Breviscapine alleviates hepatic injury and inhibits PKC-mRNA and its protein expression in brain-dead BA-Ma mini pigs

BACKGROUND: Brain-dead donors are the main sources for organ transplantation, but many studies show that brain-death affects the organ's function after transplantation. This study was undertaken to investigate liver injury after brain-death in BA-Ma mini pigs and the protective effects of breviscapine on hepatic function and on PKC-alpha mRNA and its protein expression. METHODS: Fifteen BA-Ma mini pigs were equally divided into 3 groups at random: brain-dead (group B), breviscapine pretreated (group P), and control (group Q. The brain-dead model was established by increasing intracranial pressure in a modified, slow and intermittent way. At 3, 6, 12, 18 and 24 hours after the initial brain-death, the levels of serum AST, ALT, TNF-alpha, IL-1 beta, and IL-6 were determined. The changes in hepatic tissues were assessed, and the expression of PKC-alpha and PKC-alpha mRNA was detected by immunohistochemistry and RTPCR, respectively. RESULTS: The levels of AST and ALT in groups B and P began to increase 12 hours after brain-death, while the values in group P were lower than those in group B (P<0.05). The levels of IL-1 beta, IL-6, and TNF-alpha in groups B and P at 3, 6, 12 and 18 hours were lower than those in group B (P<0.05). At 6, 12 and 24 hours, the expressions of PKC-a mRNA and PKC-a protein in group P were lower than those in group B (P<0.05). The degree of injury to hepatic cells in group P was milder than that in group B. CONCLUSIONS: Breviscapine inhibits the degree of PKC-alpha mRNA transcription and its protein translation, decreases the release of inflammatory factors, and thus alleviates hepatic injury during brain-death.

Dietary proline supplementation alters colonic luminal microbiota and bacterial metabolite composition between days 45 and 70 of pregnancy in Huanjiang mini-pigs

Background: Pregnancy is associated with important changes in gut microbiota composition. Dietary factors may affect the diversity, composition, and metabolic activity of the intestinal microbiota. Among amino acids, proline is known to play important roles in protein metabolism and structure, cell differentiation, conceptus growth and development, and gut microbiota re-equilibration in case of dysbiosis.Results: Dietary supplementation with 1% proline decreased(P < 0.05) the amounts of Klebsiella pneumoniae,Peptostreptococcus productus, Pseudomonas, and Veillonella spp. in distal colonic contents than that in the control group. The colonic contents of Butyrivibrio fibrisolvens, Bifidobacterium sp., Clostridium coccoides, Clostridium coccoides-Eubacterium rectale, Clostridium leptum subgroup, Escherichia coli, Faecalibacterium prausnitzii,Fusobacterium prausnitzii, and Prevotella increased(P < 0.05) on d 70 of pregnancy as compared with those on d 45 of pregnancy. The colonic concentrations of acetate, total straight-chain fatty acid, and total short-chain fatty acids(SCFA) in the proline-supplemented group were lower(P < 0.05), and butyrate level(P = 0.06) decreased as compared with the control group. Almost all of the SCFA displayed higher(P < 0.05) concentrations in proximal colonic contents on d 70 of pregnancy than those on d 45 of pregnancy. The concentrations of 1,7-heptyl diamine(P = 0.09) and phenylethylamine(P < 0.05) in proximal colonic contents were higher, while those of spermidine(P = 0.05) and total bioamine(P = 0.06) tended to be lower in the proline-supplemented group than those in the control group. The concentrations of spermidine, spermine, and total bioamine in colonic contents were higher(P < 0.05) on d 70 of pregnancy than those measured on d 45 of pregnancy. In contrast, the concentration of phenylethylamine was lower(P < 0.05) on d 70 than on d 45 of pregnancy.(Continued on next page)(Continued from previous page)Conclusion: These findings indicate that L-proline supplementation modifies both the colonic microbiota composition and the luminal concentrations of several bacterial metabolites. Furthermore, our data show that both the microbiota composition and the concentrations of bacterial metabolites are evolving in the course of pregnancy. These results are discussed in terms of possible implication in terms of luminal environment and consequences for gut physiology and health.

Development of a human rotavirus induced diarrhea model in Chinese mini-pigs

AIM: to establish a new animal model for the research of human rotavirus(HRV) infection, its pathogenesis and immunity and evaluation of potential vaccines.METHODS: 5-d, 30-d and 60-d-old Chinese mini-pigs, Guizhou and bamma, were inoculated with a single oral dose of attenuated strain Wa, G1, G3 of HRV, and PbS(control), respectively, and fecal samples of pigs from 0 to 7 d post infection(DPI) were collected individually. Enzyme linked immunosorbent assay was used to detect HRV antigen in feces. the HRV was tested by real-time PCR(Rt-PCR). the sections of the intestinal tissue were stained with hematoxylin and eosin to observe the morphologic variation by microscopy. Immunofluorescence was used to determine the HRV in intestinal tissue. HRV particles in cells of the ileum were observed by electron micrography.RESULTS: When inoculated with HRV, mini-pigs younger than 30 d developed diarrhea in an agedependent manner and shed HRV antigen of the sameinoculum, as demonstrated by Rt- PCR.Histopathological changes were observed in HRV inoculated mini-pigs including small intestinal cell tumefaction and necrosis. HRV that was distributed in the small intestine was restricted to the top part of the villi on the internal wall of the ileum, which was observed by immunofluorescence and transmission electron microscopy. Virus particles were observed in Golgi like follicles in HRV-infected neonatal minipigs. Guizhou mini-pigs were more sensitive to HRV than bamma with respect to RV antigen shedding and clinical diarrhea.CONCLUSION: these results indicate that we have established a mini-pig model of HRV induced diarrhea. Our findings are useful for the understanding of the pathogenic mechanisms of HRV infection.

Correlation between the Polymorphism of PPARγ-2 gene and the Susceptibility of Type 2 Diabetes Mellitus in Guangxi Bama Mini-pigs

[ Objective] The research aimed to discuss the relationship between the polymorphism of PPARy.2 gene and the susceptibility of type 2 diabetes mellitus （T2DM） in Guangxi Bama mini-pigs. [ Method] 24 Guangxi Bama mini-pigs were fed with high-fat and high-sucrose diet, and partial sequences of exon 2 of PPARy-2 gene were amplified by using PCR method. In addition, the contents of fasting blood glucose and insulin （INS） in Guangxi Bama mini-pigs were determined, and the glucose tolerance test （GTT） was also carried out. [ Result] There was one SNP site （19813A/G） Jn partial sequence of exon 2 of the cloned PPAFly-2 gene, and AA （7 pigs） and AG （17 pigs） genotype were detected. The contents of fasting insulin and 60-min blood glucose in GTT in AG-genotype Guangxi Bama mini-pigs were significantly higher than those of AA genotype （ P 〈0.05）, while the incidence of T2DM in AG-genotype Guangxi Bama mini-pigs （71.4%） was obviously higher than that of AA gen- otype （5.9%）. [ Conclusion] The polymorphism of 19813A/G in exon 2 of PPARy-2 gene was related with the susceptibility of T2DM in Guangxi Bama mini-pigs.

Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting

Unbalanced brain serotonin(5-HT) levels have implications in various behavioral abnormalities and neuropsychiatric disorders. The biosynthesis of neuronal 5-HT is regulated by the rate-limiting enzyme, tryptophan hydroxylase-2(TPH2). In the present study, the clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas) system was used to target the Tph2 gene in Bama mini pig fetal fibroblasts. It was found that CRISPR/Cas9 targeting efficiency could be as high as 61.5%, and the biallelic mutation efficiency reached at38.5%. The biallelic modified colonies were used as donors for somatic cell nuclear transfer(SCNT) and 10 Tph2 targeted piglets were successfully generated. These Tph2 KO piglets were viable and appeared normal at the birth.However, their central 5-HT levels were dramatically reduced, and their survival and growth rates were impaired before weaning. These Tph2 KO pigs are valuable large-animal models for studies of 5-HT deficiency induced behavior abnomality.

Advantages of a miniature pig model in research on human hereditary hearing loss

In medical laboratory animals, the pig is the closest species to human in evolution, except for primates. As an animal model, the pig is highly concerned by many scientists, including comparative biology, developmental biology, medical genetics. Rodents as animal model for human hearing defects has are poor producibility and reliability, due to differences in anatomical structure, evolutionary rate and metabolic rate, but these happens to be the advantages of the pig model. In this paper, we will summarize the application of miniature pig in the study of human hereditary deafness.

Effects of dietary protein/energy ratio on growth performance,carcass trait,meat quality,and plasma metabolites in pigs of different genotypes

Background： The protein/energy ratio is important for the production performance and utilization of available feed resources by animals. Increased protein consumption by mammals leads to elevated feed costs and increased nitrogen release into the environment. This study aimed to evaluate the effects of dietary protein/energy ratio on the growth performance, carcass traits, meat quality, and plasma metabolites of pigs of different genotypes. Methods： Bama mini-pigs and Landrace pigs were randomly assigned to two dietary treatment groups （Chinese conventional diet with low protein/energy ratio or National Research Council diet with high protein/energy ratio; n = 24 per treatment） in a 2 x 2 factorial arrangement. Blood and muscle samples were collected at the end of the nursery, growing, and finishing phases. Results： We observed significant interactions （P 〈 0.05） between breed and diet for total fat percentage, intramuscular fat （IMF） content, protein content in biceps femoris （BF） muscle, and plasma urea nitrogen （UN） concentration in the nursery phase; for average daily gain （ADG）, average daily feed intake （ADFI）, dry matter, IMF content in psoas major （PM） muscle, and plasma total protein and albumin concentrations in the growing phase; and for drip loss and plasma UN concentration in the finishing phase. Breed influenced （P 〈 0.05） growth performance, carcass traits, and meat quality, but not plasma metabolites. Throughout the trial, Landrace pigs showed significantly higher （P 〈 0.0_5） ADG, ADFI, dressing percentage, lean mass rate, and loin-eye area than did Bama mini-pigs, but significantly lower （P 〈 0.0.5） feed/gain ratio, fat percentage, backfat thickness, and IMF content. Dietary protein/energy ratio influenced the pH value, chemical composition of BF and PM muscles, and plasma activities of glutamic-pyruvic transaminase and gamma-glutamyl transpeptidase, and plasma concentration of UN. Conclusions： Compared with Landrace pigs, Bama mini-pigs showed slower growth and lower carcass performance, but had better meat quality. Moreover, unlike Landrace pigs, the dietary protein/energy ratio did not affect the growth performance of Bama mini-pigs. These results suggest that, in swine production, low dietary protein/energy ratio may be useful for reducing feed costs and minimizing the adverse effects of ammonia release into the environment.

A new approach to endocochlear potential and potassium ion concentration measures in mini pig models

Mini pig models are large mammals and their ears are more similar with human beings in structure and development than other animals.However,the study on porcine ears is still in the initial stage and there is no description of an ideal operation approach to endocochlear potential and potassium ion concentration measurements.In this article,we describe a pre-auricular surgical approach to access the middle and inner ear for endocochlear potential and potassium ion concentration measures in mini pig models.Ten one-week old normal mini pigs were used in the study.The bulla of the temporal bone was accessed via a pre-auricular approach for endocochlear potential and potassium ion concentration measurements.The condition of the animals during the first post—experiment 24 h was observed.One animal died during surgery.The preauricular approach improved protection and preservation of relevant nervous and vascular elements including the facial nerve and carotid artery.So,the pre-auricular approach can be used for endocochlear potential and potassium ion concentration measurements with improved nerve and artery preservation mini pigs.

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury

We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases,neurotrauma,and stroke.In this study,using a mini pig model of spinal cord injury,we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy.Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector(Ad5/35 F)that carried an enhanced green fluorescent protein(EGFP)reporter gene in the plastic blood bag.The day after blood donation,the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate.A week after gene-modified leucoconcentrate therapy,fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury.In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed.In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes.Thus,the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions.This paper presents a proof-of-concept of simple,safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.5)on May 27,2014.

基于NOX5-ERK1/2信号通路探讨健脾祛痰化瘀方对动脉粥样硬化小型猪炎症反应的影响

目的 观察健脾祛痰化瘀方对动脉粥样硬化(AS)小型猪氧化应激和炎症反应的影响,基于NOX5-ERK1/2信号通路探讨其作用机制。方法 将12只巴马小型猪随机分为对照组、模型组和健脾祛痰化瘀方低、高剂量组,每组3只。采用高脂饮食饲养24周构建动脉粥样硬化模型,给药组同时在饲料中添加健脾祛痰化瘀方。分别于给药0、16、24周检测小型猪一般体征(体长、腹围、体质量、食物摄入量和粪便含水量),HE染色观察主动脉形态,油红O染色观察主动脉和心肌组织脂质沉积,透射电镜观察胸主动脉组织超微结构,全自动生化分析仪检测血清总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量,ELISA检测血清活性氧(ROS)、白细胞介素(IL)-6、IL-10、肿瘤坏死因子(TNF)-α、超敏C反应蛋白(hs-CRP)、血管细胞黏附分子-1(VCAM-1)、细胞间黏附分子-1(ICAM-1)含量,Western blot检测主动脉组织NADPH氧化酶5(NOX5)、细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、VCAM-1、增殖细胞核抗原(PCNA)蛋白表达。结果 与正常组比较,模型组小型猪16、24周腹围、体质量、食物摄入量增加(P<0.01),主动脉内膜明显增厚,内皮细胞破坏,脂质沉积,平滑肌细胞水肿,线粒体肿胀明显,血清TC、LDL-C含量及ROS、IL-6、IL-10、TNF-α、hs-CRP、VCAM-1、ICAM-1含量升高,HDL-C含量降低(P<0.01);主动脉组织NOX5、p-ERK1/2、VCAM-1、PCNA蛋白表达升高(P<0.01)。与模型组比较,健脾祛痰化瘀方低、高剂量组16、24周腹围、体质量、食物摄入量减少(P<0.05,P<0.01),斑块面积和脂质沉积减少,内皮细胞破坏减轻,血清TC、LDL-C含量及ROS、IL-6、IL-10、TNF-α、hs-CRP、VCAM-1、ICAM-1含量降低,HDL-C含量升高(P<0.01,P<0.05);主动脉组织NOX5、p-ERK1/2、VCAM-1、PCNA蛋白表达降低(P<0.01,P<0.05)。结论 健脾祛痰化瘀方可减轻小型猪AS,其机制可能与抑制NOX5-ERK1/2信号通路激活、减轻氧化应激诱导的炎症反应有关。

猪睾丸Dickkopf样顶体蛋白1基因的转录调控分析

【目的】Dickkopf样顶体蛋白1(DKKL1)是一种重要的顶体蛋白,为发掘其重要功能及潜在的临床价值,以版纳微型猪近交系(BMI)为对象,研究其睾丸组织中DKKL1的分子结构、转录调控特征和蛋白质功能。【方法】通过全转录组测序获得BMI DKKL1的表达量,使用逆转录聚合酶链反应(RT-PCR)获得DKKL1编码区序列并分析其基因结构和蛋白质特征,使用UniProt数据库对DKKL1进行功能注释并分析DKKL1与微小RNA(miRNA)、长链非编码RNA(lncRNA)间的竞争性内源RNA(ceRNA)转录调控。【结果】获得的BMI DKKL1编码区全长为702 bp,位于基因第6号染色体上,有5个外显子和4个内含子;蛋白质功能分析表明,DKKL1包含233个氨基酸,具有疏水性,含磷酸化位点、信号肽和较多的无规则卷曲亚结构;系统进化和同源性分析表明,DKKL1氨基酸序列在哺乳动物间高度保守;蛋白互作分析显示,DKKL1与含螺旋结构域的蛋白155(CCDC155)、转录增强缔合域蛋白2(TEAD2)、RAS癌基因家族成员11B(RAB11B)等多种蛋白互作;基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析表明,这些蛋白富集在典型Wnt信号等通路上;GO功能注释表明,DKKL1在睾酮生物合成过程的负向调控、透明带穿透、顶体泡生成等方面具有重要功能;ceRNA转录调控网络分析表明,DKKL1被miR-15a和miR-15b靶向调控。【结论】本研究获得了BMI睾丸全转录数据,阐明了DKKL1的分子特征、蛋白质功能并构建了转录调控网络。

Assessment of Economic Viability of Mini-livestock Production in Delta State,Nigeria:Implication for Extension Delivery Services

The study examined the popularity, role and economic viability of mini-livestock keeping in Delta State, Nigeria. The specific objectives were to describe the socio-economic characteristics of mini-livestock farmers, identify the types of animals reared as mini-livestock in the area, ascertain the reasons for keeping mini-livestock by farmers, determine the benefit level of mini- livestock farmers, and identify the problems facing mini-livestock farmers in the study area. Using snowball sampling technique, 674 respondents were sampled with the aid of a structured questionnaire. Various descriptive and inferential statistics were used for data analyses. The results of the study indicated various types of mini-livestock reared in the study area, mainly snail farming or heliciculture （about 90%） and rearing of cane rat （84.27%）; the most important reasons for keeping mini-livestock were income （99.6%）, employment （99.26%） and food （94.96%）. The result further indicated that protein consumption and improved income were the most important benefits to respondents. In spite of these benefits, the respondents faced some constraints, amongst which were lack of technical information （X=4.70）, lacking of enough space for expansion （X=4.58）, inadequate training （X=4.20）, poor extension delivery services （X=4.20） and problems of feed availability （X=3.86）. In spite of these constraints, the production of mini-livestock was found to be profitable in the study area. Based on the t＇mdings, it was recommended, among others, that mini-livestock farmers in the study area should be trained on various aspects of the management of mini-livestock especially by organising workshops for them.

母猪添加益生菌和合生元对子代巴马香猪肌肉脂肪酸组成及相关基因表达的影响

本试验旨在研究母体添加益生菌和合生元对子代肌肉脂肪酸组成及相关基因表达的影响。选用64头妊娠巴马香猪,随机分为对照组(基础饲粮)、抗生素组(50 g·t^(-1)维吉尼亚霉素)、益生菌组(200 mL·d^(-1)益生菌发酵液)和合生元组(500 g·t^(-1)低聚木糖+200 mL·d^(-1)益生菌发酵液),整个妊娠和哺乳期饲喂相应试验饲粮。断奶后每窝选取2头接近平均体重的仔猪,每组32头,饲喂基础饲粮。于125日龄每组选取8头,采集股二头肌和腰大肌样品,测定中长链脂肪酸组成和相关基因的表达。结果表明:与对照组相比,抗生素组股二头肌二十碳二烯酸的含量显著增加(P<0.05)、硬脂酰辅酶A去饱和酶(SCD)和胆固醇调节原件结合蛋白1(SREBP^(-1))表达显著上调(P<0.05);益生菌组股二头肌和腰大肌十七碳酸的含量显著减少(P<0.05),股二头肌激素敏感脂酶(HSL)和腰大肌过氧化物酶体增殖物激活受体γ(PPARγ)表达显著上调(P<0.05);合生元组股二头肌亚油酸、二十碳一烯酸和n-6多不饱和脂肪酸的含量显著减少(P<0.05),腰大肌过氧化物酶体增殖物激活受体α(PPARα)表达显著上调(P<0.05);抗生素和益生菌组股二头肌二十碳酸的含量显著减少(P<0.05),股二头肌PPARα、腰大肌脂肪甘油三酯脂酶(ATGL)和脂肪酸结合蛋白4(FABP 4)表达显著上调(P<0.05);益生菌和合生元组腰大肌肉碱棕榈酰转移酶1(CPT^(-1))和脂蛋白脂酶(LPL)表达显著上调(P<0.05);三个试验组股二头肌反式油酸的含量显著减少(P<0.05),ATGL、FABP 4和LPL表达显著上调(P<0.05)。综上所述,母体添加益生菌和合生元可改变子代肌肉的中长链脂肪酸组成、调控脂质代谢相关基因表达,有利于猪肉营养价值和风味的改善;添加抗生素可改善肌肉脂肪酸的组成。

宫内发育迟缓对环江香猪哺乳仔猪IGF发育模式的影响

本文旨在探讨宫内发育迟缓(IUGR)发生与胰岛素样生长因子(IGF)发育模式的联系。选用20头体况相近的环江香猪母猪,分娩后从每窝挑出1头体重最大(定义为正常出生重,NBW)仔猪和1头体重最小仔猪(定义为IUGR),分为NBW组和IUGR组,每组20头。分别于0、7、14和21日龄时,从各组随机选取5头仔猪,前腔静脉采血,离心分离血浆,检测IGF-1浓度;采集肝和背最长肌样品,检测IGF-1、IGF-1R、IGFBP-3和IGFBP-5的基因表达水平。结果表明:与NBW仔猪相比,0~21日龄IUGR仔猪体重和血浆IGF-1含量均显著降低(P<0.05);0~14日龄IUGR仔猪的体重逐渐增加(P<0.05),而21日龄IUGR仔猪的体重与14日龄差异不显著(P>0.05)。0日龄IUGR仔猪肌肉IGF-1R、IGFBP-3和IGFBP-5、肝IGFBP-5的表达显著高于NBW仔猪(P<0.05),7日龄IUGR仔猪肌肉和肝的IGF-1表达均显著低于NBW仔猪(P<0.05),21日龄IUGR仔猪肌肉IGF-1R的表达水平显著高于NBW仔猪(P<0.05)。7日龄NBW仔猪肝和肌肉IGF-1以及IUGR仔猪肝IGF-1的表达水平显著高于其他日龄(P<0.05);0、14日龄IUGR仔猪肌肉IGF-1的表达水平显著高于7和21日龄(P<0.05);7日龄NBW仔猪肝和肌肉IGFBP-5的表达水平显著高于其他日龄(P<0.05);IUGR仔猪肝IGFBP-3的表达水平在14日龄显著上调,IGFBP-5的表达水平在0日龄显著高于其他日龄(P<0.05)。综上,IUGR可能通过降低哺乳环江香猪IGF-1的表达、调节IGF-1受体及结合蛋白的表达,导致机体内IGF-1含量降低,进而延缓其生长发育。

饲粮添加益生菌和合生元对妊娠-哺乳期母猪免疫功能与抗氧化能力的影响

本试验旨在研究饲粮添加益生菌和合生元对妊娠-哺乳期母猪免疫功能与抗氧化能力的影响。选用64头妊娠巴马香猪,随机分为对照组(饲喂基础饲粮)、抗生素组(在基础饲粮中添加50 g/t维吉尼亚霉素)、益生菌组[在基础饲粮中添加200 mL/(头·d)益生菌发酵液]和合生元组[在基础饲粮中添加200 mL/(头·d)上述益生菌发酵液和500 g/t低聚木糖],每组16头,单栏饲喂。分别于妊娠第45、75和105天以及分娩后第7和21天,每组选取6头母猪,前腔静脉采血,离心分离血浆,测定免疫球蛋白、免疫细胞因子含量和氧化-抗氧化指标。结果表明:与对照组相比,1)饲粮添加益生菌显著降低了哺乳第7天免疫球蛋白G(IgG)含量(P<0.05);饲粮添加合生元显著降低了妊娠第45和75天以及哺乳第7天免疫球蛋白M(IgM)含量(P<0.05);饲粮添加抗生素显著降低了妊娠第45、75和105天以及哺乳第7天IgM含量(P<0.05);饲粮添加益生菌和合生元显著提高了哺乳第21天IgM含量(P<0.05)。2)饲粮添加益生菌显著降低了妊娠第105天干扰素-γ(IFN-γ)含量以及哺乳第7天白细胞介素-2(IL-2)、IFN-γ和肿瘤坏死因子-α(TNF-α)含量(P<0.05),显著提高了哺乳第7天白细胞介素-10(IL-10)含量(P<0.05);饲粮添加合生元显著降低了妊娠第45天促炎细胞因子[(IL-2、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、IFN-γ和TNF-α]含量,妊娠第75天IFN-γ和TNF-α含量,妊娠第105天IL-10、IFN-γ和TNF-α含量,哺乳第7天促炎细胞因子(IL-2、IL-8、IFN-γ和TNF-α)含量以及哺乳第21天IL-8含量(P<0.05);饲粮添加抗生素显著降低了各时间点IL-8含量,妊娠第45和75天及哺乳第7和21天IFN-γ含量以及妊娠第45、75和105天TNF-α含量(P<0.05)。3)饲粮添加益生菌和合生元显著提高了哺乳第7和21天过氧化氢(H2O2)含量,以及哺乳第21天还原型谷胱甘肽(GSH)含量(P<0.05)。综上所述,饲粮添加益生菌和合生元可降低妊娠-哺乳期母猪血浆促炎细胞因子含量,增强妊娠至哺乳阶段母猪体液免疫功能,改善哺乳后期母猪抗氧化能力,这有利于母猪的机体健康。

西藏小型猪颈动脉粥样硬化模型的建立及评价研究

目的采用高脂饮食联合左侧颈动脉球囊损伤术建立西藏小型猪颈动脉粥样硬化(carotid atherosclerosis,CAS)模型,观察西藏小型猪CAS模型的血液生化、影像学及病理学特点。方法10只雄性西藏小型猪随机分为两组,正常组饲喂正常饲料,模型组持续饲喂高脂饲料且2周后进行左侧颈动脉球囊损伤术,造模周期为12周。分别在高脂饲喂前(-2周)、球囊损伤(0周)及术后2周、6周、10周检测其体重和血液生化等指标,并在术后10周进行超声、血流多普勒、血管造影等影像学观测。动物安乐死后取左侧颈动脉进行HE、Masson和油红“O”染色,观察其形态、炎症、纤维化和脂质沉积等情况,分析西藏小型猪的病变特点。结果西藏小型猪CAS模型的多项血液生化指标与临床相似;影像学结果显示CAS模型左颈动脉血管显著狭窄且阻力增大,导致血流速度减小;病理观察有明显炎症细胞浸润、胶原纤维增生、血管壁增厚和脂质沉积等,与临床表现近似。结论西藏小型猪CAS模型与临床特点相似,模型易感且稳定,是适用于研究CAS的良好的理想模型。

脂肪间充质干细胞与甲泼尼龙联合用药对小型猪异体皮肤移植的影响

旨在探究脂肪间充质干细胞(adipose derived mesenchymal stem cells,ADSCs)与甲泼尼龙(methylprednisolone,MP)联合用药对小型猪异体皮肤移植(allogeneic skin graft,ASG)所产生的免疫排斥反应与创口愈合的影响。对12头小型猪建立同种异体皮肤移植模型,根据术后对异体皮肤移植皮片不同的干预方式,将小型猪随机分为4组:异体皮肤移植组(ASG)、甲泼尼龙组(MP)、脂肪间充质干细胞组(ADSCs)、脂肪间充质干细胞与甲泼尼龙联合用药组(ADSCs+MP)。术前与术后7、14 d采集血液和组织样本,术后即刻及术后7、14、21 d拍照记录移植表皮临床变化情况。通过对移植皮片表观检查、病理组织学检测,血常规以及血清生化,移植皮片组织氧化应激、炎症、再生的指标检测,综合评价4组不同干预方式对小型猪异体皮肤移植所产生的免疫排斥反应与创口愈合方面的影响。从皮片表观与病理学分析可知,ASG组移植皮片存活时间在7 d左右、MP组移植皮片存活时间在7~14 d、ADSCs组与ADSCs+MP组存活时间为14~21 d。并且在7、14 d时ADSCs+MP组的GSH、SOD的表达量显著升高,在免疫排斥方面,ADSCs+MP组的CD4^(+)T细胞的数量,NF-κB的蛋白与基因表达量,IL-2、IFN-γ、TNF-α、IL-1β、IL-6促炎因子的表达量相对于ASG组显著降低。在创口愈合方面,ADSCs+MP组相对于ASG组VEGF、TGF-β的蛋白表达,SDF-1、CXCR4的表达显著上升。并且ADSCs+MP组相对于ASG组,PI3K、AKT、11β-HSD1的蛋白与基因的表达量显著降低。综上所述:ADSCs+MP组相对于ASG组与MP组,可以延长移植皮片存活时间。并且ADSCs+MP可以有效通过促进GSH、SOD的表达抑制氧化应激。在排异方面,ADSCs+MP可以通过抑制NF-κB的表达抑制IL-2、IFN-γ、IL-6、TNF-α等炎性因子的表达,从而抑制移植过后的免疫排斥反应。并且ADSCs+MP可以通过促进VEGF、TGF-β等愈合因子的表达促进创口愈合。进一步研究发现ADSCs可能通过PI3K/AKT来影响11β-HSD1的表达,进而抑制皮肤皮质醇含量,并促进TGF-β、VEGF等生长因子的表达来促进创口肉芽组织再生与血管迁移。

猪DAZL启动子克隆、转录表达及蛋白功能特性分析

【目的】研究猪类无精症缺失基因DAZL在版纳微型猪近交系(Banna mini-pig inbred line,BMI)中的启动子特征、转录表达及蛋白功能。【方法】使用PCR技术克隆DAZL启动子序列,并分析其结构;使用RNA测序技术获得BMI睾丸组织中DAZL的表达量,并构建转录调控网络;分析DAZL的氨基酸同源性与蛋白功能特性,并构建互作蛋白网络。【结果】成功扩增了DAZL长度为2378 bp的启动子序列,含1个CpG岛、3个核心启动子以及Oct-1、Sp1、Pit-1a等10种转录因子结合位点;DAZL在BMI睾丸组织中有较高的正表达;ceRNA调控网络分析表明DAZL被sscmiR-199a-5p、ssc-miR-199b-5p、ssc-miR-17-5p、ssc-miR-103、ssc-miR-24-3p和ssc-miR-107等6个miRNAs靶向调控;功能注释发现其主要在精子发生、生殖细胞发育、翻译调控中发挥重要功能。DAZL及其邻近基因分析发现其在各物种中高度保守,且具有较为保守的邻近基因;蛋白互作网络、GO和KEGG富集分析显示DAZL与PUM2、DAZAP2和SYCE1等多个蛋白互作,主要参与精子发生、卵子发生、有性生殖等重要生殖相关通路;转录组数据与蛋白编码基因之间的表达量相关性显示DAZL的表达量与SOHLH1显著相关。【结论】本研究从DNA、RNA和蛋白质角度全面分析了猪DAZL的启动子结构、转录表达和蛋白功能,结果为更进一步研究DAZL在精子发生中的作用奠定了基础。

一种微创制备巴马小型猪急性心肌梗塞模型的技术及评价方法

目的采用微创冠状动脉内球囊封堵法制备巴马小型猪急性心肌梗塞(acute myocardial infarction,AMI)模型,并采用血液学、功能学与病理学方法综合评价模型建立情况。方法巴马小型猪12只采集基线数据后,全麻下行股动脉穿刺,利用经皮冠状动脉内球囊封堵法阻断左前降支(第一对角支下2~5 mm)血流90 min,术中行动态心电图监测,并于术后定期采血测定心肌损伤标志物,同时行超声心动图检查评估心功能,术后第28天取材后通过大体观察、Masson染色及天狼星红染色评估心肌梗塞面积与程度。结果12只小型猪中有10只完成封堵,另2只因术中频发恶性心律失常未达到封堵时间。在完成封堵的动物中有7只存活至28 d,其余3只在实验终点前意外死亡。在造模成功个体中观察到封堵后心电图ST-T呈动态变化,术后4 h心肌损伤标志物较术前升高最明显,差异有统计学意义(P<0.01)。术后第7天超声心动图即示左室射血分数(LVEF)、左室短轴缩短率(LVFS)、左室舒张末期容积(LVEDV)与左室收缩末期容积(LVESV)较术前相比有显著性改变(P<0.05)。心脏大体观察示梗塞心肌呈灰白色,主要位于左前壁;病理染色示梗塞区累及心室壁全层。结论本研究可成功建立巴马小型猪急性心肌梗塞模型,并对模型有效性做出综合评价。

精氨酸对妊娠环江香猪胎儿生长发育的影响

【目的】研究精氨酸对妊娠环江香猪胎儿生长发育的影响。【方法】试验选取体重35—40 kg的环江后备母猪10头,单栏饲养,每栏1头。配种15 d后,随机分为2组,每组5头。试验组在基础日粮中添加0.83%L-精氨酸,对照组添加1.71%L-丙氨酸(等氮对照)。配种第45天,称取母猪体重,颈动脉放血处死母猪,收集血液,肝素抗凝,离心分离血浆;解剖母猪,记录胎儿数量、重量和存活率,采集羊水、尿囊液和胎盘,-20℃保存,用于实验室分析。【结果】结果表明,精氨酸组与丙氨酸组相比,胎儿平均个体重和黄体数分别提高14.4%(P<0.05)和6.0%(P>0.05),胎盘粗脂肪含量降低14.7%(P<0.05),羊水和尿囊液中精氨酸的浓度分别提高65.0%和79.1%(P<0.05),尿囊液中赖氨酸浓度降低28.3%(P<0.05);血浆和尿囊液中NO的含量分别提高22.9%和66.3%(P<0.05),胎盘中总NO合酶、诱导型NO合酶和结构型NO合酶的活性分别提高75.5%、61.7%和84.5%(P<0.05)。【结论】提示精氨酸通过促进胎盘一氧化氮的生成促进妊娠母猪早期胎儿的生长发育。