脂多糖和维生素D对鸡肠上皮细胞β-防御素表达的影响

发布时间：2018-01-21 23:56

本文关键词： 鸡肠上皮细胞维生素D 脂多糖 β-防御素　出处：《四川农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：抗菌肽(Antimicrobial peptides, AMPs)是先天免疫反应的效应分子,其在保护机体免受病原菌威胁的过程中作用显著。AMPs具有广谱的抗细菌、真菌、病毒以及支原体活性,其表达分为固定表达和诱导表达且受免疫应激类因子和营养因子类调控,如脂多糖(Lipopolysaccharide, LPS)和维生素D。家禽主要包含两大类AMPs分子,即β-防御素和cathelicidins,其中β-防御素14种、cathelicidins4种。在人、小鼠以及牛等哺乳动物的研究中发现维生素D对AMPs有调控作用,提示可通过营养因素提高机体β-防御素的表达从而提高机体对外界病原的抵抗力进而达到减少抗生素在畜禽养殖中的应用的目的。家禽中是否存在这种调节机制尚未见报道。肠道在担负消化和吸收功能的同时还具有免疫功能,是研究维生素D在免疫应激条件下对其β-防御素基因表达的影响的理想模型。本研究中我们优化了鸡肠上皮原代细胞分离、培养的条件。结果表明：15日龄的鸡胚的肠道组织更适用于肠上皮细胞的分离、培养；采用多次少时的消化策略得到的健全的肠隐窝集团更多、活力更强,对鸡胚的利用率大大提高；采用细胞筛区分隐窝和单个细胞从而对肠上皮细胞进行纯化所耗时间大为减少、纯度更高,纯度可达到85%以上；肠上皮原代细胞培养24 h-48 h进入对数增长期,72 h后细胞界限模糊,开始出现凋亡。设置不同浓度的LPS (0.50、100、200、400、800 μg/mL)和维生素D(00.2、2、20、200 ng/mL)处理对数生长期的肠上皮原代细胞。结果表明：低浓度的LPS不能引起肠上皮细胞的免疫反应,800 μg/mL的LPS能显著提高TLR4、IL-1和IL-6的表达,即选取800 μg/mL的LPS处理作为激活鸡肠上皮细胞免疫反应的有效浓度;单独添加维生素D对肠上皮细胞表达VDR、IL-1和IL-6的表达无显著作用,但在添加LPS的基础上,其能显著提高VDR的表达、显著降低IL-I和IL-6的表达,表明维生素D能抑制由LPS引起的炎性反应,且这种抑制作用随维生素D的浓度升高而加强。综合考虑,选取20μg/mL为维生素D的处理浓度。以筛选的LPS浓度和VD浓度处理鸡肠上皮原代细胞。结果表明,LPS能显著提高GAL-1、GAL-3、GAL-4、GAL-5、GAL-6、GAL-9、GAL-10、GAL-12和GAL-13的表达；维生素D能显著提高GAL-3、GAL-4、GAL-5、GAL-6和GAL-9的表达,但这种作用仅在免疫应激条件下才有所体现；对14种鸡β-防御素基因启动子区域进行分析发现有7种含有VDRE位点,其中6种受到维生素D的调控。总之,维生素D在肠上皮细胞处于免疫应激条件下能提高β-防御素的表达,且维生素D对β-防御素的调控作用可能与其启动子区的VDRE有关,但具体机制还需要进一步验证。
[Abstract]:Antimicrobial peptide (AMPs) is the effector of innate immune response. AMPs has a broad spectrum of antimicrobial, fungal, viral and mycoplasma activities. Its expression can be divided into fixed expression and induced expression, which is regulated by immune stress factor and nutrition factor, such as lipopolysaccharide. Poultry consists of two main classes of AMPs molecules, 尾 -defensin and cathelicidins, of which 14 are 尾 -defensins. Cathelicidins4 species. Vitamin D has been found to regulate AMPs in mammals such as humans, mice and cattle. It is suggested that the expression of 尾 -defensin can be enhanced by nutritional factors, thus increasing the resistance of the body to external pathogens, thus reducing the use of antibiotics in livestock and poultry breeding. Whether there is such a regulator in poultry? The intestinal tract has the function of digestion and absorption, but also has immune function. It is an ideal model to study the effect of vitamin D on the expression of 尾 -defensin gene under immune stress. In this study, we optimized the isolation of chicken intestinal epithelial primary cells. The results showed that the intestinal tissue of chicken embryo at 15 days old was more suitable for the isolation and culture of intestinal epithelial cells. The number of intestinal crypt groups was more and the utilization rate of chicken embryo was improved greatly by adopting the digestion strategy of several times and little time. The cell sieve was used to distinguish crypt from single cell so that the time for purification of intestinal epithelial cells was greatly reduced and the purity was higher than 85%. After 24 h-48 h of primary culture of intestinal epithelial cells entered the logarithmic growth phase, the cell line was blurred and apoptosis began to appear. 400,800 渭 g / mL) and vitamin D 00.2g / mL20. The results showed that low concentration of LPS could not induce the immune response of intestinal epithelial cells. LPS at 800 渭 g / mL could significantly increase the expression of IL-1 and IL-6. In other words, 800 渭 g / mL of LPS was selected as the effective concentration to activate the immune response of chicken intestinal epithelial cells. Vitamin D alone had no significant effect on the expression of IL-6 and IL-1 in intestinal epithelial cells, but on the basis of adding LPS, it could significantly increase the expression of VDR. A significant decrease in the expression of IL-I and IL-6 suggests that vitamin D can inhibit the inflammatory response induced by LPS, and this inhibition is enhanced with the increase of vitamin D concentration. The concentration of vitamin D was 20 渭 g / mL. The primary cells of chicken intestinal epithelium were treated with the selected concentration of LPS and VD. The results showed that LPs could significantly increase the concentration of GAL-1 and GAL-3. The expression of GAL-12 and GAL-13 in the GAL-10 of GAL-9 of GAL-4 and GAL-5; Vitamin D could significantly increase the expression of GAL-6 and GAL-9 in GAL-5 of GAL-3, but only under the condition of immune stress. The analysis of the promoter region of 尾 -defensin gene in 14 species of chickens revealed that 7 species contained VDRE loci, 6 of which were regulated by vitamin D. Vitamin D can increase the expression of 尾 -defensin in intestinal epithelial cells under immune stress, and the regulation of vitamin D on 尾 -defensin may be related to the VDRE of its promoter region. But the specific mechanism still needs further verification.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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