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.

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.

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.

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.

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.

母猪饲粮添加益生菌和合生元对子代巴马香猪肌肉氨基酸组成及生长发育相关基因表达的影响

[目的]本文旨在研究母猪饲粮添加益生菌和合生元对子代巴马香猪肌肉氨基酸组成及相关基因表达的影响。[方法]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),每组16头母猪,单栏饲养。母猪在整个妊娠期和哺乳期饲喂相应试验饲粮;仔猪断奶后,每窝选取2头仔猪,每组32头(8栏,每栏4头),饲喂基础饲粮。分别于65、95和125日龄每组选取8头采集股二头肌和腰大肌样品,测定其水解氨基酸组成和相关基因的表达量。[结果]与对照组相比,合生元组子代巴马香猪股二头肌粗蛋白、Gly、Ser、Ala、Asp、Glu、Pro和Ile含量,腰大肌Gly、Glu、Ser和His含量均显著增加(P<0.05);益生菌组子代巴马香猪股二头肌肌肉萎缩F-box蛋白32和生肌调节因子(MyoG)、腰大肌生肌因子5(Myf5)的表达水平显著上调(P<0.05),腰大肌肌球蛋白重链Ⅱb(MyHCⅡb)、股二头肌和腰大肌肌肉生长抑制素(MSTN)的表达水平显著下调(P<0.05);合生元组子代巴马香猪股二头肌Myf6、生肌分化因子和MyoG以及腰大肌Myf5和MyoG的表达水平显著上调(P<0.05),股二头肌MSTN的表达水平显著下调(P<0.05);益生菌组和合生元组子代巴马香猪股二头肌MyHCⅡx的表达水平显著下调(P<0.05);抗生素组子代巴马香猪腰大肌MyHCⅡx的表达水平显著上调(P<0.05),而股二头肌MyHCⅠ和腰大肌MyHCⅡa的表达水平显著下调(P<0.05)。[结论]母猪饲粮添加益生菌和合生元可改变子代巴马香猪肌肉氨基酸组成,调控生肌因子相关基因表达,上调氧化型肌纤维基因的表达、下调酵解型肌纤维基因的表达,从而有利于肉品质的改善。

巴马小型猪仰卧位的新型固定方法

目的 研究广西巴马小型猪仰卧位的固定方法以便于动物实验的开展。方法 选择行胸腔器官获取的广西巴马小型猪12头,接受全身麻醉后采用专用的固定装置进行仰卧位固定。结果 12头广西巴马小型猪在手术中体位得到很好的保持,未发生侧翻、移位等现象。结论 采用专用的固定装置对广西巴马小型猪进行仰卧位固定是一种简单有效的方法。

基于16S rRNA测序分析敲除基因ZBED6后巴马猪肠道微生物菌群的变化

旨在探究在巴马猪中ZBED6基因敲除之后肠道菌群的变化和肌肉表型的关联。本研究以5月龄野生型和ZBED6敲除型广西巴马小型猪为研究对象,按性别和基因型分成3组进行比较,分别是野生型公猪比野生型母猪(公WT:母WT)、野生型母猪比敲除型母猪(母WT:母KO)、野生型公猪比敲除型公猪(公WT:公KO),利用16S rRNA基因的高通量测序技术分析各肠段微生物组成。结果表明:1)在巴马猪中,大肠的菌群多样性显著高于小肠;2)与雌性野生型猪相比,雄性野生型猪瘦肉率更高,回肠苏黎世杆菌属、盲肠链球菌属、直肠消化链球菌属在雄性野生型猪中显著富集,回肠乳杆菌属在雌性野生型猪中显著富集;3)敲除ZBED6之后,猪的肌肉生长增加。肠道菌群组成显示,母猪的回肠中乳杆菌含量显著下降,盲肠中SMB53的含量显著上升。敲除ZBED6后,公猪直肠中普雷沃菌属和瘤胃球菌属的含量显著上升,盲肠中SMB53的含量显著上升。结果提示,苏黎世杆菌属、链球菌属、消化链球菌属和乳杆菌属在肠道内的差异可能会影响能量代谢从而促进雄性野生型猪的肌肉生长;敲除了ZBED6之后,猪的盲肠中SMB53丰度的显著增加可能是导致肌肉增多的一个因素。

含血必净注射液的供心保存灌注液对ECMO支持下离体空跳猪心心肌保护作用观察

目的观察含血必净注射液的供心保存灌注液对体外膜肺氧合(ECMO)支持下离体空跳猪心的心肌保护作用,并探讨其可能的机制。方法选取健康成年广西巴马小型猪12只,随机分成血必净组和对照组,每组6只。血必净组和对照组均采用ECMO支持猪离体空跳心脏,分别用含50 mL血必净注射液、生理盐水的去白细胞氧合血连续灌注,维持心脏空跳8 h。灌注0 h(T1)、2 h(T2)、4 h(T3)、6 h(T4)、8 h(T5),记录两组心率、灌注压、灌注流量,采用ELISA法检测各时间点灌注液炎症相关指标白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平。使用全自动生化分析仪检测两组全身肝素化后且灌注前(T0)及T1~T5时间点灌注液心肌损伤标志物cTnT(cTnT)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)水平。透射电镜下观察两组T5时间点的心肌组织超微结构。结果两组T1~T5时间点心率、灌注压、灌注流量比较差异均无统计学意义(P均>0.05)。与同组T1时间点比较,对照组T3~T5时间点灌注液IL-1β、IL-6、TNF-α、cTnT、CK、CK-MB、LDH水平均升高,血必净组T4~T5时间点灌注液cTnT、CK、CK-MB、LDH水平均升高(P均<0.05);血必净组T3~T5时间点灌注液IL-1β、IL-6、TNF-α、cTnT水平均低于对照组,T2~T5时间点灌注液CK、CK-MB、LDH水平均低于对照组(P均<0.05)。心肌组织超微结构:对照组心肌组织中重度退行性变、脂肪变,细胞内基质大面积溶解,肌纤维结构排列紊乱、大量断裂,线粒体大多肿胀严重、大小不均,细胞膜溶解、破损;与对照组比较,血必净组细胞结构损伤相对较轻,心肌细胞轻度肥大,线粒体轻度肿胀、大小均一,肌纤维粗细均匀、排列整齐。结论含血必净注射液的供心保存灌注液对ECMO支持下离体空跳猪心具有心肌保护作用,其机制可能与抑制炎症反应、减轻心肌细胞线粒体损伤有关。

巴马小型猪肝移植无肝期动物模型的建立

目的构建重复性和稳定性好的巴马小型猪肝移植无肝期动物模型。方法选用5只巴马小型猪,构建肝移植无肝期模型,为避免出现无肝期肠道淤血、循环衰竭,采用静脉-静脉转流模型,维持无肝期血压稳定以及加强术中麻醉与体液管理。观察并记录手术时间、出血量、补液量、转流流量及实验动物在血管阻断前、血管阻断时、静脉-静脉转流(venovenous bypass,VVB)开始后30 min、60 min、90 min和血管开放后30 min共6个时间点的血流动力学、肠损伤指标、肾损伤指标及内环境指标的变化情况。结果构建的小型猪肝移植无肝期静脉-静脉转流动物模型,静脉引流均较充分,主要表现为5只巴马小型猪均存活,且术中肠管颜色红润、蠕动正常,尿量正常。手术时间为(160±8)min,出血量约(158±32)ml,补液量为(1760±251)ml。与血管阻断前相比,血管阻断时动物心率明显加快,平均动脉压(mean arterial pressure,MAP)明显降低至(44±15)mmHg,血液pH值下降、乳酸升高。转流开始后巴马小型猪MAP上升,心率变慢,而血液pH值、乳酸变化不明显。整个实验期间,实验动物电解质变化不明显,而肌酐、肠脂肪酸结合蛋白缓慢升高。结论构建的巴马小型猪肝移植无肝期动物模型稳定且安全,能为进一步的静脉-静脉转流装置的验证等前临床实验提供有效的动物模型。

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。

舒泰复合右美托咪定两种给药方案对巴马小型猪麻醉效果观察

【目的】探究不同舒泰复合右美托咪定给药方案对巴马小型猪麻醉效果的影响。【方法】将8头巴马小型猪随机均分为静脉注射(Ⅳ)组和肌内注射(IM)组,麻醉间隔期为14 d。Ⅳ组耳缘静脉注射舒泰3.3 mg/kg+右美托咪定8μg/kg;IM组耳后颈部肌内注射舒泰4.6 mg/kg+右美托咪定11.2μg/kg。在麻醉过程中对巴马小型猪的麻醉时期、麻醉效果、生理指标及生物反射进行监测。【结果】Ⅳ组麻醉诱导时间、维持时间、苏醒时间分别为3.75、85.75和30.25 min。IM组麻醉诱导时间、维持时间、苏醒时间分别为7.13、107.00和41.00 min。Ⅳ和IM组分别可维持50和65 min外科麻醉期。IM组80~110 min时麻醉效果评分显著高于Ⅳ组(P<0.05)。麻醉过程中两种方案的体温(T)下降,心率(HR)和血氧饱和度(SpO_(2))先降低后升高,呼吸频率(RR)、收缩压(SBP)、舒展压(DBP)和平均动脉压(MAP)先升高后降低。IM组50~80 min时RR显著高于Ⅳ组(P<0.05)。IM组10~15 min时SBP显著低于Ⅳ组(P<0.05)。Ⅳ和IM组生物反射完全抑制时间分别为45和50 min。【结论】舒泰复合右美托咪定对巴马小型猪麻醉效果良好,诱导迅速,维持时间长,苏醒平稳,对巴马小型猪生理指标和生物反射影响较小,其中IM组麻醉方案比Ⅳ组更适合临床外科手术和科研教学使用。

智能针灸机器人针刺可行性及安全性评价实验动物平台建立的探索

目的本研究旨在探索智能针灸机器人针刺可行性及安全性评价的实验动物平台,为后续进一步开展研究奠定基础。方法将6只2月龄广西巴马小型猪作为实验对象,进行麻醉后针刺验证。首先进行人工针刺验证,每只实验动物选取6个穴位,行提插捻转手法后留针20 min,并进行自身前后对照。实验共进行28 d,每2 d进行1次实验,共10次。人工针刺验证后在已选6个穴位的基础上每个穴旁10 mm处取一点,共12点,使用针灸机器人智能针刺模块对实验动物进行不同频率及角度的针刺操作进一步验证动物平台的稳定性及可行性。结果血常规中安全性相关指标均无异常,较自身前后对比稳定。血生化指标无异常,较自身前后对比稳定。在实验全程监测中动物的平均心率为每分钟124次。动物的平均血压为87/36 mmHg。巴马小型猪在室温25℃下的平均体温为36℃。体温在实验过程中及实验前后均无明显的变化。在搭建的实验平台基础上,针灸机器人智能针刺模块针刺手法操作预实验均顺利完成,实验过程中未出现针刺相关异常情况如弯针、断针、滞针等,实验动物未出现明显异常。结论本研究借鉴了已有的小型猪实验方法,初步建立起了稳定的智能针灸机器人针刺可行性及安全性评价实验动物平台,为进一步智能针灸机器人的相关研究开展奠定基础。

巴马小型猪血虚证模型的建立

目的采用乙酰苯肼(acetylphenylhydrazine,APH)联合环磷酰胺(cyclophosphamide,CTX)建立巴马小型猪血虚证模型。方法12只巴马小型猪随机分为模型组和空白组,每组6只,常规喂养7 d后,空白组不做特殊处理,模型组采用实验第1天、第4天皮下注射APH 50 mg/kg,第5~8天腹腔注射CTX 20 mg/kg的方法建立巴马小型猪血虚证模型,观察两组巴马小型猪一般体征,测定红细胞计数(red blood cell count,RBC)、平均红细胞血红蛋白(mean corpuscular hemoglobin,MCH)、红细胞比容(hematocrit,HCT)、血红蛋白(hemoglobin,HGB)、白细胞计数(white blood cell count,WBC)、单核细胞数量(number of monocytes,MON)、淋巴细胞数量(lymphocyte number,LYM)、中性粒细胞数量(number of neutrophil,NEU)以评价模型是否建立成功。结果模型组巴马小型猪出现精神萎靡倦怠,鼻唇、四肢等部位淡白失泽,被毛蓬乱、干燥粗糙等一系列血虚症状,与空白组比较,模型组RBC、HCT、HGB、WBC、MON、LYM均降低,差异有统计学意义(P<0.05)。结论第1天、第4天皮下注射APH 50 mg/kg,第5~8天腹腔注射环磷酰胺CTX 20 mg/kg的方法可以成功建立巴马小型猪血虚证模型。

中华眼镜蛇毒致广西巴马小型猪坏死组织渗出液中的蛇毒成分分析

目的:探究中华眼镜蛇毒致广西巴马小型猪坏死组织渗出液中的蛇毒成分及其蛋白表达的动态变化。方法:给予广西巴马小型猪注射中华眼镜蛇毒,构建蛇伤中毒的局部组织坏死模型,在注射毒素后的6 h、12 h、24 h、36 h和48 h取小猪坏死组织的渗出液,采用无标记的蛋白质组学技术鉴定分析渗出液中蛇毒蛋白质组成及动态变化,通过对所鉴定出的蛋白进行KEGG及GO通路富集分析,以深入了解蛇毒蛋白相关的生物学功能。结果:通过蛇伤中毒后广西巴马小型猪的生物学行为、局部肌肉组织的病理结果、注射部位的伤口变化来评判动物模型,广西巴马小型猪中毒后出现呼吸急促、肌肉组织坏死、局部伤口溃烂等症状,均符合临床特征的实际情况,表明成功构建了中华眼镜蛇毒导致局部组织坏死的广西巴马小型猪模型。在广西巴马小型猪的局部组织坏死渗出液中鉴定到40种蛇毒蛋白质,涵盖三指毒素、磷脂酶A2、富含半胱氨酸分泌蛋白、蛇毒金属蛋白酶、核苷酸焦磷酸/磷酸二酯酶等蛇毒蛋白家族,并呈现出动态性数量变化。结论:在中华眼镜蛇毒致广西巴马小型猪过程中,不同时间段的组织渗出液中均鉴定出多种三指毒素、磷脂酶A2等蛇毒蛋白,其可能在局部组织坏死中发挥着重要作用。

广西巴马小型猪21个微卫星座位的DNA多态性分析

利用 2 1个微卫星座位统计了广西巴马小型猪品系内的等位基因组成 ,计算了各位点的基因纯合率和多态性信息含量 (PIC)及平均杂合度。结果 ,2 1个位点的平均基因纯合率为 5 5 .5 % ,平均PIC为 0 .3 5 0 1,平均杂合度为 0 .3 881。结果提示 ,广西巴马小型猪具有一定的遗传稳定性 ,已成为一个稳定的遗传群体 。