Whole-genome sequencing study to identify candidate markers indicating susceptibility to type 2 diabetes in Bama miniature pigs

Background:Hundreds of single-nucleotide polymorphism(SNP)sites have been found to be potential genetic markers of type 2 diabetes mellitus(T2DM).However,SNPs related to T2DM in minipigs have been less reported.This study aimed to screen the T2DM-susceptible candidate SNP loci in Bama minipigs so as to improve the success rate of the minipig T2DM model.Methods:The genomic DNAs of three Bama minipigs with T2DM,six sibling lowsusceptibility minipigs with T2DM,and three normal control minipigs were compared by whole-genome sequencing.The T2DM Bama minipig-specific loci were obtained,and their functions were annotated.Meanwhile,the Biomart software was used to perform homology alignment with T2DM-related loci obtained from the human genome-wide association study to screen candidate SNP markers for T2DM in Bama miniature pigs.Results:Whole-genome resequencing detected 6960 specific loci in the minipigs with T2DM,and 13 loci corresponding to 9 diabetes-related genes were selected.Further,a set of 122 specific loci in 69 orthologous genes of human T2DM candidate genes were obtained in the pigs.Collectively,a batch of T2DM-susceptible candidate SNP markers in Bama minipigs,covering 16 genes and 135 loci,was established.Conclusions:Whole-genome sequencing and comparative genomics analysis of the orthologous genes in pigs that corresponded to the human T2DM-related variant loci successfully screened out T2DM-susceptible candidate markers in Bama miniature pigs.Using these loci to predict the susceptibility of the pigs before constructing an animal model of T2DM may help to establish an ideal animal model.

Single-cell profiling of the pig cecum at various developmental stages

The gastrointestinal tract is essential for food digestion,nutrient absorption,waste elimination,and microbial defense.Single-cell transcriptome profiling of the intestinal tract has greatly enriched our understanding of cellular diversity,functional heterogeneity,and their importance in intestinal tract development and disease.Although such profiling has been extensively conducted in humans and mice,the single-cell gene expression landscape of the pig cecum remains unexplored.Here,single-cell RNA sequencing was performed on 45572 cells obtained from seven cecal samples in pigs at four different developmental stages(days(D)30,42,150,and 730).Analysis revealed 12 major cell types and 38 subtypes,as well as their distinctive genes,transcription factors,and regulons,many of which were conserved in humans.An increase in the relative proportions of CD8^(+)T and Granzyme A(low expression)natural killer T cells(GZMA^(low)NKT)cells and a decrease in the relative proportions of epithelial stem cells,Tregs,RHEX^(+)T cells,and plasmacytoid dendritic cells(pDCs)were noted across the developmental stages.Moreover,the post-weaning period exhibited an up-regulation in mitochondrial genes,COX2 and ND2,as well as genes involved in immune activation in multiple cell types.Cell-cell crosstalk analysis indicated that IBP6^(+)fibroblasts were the main signal senders at D30,whereas IBP6^(−)fibroblasts assumed this role at the other stages.NKT cells established interactions with epithelial cells and IBP6^(+)fibroblasts in the D730 cecum through mediation of GZMA-F2RL1/F2RL2 pairs.This study provides valuable insights into cellular heterogeneity and function in the pig cecum at different development stages.

Improved methodology for efficient establishment of the myocardial ischemia-reperfusion model in pigs through the median thoracic incision

To investigate the feasibility and effectiveness of establishing porcine ischemia-reperfusion models by ligating the left anterior descending(LAD)coronary artery,we first randomly divided 16 male Bama pigs into a sham group and a model group.After anesthesia,we separated the arteries and veins.Subsequently,we rapidly located the LAD coronary artery at the beginning of its first diagonal branch through a mid-chest incision.Then,we loosened and released the ligation line after five minutes of pre-occlusion.Finally,we ligated the LAD coronary artery in situ two minutes later and loosened the ligature 60 min after ischemia.Compared with the sham group,electrocardiogram showed multiple continuous lead ST-segment elevations,and ultrasound cardiogram showed significantly lower ejection fraction and left ventricular fractional shortening at one hour and seven days post-operation in the model group.Twenty-four hours after the operation,cardiac troponin T and creatine kinase-MB isoenzyme levels significantly increased in the model group,compared with the sham group.Hematoxylin and eosin staining showed the presence of many inflammatory cells infiltrating the interstitium of the myocardium in the model group but not in the sham group.Masson staining revealed a significant increase in infarct size in the ischemia/reperfusion group.All eight pigs in the model group recovered with normal sinus heart rates,and the survival rate was 100%.In conclusion,the method can provide an accurate and stable large animal model for preclinical research on ischemia/reperfusion with a high success rate and homogeneity of the myocardial infarction area.

Genomic and transcriptomic analyses reveal selection of genes for puberty in Bama Xiang pigs

The Bama Xiang pig （BMX） Chinese indigenous breed is a famous early-maturing with a two-end black coat To uncover the genetic basis of the BMX phenotype, we conducted comparative genomic analyses between BMX and East Asian wild boars and Laiwu pigs, respectively. Genes under positive selection were enriched in pathways associated with gonadal hormone and melanin synthesis, consistent with the phenotypic changes observed during development in BMX pigs. We also performed differentially expressed gene analysis based on RNA-seq data from pituitary tissues of BMX and Large White pigs. The CTTNBP2NL, FRS2, KANK4, and KATNAL1 genes were under selection and exhibited expressional changes in the pituitary tissue, which may affect BMX pig puberty. Our study demonstrated the positive selection of early maturity in the development of BMX pigs and advances our knowledge on the role of regulatory elements in puberty evolution in pigs.

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity ofgene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 （TLR4） in Bama miniature pigs （Sus scrofa domestica） following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a non- classical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction （qRT-PCR）. The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 （RPL4） and TATA box-binding protein （TBP） were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear ceils （PBMCs） during heat shock. The mRNA level of the TLR4 gene （both classical and spliced） in stressed pigs increased significantly （P〈0.05）. Further, the expression levels of the alternative spliced variant of TLR4 （TLR4-ASV） showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

Nucleus Transfer Efficiency of Ear Fibroblast Cells Isolated from Bama Miniature Pigs at Various Ages

Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a pan- city of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer （NT）. The present study examined the NT efficiency of ear fibroblasts from fetal, new- born, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells ini- tially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were ＂S-shaped＂ in different age groups. The cell proliferation of postnatal ear fi- broblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts （P〈0.05 or P〈0.01）. Two-month- and 4-month-old ear fibroblasts had a sig- nificantly higher proportion of G1 stage cells （85% to 91%） than those at 6 and 12 months （66% to 74%, P〈0.01）. The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group （8.06%）. It was concluded that 〈4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

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.

基于转录组测序的巴马香猪和杜洛克猪骨骼肌差异lncRNA分析

【目的】通过对巴马香猪和杜洛克猪背最长肌组织进行转录组测序鉴定差异表达lncRNA,筛选与骨骼肌发育形成相关的候选基因,为明确lncRNA对骨骼肌生长发育的调控机制提供理论基础。【方法】采集12月龄的巴马香猪和杜洛克猪背最长肌组织进行转录组测序,以错误发现率(FDR)<0.05且|log2Fold Change|>1为标准筛选差异表达基因(DEGs)和差异表达lncRNA,并进行实时荧光定量PCR验证,预测差异表达lncRNA靶基因并进行GO功能注释和KEGG信号通路富集分析,选取表达差异最大的lncRNA构建其与靶基因互作网络。【结果】在巴马香猪和杜洛克猪背肌间共鉴定出6316个DEGs和675个差异表达lncRNA;GO功能注释分析结果表明,差异表达lncRNA靶基因主要涉及代谢过程、肌肉组织发育及骨骼肌细胞增殖和分化等生物过程;KEGG信号通路富集分析结果表明,差异表达lncRNA靶基因在Hippo和Wnt信号通路显著富集(P<0.05),说明差异表达lncRNA与骨骼肌细胞的增殖、自噬和分化相关;对表达差异最大的lncRNA-MSTRG.16703进行实时荧光定量PCR验证,发现其在巴马香猪中的相对表达量极显著高于杜洛克猪(P<0.01),与转录组测序结果一致。lncRNA-MSTRG.16703与靶基因互作网络分析结果表明,上调靶基因包括QKI、MBNL1和YBX2,QKI和MBNL1在均与可变剪接相关。【结论】在巴马香猪和杜洛克猪间发现的差异表达lncRNA是调控骨骼肌发育的候选基因,其靶基因主要富集在Hippo和Wnt等骨骼肌发育相关信号通路。lncRNA-MSTRG.16703的表达差异最大且在巴马香猪中上调,其靶基因在骨骼肌细胞增殖分化和肌纤维形成中有重要调控作用。

“酶、植物提取物和益生菌”复方制剂对巴马香猪生长性能、血清生化指标和抗氧化指标的影响

探讨“酶、植物提取物和益生菌”复方制剂对巴马香猪生长性能、血清生化指标和抗氧化指标的影响,为“酶、植物提取物和益生菌”复方制剂的科学应用提供参考。试验选取健康状况相近的3月龄巴马香猪60头,平均体重15 kg左右,公母随机,分为对照组、试验1组和试验2组,每组20头巴马香猪,每组4个重复,每个重复5头。其中对照组饲喂基础日粮,试验1组饲喂基础日粮+100 g/kg植物提取物,试验2组饲喂基础日粮+100 g/kg“酶、植物提取物和益生菌”复方制剂,试验为期28 d。测定各组猪只的生长性能、血清生化指标和抗氧化指标。结果表明,“酶、植物提取物和益生菌”复方制剂能提高巴马香猪的生长性能、机体免疫力和抗氧化能力。

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.

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。

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。

1例离断肢体建立异种交叉循环的初步观察

背景异种交叉循环在器官保存领域的应用已取得一定的成果,但其在肢体组织保存方面效果仍未明确。目的探究异种交叉循环术保存断足的效果。方法将因骨肉瘤行截肢手术的断肢再次处理,自踝关节上方约5 cm截断,随后将不含肿瘤的断足主要血管连接至巴马猪股动脉和股静脉,通过趾尖血氧饱和度、趾尖脉率、全血细胞计数和断足肌肉组织形态评估保存效果。结果成功实施了异种交叉循环术,维持断足肌肉组织正常形态超过4 h。趾尖血氧饱和度64%~99%,趾尖脉率45~106/min。白细胞计数(13.3~19.7)×10^(9)/L,淋巴细胞计数(2.1~4.36)×10^(9)/L,血小板计数(242~1534)×10^(9)/L。0 h和4 h断足肌纤维形态正常,8 h断足肌纤维排列紊乱,失去部分组织形态。0 h肌间隙血管未充盈,4 h肌间隙血管有红细胞通过,8 h肌间隙血管内形成血栓。结论异种交叉循环术可维持断足肌肉组织正常形态超过4 h,但如何减少免疫排斥反应仍需进一步研究。

三种动物胰腺组织单细胞悬液制备效果比较

目的比较巴马小型猪、SD大鼠、C57BL/6小鼠三种动物胰腺组织单细胞解离的效率和效果。方法分别将巴马小型猪、SD大鼠、C57BL/6小鼠麻醉后取新鲜胰腺组织,进行单细胞解离制备单细胞悬液。然后采用吖啶橙/碘化丙啶染色分别标记所有细胞核酸及死细胞核酸,应用全自动细胞荧光分析仪采集图像,观察细胞形态,计算细胞平均直径及平均圆度;根据吖啶橙/碘化丙啶染色情况,计算细胞活率、总细胞浓度和活细胞浓度。并对三种动物胰腺组织单细胞悬液各参数进行比较。结果三种动物胰腺组织单细胞悬液细胞形态完整、边界清楚,均有一定程度的结团。SD大鼠胰腺细胞的平均直径最大,C57BL/6小鼠细胞平均圆度最大。巴马小型猪和SD大鼠细胞活率较高,巴马小型猪总细胞浓度和活细胞浓度最高。结论成功制备了巴马小型猪、SD大鼠、C57BL/6小鼠三种动物胰腺组织单细胞悬液,为后续单细胞测序的开展奠定了基础。

高脂高糖饮食对巴马小型猪肠道菌群的影响

目的探讨高脂高糖饮食8个月对巴马小型猪血脂和粪便中肠道菌群的影响。方法将10头巴马小型猪随机分为对照组(n=3)和实验组(n=7),对照组小型猪正常喂养,实验组给予高脂高糖饮食。各组动物分别于实验前及实验8个月后进行空腹采血,检测三酰甘油、总胆固醇、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇等血脂指标。实验8个月后无菌采集各动物粪便,采用16S rRNA测序技术检测肠道菌群组成。结果高脂高糖饮食8个月后,实验组粪便中肠道菌群物种数量未见明显改变,厚壁菌门相对丰度增加,拟杆菌门和变形菌门丰度降低(P<0.05)。实验组小型猪体质量、三酰甘油、总胆固醇、低密度脂蛋白胆固醇水平均显著高于对照组(P<0.05)。结论高脂高糖饮食可以增加巴马小型猪粪便中厚壁菌门相对丰度,降低拟杆菌门和变形菌门相对丰度,提示这些肠道菌群的失调可能参与调节了宿主高脂血症的发生。

微血流成像联合超声造影诊断猪肾创伤伴活动性出血的实验研究

目的探讨微血流成像(MFI)联合超声造影(CEUS)在猪肾创伤伴活动性出血诊断及定位中的应用价值。方法选取健康巴马小型猪10只,建立肾创伤伴活动性出血模型。模型建立成功后进行CEUS及MFI检查,比较MFI联合CEUS与CEUS诊断及定位活动性出血的准确率;应用MFI联合CEUS测量活动性出血受损血管破口内径。结果1只巴马小型猪由于麻醉原因在实验过程中意外死亡,剩余9只巴马小型猪共成功构建18处肾创伤伴活动性出血灶。CEUS与MFI联合CEUS诊断活动性出血的准确率分别为77.78%、94.44%,二者比较差异无统计学意义;CEUS与MFI联合CEUS定位活动性出血的准确率分别为55.56%、94.44%,二者比较差异有统计学意义(P<0.05)。MFI联合CEUS测得活动性出血受损血管破口内径为1.54~2.42 mm,平均(1.94±0.29)mm。结论MFI联合CEUS能清晰显示猪肾创伤灶内受损血管,准确诊断活动性出血,且定位活动性出血的能力优于CEUS。

巴马小型猪ABCD 4基因序列分析、真核表达载体构建及组织表达分析

【目的】获取巴马小型猪ATP结合盒D亚科成员4(ATP binding cassette subfamily D member 4,ABCD4)基因CDS区序列并预测其编码蛋白的结构功能,构建真核表达载体,了解ABCD 4基因在巴马小型猪中的组织分布情况,为探究ABCD 4基因对巴马小型猪生长发育的作用提供理论和分子基础。【方法】以巴马小型猪皮下脂肪组织cDNA为模板,利用RT-PCR对ABCD 4基因CDS区进行扩增、测序,利用生物信息学分析软件对巴马小型猪ABCD 4基因CDS区序列与不同物种比对,构建系统进化树,并对ABCD4蛋白的理化性质、跨膜结构等进行预测。将获得的目的基因连接至pEGFP-N1载体并转染到IPEC-J2细胞,检测转染后荧光和基因表达量变化。通过实时荧光定量PCR检测巴马小型猪各组织ABCD 4基因表达量。【结果】巴马小型猪ABCD 4基因CDS区全长1818 bp,编码605个氨基酸,与Ensembl上猪参考序列(ENSSSCT00000037529)的相似性为99.7%,存在6处碱基突变和1处碱基插入。相似性比对发现,猪和人、原鸡、倭黑猩猩、马、双峰驼、牛ABCD4序列的相似性分别为87.6%、72.7%、87.6%、91.7%、88.1%和88.6%。系统进化树显示,猪与马亲缘关系最近,与原鸡亲缘关系最远。生物信息学分析显示,ABCD4蛋白分子质量为68.66 ku,理论等电点(pI)为7.13,存在5个跨膜螺旋结构,无信号肽,属于跨膜蛋白;存在58个磷酸化位点和10个N-糖基化位点;二级结构中α-螺旋、β-转角、无规则卷曲、延伸链占比分别为50.91%、3.64%、28.43%和17.02%。细胞转染试验结果显示,转染30 h后重组质粒组和空载体对照组均有荧光出现,且转染重组质粒的细胞内ABCD 4基因表达量极显著高于空载体对照组(P<0.01)。实时荧光定量PCR结果显示,ABCD 4基因在巴马小型猪皮下脂肪中表达量最高,显著高于其他组织(P<0.05);在心脏中表达量最低。【结论】本试验成功扩增获得巴马小型猪ABCD 4基因CDS区序列,ABCD4蛋白是一种疏水性跨膜蛋白,主要在巴马小型猪皮下脂肪中表达。本试验结果为探究ABCD 4基因对猪背膘厚的影响提供了分子依据。

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。

舒泰与赛拉嗪联合使用对巴马小型猪的麻醉效果及影响

试验旨在探究巴马小型猪联合使用不同剂量的舒泰和赛拉嗪的麻醉效果以及对生理指标的影响。对6只巴马小型猪间隔使用舒泰0.75 mg/kg+赛拉嗪6 mg/kg (A组)、舒泰0.75 mg/kg+赛拉嗪12 mg/kg (B组)、舒泰1.00 mg/kg+赛拉嗪8 mg/kg (C组)进行麻醉,不同剂量组麻醉间隔5 d以上,观察麻醉效果,并对肛温、呼吸频率、收缩压(SBP)、舒张压(DBP)、平均动脉压(MBP)、心电图(ECG)以及血常规和血液生化指标进行监测。结果显示,各组麻醉成功率均为100%,且麻醉状态良好;A组、B组和C组的麻醉诱导时间分别为5.17、4.17、4.67 min,差异不显著(P>0.05);麻醉维持时间分别为49.33、93.50、79.17 min,A组显著低于B组和C组(P<0.05);麻醉恢复时间分别为2.17、2.67、4.67 min,C组显著高于A组和B组(P<0.05);麻醉后各组肛温、呼吸频率、SBP、DBP、MBP在整体上呈降低趋势,且B组和C组的变化更为明显;麻醉后各组Q-T间期首先出现延长,之后逐渐波及QRS波群和P-R间期;麻醉后约24 h,各组猪血常规指标未发生明显改变,而麻醉后A组猪血清尿素氮(BUN)含量以及B组与C组猪血清谷草转氨酶(AST)活性、BUN含量显著高于麻醉前(P<0.05)。研究表明,各剂量组巴马小型猪均快速进入安全且稳定的麻醉状态,且通过调整麻醉剂用量可实现对动物麻醉时长和生理指标影响程度的控制。

不同来源猪皮与重建人类表皮模型对面膜中咖啡因透过性比较研究

目的探索评估面膜中咖啡因经皮透过性最适合的皮肤模型。方法采用两种离体小型猪皮肤(巴马小型猪和小型家猪)和重建人类表皮为皮肤模型材料,应用Franz扩散池法测定4个处方面膜(面膜1不加渗透剂,面膜2、3和4促渗剂用量分别为0.5%、1.0%和2.0%)中咖啡因的累积透过量,以HPLC法测定接收液中不同时间点咖啡因的浓度。结果面膜1中咖啡因在两种小型猪皮肤模型中120 min累积透过百分率和咖啡因在人体的实际透过率是基本一致的。面膜2中咖啡因在重建人类表皮模型中的时间-累积透过量、稳态流量(J_(SS))和渗透系数(K_(p))均显著高于两种离体小型猪皮肤模型。巴马小型猪皮肤模型对面膜2、3和4中咖啡因的时间-累积透过量具有显著区分力,但小型家猪皮肤模型对面膜3和4中咖啡因的时间-累积透过量差异无统计学意义。结论巴马小型猪皮肤是最适合于评估面膜中咖啡因皮肤透过性研究的模型。