雏鸵鸟胸腺内细胞凋亡,与对照组相比,B80组和B160组胸腺内凋亡细胞数量较少,B320组和B640组胸腺内凋亡细胞数量增多,凋亡细胞成群分布。(3)Caspase-3活化体检测发现,B320组和B640组胸腺内活化体caspase-3阳性信号显著增强,高剂量的硼诱导caspase-3的活化,诱发鸵鸟胸腺细胞凋亡。(4)racepcr克隆扩增鸵鸟foxn1基因,随机引物扩增鸵鸟foxn1基因中间保守片段,长为1477bp,5’race和3’race的长度分别为384bp和1050bp,全长为2736bp,编码654个氨基酸。鸵鸟foxn1基因是高度保守的基因,与猎隼、虎皮鹦鹉、游隼、石鸽、野鸭和鸡的foxn1基因同源性分别为92.1%,91.1%,90.8%,89.6%,88.1%和83.5%。(5)foxn1阳性信号主要分布在鸵鸟胸腺髓质部,胸腺皮质部有少量分布,主要标记胸腺上皮细胞。80mg/l的硼酸剂量组能够显著提高foxn1蛋白在鸵鸟胸腺中的表达,而640mg/l的硼酸剂量能够显著降低foxn1蛋白在胸腺中的表达。(6)bmp2和bmp4的mrna表达水平呈硼剂量依赖性,表达趋势先上升后下降,在80mg/l的硼处理组中,bmp2和bmp4在鸵鸟胸腺内显著高表达,在640mg/l的硼处理组中,bmp2和bmp4的表达水平被显著抑制。饮水中添加适量的硼促进雏鸵鸟胸腺的发育,鸵鸟胸腺正常发育;过量的硼导致雏鸵鸟胸腺组织学结构破坏,诱发雏鸵鸟胸腺内细胞凋亡,活化体caspase-3表达增强,抑制雏鸵鸟胸腺的正常发育。80mg/l硼促进bmp2,bmp4,foxn1蛋白的表达,bmp2,bmp4,foxn1蛋白表达适度增加会促进胸腺上皮细胞的分化发育,从而促进t细胞的分化发育,增强鸵鸟机体免疫抵抗力。高剂量的硼(640mg/l)显著抑制bmp2,bmp4,foxn1蛋白在鸵鸟胸腺内的表达,导致鸵鸟胸腺结构的破坏和退化,胸腺的皮髓交界处损坏严重,胸腺上皮细胞分化发育受阻,抑制t细胞的正常生长发育,降低鸵鸟机体免疫机能。2.雏鸵鸟胸腺应答硼刺激的rna-seq分析为了深入研究硼参与鸵鸟免疫应答的作用机制,本研究采用rna-seq技术,首次对微量元素硼在鸵鸟胸腺免疫应答反应中的作用进行了高通量测序分析。本试验共选取3个样本进行rna-seq分析,分别是对照组鸵鸟胸腺,b80组鸵鸟胸腺,b640组鸵鸟胸腺。rna-seq分析结果显示:(1)3个鸵鸟胸腺测序文库(control组样本文库,b80组样本文库和b640组样本文库),分别包含3220173000,3427886800和3199872000条rawdata。control组样品,b80组样品和b640组样品的cleanreads数分别为3175394800,3379919000和3157669200,cleandata占reads总数的比例分别为98.61%,98.60%和98.68%。(2)control组样品,b80组样品和b640组样品单一比对上鸵鸟参考基因组的reads数分别为11929989,12851275和11411617,占总reads的比例分别为75.14%,76.04%和72.28%。(3)基因覆盖度统计分析显示,三个文库中测序覆盖度达90-100%的基因数量占总数量的比例分别为72%,71%和69%。(4)成对差异基因统计结果显示:control组和b640组,b80组和b640组,control组和b80组差异基因比较总计分别有2044个(上调基因228个,下调基因1816个),1085个(上调基因222个,下调基因863个)和902个(上调基因309个,下调基因593个)差异基因。(5)差异基因趋势分析结果显示:本试验中所有的差异基因共分成了7种表达趋势,其中趋势0,趋势1和趋势3具有差异显著性。趋势0中共包含1290个差异基因,在3个文库中(control,b80和b640)的表达依次呈现持续下降的趋势;趋势1中共包含1030个差异基因,在3个文库中的表达依次呈现先下降后保持不变的趋势;趋势3中共包含1487个差异基因,在3个文库中的表达依次呈现先上升或不变后下降的趋势。(6)差异基因kegg信号通路富集分析结果显示:富集前10条信通路中细胞因子-受体互作通路,癌症通路,钙离子信号通路,肌动蛋白细胞骨架调节通路和mapk信号通路显著富集,这些信号通路主要参与机体的免疫应答及炎症应答过程。(7)成对样品差异基因kegg富集结果显示,除上述信号通路,硼还参与toll样受体信号通路,b细胞受体通路,t细胞受体通路和凋亡通路的调节。由此表明,硼对鸵鸟机体的炎症及免疫机能的相关信号通路的影响,主要包括mapk信号通路,钙离子通路,b细胞受体通路,t细胞受体通路,肌动蛋白调节信号通路,toll样受体信号通路,凋亡信号通路及癌症信号通路。3.硼对鸵鸟机体免疫、炎症及生长发育相关通路的影响为了进一步验证rna-seq的分析结果,并探讨不同剂量的硼对鸵鸟胸腺内免疫机能相关信号通路的影响作用,本项目对参与免疫机能调节的信号通路进行了研究,主要包括mapk通路,钙离子通路,b细胞受体通路,t细胞受体通路,toll样受体通路,癌症信号通路以及细胞凋亡通路。(1)硼对mapk通路活性的影响:共有27个差异基因富集到mapk信号通路中,control组和b80组中的大部分基因表达上升,而b640组中的基因多数表达下调,27个差异基因中有24个基因表达下降,3个基因表达上升。硼能够影响鸵鸟机体内ras/erk通路,jnk通路和p38mapk通路,但不能参与erk5通路的调节。qrt-pcr及westernblot验证结果显示,80mg/l硼促进了鸵鸟胸腺内p-erk,p-jnk和p-p38蛋白水平的表达,随着硼剂量的增加,p-erk,p-jnk和p-p38蛋白表达水平逐渐降低,在640 mg/L硼组蛋白表达水平最低。硼能够调节ERK,JNK以及p38MAPK通路中激酶的表达水平,影响MAPK信号通路的活性。(2)硼对钙离子通路活性的影响:共有11个差异基因富集到钙离子通路中,硼主要调节钙调磷酸酶(CaN)和钙调素依赖蛋白激酶(CaMK)的活性。硼对CaN的两个亚基均有调节作用,主要调控PPP3R1的活性,对PPP3CA的调节作用较小。硼能够通过调节Ca N的活性,影响CaN下游转录因子NFAT和MEF2C的表达水平,参与Ca2+-CalcineurinNFAT信号通路活性调节。(3)硼对B细胞受体信号通路和T细胞受体信号通路活性的影响:共有7个差异基因富集到B细胞受体信号通路中,共有11个差异基因富集到T细胞受体信号通路中。T、B细胞受体能够介导肌动蛋白细胞骨架,MAPK通路,PI3K-AKT通路以及Ca N-NFAT通路的活化。根据KEGG和荧光定量分析结果,显示硼能够影响PI3K激酶的活性,调控PI3K-AKT通路。硼对T、B细胞因子受体通路的影响作用与MAPK通路,钙离子通路以及PI3K-AKT通路的活性密切相关。(4)硼对Toll样受体通路活性的影响:共有11个差异基因富集到Toll样受体信号通路中,硼对Toll样受体通路的调节包括两部分,不仅能够调控MyD88依赖性信号通路,也能够调控MyD88非依赖性信号通路。并且高剂量硼对鸵鸟机体中Toll样受体信号通路的调节呈现负调控调节,抑制了Toll样受体信号通路的活化,影响机体免疫应答的机能。(5)硼对热休克蛋白家族(HSP)的影响:热休克蛋白与机体的细胞凋亡密切相关,是一种抗凋亡蛋白。热休克蛋白家族中的代表性成员是Hsp70,Hsp70的伴侣分子是Hsp40。高剂量的硼显著抑制了Hsp70和Hsp40蛋白的表达,抑制了鸵鸟机体内热休克蛋白的抗凋亡作用以及介导机体免疫应答的能力,导致鸵鸟机体抵抗力降低。(6)硼对细胞凋亡通路及癌症通路中相关基因的影响:硼主要影响TRAIL诱导的外源性细胞凋亡通路的活性,且参与凋亡抑制蛋白FLIP和IAP的活性调节,对caspase家族的调节作用较小。硼参与鸵鸟机体内癌症信号通路的调节,主要影响癌症信号通路中Wnt信号通路,MAPK信号通路,PI3K-AKT信号通路以及细胞因子-受体互作通路的活性,这些信号通路发挥着复杂精细的调控作用,共同参与雏鸵鸟机体内细胞的分化、增殖及凋亡过程。
[Abstract]:African ostrich has great economic value and scientific value. Ostrich rearing period is 3 months long. The artificial ostrich is susceptible to stress, infection and disease, which makes the ostrich morbidity and mortality rate as high as 10%-50%., the ostrich is fast growing bird, and the ostrich ostrich's individual development is different and immune function. The strength and weakness directly reflect the growth and development of ostrich in the brood stage. Thymus is an important central immune organ and lymphoid organ in the body. It is also an important immune organ for the body to resist external antigen stimulation and participate in the differentiation of T lymphocytes. Boron is a necessary trace element of the animal body and has many biological functions. Boron is used in the immune system. A moderate amount of boron is beneficial to the growth and development of immune organs. In this experiment, the 1 day old ostrich was selected as the research object, and a gradient dose of boric acid was added to the drinking water, and the thymus was killed at the age of 90 days and the thymus was slaughtered. The effect of Boron on the thymus of the ostrich central immune organ was studied to explain the immune response of boron to the ostrich body. The effect of.1. boron on the growth and development related genes of chick thymus Foxn1 participates in the differentiation and development of thymic epithelial cells, and plays a key regulatory role. BMP2 and BMP4 participate in the regulation of the development of thymus T cells. Foxn1, BMP2 and BMP4 genes are closely related to the homeostasis of the thymus environment. The effect of apoptosis and the regulation of the expression of Foxn1, BMP2 and BMP4 on the development of ostrich thymus development, to reveal the related mechanism of boron to the development of ostrich thymus development, provide reference.HE staining technique to observe the effect of Boron on the histological structure of the thymus of ostrich, RACE PCR clone ostrich Foxn1 gene sequence, immunohistochemical technique and Western blot Localization and expression of Foxn1 protein in the ostrich thymus by technique. The expression of BMP2 and BMP4 protein in the ostrich thymus was detected by immunofluorescence. The fluorescence quantitative PCR technique was used to detect the Foxn1 gene, BMP2 gene and the mRNA expression level of BMP4 gene to detect the apoptosis in the ostrich's thymus, and the immunohistochemical detection of activator caspase. The results were as follows: (1) the effects of Boron on the histological structure of the thymus of ostrich were observed as follows: (1) HE staining was used to observe the histological structure of the thymus. The morphological structure of the thymus in the high dose boron group (group B320 and B640) was incomplete, the boundary of the skin marrow junction was not clear, the appearance of "star empty" in the thymus cortex, the sharp decrease of thymus cells and the exhaustion of the thymus, and the thymus medulla In the B640 group, the thymus thymus structure was most serious, the thymus epithelial cell vacuolation and the medulla were the most obvious. The morphological structure of the thymus gland in the low dose boron group (B40 and B80) was the same as that in the control group. (2) the TUNEL technique was used to detect the apoptosis in the thymus of the ostrich, compared with the control group, the B80 and B160 groups were withered in the thymus gland. The number of dead cells was less, the number of apoptotic cells in the thymus of B320 and B640 increased and the apoptotic cells were distributed. (3) the detection of Caspase-3 activator found that the positive signal of Caspase-3 positive in the B320 and B640 groups was significantly enhanced. The high dose of boron induced the activation of Caspase-3 and induced the apoptosis of the ostrich thymocyte. (4) racepcr cloned and amplified ostrich. The bird Foxn1 gene amplified the conservative fragment of the ostrich Foxn1 gene with random primers. The length of the ostrich Foxn1 gene was 1477bp, the length of 5 'race and 3' race was 384bp and 1050bp, the whole length was 2736bp, and the 654 amino acids were encoded. The ostrich Foxn1 gene was highly conserved, and the homology of the Foxn1 genes of the Falcon, parrots, falcons, pigeons, ducks and chickens was 92.1, respectively. The positive signals of%, 91.1%, 90.8%, 89.6%, 88.1% and 83.5%. (5) Foxn1 were mainly distributed in the ostrich thymus medulla, and the thymic cortex had a small amount. The boric acid dose group that mainly labeled the thymic epithelial cell.80mg/l could significantly increase the expression of Foxn1 protein in the ostrich thymus, and the 640mg/l dose of boric acid could significantly reduce the Foxn1 protein in the thymus gland. (6) the expression level of mRNA in BMP2 and BMP4 showed a boron dose dependence and the expression trend increased first and then decreased. In the boron treatment group of 80mg/l, the expression of BMP2 and BMP4 was highly expressed in the ostrich thymus. In the boron treatment group of 640mg/l, the expression level of BMP2 and BMP4 was significantly suppressed. The addition of proper boron in drinking water to promote the hair of the chick thymus. The thymus development of ostrich is normal; excessive boron leads to the destruction of the histological structure of the thymus of ostrich, inducing apoptosis in the thymus of the ostrich, the expression of activator caspase-3 and the inhibition of the normal development of the thymus of the ostrich,.80mg/l boron promotes the expression of BMP2, BMP4, Foxn1 protein, BMP2, BMP4, and Foxn1 protein may increase the thymus epithelium fine. The differentiation and development of the cell promote the differentiation and development of T cells and enhance the immune resistance of the ostrich body. The high dose of boron (640mg/l) significantly inhibits the expression of BMP2, BMP4, Foxn1 protein in the ostrich thymus, resulting in the destruction and degradation of the ostrich thymus structure, the serious damage to the borderline junction of the thymus, the differentiation and development of the thymus epithelial cells, and the inhibition of the t. The normal growth and development of the cells, the reduction of the RNA-seq analysis of the thymus response to the thymus of ostrich.2. in order to study the mechanism of boron in the immune response of the ostrich, this study used RNA-seq technology to analyze the role of microelement boron in the ostrich thymus immune response for the first time. A total of 3 samples were selected for RNA-seq analysis, which were the control group ostrich thymus, B80 group ostrich thymus, and the b640 group ostrich thymus.Rna-seq analysis results showed: (1) 3 ostrich thymic sequence library (control sample library, B80 group library and b640 group sample library), including 32201730003427886800 and 3199872000 rawdata.c, respectively. The number of cleanreads in group ontrol, group B80 and b640 is 31753948003379919000 and 3157669200 respectively. The proportion of cleandata to reads is 98.61%, 98.60% and 98.68%. (2) control, respectively. The reads number of the ostrich reference genome of the B80 group and b640 group is 1192998912851275 and 1141161, respectively. 7, the proportion of total reads was 75.14%, 76.04% and 72.28%. (3) gene coverage statistical analysis showed that the proportion of gene number in the three library was 72%, 71% and 69%. (4), respectively: control and b640 group, B80 group and b640 group, control group and B80 group differential gene. The total total was 2044 (up - regulation 228, down gene 1816), 1085 (up - regulation 222, down - regulated gene 863) and 902 (up - regulation 309, down gene 593) differential gene. (5) difference gene trend analysis showed that all the difference genes were divided into 7 trends, trend 0, trend 1 Trend 3 has significant difference. Trend 0 contains 1290 differentially genes, and the expression in 3 libraries (control, B80 and b640) shows a trend of continuous decline; trend 1 contains 1030 differentially expressed genes, and the expression in 3 library is in turn decreasing and holding the same trend; trend 3 contains 1487 differentially genes, The expression in the 3 libraries showed a trend of first or later decline. (6) the analysis of the enrichment analysis of the difference gene KEGG signaling pathway showed that the cytokine receptor interaction pathway, the cancer pathway, the calcium ion signaling pathway, the actin cytoskeleton regulation pathway and the MAPK signaling pathway were enriched in the 10 signal pathways. The signal pathway mainly participates in the immune response and inflammatory response process of the body. (7) the results of the KEGG enrichment of differential genes in the paired samples show that boron also participates in the toll like receptor signaling pathway, the B cell receptor pathway, the T cell receptor pathway and the apoptosis pathway. The effects of related signaling pathways include MAPK signaling pathway, calcium channel, B cell receptor pathway, T cell receptor pathway, actin regulation signaling pathway, toll like receptor signaling pathway, apoptosis signaling pathway, and cancer signal pathway.3. boron on ostrich body immunity, inflammation and growth related pathways in order to further examine The results of RNA-seq analysis, and the effect of different doses of Boron on the immune function related signaling pathway in the ostrich's thymus, were studied in this project, including the MAPK pathway, calcium channel, B cell receptor pathway, T cell receptor pathway, toll like receptor pathway, and cancer signaling pathway. Road and apoptotic pathway. (1) the effect of Boron on the activity of MAPK pathway: a total of 27 different genes were enriched in the MAPK signaling pathway, most of the gene expression in group control and B80 increased, while most of the genes in the b640 group were down regulated, 24 of the 27 genes decreased and 3 genes increased. Boron could affect ostrich. The ras/erk pathway, the JNK pathway and the p38MAPK pathway in the bird's body, but not involved in the regulation of the ERK5 pathway,.Qrt-pcr and Westernblot verification showed that 80mg/l boron promoted the expression of p-ERK, p-JNK and p-p38 protein levels in the ostrich thymus. With the increase of boron dosage, p-ERK, p-JNK and protein expression level gradually decreased, in the 640 boron histone. The lowest expression level. Boron can regulate the expression level of kinase in ERK, JNK and p38MAPK pathways and influence the activity of MAPK signaling pathway. (2) the effect of Boron on the activity of calcium ion pathway: a total of 11 different genes are enriched in the calcium channel, boron mainly regulates the activity of calcineurin (CaN) and calmodulin dependent protein kinase (CaMK). Boron is to CaN The two subunits can regulate the activity of PPP3R1 and regulate the activity of PPP3CA. Boron can regulate the expression level of NFAT and MEF2C by regulating the activity of Ca N, and participate in the activity regulation of Ca2+-CalcineurinNFAT signaling pathway. (3) the boron on the B cell receptor signaling pathway and the T cell receptor signaling pathway. A total of 7 differential genes are enriched in the B cell receptor signaling pathway, and a total of 11 different genes are enriched into the T cell receptor signaling pathway.T, and the B cell receptor can mediate the actin cytoskeleton, the MAPK pathway, the PI3K-AKT pathway and the activation of the Ca N-NFAT pathway. The activity of PI3K kinase regulates the activity of the PI3K-AKT pathway. The effect of Boron on the T, B cell factor receptor pathway is closely related to the MAPK pathway, the calcium channel and the activity of the PI3K-AKT pathway. (4) the effect of Boron on the activity of Toll like receptor pathway: a total of 11 different genes are enriched in the Toll like receptor signaling pathway, and the modulation of boron to the Toll like receptor pathway The two part includes not only the regulation of the MyD88 dependent signaling pathway but also the regulation of the MyD88 non dependent signaling pathway, and the regulation of the Toll like receptor signaling pathway in the ostrich body is negatively regulated by high dose boron, which inhibits the activation of the Toll like receptor signaling pathway and affects the function of the immune response. (5) boron to heat shock. The influence of protein family (HSP): the heat shock protein is closely related to the apoptosis of the body and is an anti apoptotic protein. The representative member of the heat shock protein family is Hsp70. The Hsp70 partner is the high dose of Hsp40., which inhibits the expression of Hsp70 and Hsp40 protein, and inhibits the anti apoptosis of the heat shock protein in the ostrich body. The ability to use and mediate the immune response of the body leads to the reduction of the resistance of the ostrich body. (6) the effect of Boron on the apoptosis pathway and the related genes in the cancer pathway: Boron mainly affects the activity of the exogenous apoptosis pathway induced by TRAIL, and is involved in the regulation of the activity of FLIP and IAP, and the regulation of the caspase family is more effective. Boron is involved in the regulation of the cancer signal pathway in the ostrich body, which mainly affects the activity of Wnt signaling pathway, MAPK signaling pathway, PI3K-AKT signaling pathway and cytokine receptor interaction pathway in the cancer signaling pathway. These signaling pathways play a complex and fine regulatory role, and participate in the differentiation, proliferation and proliferation of cells in the ostrich body. The process of apoptosis.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S839
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本文编号：1872760