畜牧兽医学报  2017, Vol. 48 Issue (6): 1035-1043. DOI: 10.11843/j.issn.0366-6964.2017.06.008    PDF    
镰刀菌毒素对断奶仔猪子宫组织结构、Hsp70分布和表达的影响
孙文涛1#, 高文博1#, 戴美玲2, 陈祥兴3, 杨在宾1, 姜淑贞1, 杨维仁1, 黄丽波1     
1. 山东农业大学动物科技学院, 泰安 271018;
2. 天蓬集团有限公司, 衢州 324122;
3. 南京农业大学, 南京 210095
摘要:本研究旨在探讨镰刀菌毒素对断奶仔猪子宫的组织结构、Hsp70的分布和mRNA表达量的影响。选择35日龄体重为(8.45±0.94)kg的健康三元杂交(杜洛克×长白×大白)雌性断奶仔猪20头,仔猪随机分为2组,每组10头。对照组饲喂基础饲粮,镰刀菌毒素组饲喂含有镰刀菌毒素(玉米赤霉烯酮0.90 mg·kg-1,呕吐毒素1.43 mg·kg-1,烟曲霉毒素5.85 mg·kg-1)的试验饲粮,预试期7 d,正试期35 d,试验结束进行屠宰取出子宫。结果表明:与对照组相比,镰刀菌毒素极显著降低了仔猪的平均日增重和平均日采食量(P<0.01),引起子宫器官指数极显著增加(对照组:0.91±0.03,镰刀菌毒素组:1.90±0.10,P<0.01),子宫腺腺泡数量增多、密度增大,子宫内膜厚度极显著增加(对照组:1 002.55±101.22 μm,镰刀菌毒素组:1 343.09±104.57 μm,P<0.01)。肌层增厚一倍以上(对照组:355.58±28.26 μm,镰刀菌毒素组:779.56±40.38 μm),与对照组相比均差异极显著(P<0.01)。子宫中的Hsp70阳性细胞主要分布在子宫内膜上皮和子宫腺柱状上皮内,为胞质着色。与对照组相比,镰刀菌毒素组子宫Hsp70的免疫阳性反应明显增强(P<0.01),且内环肌层也可见有Hsp70阳性细胞存在,子宫腺上皮细胞Hsp70 IOD较对照组增加2倍多。镰刀菌毒素组子宫Hsp70 mRNA相对表达量显著高于对照组(P<0.05)。综上表明,本试验条件下,镰刀菌毒素可引起断奶仔猪子宫氧化应激反应,子宫内膜上皮和子宫腺上皮通过增加自分泌Hsp70能力,一定限度内能够对抗镰刀菌毒素对子宫正常生理机能的影响。
关键词镰刀菌毒素    子宫    Hsp70    断奶仔猪    
Effect of Dietary Fusarium Toxin on Histological Structure, Distribution and Expression of Hsp70 in the Uterus of Post-Weaning Piglets
SUN Wen-tao1#, GAO Wen-bo1#, DAI Mei-ling2, CHEN Xiang-xing3, YANG Zai-bin1, JIANG Shu-zhen1, YANG Wei-ren1, HUANG Li-bo1     
1. College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China;
2. Tian Peng Group Co. Zhejiang, Quzhou 324122, China;
3. Nanjing Agricultural University, Nanjing 210095, China
Abstract: The present study was aimed at investigating the effects of fusarium toxins on histological structure, Hsp70 distribution and mRNA expression levels in the uterus of post-weaning piglets. A total of 20 healthy post-weaning piglets (Duroc×Landrace×Large White) weaned at 35 d with an average body weight ((8.45±0.94) kg)were randomly allocated into 2 treatments with 10 in the control group and 10 in the fusarium toxins group. Piglets of the control group were fed a based diet only, and piglets of the fusarium toxins group were fed test diet containing fusarium toxin (0.90 mg·kg-1 ZEN, 1.43 mg·kg-1 DON, 5.85 mg·kg-1 FUM) for 35 d after 7 d adaptation. Results showed that the average daily feed intake and average daily gain of piglets of the fusarium group were decreased significantly (P < 0.01), however, the uterine organ index (control group: 0.91±0.03, fusarium toxins group: 1.90±0.10), the number and density of uterine glandular acini and the thickness of endometrium of postweaning piglets (control group: 1 002.55±101.22 μm, fusarium toxins group: 1 343.09±104.57 μm, P < 0.01) were increased significantly, the thickeness of the myometrium was more than doubled that of the control group (control group: 355.58±28.26 μm, fusarium toxins group: 779.56±40.38 μm, P < 0.01), these were extremly significant difference in comparison with the control group (P < 0.01). The observation result of Hsp70 immunoreactive cells revealed that Hsp70 were mainly distributed in the cytoplasm of the endometrium epithelium and columnar glandular epithelium. Compared with the control group, the Hsp70 immunoreactivity of the fusarium toxins group was enhanced obviously (P < 0.01), as well as Hsp70 positive cells of the fusarium toxins group were also found in the inner circular muscle layer of the uterus, and the IOD of Hsp70 in the uterine glandular epithelial cells was 2 times higher than that of the control group (P < 0.01). The relative expression of Hsp70 mRNA in the fusarium toxins group was significantly higher than that of the control group (P < 0.05). In conclusion, the present study found that the fusarium toxins could cause the oxidative stress of the uterus in post-weaning piglets, and this results suggested that the endometrial epithelium and columnar glandular epithelium could resist the effects of fusarium toxins on the normal physiological function of the uterus within a certein degree by incereasing its autocrine ability of HSP70 in piglet uterus.
Key words: fusarium toxins     uterus     Hsp70     postweaning piglets    

农作物在其生长收获、运输和贮存期间,因温度及湿度条件不合适容易滋生镰刀菌,在多雨潮湿季节收获或储存环境较差的农作物表现更为明显[1-2]。镰刀菌产生的镰刀菌毒素(Fusarium toxins)通过污染人类的粮食及畜禽饲料而影响人和动物的身体健康[3]。在全球范围内,谷粒中的镰刀菌毒素主要有单端孢霉烯族化合物(Trichothecenes-2,T-2)、玉米赤霉烯酮又称F-2毒素(Zearalenone,ZEN)、烟曲霉毒素(Fumonisins,FUM)和呕吐毒素(Deoxynivalenol,DON),谷物中多呈现几种毒素联合污染的情况[1, 4],在中国,被ZEN和DON污染的农作物的在黄河流域以北地区尤为普遍[4-6]

热休克蛋白70(Heat shock protein 70,Hsp70) 是热休克蛋白家族中较保守的成员,多数生物体内均有表达,而且大多数细胞都能合成[7-8]。近年来Hsp70成为研究热点,已证实其有分子伴侣功能,提高细胞对应激原的耐受性、抗细胞凋亡、减轻细胞过氧化及炎症性损伤等作用[9-12]。本课题组的前期研究证实镰刀菌毒素能够引起断奶仔猪肠道免疫细胞和上皮细胞产生过量的白介素-1β(Interleukin-1β,IL-1β)和白介素-6(Interleukin-6,IL-6),促进炎症反应发生[13];日粮添加ZEN引起断奶仔猪小肠Hsp70表达异常[14],及卵巢内原始卵泡提早发育[15-16]。本试验是在已有的研究基础上,探讨镰刀菌毒素对断奶仔猪子宫内Hsp70分布和mRNA相对表达量影响,为预防镰刀菌毒素对仔猪生殖危害、提高养猪生产效益提供理论依据。

1 材料与方法 1.1 试验动物与材料

选择35日龄三元杂交(杜洛克×长白×大白)雌性断奶仔猪20头(平均体重为(8.45±0.94) kg)作为试验动物。

霉菌毒素饲粮:本课题组已经完成对山东省16个饲料厂和21个养殖场的饲料原料样品霉菌毒素含量的检测工作,调查了山东省的霉菌毒素污染状况,从中选择毒素水平低于检测限的原料配制本试验的对照饲粮,选择自然霉变玉米和霉变玉米蛋白粉配制本研究的镰刀菌毒素饲粮[13]

1.2 试验设计

将试验仔猪随机分为对照组和镰刀菌毒素组,每组10头,组间初始体重差异不显著(P>0.05)。对照组的仔猪饲喂基础饲粮,镰刀菌毒素组的仔猪饲喂的试验饲粮是用自然霉变玉米和霉变玉米蛋白粉替代正常玉米和玉米蛋白粉进行配制,预试期7 d,试验周期35 d。试验在山东农业大学动物科技学院试验站进行。

仔猪饲养标准参考NRC(2012) 推荐饲粮配制量。配制饲粮前先进行发霉玉米和玉米蛋白粉中黄曲霉毒素(Aflatoxins,AFT)、ZEN、DON、T-2毒素和FUM含量的检测,根据毒素水平分别用50%自然发霉玉米蛋白粉和玉米替代基础饲粮中的玉米蛋白粉和玉米配制镰刀菌毒素组饲粮(镰刀菌毒素饲粮)[13]。饲粮组成及营养水平见表 1

表 1 饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of diets (air-dry basis)

在试验开始前一周, 一次性将试验所需饲粮配制完成,且在试验开始和结束分别取饲粮样品,用于进行毒素含量和粗蛋白质水平的检测[17]。采用酶联免疫吸附(ELISA)或荧光测定法检测AFL、T-2毒素、ZEN和FUM含量,高效液相色谱法(HPLC,High performance liquid chromatography)测定DON含量。几种毒素的最低检测限分别ZEN 0.1 mg·kg-1、DON 0.1 mg·kg-1、AFL 1.0 μg·kg-1、FUM 0.25 mg·kg-1和T-2毒素1.0 μg·kg-1。饲粮的毒素水平见表 1。本试验日粮中的AFL和T-2毒素均低于检测限水平。

1.3 测定指标及方法 1.3.1 样本采集

试验结束后,对仔猪进行电击后, 放血致死,剖开腹腔, 迅速取出子宫,并采集两份样品,一份置于Bouin’s液中固定,用于制备免疫组化切片,另一部分液氮中速冻,置于-80 ℃冰箱保存,用于检测Hsp70 mRNA相对表达量。

1.3.2 免疫组化(超敏二步法)

取Bouin’s液中固定好的子宫组织进行流水冲洗,经梯度乙醇逐级脱水,二甲苯透明,石蜡包埋制成石蜡组织块,切片机(LEICA RM2135,德国)切片,片厚5 μm。

免疫组化程序:1) 石蜡切片经常规脱蜡、脱苯至水;2) 0.01 mol·L-1,pH 6.0的柠檬酸缓冲液抗原热修复(微波修复),PBS(0.01 mol·L-1,pH 7.2) 洗3次,5 min·次-1(PBS,下同);3)10% H2O2孵育30 min,37 ℃,阻断内源性过氧化物酶,PBS洗3次;4)5%胎牛血清孵育1 h,37 ℃,封闭游离电荷; 5) 甩去血清,加鼠抗Hsp70多克隆抗体(1:100)(BM0368,武汉博士德生物工程有限公司),4 ℃孵育过夜,PBS洗3次;6) 二抗为抗鼠Polink-2 plus®超敏二步法免疫组化检测试剂盒(PV-9002,北京中杉金桥生物技术有限公司); 7) DAB显色(RA110,TIANGEN); 8) 苏木精复染,上行脱水、透明、封片、观察。阴性对照采用PBS代替一抗,其他步骤相同。

1.3.3 HE染色

石蜡切片经二甲苯脱蜡,梯度酒精至蒸馏水,苏木素染液中染色10 min,盐酸酒精分化5 s,自来水中蓝化15 min,伊红染液中10 s,经95%乙醇、100%乙醇脱水,二甲苯透明,中性树胶封片,明视野显微镜下观察。

1.3.4 子宫Hsp70 mRNA相对表达量检测

根据GenBank报道的猪的Hsp70和GAPDH基因序列,用Primer 6.0设计相应特异性引物,引物由华大基因合成(表 2)。

表 2 RT-PCR反应的引物序列 Table 2 Sequence of primers for real-time PCR

取50~70 mg冻存子宫组织,液氮研磨,用Trizol法提取总RNA,并用紫外分光光度计测其浓度和纯度。按照PrimeScript RT Master Mix反转录试剂盒(RR036A,TaKaRa,大连)说明进行反转录,反应体系为20 μL。

qRT-PCR反应体系为20 μL,按照SYBR Premix® Ex TaqTM(Tli RNaseH Plus)(DRR420A,TaKaRa,大连)试剂盒说明进行操作。体系组成:SYBR Primerx Ex Taq 10 μL;上游和下游引物各0.4 μL(10 μmol·L-1);ROX Reference DyeⅡ 0.4 μL;cDNA 2 μL(<100 ng),dH2O 6.8 μL。每个样品设置3个重复。使用ABI 7500 Real-time PCR仪进行扩增反应,反应条件为95 ℃预变性30 s,95 ℃变性5 s,60 ℃退火34 s,95 ℃ 15 s, 60 ℃60 s,95 ℃15 s,循环数为40。

1.4 数据分析

采用Image pro-Plus 6.0软件测量子宫截面免疫阳性反应物质的累积光密度(Integrated optic density, IOD)。每头猪子宫切片隔5张取1张,共取5张,统计3头猪数据。根据qRT-PCR试验所得CT值,以GAPDH为内参基因,运用2-△△CT方法分析Hsp70 mRNA的相对表达量[18]。数据采用SPSS17.0软件进行统计分析,结果用“平均值±标准误(Mean±SE)”表示。

2 结果 2.1 子宫器官指数及组织学结构

镰刀菌毒素可引起断奶仔猪子宫器官指数显著增加(表 3)(对照组:0.91±0.03,镰刀菌毒素组:1.90±0.10),子宫壁明显增厚肥大,子宫内膜厚度增加(对照组:1 002.55±101.22 μm,镰刀菌毒素组:1 343.09±104.57 μm),与对照组相比差异极显著(P<0.01)。子宫肌层厚度与对照组相比增厚一倍以上(对照组:355.58±28.26 μm,镰刀菌毒素组:779.56±40.38 μm,P<0.01),组织学结构的观察显示子宫内膜上皮内胞质淡染呈灰白色的细胞增多,似有分泌物存在,子宫腺腺泡数量增多、密度增大(图 1FE),子宫内膜皱褶变高(图 1BA),肌层的平滑肌细胞数量增加明显(图 1HG)。

表 3 镰刀菌毒素对断奶仔猪生长性能和子宫的影响 Table 3 Effects of fusarium toxins on growth performance and uterus in piglets
A、C、E、G.对照组;B、D、F、H.镰刀菌毒素组。A、B.子宫(比例尺:200 μm);C、D.子宫内膜上皮(比例尺:20 μm);E、F.子宫腺(比例尺:20 μm)。箭头.肌层厚度;EE.子宫内膜上皮;GG.子宫腺;M.肌层;V.血管;LP.固有层。下同。G、H.子宫肌层(比例尺:100 μm) A, C, E and G.The uterus of the control group; B, D, F and H.The uterus of the fusarium toxins group.A and B.The uterus (The scale bars is 200 μm); C, D.The endometrium epithelium (The scale bars is 20 μm); E, F.The uterine gland (The scale bars is 20 μm).The arrow indicate the thickness of myometrium.EE.Endometrium epithelium; GG.Uterine gland; M. Myometrium; V.Vessel; LP. Lamina propria.The same as below.G, H.The myometrium (The scale bars is 100 μm) 图 1 镰刀菌毒素对子宫组织学结构的影响(HE染色方法) Figure 1 Effects of fusarium toxins on the histological structure of uterus in piglets(hematoxylin-eosin method)

镰刀菌毒素导致断奶仔猪生长性能降低,与对照组相比平均日采食量(ADF)和平均日增重(ADG)极显著降低(P<0.01,表 3) [13]

2.2 子宫的Hsp70的分布及IOD的分析

免疫组化结果显示, 在断奶仔猪的子宫中均有Hsp70阳性细胞分布,阳性反应物质呈黄色、棕黄色,且阴性对照不着色(图 2G)。说明本试验采用的一抗及免疫组化方法具有免疫反应的特异性。子宫中的Hsp70阳性细胞主要分布在子宫内膜上皮和子宫腺上皮细胞内,为胞质着色,但镰刀菌毒素组的免疫阳性反应(图 2B)明显强于对照组(图 2A)。镰刀菌毒素组的Hsp70免疫阳性反应物质位于子宫内膜上皮和子宫腺上皮细胞核上方的胞质内,呈团块状(图 2DF),而对照组分布均匀(图 2CE)。镰刀菌毒素组的内环肌层也可见有Hsp70阳性细胞存在(图 2HB),但外环肌层为阴性(图 2B),对照组肌层呈阴性反应(图 2A)。子宫内膜中的Hsp70阳性反应的IOD的分析结果显示(表 4),镰刀菌毒素组子宫内膜上皮和子宫腺上皮细胞Hsp70的IOD均极显著高于对照组(P<0.01)。腺上皮细胞Hsp70的IOD较对照组增加2倍多。Hsp70的IOD统计结果显示子宫腺上皮细胞免疫阳性反应明显强于内膜上皮。

箭头.子宫内Hsp70免疫阳性细胞;G.阴性对照;H.肌层Hsp70免疫阳性细胞 The arrow indicate the Hsp70 positive cells in the uterus; G. Negative control; H.The Hsp70 positive cells of the myometrium 图 2 镰刀菌毒素对Hsp70在子宫分布的影响 Figure 2 Effect of fusarium toxins on the distribution of Hsp70 positive cells in the uterus
表 4 子宫内膜中的Hsp70阳性反应的IOD的结果 Table 4 Result of IOD of Hsp70 positive reaction in the endometrium
2.3 子宫Hsp70的mRNA相对表达量

qRT-PCR产物经1.5%琼脂糖凝胶电泳,结果显示, 目的基因扩增片段长度与引物设计的产物大小一致(图 3);统计结果表明, 镰刀菌毒素组子宫Hsp70的mRNA相对表达量显著高于对照组(P<0.05)

A.柱形图;B.电泳图。*.P<0.05 A.Column diagram; B.Electrophoretogram. *.P < 0.05 图 3 镰刀菌毒素对子宫的Hsp70 mRNA相对表达量的影响 Figure 3 Effects of fusarium toxins on the mRNA relative expression of Hsp70 in the uterus

综上,子宫的Hsp70的IOD水平和mRNA相对表达量的统计结果与免疫组化观察结果相一致。

3 讨论

中国对于饲料中FUM的含量没有制定相应的限量标准,但限定ZEN和DON含量分别不能高于0.5和1.0 mg·kg-1,而欧盟规定在仔猪饲粮中的DON、ZEN以及FUM最高限量要小于0.9、0.1和5.0 mg·kg-1 [19-20]。本试验是与陈祥兴等[13]的研究共用的断奶仔猪,试验结果发现对照组基础日粮内仍然可检测到DON(0.17 mg·kg-1)和FUM(0.34 mg·kg-1),虽然含量远低于中国饲料卫生标准规定,试验结果中忽略了对照组中毒素含量对统计结果影响,但同时也反映出中国粮食作物镰刀菌毒素污染的普遍性。

镰刀菌毒素的ZEN、DON以及AFT给养猪生产带来了的负面影响, 现已成为全球关注的热点问题之一。镰刀菌侵染的作物种类较多,包括玉米、小麦、大豆、油菜等各种作物,其毒性作用尤其以猪最易感[21]。有关镰刀菌毒素对人、家畜及家禽影响的研究报道很多。ZEN可影响饲料转化效率[13, 15],增加子宫的体积、重量及器官指数[15]、促进卵巢内原始卵泡的提前发育[16],而本研究结果发现, 饲喂自然霉变玉米和霉变玉米蛋白粉同样使子宫体积增大,器官指数增加,并且较单纯添加ZEN的增加效果明显[15]。从子宫组织结构变化的观察结果发现, 主要源于子宫内膜腺体细胞和肌层细胞大量增殖的结果。

热休克蛋白本质上是生物体为了保护细胞应对各种因素的应激而不受应激性损伤所产生的特殊蛋白质。Hsp70作为重要的分子伴侣,在蛋白质构象的维持、促进细胞器蛋白的跨膜运输和肽链折叠等方面起重要作用[22-23],并能减轻细胞过氧化及炎症性损伤。当给予培养的大鼠肠上皮细胞缺氧再复氧处理后,上皮细胞内Hsp70基因呈现过度表达,而凋亡、死亡的细胞减少,细胞增殖能力增强,细胞骨架损伤减轻[24]。本研究结果表明,镰刀菌毒素可引起断奶仔猪子宫的Hsp70表达量显著增加,可能是镰刀菌毒素对仔猪子宫内膜细胞产生了损伤,导致了子宫为对抗毒素的应激而过表达了Hsp70,这与Y.Tsukimi等[25]发现肠道在受到各种应激因素(如毒性物质、热应激等)作用时,会诱导产生Hsp70过表达的结果相类似,而且Hsp70在动物机体受到热应激及烫伤的损害时,可增强细胞对损伤的耐受力、减少细胞的凋亡、维持细胞的正常功能、减轻应激带来的损伤[26]。综上表明,镰刀菌毒素诱导子宫内膜细胞内Hsp70基因的过度表达,是细胞抵抗毒素对子宫细胞氧化损伤的结果。

镰刀菌毒素成分之一的ZEN对生殖器官影响最为明显,而且以猪最为敏感。已有的研究发现, 不同剂量的ZEN会对猪机体造成不同程度的影响,如延长母猪的发情间期的时间,使妊娠母猪的繁殖率降低,假孕发病率增加,窝产仔数下降、卵巢的卵泡和颗粒细胞的闭锁和凋亡[27-29],当试验日粮中含有ZEN 0.90 mg·kg-1、DON 1.43 mg·kg-1、FUM 5.85 mg·kg-1的毒素物质被仔猪吸收后,可引起断奶仔猪的子宫体积增大,子宫内膜增厚、器官指数增加,子宫内膜细胞内Hsp70基因的过度表达。结果表明,若是性成熟或妊娠母猪同样也会引起子宫的氧化应激损伤,改变或破坏子宫的微环境,而胚胎着床和妊娠的维持对子宫的微环境稳态要求十分严格。本试验结果对于解释ZEN使母猪出现假孕、不孕不育、降低产仔数、胎儿畸形或被吸收现象以及影响胚胎定植、胎儿发育和新生儿活力[29]的结果提供理论依据。

4 结论

本试验条件下,镰刀菌毒素显著影响断奶仔猪子宫组织学结构及Hsp70阳性分布和mRNA相对表达量,说明镰刀菌毒素饲粮(0.90 mg·kg-1 ZEN,1.43 mg·kg-1 DON,5.85 mg·kg-1 FUM)可引起子宫内膜细胞的氧化损伤,改变或破坏子宫的微环境,激发Hsp70的过表达。

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