【作者】 张顺华；

【导师】 李学伟；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 乳汁是哺乳动物新生儿最重要的营养和被动免疫来源,富含多种免疫成分,能够满足婴幼儿大脑发育、生长以及健康免疫系统的各种需求。动物体液中的microRNA (miRNA)通常被选择性地包裹在exosome中。Exosome是由很多种类的细胞所分泌的一种带膜囊泡,这些exosome中的miRNAs能够被主动地定向输送到受体细胞,并调节靶基因的表达和受体细胞的功能。本研究中,利用原子力显微镜(Atomic force microscope, AFM)证实猪乳汁中大量存在exosome的基础上,构建了约克夏猪整个泌乳期内(从初生到28日龄)6个不同泌乳阶段(出生后0、3、7、14、21和28d)乳汁exosome small RNA文库,以出生后0d和3d的乳汁作为初乳,其他日龄的乳汁作为常乳进行对比性分析,用高通量测序的方法分析和注解了初乳(0d和3d)和常乳(7、14、21和28d)exosome中的miRNA表达谱,筛选与免疫密切相关的miRNA,分析其在不同环境下的耐受(稳定)性；对免疫相关的miRNA在仅初乳和仅常乳喂养的仔猪体内的差异表达进行研究，通过仔猪活体模型研究以评估其在仔猪生长发育及免疫系统形成过程中的功能。结果表明：以超高速离心的方法从猪乳汁中分离到含量较丰富的exosome。通过AFM在纳米水平观察到猪乳汁中exosome平均宽度约100nm,高度约12nm。Small RNA文库中没有检测到18S和28S核糖体RNA,但大量存在长度小于300nt的small RNAso Illumina Genome Analyzer Ⅱ(GA Ⅱ)高通量测序结果表明,猪乳汁exosome中存在丰富的miRNAs,绝大部分(91.97±3.18%)可“定位”(即可map到猪基因组序列中)的读数长度在21-24nt之间,其中,长度为22nt的读数达到(67.93±4.81)%。长度为23nt的读数占(15.92±2.27)%,24nt占(4.71±1.65)%,21nt占(3.42±1.77)%。这些读数的长度都是典型的Dicer酶加工产物的长度,符合miRNA的基本结构特征。高通量测序数据的生物信息学分析表明,在8个猪乳汁exosome small RNA文库中共检测到180种pre-miRNAs。其中,140种(77.78%)是已知的猪pre-miRNAs,另外40种(22.22%)是新发现的猪pre-miRNAs。新发现的40种猪pre-miRNAs全部与miRBase18.0中已报道的人pre-miRNAs具有同源性,并能够被定位到猪基因组上。这180种pre-miRNAs共可编码237种成熟miRNAs,对应234种特异性]niRNAs。基于Pathway Central Database (SABiosciences, MD, USA)的注解,在niRBase(Version18.0)数据库中注册的1527种已知人的pre-miRNAs中,存在84种(5.50%)与淋巴细胞和其他免疫细胞功能相关的免疫性pre-miRNAs。以此为基础的比对结果表明,本研究的8个猪乳汁exosomesmall RNA文库中,共检测到58种(69.05%)存在并富集于各阶段猪乳汁exosome的miRNA文库中(P＜10-16,χ2test)。并且,各个泌乳阶段与免疫相关的pre-miRNAs在总乳汁exosome miRNA文库中的比例都超过了30%,极显著地高于人miRNA文库中免疫性相关pre-miRNAs5.50%的比例。此外,在出生后前3天的初乳中,与免疫相关的pre-miRNAs均达到52种以上,显著地高于常乳(P<0.05)。进入常乳期后,则降低到40种左右,这暗示着乳汁在仔猪免疫系统功能中发挥着重要功能。在乳汁exosome的miRNA转录本中,绝大多数miRNAs转录子来自于很少几种miRNAs。在234种特异性miRNAs中,以reads数计算,表达水平最高的前10种miRNAs的含量超过总量的87.25%。6个泌乳阶段里表达水平最高的前10种高丰度miRNAs一共包括有13种特异性miRNAs。其中有7种miRNAs (miR-148a-3p, miR-182-5p, miR-200c-3p, miR-25-3p, miR-30a-5p, miR-30d-5p和miR-574-3p)在6个泌乳阶段miRNA文库里的表达量都排在前10位,它们在不同的免疫和病理状态下承担着重要的功能。另外6种miRNAs(let-7a-1-5p, miR-30c-2-5p and-1-5p, miR-191-5p, miR-375-3p, miR-21-5p和miR-27b-3p)至少在某一个泌乳阶段的miRNA文库里的表达量位于前10位,它们也无一例外地都和不同的免疫与病理学反应相关。猪乳汁exosome中miRNA的表达谱呈现典型的泌乳特异性模式,初乳和常乳间存在明显差异,表现为免疫相关miRNA在初乳中的含量显著高于常乳。定量分析结果表明,13种在6个泌乳阶段里表达量最高的与免疫相关的高丰度miRNAs中,除miR-148a-3p外,其它12种特异性miRNAs在初乳中的表达量都高于常乳,而在6个阶段里所有高丰度miRNAs中表达水平都为最高的niR-148a-3p的表达量则是随着泌乳期而增加。这些免疫相关的miRNAs都会功能性地靶向编码细胞活素和其它免疫调节蛋白,以及免疫反应信号通路上的其它组分等特异性转录本而行使其免疫调控功能。与外源添加的秀丽隐杆线虫(C. elegans)特异性miRNAs (cel-lin-4-5p, cel-miR-2-3p和cel-miR-39-5p)和拟南芥(Arabidopsis thaliana)特异性miRNA (ath-miR-159a-3p)相比,5种内源性乳汁exosome来源的高表达：niRNAs (miR-148a-3p, miR-30c-5p, miR-574-3p, miR-21-5p和miR-27b-3p)对不同的极端理化条件(低温、高温、低pH、反复冻融、外源RNA酶等)都表现出较强的抗降解能力,说明乳汁exosome中的miRNAs是以一种非常稳定的状态存在,可以保护其在不同极端条件下免受内源性RNA酶的降解。不同乳汁饲喂实验表明,仅初乳和仅常乳喂养的仔猪,其血清中的7种代表性血液循环代谢生化指示物的含量在两组仔猪血清中相近,没有发生显著性变化(P>0.05)。但13种特异性免疫相关的高丰度miRNAs在不同饲喂组间的血清表达量呈现出明显的差异。表现为：在只喂初乳的仔猪中,除了miR-148a-3p外的其它12种miRNAs的血清表达量都显著地高于只饲喂常乳的仔猪。而miR-148a-3p的表达量在饲喂常乳组仔猪血清中的表达量则显著地高于饲喂初乳组(P<0.05)。仔猪血清中各免疫相关高丰度miRNAs的总体表达趋势与之前乳汁实验中关于初乳和常乳中13种免疫相关高丰度miRNAs的表达规律一致。这些猪乳exosome中与免疫相关的miRNA可能是通过消化道而直接进入仔猪的体内发挥其调控功能,从而潜在影响着仔猪免疫系统的发育。上述研究结果为深入研究乳汁在婴儿免疫系统发育过程中的重要功能提供了动物模型基础数据,证明猪是人类泌乳医学和免疫疾病研究理想的模式生物。更多还原

【Abstract】 To newborn mammals, breast milk is the primary source of nutrition and immune. Breast milk is rich in immunological components, can meet all the requirements of brain development, growth and immunology system. MicroRNAs (miRNAs) are widely exist in various kinds of body fluids and tissues, and are selectively packaged inside the exosomes. Exosome is a type of membrane vesicles, secreted by many kinds of cell types. These miRNAs packaged in exosomes could be actively delivered into the recipient cells, and regulate the expression of target genes and the function of recipient cells. In this study, exosomes are confirmed to present in Yorkshire breast milk observating with atomic force microscope (AFM), and we constructed8milk exosomal small RNA libraries from6typical time points (0day,3days,7days,14days,21days and28days after birth) across the entire lactation period (from newborn to28days after birth). The breast milk of0day and3days after birth were classed into colostrum, and the others were mature milk. Utilizing the method of deep sequencing, the expression profiles of these lactation-related exosomal miRNAs in porcine milk were analyzed,we screened mmune-related miRNAs are present and enriched in breast milk exosomes and are generally resistant to relatively harsh conditions. These immune-related miRNAs are present in higher numbers in the colostrum compared with the mature milk or the blood of colostrum-only fed piglets compared with the mature milk-only fed piglets. Taken together, We got the following results:By the method of high speed ultracentrifugation, we isolated abundant of exosome-like vesicles from porcine breast milk. The morphology and size of exosomes were observed by (AFM at the nanometer-scale. The size of these exosome-like vesicles following normal distribution, and the average width was approximate100nm and the height was about12nm. All the morphological characters of these exosome-like vesicles from porcine breast milk were similar with the reported exosomes in human saliva and breast milk.No18S and28S ribosomal RNA were found in the milk exosomes, but contain large number of small RNAs that were below300nt in length. Eight small RNA libraries were subjected to single-end sequencing in36nt reads using an Illumina Genome Analyzer Ⅱ. The analysis of sequencing data confirmed the enrichment of miRNAs in porcine breast milk exosomes. Of these mappable reads in the eight libraries, the overwhelming majority are21-24nt in length (91.97±3.18%). More than half of the reads are22nt in length (67.93±4.81%), followed by23nt (15.92±2.27%),24nt (4.71±1.65%), and21nt (3.42±1.77%), which are typical sizes of Dicer-processed products, confirming the presence of miRNAs in milk exosomes.The bioinformatics pipeline for miRNA discovery and expression profiling discovered180pre-miRNAs in the8exosomal small RNA libraries, of which140(77.78%) are known porcine pre-miRNAs and40are novel porcine pre-miRNAs. All of these40novel porcine pre-miRNAs are homologous to human pre-miRNAs deposited in miRBase18.0and could be mapped to the pig genome. These180pre-miRNAs encode237mature miRNAs, corresponding to234unique miRNAs.Based on the annotation of the Pathway Central Database (SABiosciences, MD, USA), among these1527known human pre-miRNAs,84(5.50%) are immune-related pre-miRNAs.Out of these84immune-related pre-miRNAs,58(69.05%) are present and enriched in each milk exosomal miRNA library (P＜10-16,χ2test). And in the8porcine milk exosomal miRNA libraries, the immune-related pre-miRNAs account for more than30%of the total pre-miRNAs, which was significantly higher than5.5%, the percentage in the human miRNA library. In the colostrum libraries (0day and3days after borth), there are more than52immune-related pre-miRNAs, significantly higher than40, the number of immune-related pre-miRNAs in the mature milk libraries (P<0.05).The enrichment of immune-related pre-miRNAs in the milk exosome suggested the important role of milk in the development of immune system.In the breast milk exosomal miRNA transcriptome, the majority of abundant miRNAs are from few miRNAs. The top10unique miRNAs with the highest expression level account for more than87.25%, by total counts, of all the234unique miRNAs. The unified set of top10unique miRNAs over six lactation stages corresponds to13kinds of unique miRNAs. Among these13miRNAs,7miRNAs (miR-148a-3p, miR-182-5p, miR-200c-3p, miR-25-3p, miR-30a-5p, miR-30d-5p, and miR-574-3p) are present in the top10miRNAs in all six libraries, which suggests essential roles in various immune and pathological conditions. The other6miRNAs (let-7a-1-5p, miR-30c-2-5p and-1-5p, miR-191-5p, miR-375-3p, miR-21-5p, and miR-27b-3p), which are present in the top10miRNAs in at least one of the6lactation stages, are all related to various immune and pathological responses. The miRNA expression profiles show typical lactation-specific pattern. The immune-related miRNAs exist in the colostrum are significantly higher than those in the mature milk. By quantitative analyzing, apart from miR-148a-3p, almost all the other12unique immune-related miRNAs exhibited higher abundances in the early lactation periods (0and3days) than in the later lactation periods (7,14,21and28days). And the expression level of miR-148a-3p, the top ranking miRNA across6lactation stages by counts, increased throughout the entire porcine lactation stage.All of these immune-related miRNAs have been shown to functionally target specific transcripts encoding cytokines, other immunological regulatory proteins, and immune response signaling pathway components.When the milk was subjected to various harsh conditions (low temperature, high temperature, low pH, multiple freeze-thaw cycles, and exogenous RNase), the exogenous synthetic worm-specific C. elegans miRNAs (cel-lin-4-5p, cel-miR-2-3p and cel-miR-39-5p) and Arabidopsis thaliana miRNA (ath-miR-159a-3p) were sharply degraded. However, the5endogenous breast milk exosome-derived high expression miRNAs (miR-148a-3p, miR-30c-5p, miR-574-3p, miR-21-5p and miR-27b-3p) exhibited resistance to degradation and high stability to harsh conditions. These results suggest that milk-derived miRNAs are selectively packaged into exosomes and exist in a remarkable stable form that is protected from endogenous RNase activity across various harsh conditions.After feeding with different milk, the colostrum-only and mature milk-only fed piglets have similar levels of7representing circulating metabolic indicators in their serum (P＞0.05). Nonetheless, the expression level of the above13immune-related abundant miRNAs in the serum changed significantly. Out of the13immune-related mRNAs, apart from miR-148a-3p, all the other12miRNAs exhibited higher abundance in the colostrum-fed piglets compared with the mature milk-fed piglets (P＜0.01). And the expression level of miR-148a-3p was significantly higher in the mature milk-fed piglets (P＜0.05). The expression pattern of above mentioned immune-related miRNAs in the serum of piglets kept the same trend as the results in the porcine colostrum and mature milk. It is reasonable to hypothesis that these immune-related exosomal miRNAs in breast milk may be transferred into the piglet body via the digestive tract under the protection of exosomes.The results in this study provide basic information for further research of the important roles of breast milk in the development of infant’s immune system. The study also proved that pig can be used as an ideal model for studying human lactation medicine and immune diseases.更多还原