【作者】 刘芳宁；

【导师】 张彦明； Li Juan Yuan；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2010， 博士

【摘要】 使用益生菌预防或治疗轮状病毒(Rotavirus,RV)感染和腹泻是临床中常用的方案,但具体机制目前还不清楚。在本研究中我们分别在体外RV感染的猪小肠上皮细胞(Porcine small intestinal epithelial cell line,IPEC-J2)模型和体内RV感染的悉生(Gnotobiotic,Gn)猪模型上探索了益生菌对RV感染和腹泻的保护机制。体外研究中,嗜酸乳杆菌(Lactobacillus acidophilus,LA)或者鼠李糖乳酸杆菌(Lactobacillus rhamnosus GG,LGG)在猪轮状病毒(Porcine rotavirus, pRV) OSU或人轮状病毒(Human rotavirus, hRV) Wa接种IPEC-J2细胞前24 h和接种细胞后1 h被用来处理细胞。我们发现pRV在IPEC-J2细胞上的感染力明显高于hRV。LA或LGG处理细胞并不减少RV在细胞上的繁殖和复制。pRV感染细胞增加了MUC3黏蛋白的分泌。LGG可降低pRV感染细胞中分泌型MUC3黏蛋白产量;但可增加正常细胞中膜结合型MUC3黏蛋白产量。病毒感染前24 h用LA处理细胞可显著增加pRV在细胞上的繁殖并显著提高细胞的IL-6反应,这与LA的佐剂效果一致。病毒感染后用LGG处理细胞24 h可下调细胞的IL-6反应,这与LGG的抗炎效果相符。IPEC-J2细胞表达套样受体(Toll-like receptor,TLR) 2、TLR3和TLR9。LGG和肽聚糖(peptidoglycan,PGN)上调TLR2在细胞中的表达与LGG下调IL-6的结果相呼应,说明LGG的保护作用与其上调TLR2在细胞中的表达有关。pRV感染可使IPEC-J2细胞单层跨膜电阻(Trans-epithelial electrical resistance,TEER)在感染后6-18 h显著下降,但在接种后24 h恢复到正常水平,这和RV感染在体内引起的病程相似,进一步说明了IPEC-J2细胞功能接近正常猪小肠上皮细胞。LA可以有效阻止pRV感染造成的细胞单层TEER下降,但LGG对pRV感染造成的细胞单层TEER变化没有明显影响。在病毒接种后24 h,LA或LGG处理或/和pRV感染细胞对细胞的通透性和紧密连接(Tight junction,TJ)蛋白(occludin、ZO-1、claudin-1、claudin-3和claudin-4)及粘合连接(Aherents junction,AJ)蛋白(α-catenin和β-catenin)在细胞上的分布没有明显影响,这可能与pRV感染后采样时间过晚有关。LGG明显提高正常肠上皮细胞的TEER及AJ蛋白α-catenin和β-catenin的表达量,说明LGG对肠屏障功能有维持和增强作用。但在LA或LGG处理和/或pRV感染期间(病毒接种后6~18 h),随着TEER的变化,细胞单层通透性和细胞中TJ和AJ蛋白数量和分布是否随时间而改变,还需进一步的研究检测。pRV感染的IPEC-J2细胞模型是一个完全同源模型,这个模型对研究益生菌对RV感染肠上皮细胞的先天性免疫反应和肠屏障功能的影响非常有用。体内研究中,Gn猪被分为4组:(1)接种LGG和hRV(LGG+hRV+),(2)只接种hRV(LGG-hRV+),(3)只接种LGG(LGG+hRV-),(4)空白对照(LGG-hRV-)。从3日龄起,给LGG+组的猪口服剂量10倍递增的LGG (103~1012 cfu[colony forming units]/mL)直到猪被宰杀。在LGG饲喂9 d后,给hRV+组猪口服接种hRV并在病毒接种后3-6 d宰杀猪取样检查。结果表明LGG在Gn猪体内定植的数量保持在一定范围(107～108 cfu/mL),并不随体外摄入的LGG剂量的增加而增加,而且hRV感染明显提高了LGG在Gn猪肠道中定植的数量(108～109 cfu/mL)。LGG对hRV感染和腹泻有一定程度的保护和缓解作用,可使hRV腹泻出现的天数明显推迟,hRV从粪便开始排出的天数推迟、从粪便中排出病毒的天数和病毒血症出现的天数均缩短、累计的平均腹泻指数和可测病毒的最高滴度降低并使有腹泻猪的百分率降低。hRV感染Gn猪后有这样一个趋势:在病毒接种后3~6 d,腹泻越严重的猪回肠上皮细胞之间的连接越紧密,回肠上皮中可检测到的TJ和AJ蛋白的产量越高。这种变化是由于RV感染后损伤的肠上皮细胞补偿性增生造成的。LGG明显降低了hRV感染猪回肠组织中TJ蛋白(occludin、claudin-1和claudin-3)和AJ蛋白β-catenin的数量,这间接说明LGG明显降低了hRV感染造成的肠上皮损伤和增生,这是LGG对RV感染和腹泻保护性调节的一个重要机制。但在hRV接种的最初72 h内,TJ和AJ蛋白在回肠上皮细胞中的分布和产量在hRV接种后不同时间点的变化有待于进一步研究检测。另外LGG可通过增加肠道中黏蛋白水平、明显上调抗炎细胞因子TGF-β反应和维持促炎细胞因子TNF-α、IFN-γ和IL-8反应来调节肠的屏障功能和RV感染引起的炎性反应,进而调节或保护hRV感染和腹泻。Gn猪肠上皮细胞结构、生理功能和免疫功能和人非常相近,hRV感染的新生Gn猪模型为我们提供了一个了解LGG保护RV感染和腹泻机制的理想模型。更多还原

【Abstract】 Specific beneficial effects of probioics have been documented in a large number of clinical trials for rotavirus infection and diarrhea. However, mechanisms behind such effects are not well understood. In this study, we first evaluated a non-transformed porcine jejunum epithelial cell line (IPEC-J2) as an in-vitro model of rotavirus infection and probiotic treatment. We then studied the impact of LGG colonization on intestinal epithelial barrier function and cytokine responses in the gnotobiotic (Gn) pig model of rotavirus gastroenteritis.In the study of in-vitro IPEC-J2 cell model, porcine rotavirus (pRV) OSU strain or human rotavirus (hRV) Wa strain, along with Lactobacillus acidophilus (LA) or Lactobacillus rhamnosus GG (LGG) were used to inoculate IPEC-J2 cells. LA or LGG treatment was applied pre- or post-rotavirus infection. We demonstrated that IPEC-J2 cells were productively infected by pRV. LA or LGG treatment of the cells did not reduce virus replication. MUC3 mucin secretion was increased after pRV infection on the cells. LGG treatment post-rotavirus infection reduced the mucin secretion response induced by pRV; LGG alone increased the production of membrane-associated MUC3 mucin. LA treatment prior to rotavirus infection significantly increased pRV replication and the IL-6 response to pRV infection, which is consistent with the adjuvant effect of LA. LGG treatment after rotavirus infection down-regulated the IL-6 response, which confirmed the anti-in?ammatory effect of LGG. IPEC-J2 cells expressed TLR2, TLR3, and TLR9 constitutively. TLR2 expression was upregulated by LGG and peptidoglycan, corresponding to the decreased IL-6 response, indicating that the protective effect of LGG is associated with upregulation of TLR2 expression on intestinal epithelial cells. Trans-epithelial electrical resistance (TEER) decreased significantly at post-rotavirus inoculation 6~18 h; however, the TEER recovered to the same level as the mock-infected cells at post-rotavirus inoculation 24 h, which is consistent with the course of rotavirus infection and diarrhea in-vivo. LA, but not LGG, significantly inhibited the TEER reduction caused by pRV infection. LA or LGG treatment and/or pRV infection did not change the permeability of IPEC-J2 monolayer and the distribution of tight junction (TJ) proteins (occludin, ZO-1, claudin-1, claudin-3 and claudin-4) and aherents junction (AJ) proteins (α-catenin andβ-catenin) on the cells at post-rotavirus inoculation 24 h. However, during LA or LGG treatment and/or RV infection, whether cell monolayer permeability and TJ or AJ proteins change over the time course (post-rotavirus inoculation 6~18 h) or not need to be studied. Nevertheless, LGG significantly increased the TEER and the production ofα-catenin andβ-catenin in mock cells, indicating the protective effect of LGG on intestinal epithelia barrier. The IPEC-J2 cell model of pRV infection is a completely homologous system. It is a valuable model for studying the interactions among rotavirus-host-probiotics, and the mechanisms behind the immunomodulating effect of probiotic bacteria on innate immune responses and intestinal epithelial barrier function.In the study of in-vivo Gn pig model, we used 4 Gn pigs in each group as follows: (1) LGG colonization plus hRV inoculation (LGG+hRV+), (2) hRV only (LGG-hRV+), (3) LGG only (LGG+hRV-) or (4) mock control (LGG-hRV-). Before hRV inoculation, pigs were fed with LGG with a daily dose increase of 10-fold (103~1012 ffu [colony forming units]/mL). The intestinal LGG counts remained in the rage of 107~108 cfu/ml and did not increase with the increasing intake amount of probiotics. hRV infection significantly increased the LGG counts in intestine (108~109 cfu/ml).The mean duration of rotavirus diarrhea, fecal virus shedding and viremia was shorter, mean cumulative score of diarrhea and peak virus titer were lower, and the percentage of pigs with diarrhea was less in the LGG+hRV+ group than in the LGG-hRV+ group. Furthermore, the pigs in the LGG+hRV+ group had a delayed days-to-onset of fecal virus shedding or diarrhea compared to the LGG-hRV+ group. These results demonstrated a clear and moderate protective effect of LGG on hRV-induced diarrhea and virus replication in Gn pigs. There was a trend that pigs with diarrhea had tighter junction and more TJ and AJ proteins in intestinal epithelium than the mock control pigs at post-inoculation 3~6 d. This difference may be the compensatory effect observed after rotavirus infection. A time course study of the intestinal TJ and AJ protein expression and distribution during the first 72 h after rotavirus inoculation is needed to address the question. LGG-feeding significantly decreased the production of TJ proteins (occludin, claudin-1 and claudin-3) and AJ proteinβ-catenin in ileum from LGG+hRV+ pigs compared to LGG-hRV+ pigs, indirectly indicating that LGG colonization reduced the lesion and proliferation of intestinal epithelial cells cuased by hRV infection. Additionally, The protective effect of LGG on hRV-induced diarrhea and virus replication may be attributed to the increased MUC3 mucin production, significantly enhanced anti-inflammatory TGF-βcytokine responses to hRV infection and maintained pro-inflammatory cytokine TNF-α, IFN-γand IL-8 response. The neonatal Gn pig model of human rotavirus infection and disease provided an excellent model system for these studies because this model closely mimics the human intestinal epithelial structure, physiology and immune responses.更多还原