淋巴毒素β受体调节猪肠道免疫和肠道微生物的功能研究

The death of piglets before weaning caused by enteric diseases due to low immunity makes the huge loss for the pig industry. Gut immune system is composed by immune cells, immunoglobin A (IgA) and intestinal microbes.Lmyphotoxin plays the key role in gut immunity by regulation of the IgA production. It has been already shown that the LTβR-/- mice are sensitive to bacterial infection due to the absense of lymphoid organs in these mice, and LTβR signaling in intestinal epithelial cells was required for recruitment of neutrophils to the infection site early during infection via production of CXCL1, and CXCL2 chemokines. And, LTβR-/- mice had the different intestinal microbes compared to the heterozygotes. All these data make us speculate that LTβR plays the important role in porcine gut immunity and intestinal microbes component. In this project, we will isolate porcine lymphotoxin β receptor (LTβR) gene and identify its expression pattern in different parts of intestines. Furthermore, we will prepare LTβR-/- IPEC-J2 cells by Crisper/Cas9 technique and detect its function in migration and apoptosis of IPEC-J2 cells. More important, the potential role of LTβR mediated signaling pathways in porcine gut immunity and gut microbiota component will be explored by blocking its function using fusing protein (LTβR-IgG) injection to weaning pigs. Our research findings would not only be helpful to better understand the mechanisms of LTβR-mediated signaling pathway in porcine gut immunity, but also be useful in improving pig disease resistance traits.

仔猪断奶期间肠道免疫功能低下导致的肠道疾病给养猪业造成了巨大的损失。肠道免疫系统是由免疫细胞、免疫球蛋白（IgA）及肠道微生物组成；淋巴毒素及其受体通过对IgA的调控在肠道免疫中起关键作用。研究表明，淋巴毒素β受体基因（LTβR）敲除小鼠体内缺少淋巴组织，IgA含量下降，细菌感染后死亡率增高；相反，LTβR信号通路激活能发挥抗细菌感染的作用，同时也能改变肠道菌群的组成。由此我们推断LTβR介导的信号通路被阻断后，通过影响IgA的产生而调控猪的肠道免疫水平和肠道菌群组成。因此，本项目将克隆猪LTβR基因，利用Crisper/Cas9技术在猪空肠细胞内实现体外基因表达沉默、给断奶仔猪注射融合蛋白LTβR-IgG实现体内信号通路阻断，并结合宏基因组学的研究手段，系统研究LTβR及其介导的信号通路对猪肠道免疫和肠道微生物组成的调控机制，为培育高免疫力的猪品种提供理论基础和可能的候选基因。

LTβR基因在哺乳动物淋巴结的发育和功能维持方面、在维持机体正常肠道功能方面均起着非常重要的作用，然而其在猪肠道中的功能还是一片空白。本项目以猪空肠细胞系细胞系IPEC-J2为材料，对LTβR的功能进行了研究。我们首先克隆了猪LTβR基因，该基因包含10个外显子，编码421个氨基酸，在物种间高度保守。LTβR基因在不同肠段的表达水平不一致，在猪十二指肠中的表达量最低，在空肠的表达量最高。利用Crisper/Cas9技术在猪空肠细胞系IPEC-J2中成功敲除了LTβR基因，双等位基因打靶效率为10.4%（KO细胞）。利用基因敲除细胞研究了LTβR及其介导的信号通路对猪空肠细胞系IPEC-J2的增殖和凋亡的影响。LTβR敲除后显著降低了IPEC-J2细胞的增殖，究其原因可能是有更多的细胞被阻滞在细胞周期S期。激光全息显微镜技术检测到LTβR基因敲除促进了IPEC-J2细胞的凋亡。进一步，我们比较了流行性仔猪腹泻病毒感染后KO细胞和WT细胞的PDEV病毒载量。结果发现，LTβR基因敲除使得IPEC-J2细胞对PDEV感染更为敏感。而KO细胞在感染病毒之后，NFκB靶标基因IL-6和IL-8的表达均显著下降，LTβR下游基因，也是肠道粘膜完整性相关基因，VCAM1和IL-22的表达也显著下调，这可能是KO细胞对仔猪腹泻病毒更加敏感的原因。另外，在LTβR基因的第二内含子中存在一个G-A的突变，此突变的功能还在进一步分析中。本项目为深入了解LTβR基因在猪肠道免疫中的调节作用提供理论基础，为猪的抗病育种，特别是宿主对流行性病毒感染后应答的研究提供了新的素材。