【作者】 陈丽颖；

【导师】 王艳玲；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2008， 博士

【摘要】 仔猪成活率一直是影响养猪业发展的重要因素之一,而母乳的免疫球蛋白水平及其向仔猪的转运对仔猪早期存活至关重要。刚出生的仔猪体内抗体基本为零,新生仔猪的免疫主要来自初乳中的母源抗体提供的被动免疫。IgG是家畜初乳中含量最丰富的免疫球蛋白成分,IgG由乳腺向乳汁中的分泌以及新生动物对乳中母源IgG的摄取均需要穿越上皮细胞屏障,即进行细胞转运作用,这一过程是由受体介导的,而特异性转运IgG的唯一受体是新生儿Fc受体（neonatal Fc receptor,FcRn）。研究表明,FcRn具有独特的重要功能,它不仅在IgG从母体到仔畜的转运中发挥着极其重要的作用,而且在成年动物体内终生表达,维持血液IgG和清蛋白水平的相对平衡和稳定,FcRn还与动物的生长发育和免疫应答密切相关。对该受体的研究可以揭示机体免疫球蛋白的转运机制,从而对临床应用和生产实践具有重要的理论指导价值。本研究以大白和长白二元杂交仔猪作为研究对象,分别采集从出生当天至断奶后共12个日龄仔猪血液和肝、肾、脾、心脏、胃以及肠道各段组织,进行了如下方面的试验。1.应用RT-PCR方法研究了FcRn在仔猪体内不同组织的表达分布及不同日龄仔猪胃肠道FcRn的表达变化。结果表明,FcRn在仔猪肝、肾、脾、心脏以及胃肠道各段组织中均有表达。经对FcRn/GAPDH电泳条带密度扫描分析,仔猪胃肠道中以十二指肠后段和空肠前段表达量最高,在十二指肠和空肠段,从一出生就有较高水平的FcRn转录,吮乳后至出生后1d达到峰值,4d后FcRn mRNA转录水平明显降低（P＜0.05）,至断奶时降到最低。回肠段FcRn表达水平较十二指肠和空肠段低,表达量变化不明显。胃与结肠部位FcRn的表达最低,且不同日龄间表达比较稳定。FcRn在仔猪十二指肠后段和空肠前段的高表达水平提示仔猪十二指肠、审肠是FcRn转运初乳中IgG的主要部位。2.应用实时荧光定量PCR方法,对仔猪肝脏和胃肠道组织中FcRn的表达进行了定量检测。根据GenBank公布的猪FcRn重链mRNA基因保守序列自行设计PCR引物和荧光探针,并以β-actin为管家基因,建立测定仔猪FcRn mRNA表达的实时荧光定量PCR方法。检测结果表明,FcRn在仔猪体内不同组织中表达水平有显著差异。其中,在肝脏中表达水平最高（△Ct=4.95）;其次是在十二指肠远端和空肠近端;十二指肠近端和空肠远端的表达量次之;在仔猪回肠和结肠部位均为低表达,且表达量极为接近;FcRn在仔猪胃内也有表达,但表达量最低（△Ct=6.91）。FcRn mRNA表达量随日龄的变化规律为:出生当天表达水平较低,未吃初乳（0w）和已吃初乳（0y）没有显著差异（P＞0.05）;1d时表达水平显著升高（P＜0.05）,随后维持在较稳定的水平直至21 d时有所下降;28 d时FcRn在胃肠道各部位表达均有显著升高（P＜0.05）;35 d时则普遍回落到与前期相近的水平。3.建立了检测仔猪小肠FcRn表达的地高辛标记探针原位杂交方法。根据GenBank公布的猪FcRn重链（α链）mRNA基因保守序列设计并合成带地高辛标记的原位杂交试验用cDNA探针。经仔猪小肠冰冻组织切片最佳厚度选择,以及H₂O₂溶液祛除小肠组织中内源性辣根过氧化物酶作用对杂交结果的影响,选择合适的固定剂、细胞膜蛋白消化剂、杂交液和确定杂交反应的温度、时间以及杂交后处理等一系列试验条件优化,建立了检测仔猪小肠FcRn mRNA的原位杂交方法。结果显示,原位杂交的最佳反应条件为:冰冻组织切片厚度为14μm;固定液采用4%多聚甲醛溶液,固定时间为1 h:杂交前用1%的Triton X-100处理切片15 min;采用含有50%去离子甲酰胺的Yeast RNA杂交液,每张切片滴加30μL～50μL:探针浓度为1.5μ/mL;杂交温度为46.8℃,时间为18 h;与辣根过氧化物酶标记的抗地高辛抗体（anti-DIG-HRP）于4℃作用16 h后,用DAB显色10～15 min;切片最后用苏木素染色液复染。4.应用原位杂交方法研究了FcRn mPNA在仔猪小肠各段的表达分布特点,并利用相应软件进行了表达量分析。结果表明,在仔猪十二指肠、空肠和回肠组织的小肠上皮细胞和黏膜下层等部位均检测到清晰的杂交信号。其中,在小肠黏膜上皮检测到大量的FcRn mRNA阳性信号,尤其是空肠部位表达信号最强,其次是十二指肠远端,而在十二指肠近端和回肠段表达信号相对较弱;而小肠黏膜固有层的阳性信号主要集中在固有层内小血管周围,且以空肠部位为主;在十二指肠、空肠和回肠部位的黏膜下层均检测到FcRn mRNA阳性信号,其中在空肠黏膜下层表达最强。仔猪FcRn的表达动态为:出生3 d内FcRn在仔猪十二指肠、空肠和回肠部位均为高表达,3 d时出现显著下降,随后表达水平基本稳定,直至21 d后才出现回升。其中,十二指肠部位从仔猪一出生即高表达FcRn,而回肠部位FcRn的表达从3d开始直至断奶后一直维持在低水平。5.进行了仔猪血清IgG水平动态的检测,并分析了与小肠FcRn表达的相关性。利用放射免疫测定（RIA）检测了从初生到断奶阶段仔猪血液免疫球蛋白IgG的水平及其变化动态。结果表明,在出生后2 d内仔猪血液IgG抗体水平的变化与小肠FcRn表达量具有高度相关性,提示FcRn在新生仔猪肠道转运IgG过程中的主要作用。其中在仔猪刚出生时IgG含量为0,吸吮初乳后（0.5 d）水平迅速上升,至1d时达到峰值,而在3 d时IgG水平出现急剧下降,IgG水平在7d时达到第2次高峰,但从10d显著下降随后基本保持稳定,至35d时血液IgG水平又出现大幅度升高,达到4.74 mg/mL。对仔猪FcRn表达的定性、定位和定量检测均表明,FcRn mRNA在十二指肠远端和空肠近端呈现高水平表达,这与仔猪小肠的营养吸收特点相符,提示FcRn参与肠道IgG转运,在小肠吸收IgG过程中发挥转运受体作用。而FcRn mRNA在仔猪出生后3d内的高表达与新生仔猪吸收初乳的高峰时间及“肠关闭”时间相吻合,对仔猪血清IgG水平的检测也显示了新生仔猪IgG含量峰值的出现与FcRn在小肠的高表达存在一致性,说明了FcRn对新生动物获取足够被动免疫的重要性。更多还原

【Abstract】 Piglet survival rate is one of the most important factors that influence the development of hog industry,and the immunoglobulin level in sow milk and its transportation to the newborn are crucial to piglet survival.The antibody level in piglet is nearly null at birth,therefore the immunity of infant pig predominantly depends on passive protection provided by the maternal antibodies from colostrum.Being the most abundant colostral Ig component in livestock animals, IgG must be transported across epithelial barriers through transcytosis when it is secreted from mammary gland to milk and when it is absorbed by the newborn animals,through a specific IgG transport receptor,namely the neonatal Fc receptor（FcRn）.Research has shown that FcRn performs unique physiological functions,which not only plays a critical role in IgG transcytosis from mother to baby,but presents in adult through lifetime mentaining the homeostasis of serum IgG and albumin proteins as well,and it is also closely related with animal growth and development and immune response.Research on this receptor helps reveal IgG transfer mechnism and thus provides practical and theoretical guidance on piglet husbandry.Targeted on Landrace-Large White crossbred piglets from age of at birth to postweaning, tissues of liver,kidney,spleen,heart,slomach and different parts of intestine were collected at 12 age points and experiments were performed as follows.1.Expression of FcRn mRNA in different tissues of piglets was detected by RT-PCR and relative quantitation was determined and analvsed.The results indicated that FcRn was expressed in the tissues of liver,kidney,spleen,heart,stomach and all parts of intestine.The electrophoresis band density scanning analysis of FcRn/GAPDH showed that among the gastroinstestinal（GI） tract tissues the strongest signals existed at duodenum and jejunum;high level of FeRn gene transcription was detected in duodenum and jejunum from piglets at birth and reached its peak on 1d,decreased sharply from 4d on,and reached the lowest at pre-weaning（21d）.At the ileum, FcRn expression was relatively low and no distinct changes observed.The minimum of FcRn expression was detected at colon and stomach,being relatively stable.The high level of FcRn expression in distal duodenum and adjacent jejunum implies that these two parts are the major sites of milk IgG transport to swine intestine.2.The quantitation of FcRn levels in pgilet liver and GI tract tissues was performed by quantitative real-time RT-PCR（RT-FQ-PCR） with primers and a fluorecent probe for FcRn heavy chain gene,withβ-actin chosen as house-keeping gene.The results showed a significant difference of FcRn levels in swine tissues.The most abundance of FcRn was present in liver（△Ct =4.95）,followed by distal duodenum and adjacent jejunum,and then adjacent duodenum and distal jejunum,low expression levels were revealed in ileum and colon,whilst the lowest level was in stomach（△Ct =6.91）.The trend of FcRn mRNA expression in GI tract was present accordly to age:Low level of FcRn was foound at birth with no significant difference between non-suckling（0 w） and suckling（0 y） piglets（P＞0.05）,the expression markedlv increased on 1 d （P＜0.05）,remained relatively stable and decreased at 21d,thereafter it increased sharply on 28d but dropped to previous level at 35d of age.3.The method of in situ hybridization（ISH） was effectively established for the expression of FcRn in the small intestine of piglets.A digoxin-labeled single-strand cDNA probe for detection of swine FcRn mRNA was plotted according to the conserved region of the gene for a chain of porcine FcRn published in GenBank.The ISH method was optimized through a series of trials such as the elimination of endogenous HRP,and fixation agent selection.Best procedureincluded:tissue slices with thickness of 14μm,fixed with 4%polyformal solution for 1h,pre-treated with 1%Triton X-100,hybridized with 1.5μg/mL of probe at 46.8℃for 18 hrs,colorated with DAB after being incubated with anti- DIG- HRP fragment at 4℃for 16 hrs,and finally the slices were counter-stained with hematoxylin.4.FcRn mRNA localization in swine intestinal sections was analysed by means of ISH and the change of expression level was depicted.Limpid hybridization signals were detected in the small intestinal epithelial cells and submucosa of duodenum,jejunum and ileum,among which large amount of positive signals were detected in the intestinal epithelia with the most predominant in jejunum,followed by distal duodenum,whereas the expression in adjacent duodenum and ileum was faint.The relative quantitation results of swine intestinal FcRn kinetics showed a high consistency with that of RT-FQ-PCR detection.5.Titration of serum IgG levels in piglets was taken by radioimmunoassay（RIA）.The RIA results of IgG level changes in swine showed that the piglets at birth presented no IgG in the body before suckling whereas the lgG level was promptly increased after having sucked the colostrum, reaching peak concentration at 1d,dropping sharply at 3d,but recovering to the 2^nd peak at 10d, then a distinct decrease occurred at 10d followed by a period of relative stable levels,and then the IgG titer reached maximum at 35d with a concentration of 4.74 mg/mL.Generally,both the qualitative,quantitative and the positioning detection of piglet FcRn expression indicated that FcRn mRNA was predominantly expressed at distal duodenum and adjacent jejunum,consistence with the primary nutrition absorption sites.Therefore it mplied that FcRn was involved in intestinal IgG transportation and played a role of transfer receptor for IgG. The high performance of FcRn mRNA in piglets within 3d age was consistant with the timing of their peak absorption of colostrum and that of "gut-closure",and was also testified by the detection of changes of serum IgG level in piglet showing a close correlation with FcRn expression,which illustrated the critical importance of FcRn presence to the acquisition of effective passive immunity in newborn animals.更多还原