發(fā)布時間：2018-04-24 05:14

  本文選題：抗菌肽CWA + 抗生素��； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：斷奶造成仔豬腸道功能紊亂,導(dǎo)致腹瀉、生長性能下降甚至死亡,嚴重影響?zhàn)B豬業(yè)的健康發(fā)展。雖然腹瀉的影響因素較多,但多數(shù)研究表明腸道炎癥和腸道屏障功能損傷是斷奶仔豬易發(fā)生腹瀉的主要內(nèi)在原因。大量抗生素用于防治斷奶仔豬腹瀉,引發(fā)細菌耐藥性、肉產(chǎn)品中藥物殘留及環(huán)境污染等嚴重問題；同時抗生素濫用會損害機體腸道屏障功能,破壞腸道穩(wěn)態(tài)。抗菌肽是重要的先天免疫分子,因其廣譜抗菌活性、破膜殺菌機制以及不易產(chǎn)生耐藥性等特點而受到關(guān)注。最新研究表明,抗菌肽不僅具有免疫調(diào)節(jié)作用,還具有腸道上皮屏障保護功能。然而,抗菌肽應(yīng)用于防治斷奶仔豬腹瀉的效果及其對斷奶仔豬腸道炎癥和腸道上皮屏障功能的作用如何還不得而知。因此,在前期獲得了抗菌活性高、安全性好的抗菌肽CWA的基礎(chǔ)上,本研究開展了抗菌肽CWA對斷奶仔豬腹瀉的防治效果試驗,同時闡明了抗菌肽CWA對腸道炎癥和腸道屏障功能的作用；進一步在小鼠上探究了抗菌肽CWA對細菌感染導(dǎo)致的腸道炎癥和腸道屏障功能損傷的作用；最后體外細胞試驗初步探討了抗菌肽CWA緩解腸道炎癥和保護腸道屏障功能的作用機制。主要研究結(jié)果如下：試驗一抗菌肽CWA對斷奶仔豬腸道炎癥和腸道屏障功能的作用(1)抗菌肽CWA對斷奶應(yīng)激仔豬腸道炎癥的作用選擇18頭健康的杜長大仔豬,21日齡斷奶后分別腹腔注射3次生理鹽水(對照組)和抗菌肽CWA(抗菌肽CWA組),后續(xù)飼養(yǎng)7天,研究抗菌肽CWA對仔豬斷奶應(yīng)激腹瀉及其腸道炎癥的作用。結(jié)果表明,抗菌肽CWA降低了斷奶仔豬腹瀉指數(shù)(P0.05),提高了斷奶仔豬ADFI (P0.05)和ADG (P0.05)。同時,抗菌肽CWA降低了血清IgG、TNF-α、IL-6、IL-22含量(P0.05),且提高了血清中TGF-β含量(P0.05)。在腸道形態(tài)方面,抗菌肽CWA提高了空腸的絨毛高度、絨毛高度/隱窩深度比值(P0.05),提高了回腸的絨毛高度/隱窩深度比值(P0.05),降低了回腸的隱窩深度(P0.05)。在腸道炎癥方面,抗菌肽CWA降低了腸道中促炎因子的基因表達,其中對空腸的效果最為顯著；同時抗菌肽CWA減少了腸道嗜中性粒細胞和巨噬細胞浸潤；提示抗菌肽CWA能夠緩解斷奶仔豬腸道炎癥。在腸道炎癥信號通路蛋白表達方面,抗菌肽CWA顯著降低空腸組織炎癥信號通路中關(guān)鍵蛋白NF-κB、IκB-α的磷酸化水平,提示抗菌肽CWA可能通過下調(diào)NF-κB信號通路緩解腸道炎癥。(2)抗菌肽CWA對腹瀉斷奶仔豬腸道炎癥和腸道屏障功能的作用選擇108頭腹瀉的杜長大斷奶仔豬,分別腹腔注射4次生理鹽水(對照組)、恩諾沙星(抗生素組)、抗菌肽CWA(抗菌肽CWA組),研究抗菌肽CWA對腹瀉斷奶仔豬腸道炎癥和腸道屏障功能的作用。結(jié)果表明,抗菌肽CWA和抗生素均降低了斷奶仔豬的腹瀉指數(shù)(P0.05)和腹瀉率(P0.05),均提高了斷奶仔豬ADFI (P＜0.05)和ADG (P＜0.05),且兩者無顯著差異。在腸道形態(tài)方面,抗菌肽CWA和抗生素對小腸絨毛形態(tài)損傷均有緩解作用,其中對空腸的作用最顯著；掃描電鏡和透射電鏡觀察發(fā)現(xiàn),抗菌肽CWA和抗生素改善了空腸微絨毛數(shù)量與長度,且抗菌肽CWA的改善作用優(yōu)于抗生素。在腸道炎癥方面,抗菌肽CWA組和抗生素組均降低了空腸中促炎因子IL-6、IL-8、IL-22的含量(P0.05),對血清也有類似規(guī)律；同時,抗菌肽CWA和抗生素均緩解了腹瀉斷奶仔豬空腸嗜中性粒細胞浸潤；進一步,抗菌肽CWA組和抗生素組均降低了空腸組織中炎癥相關(guān)蛋白NF-κB、IκB-α、AKT的磷酸化水平及上游調(diào)控關(guān)鍵蛋白TLR4、MyD88的表達；且抗菌肽CWA緩解腸道炎癥的作用與抗生素相當。在腸道上皮屏障功能方面,抗生素組不僅降低了空腸粘液素和防御素的表達,而且降低了空腸中緊密連接蛋白ZO-1和Occludin的表達(P0.05)；然而,抗菌肽CWA卻提高了空腸中ZO-1和Occludin的表達(P0.05)；提示抗菌肽CWA可能提高腸道上皮屏障功能而抗生素卻損害腸道上皮屏障功能。在腸道微生物菌群及SCFAs含量方面,與對照組相比,抗菌肽CWA組和抗生素組均降低了糞便中大腸桿菌比例(P0.05),均提高了糞便中乳酸桿菌/大腸桿菌比值(P0.05)；然而,抗菌肽CWA提高了糞便中乳酸桿菌比例(P0.05),抗生素卻降低了糞便中乳酸桿菌比例(P0.05)；進一步,抗菌肽CWA提高了糞便中乙酸、丙酸及丁酸的濃度(P0.05),而抗生素降低了糞便中丁酸的濃度、丁酸/乙酸比值及丁酸/丙酸比值(P0.05)。試驗二 抗菌肽CWA對細菌感染小鼠腸道炎癥和腸道屏障功能的作用在發(fā)現(xiàn)抗菌肽CWA緩解斷奶仔豬腸道炎癥和腸道屏障功能損傷的基礎(chǔ)上,進一步在小鼠上研究抗菌肽CWA對細菌感染引起的腸道炎癥和腸道屏障功能損傷的影響。選擇36只斷奶小鼠分為對照組、E.coli組、E.coli+Enro組、E.coli+CWA組,EHEC O157:H7感染后分別腹腔注射生理鹽水、恩諾沙星(Enro)、抗菌肽CWA。結(jié)果表明,抗菌肽CWA和恩諾沙星均提高了染菌小鼠的存活率,緩解了染菌導(dǎo)致的小鼠體重下降。在腸道形態(tài)方面,抗菌肽CWA和恩諾沙星均緩解了染菌導(dǎo)致的小鼠空腸絨毛萎縮、隱窩增生和結(jié)腸粘膜上皮破損、杯狀細胞數(shù)量增加。在炎癥方面,E.coli+Enro組和E.coli+CWA組均緩解了染菌導(dǎo)致的小鼠血清和結(jié)腸中TNF-α、IL-6、IL-10含量升高(P0.05)；同時均緩解了染菌導(dǎo)致的小鼠空腸中IL-6含量的升高(P0.05)；抗菌肽CWA在緩解炎癥方面與恩諾沙星無顯著差異。在腸道抗菌蛋白表達方面,E.coli+Enro組和E.coli+CWA組均緩解了染菌導(dǎo)致的空腸中REG3y基因表達升高和結(jié)腸中Remlβ基因表達升高(P0.05)。然而,抗菌肽CWA組了小鼠空腸和結(jié)腸中TFF3基因表達水平(P0.05)。同時,E.coli+Enro組和E.coli+CWA組均緩解了染菌導(dǎo)致的小鼠空腸中杯狀細胞數(shù)量減少、粘液層厚度降低和結(jié)腸中杯狀細胞數(shù)量增加。進一步,Western blot和免疫熒光的結(jié)果表明,E.coli+Enro組和E.coli+CWA組均緩解了染菌導(dǎo)致的小鼠空腸MUC-2表達的降低和結(jié)腸MUC-2表達的升高(P0.05)。在腸道上皮屏障功能方面,E.coli+CWA組緩解了染菌導(dǎo)致的小鼠空腸中ZO-1和Occludin蛋白表達的下降(P0.05),而E.coli+Enro組僅緩解了ZO-1蛋白表達的下降(P0.05),抗菌肽CWA的效果優(yōu)于抗生素。在腸道微生物菌群及SCFAs濃度方面,不同處理小鼠盲腸內(nèi)容物中總菌、雙歧桿菌、芽孢桿菌的數(shù)量無顯著差異,E.coli+Enro組和Ecoli+CWA組均緩解了染菌導(dǎo)致的盲腸中乳酸桿菌數(shù)量的降低和大腸桿菌數(shù)量的升高(P0.05),同時Ecoli+CWA組緩解了染菌導(dǎo)致的盲腸中乳酸桿菌/大腸桿菌比值的下降(P0.05)。同時,Ecoli+CWA組和E.coli+Enro組均對染菌導(dǎo)致的SCFAs濃度的降低具有一定程度的緩解作用。以上結(jié)果進一步證明了抗菌肽CWA能夠緩解腸道炎癥和改善腸道屏障功能,與斷奶仔豬的試驗結(jié)果一致。試驗三抗菌肽CWA對腸道炎癥和腸遒上皮屏障功能的調(diào)控機制(1)抗菌肽CWA緩解腸道炎癥的作用機制利用LPS誘導(dǎo)的豬巨噬細胞炎癥模型發(fā)現(xiàn),恩諾沙星對LPS導(dǎo)致的炎癥反應(yīng)無緩解作用,而抗菌肽CWA可以緩解LPS導(dǎo)致的炎癥反應(yīng)且呈濃度依賴效應(yīng)。同時,抗菌肽CWA緩解了LPS導(dǎo)致的IL-6、IL-8、IL-22表達升高和NF-κB、IκB-α磷酸化水平升高(P0.05)。利用siRNA技術(shù)沉默豬巨噬細胞TLR4和MyD88后,抗菌肽CWA緩解炎癥的作用受到抑制。利用FITC標記的葡聚糖和流式細胞檢測技術(shù)發(fā)現(xiàn),抗菌肽CWA可以增強巨噬細胞吞噬FITC-葡聚糖的能力,而使用STAT-1抑制劑后,抗菌肽CWA對巨噬細胞吞噬功能的增強作用受到抑制。以上結(jié)果表明,抗菌肽CWA緩解腸道炎癥可能是通過TLR4-MyD88-NF-KB信號通路調(diào)控炎癥反應(yīng)和通過STAT-1途徑增強巨噬細胞吞噬功能等機制發(fā)揮作用。(2)抗菌肽CWA改善腸道上皮屏障功能的作用機制利用Caco-2細胞刮傷模型發(fā)現(xiàn),抗菌肽CWA提高了刮傷細胞的愈合速度,改善受損腸道上皮細胞的修復(fù)功能。利用豬空腸上皮細胞(IPEC-J2)屏障功能損傷模型發(fā)現(xiàn),抗菌肽CWA緩解了LPS導(dǎo)致的IPEC-J2單層細胞TER值的下降,同時緩解了LPS導(dǎo)致的腸道上皮細胞中緊密連接蛋白ZO-1和Occludin蛋白表達的下降。單獨添加抗菌肽CWA提高了IPEC-J2細胞中ZO-1和Occludin的表達,而添加Rac1抑制劑NSC 23766時,抗菌肽CWA對ZO-1和Occludin的提高作用受到抑制,且抗菌肽CWA對粘附IPEC-J2細胞的減少EHEC O157:H7作用也被抑制。以上結(jié)果表明,抗菌肽CWA能夠改善受損腸道上皮細胞的修復(fù)功能,通過Racl途徑調(diào)控腸道上皮細胞緊密連接蛋白表達,增強腸道上皮屏障功能。上述研究不僅為抗菌肽應(yīng)用于防治斷奶仔豬腹瀉,實現(xiàn)減少抗生素在畜禽養(yǎng)殖中的使用提供了重要的基礎(chǔ)數(shù)據(jù)與指導(dǎo)意義；而且為由腸道炎癥和腸道屏障功能損傷引起人類或者動物的多種腸道疾病提供了新的防治策略。
[Abstract]:Weaning caused intestinal dysfunction in piglets, caused diarrhea, decreased growth performance or even death, which seriously affected the healthy development of pig industry. Although there were many factors affecting diarrhea, most studies showed that intestinal inflammation and intestinal barrier function injury were the main internal causes of diarrhea in weanling piglets. A large number of antibiotics were used to prevent and control weaning. Diarrhea, bacterial resistance, drug residue and environmental pollution in meat products are serious problems, and the abuse of antibiotics will damage the intestinal barrier function and destroy the intestinal homeostasis. Antimicrobial peptides are important innate immune molecules, and are affected by their broad-spectrum antimicrobial activity, membrane breaking mechanism and resistance to resistance to drug resistance. Note. The latest research shows that antibacterial peptides not only have immune regulation, but also have protective function of intestinal epithelial barrier. However, the effect of antimicrobial peptides on the prevention and treatment of weanling piglets' diarrhea and its effect on intestinal inflammation and intestinal barrier function of weanling piglets are not well known. Therefore, the antibacterial activity is high and safe in the early stage. On the basis of a good antibacterial peptide CWA, the effect of antimicrobial peptide CWA on the diarrhea of weanling piglets was tested, and the effect of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function was clarified, and the effect of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function damage caused by bacterial infection was further explored in mice. Finally, the mechanism of antibacterial peptide CWA in alleviating intestinal inflammation and protecting intestinal barrier function was preliminarily studied in vitro. The main results were as follows: test the effect of antibacterial peptide CWA on intestinal inflammation and intestinal barrier function of weanling piglets (1) the effect of antibacterial peptide CWA on intestinal inflammation of weanling stress piglets was selected healthy. After 21 days of weaning, 3 physiological saline (control group) and antibacterial peptide CWA (antibacterial peptide CWA group) were intraperitoneally injected after 21 days of weaning. The effect of antimicrobial peptide CWA on weanling stress diarrhea and intestinal inflammation was studied. The results showed that antimicrobial peptide CWA reduced the diarrhea index of weaned piglets (P0.05) and increased ADFI (P0.05) in weanling piglets. And ADG (P0.05). At the same time, antibacterial peptide CWA reduced serum IgG, TNF- alpha, IL-6, IL-22 content (P0.05), and increased the content of TGF- beta in serum (P0.05). In the form of intestinal tract, antimicrobial peptides CWA increase the height of the villus, the ratio of the villi height / recess depth (P0.05), increase the ratio of the villus height / recess depth of the ileum, and reduce the return of the ileum. Intestinal fossa depth (P0.05). In intestinal inflammation, antimicrobial peptide CWA reduces the gene expression of proinflammatory factors in the intestinal tract, which is the most significant for jejunum; at the same time, antimicrobial peptide CWA reduces the infiltration of neutrophils and macrophages in the intestinal tract, suggesting that antimicrobial peptide CWA can relieve intestinal inflammation in weanling piglets. In the expression of road protein, antibacterial peptide CWA significantly decreased the phosphorylation level of key protein NF- kappa B and I kappa B- alpha in the inflammatory signaling pathway of jejunum tissue, suggesting that antibacterial peptide CWA may alleviate intestinal inflammation by down regulation of NF- kappa B signaling pathway. (2) the effect of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function of weanling piglets of diarrhoea is selected for 108 diarrhoea. The piglets were intraperitoneally injected with 4 normal saline (control group), enrofloxacin (antibiotic group) and antibacterial peptide CWA (antibacterial peptide CWA group). The effects of antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function of weanling piglets were studied. The results showed that the diarrhea index (P0.05) and diarrhea rate of weanling piglets were reduced by antimicrobial peptide CWA and antibiotics. (P0.05), both ADFI (P < 0.05) and ADG (P < 0.05) were increased in weanling piglets, and there was no significant difference between them. In intestinal morphology, antibacterial peptide CWA and antibiotics had a relieving effect on the morphological damage of small intestinal villi, and the most significant effect was on jejunum. The scanning electron microscopy and transmission electron microscopy showed that antibacterial peptide CWA and antibiotics improved jejunum. The number and length of microvilli, and the improvement of antimicrobial peptide CWA is better than that of antibiotics. In the field of intestinal inflammation, the CWA and IL-22 in the jejunum, the content of IL-6, IL-8, IL-22 (P0.05) in the jejunum are reduced, and the serum also has a similar rule. At the same time, the antibacterial peptide CWA and antibiotics all alleviate the neutrophils of the weanling piglets of diarrhea. Further, both the antibacterial peptide CWA group and the antibiotic group reduced the inflammation related protein NF- kappa B, the phosphorylation level of I kappa B- alpha, AKT and the expression of the key protein TLR4, MyD88, and the effect of antimicrobial peptide CWA on intestinal inflammation was equivalent to that of antibiotics. In the intestinal epithelial barrier function, antibiotic group is not only The expression of mucin and defensins in jejunum decreased and the expression of close connexin ZO-1 and Occludin in jejunum (P0.05) was reduced; however, antimicrobial peptide CWA increased the expression of ZO-1 and Occludin in jejunum (P0.05), suggesting that antimicrobial peptide CWA may improve intestinal barrier function while antibiotics damage intestinal epithelial barrier function. In terms of intestinal microbial flora and SCFAs content, compared with the control group, both the antibacterial peptide CWA group and the antibiotic group reduced the proportion of Escherichia coli in the feces (P0.05), and increased the ratio of lactobacilli / Escherichia coli (P0.05) in the feces. However, the antimicrobial peptide CWA increased the proportion of Lactobacillus in the feces (P0.05), but the antibiotic decreased the lactic acid in the feces. Bacilli ratio (P0.05); further, antibacterial peptide CWA increased the concentration of acetic acid, propionic acid and butyric acid (P0.05) in feces, while antibiotics reduced the concentration of butyric acid in feces, the ratio of butyric acid / acetic acid and the ratio of butyric acid / propionic acid (P0.05). The effect of two antimicrobial peptide CWA on intestinal inflammation and intestinal barrier function in bacteria infected rats was found in the discovery of antimicrobial peptides On the basis of CWA alleviating intestinal inflammation and intestinal barrier function damage in weanling piglets, the effect of antibacterial peptide CWA on intestinal inflammation and intestinal barrier function damage caused by bacterial infection was further studied in mice. 36 weanling mice were divided into control group, E.coli group, E.coli+Enro group, E.coli+CWA group, EHEC O157:H7 infection and abdominal injection respectively. The results of physiological saline, enrofloxacin (Enro) and antimicrobial peptide CWA. showed that both antimicrobial peptide CWA and enrofloxacin increased the survival rate of the infected mice and alleviated the loss of weight of mice caused by the infection. In the form of intestinal tract, both the antibacterial peptide CWA and enrofloxacin both alleviated the atrophy of the jejunum villus, the recess hyperplasia and the colonic mucosa caused by the infected bacteria. The skin was damaged and the number of goblet cells increased. In inflammation, both E.coli+Enro and E.coli+CWA groups relieved the increase of TNF- alpha, IL-6 and IL-10 (P0.05) in the serum and colon of mice infected with bacteria, and alleviated the increase of IL-6 content in the jejunum caused by the infection (P0.05), and the antimicrobial peptide CWA was not associated with enrofloxacin in inflammation. Significant differences. In the expression of intestinal antibacterial protein, both E.coli+Enro and E.coli+CWA groups relieved the increase of REG3y gene expression in the jejunum and the increase of Reml beta gene expression in the colon (P0.05). However, the expression level of TFF3 gene in the jejunum and colon of mice (P0.05) was found in the group of antibacterial peptide CWA (P0.05). Both E.coli+Enro and E.coli+CWA groups were both. The decrease in the number of goblet cells in the jejunum, the decrease in the thickness of the mucous layer and the increase in the number of goblet cells in the colon were alleviated. Further, the results of Western blot and immunofluorescence showed that both E.coli+Enro and E.coli+CWA groups alleviated the decrease of MUC-2 expression in jejunum and the increase of MUC-2 expression in colon (P0.05). In the intestinal epithelial barrier function, the E.coli+CWA group relieved the decrease of ZO-1 and Occludin protein expression in the jejunum of the infected mice (P0.05), while the E.coli+Enro group only alleviated the decrease of the expression of ZO-1 protein (P0.05), and the effect of the antibacterial peptide CWA was better than that of the antibiotic. In the intestinal tract microbial flora and the concentration of SCFAs, the mice were treated differently. There was no significant difference in the number of total bacteria, bifidobacteria and Bacillus in intestinal contents. Both E.coli+Enro and Ecoli+CWA groups alleviated the decrease of lactobacilli in the cecum and the increase of Escherichia coli (P0.05), and the Ecoli+CWA group alleviated the decrease of the ratio of lactobacilli / Escherichia coli in the cecum caused by dyed bacteria (P0.05 At the same time, both the Ecoli+CWA group and the E.coli+Enro group had some alleviating effect on the decrease of the SCFAs concentration caused by the infected bacteria. The above results further demonstrated that the antibacterial peptide CWA could alleviate intestinal inflammation and improve the intestinal barrier function, which was consistent with the test results of weanling piglets. Test three antibacterial peptide CWA for intestinal inflammation and intestinal epithelium screen. Regulatory mechanism of barrier function (1) the mechanism of antibacterial peptide CWA alleviating intestinal inflammation using LPS induced macrophage inflammation model, enrofloxacin has no relieving effect on LPS induced inflammatory response, and antimicrobial peptide CWA can alleviate the inflammatory response caused by LPS and be dependent on the concentration dependent effect. At the same time, antimicrobial peptide CWA relieves IL-6 caused by LPS. IL-8, IL-22 expression increased and NF- kappa B, I kappa B- alpha phosphorylation level increased (P0.05). After siRNA technology was used to silence the TLR4 and MyD88 of pig macrophages, the effect of antimicrobial peptide CWA on inflammation was inhibited. The effect of antibacterial peptide CWA on macrophage phagocytosis was inhibited after the use of STAT-1 inhibitors. The above results suggest that antimicrobial peptide CWA may play a role in regulating inflammatory response through the TLR4-MyD88-NF-KB signaling pathway and enhancing macrophage phagocytosis through the STAT-1 pathway. (2) improvement of antimicrobial peptide CWA The mechanism of intestinal epithelial barrier function using Caco-2 cell scraping model shows that antibacterial peptide CWA improves the healing speed of the scraped cells and improves the repair function of the damaged intestinal epithelial cells. Using the porcine jejunum epithelial cell (IPEC-J2) barrier function damage model, the antibacterial peptide CWA alleviates the TER value of IPEC-J2 monolayer cells caused by LPS. The decrease of the expression of close connexin ZO-1 and Occludin protein in intestinal epithelial cells caused by LPS was reduced. The expression of ZO-1 and Occludin in IPEC-J2 cells was enhanced by the addition of antimicrobial peptide CWA alone. When the Rac1 inhibitor NSC 23766 was added, the effect of antimicrobial peptide CWA on ZO-1 and Occludin was inhibited. The effect of the decrease of EHEC O157:H7 with IPEC-J2 cells was also suppressed. The results showed that the antibacterial peptide CWA could improve the repair function of the damaged intestinal epithelial cells, regulate the expression of close connexin in the intestinal epithelial cells by Racl pathway and enhance the intestinal epithelial barrier function. It provides important basic data and guidance for reducing the use of antibiotics in livestock and poultry breeding, and provides a new prevention strategy for many kinds of intestinal diseases caused by intestinal inflammation and intestinal barrier function damage.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S858.28

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1 易宏波;抗菌肽CWA對斷奶仔豬腸道炎癥和腸道屏障功能的作用及其機制[D];浙江大學(xué);2016年

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本文編號：1795301