腐胺和脯氨酸对哺乳期仔猪空肠绒毛-隐窝轴上皮细胞的多胺代谢及Wnt信号通路的影响

【目的】研究腐胺和脯氨酸对哺乳期仔猪空肠绒毛-隐窝轴上皮细胞的多胺（腐胺、亚精胺和精胺）代谢、Wnt信号通路关键基因的mRNA相对表达量的影响。【方法】选取18头0日龄的刚出生的三元杂交（长白×大白×杜洛克）仔猪，随机配对分成3组，每组6个重复，每个重复1头猪，对照组，腐胺组和脯氨酸组，分别灌喂等体积的生理盐水，5 mg·kg^-1体重添加剂量的腐胺和25 mg·kg^-1体重添加剂量的脯氨酸，到14日龄断奶，断奶后3 d屠宰，分离空肠绒毛-隐窝轴的3个不同分化程度（绒毛顶端，绒毛中段和隐窝）的细胞，分别为F1，F2，F3。高效液相色谱法测定F1，F2，F3中的多胺浓度，RT-PCR测定多胺代谢途径中的相关基因以及Wnt信号通路中关键基因的mRNA相对表达量。【结果】1、在绒毛顶端细胞F1中，与对照组相比，脯氨酸组的腐胺、亚精胺、精胺的浓度均显著增加（P＜0.05），而腐胺组的多胺均无显著差异（P＞0.05）；在绒毛中段细胞F2中，除了腐胺的含量无显著差异外，脯氨酸组和腐胺组的亚精胺、精胺的浓度均显著高于对照组（P＜0.05），且脯氨酸组的亚精胺和精胺的浓度均显著高于腐胺组（P＜0.05）；在隐窝底端细胞F3中，腐胺组和脯氨酸组的腐胺、亚精胺、精胺浓度与对照组的浓度差异均不显著（P＞0.05）。2、鸟氨酸脱羧酶(ODC)基因在绒毛顶端上皮细胞F1中的mRNA表达量，腐胺组显著高于脯氨酸组和对照组（P＜0.05）；精氨酸酶（arginase）在F2中的mRNA表达量，脯氨酸组显著高于腐胺组和对照组（P＜0.05）；sFRP3在绒毛中段上皮细胞F2中的mRNA表达量，脯氨酸组的显著高于腐胺组（P＞0.05）；sFRP4在隐窝上皮细胞F3中的mRNA表达量，腐胺组显著高于对照组和脯氨酸组（P＜0.05）。【结论】添加了外源腐胺和脯氨酸，促进了哺乳期仔猪的空肠绒毛-隐窝轴上皮细胞的分化细胞的多胺浓度及促进了多胺的代谢，而对未分化的隐窝细胞没显著影响，同时通过Wnt信号通路调控空肠绒毛-隐窝轴细胞的分化成熟。

Impact of Putrescine and Proline on Suckling Piglet Jejunum Villus - crypt Axis Epithelial Polyamine Metabolism and Wnt Signal Pathway

【Objective】The aim of this paper is to study the effects of putrescine and proline on polyamines (putrescine, spermidine and spermine) metabolites, relative mRNA expression levels of Wnt signaling molecules along the villus-crypt axis in the jejunum of sucking piglet. 【Method】 Eighteen 0-day old newborn crossbred (Landrace × Yorkshire × Duroc) piglets were randomly divided into three groups: control group, putrescine group and proline group. The piglets in control group were administered an equal volume of saline, and that in other two groups received putrescine (5 mg·kg^-1 body weight ) and proline (25 mg·kg^-1 body weight ), respectively. Piglets were weaned at 14-day old and slaughtered on 3 days postweaning. The jejuna epithelial cells along the crypt-villus axis were isolated by Krebs Henseleit (KH) solution and yield 3 “cell fractions” (Fraction 1, 2 and 3). The concentration of polyamine in F1, F2 and F3 was determined by high performance liquid chromatography, and relative mRNA expression of related genes of polyamine metabolic and wnt signaling pathways were measured by real time PCR. 【Result】 The concentration of putrescine, spermine and spermidine in F1 were significantly increased (P＜0.05) in proline group compared to control group, while there was no significant difference in putrescine group (P＞0.05). The concentrations of spermine and spermidine in F2 of proline or putrescine groups were significantly higher than that of control group (P＜0.05). the concentrations of putrescine, spermine and spermidine in F3 of three group were not changed significantly (P＞0.05). The expression of ornithine decarboxylase (ODC) gene in F1 of putrescine group was significantly higher than that of proline group and control group (P＜0.05). The expression of arginase in F2 of proline group was significantly higher than that of putrescine group and control group (P＜0.05). The expression of SFRP3 in F2 of proline group was significantly higher than that of putrescine group (P＜0.05); the expression of sFRP4 in F3 of putrescine group is significantly higher than that of control group and proline group (P＜0.05). 【Conclusion】 Exogenous putrescine and proline increased the concentration of polyamine in differentiated epithelial cells of jejunum in sucking piglet and promoted the polyamine metabolism, however, no statistically significant effect was observed on crypt cell. In addition, exogenous putrescine and proline promote differentiation in the intestinal epithelial cells by Wnt signaling pathways.