Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

滇南小耳猪肌源性因子5(MYF5)基因多态性研究

肌源性因子5(myogenic factor 5,MYF5)被认为是猪肌肉生长发育的重要基因,为探究MYF5基因的遗传变异对滇南小耳猪生长性状的影响,以期为优质滇南小耳猪肉新品种(系)培育提供有效的分子遗传标记,本试验以MYF5为候选基因,杜洛克猪(n=40)为对照组,筛选滇南小耳猪(n=18)的优势SNP位点,并对筛选的优势SNP与其主要胴体及肉质性状进行关联分析。结果显示,在滇南小耳猪MYF5基因检出的16个SNP位点中,1个位于编码区,为同义突变,并筛选到4个优势SNP。关联分析结果表明,滇南小耳猪MYF5基因1486、1574、1629以及1701位点间突变频率一致且有相近的遗传图距,连锁紧密。A1486G位点分别为GG、AG、AA基因型;G1574A位点分别为AA、GA、GG基因型;C1629T位点分别为TT、CT、CC基因型;T1701C位点分别为CC、TC、TT基因型。就肌内脂肪含量而言,MM基因型极显著高于MN基因型(P<0.01),MM基因型显著高于NN基因型(P<0.05),NN基因型显著高于MN基因型(P<0.05)。就大理石纹评分而言,MM基因型极显著高于NN基因型(P<0.01),MM基因型极显著高于MN基因型(P<0.01),MN基因型极显著高于NN基因型(P<0.01)。因此,本研究可基本推断在有关作用于滇南小耳猪种肉质性状的基因中,MYF5基因较为关键。该试验结论可为滇南小耳猪育种中改善肉品质等性状给予分子标记支撑。

Cloning and Sequence Analysis of Adiponectin Receptor 1 and Receptor 2 cDNA from Guangxi Bama Mini-pig

[ Objective] To clone and analyze the sequence of Adiponectin receptor 1 （ AdipoR1 ） and receptor 2 （AdipoR2） cDNA of Guangxi Bama mini-pig. [Method] The Adiponectin receptors cDNAs were amplified by RT-PCR using skeletal muscle total RNA as template and then ligated into pMD18-T vector after purification. The recombinant pMD18-T vector was transformed into the E. coil DH5α for identification and sequencing. And the results were compared with the cDNA sequence from other species. [Result] The fragments, 1 128 bp and 1 161 bp in size, were amplified by RT-PCR and respectively consistent with the coding sequence of AdipoR1 gene and AdipoR2 gene. The homology analysis showed that the sequences of AdipoR1 gene and AdipoR2 gene were respectively 99.8% and 99.7% homologous to the sequence of domestic pig reported in GenBank with one base and three base missense mutations correspondingly. [ Conclusion] The AdipoR1 gene and AdipoR2. gene were successfully amplified from Guangxi Bama mini-pig, laying the foundation for the further study of the biological function of AdipoR genes and the design of novel drugs with AdipoR as target.

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.

16S rRNA gene sequencing analysis of gut microbiome in a mini-pig diabetes model

Background:Currently,increasing attention is being paid to the important role of intestinal microbiome in diabetes.However,few studies have evaluated the characteristics of gut microbiome in diabetic miniature pigs,despite it being a good model animal for assessing diabetes.Methods:In this study,a mini-pig diabetes model(DM)was established by 9-month high-fat diet(HFD)combined with lowdose streptozotocin,while the animals fed standard chow diet constituted the control group.16S ribosomal RNA(rRNA)gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini-pigs.Results:The results showed that microbial structure in diabetic mini-pigs was altered,reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1,which were positively correlated with diabetes,and decreases in levels of the bac-teria Rikenellaceae,Clostridiales_vadinBB60_group,and Bacteroidales_RF16_group,which were inversely correlated with blood glucose and insulin resistance.Moreover,PICRUSt-predicted pathways related to the glycolysis and Entner-Doudoroff superpathway,enterobactin biosynthesis,and the L-tryptophan biosynthesis were significantly elevated in the DM group.Conclusion:These results reveal the composition and predictive functions of the intestinal microbiome in the mini-pig diabetes model,further verifying the relationship between HFD,gut microbiome,and diabetes,and providing novel insights into the application of the mini-pig diabetes model in gut microbiome research.

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.

浅析鹤庆甸南白族童帽的装饰艺术特征

鹤庆甸南白族童帽种类繁多、内涵丰富、装饰性强,兼具民俗审美和民俗文化,是极具特色的白族服饰之一。文章通过田野调查,对鹤庆甸南地区白族童帽的装饰题材、装饰纹样、装饰造型、装饰色彩等方面进行探究分析,进而挖掘了装饰特征背后蕴含的地域性文化特征,以期推动少数民族文化的保护与创新传承。

刘以敏教授应用《滇南本草》中药物治疗儿童免疫性血小板减少性紫癜初析

《滇南本草》作为区域性医药的一大代表作,对当地传统医药的诊疗影响深远。文章总结云南名老中医刘以敏教授临床应用《滇南本草》中所记载药物,如老鹳草、仙鹤草、山土瓜、兰花参、墨旱莲,治疗儿童免疫性血小板减少性紫癜的相关经验,并附验案一则,分析处方用药,以为传统医药治疗儿童疾患提供参考。

封闭群滇南小耳猪体重和脏器重量的测定及其相关性分析

目的测定5～6月龄滇南小耳猪体重、脏器重量,并对其进行相关分析.方法选用5～6月龄封闭群滇南小耳猪24只,雌雄各半,分别测定体重和17个脏器和腺体重量,计算脏器系数,通过SPSS软件进行脏器系数的性别间比较和脏器重量与体重间的相关性与回归分析.结果封闭群小耳猪脏器重量雌雄间仅肺脏差异有统计学意义(P<0.05);脏器系数雌雄间仅有肺系数和颌下腺系数差异有统计学意义(P<0.05).体重和脏器重量相关分析表明,雄性滇南小耳猪体重与心、脾、胰腺、腮腺有相关性(P<0.05);雌性的体重与心、膀胱有相关性(P<0.05),进一步的回归分析显示,雄性的心、脾、胰腺、腮腺,雌性的心、膀胱与体重呈线性相关(P<0.05).结论封闭群滇南小耳猪主要脏器系数性别间差异小,其体重与某些脏器重量存在一定的线性关系.

滇南小耳猪细胞生成素(MyoG)基因多态性的研究

以猪肌细胞生成素(MyoG)基因序列设计引物,采用PCR产物直接测序技术对48头滇南小耳猪和40头杜洛克的MyoG基因外显子序列进行测定和分析。发现滇南小耳猪MyoG基因有12个多态位点,其中3个位于编码区属于同义突变,有7个突变位点为优势突变。与屠宰测定的结果进行关联分析,结果表明:滇南小耳猪MyoG基因2 041,2 047位点多态性对瘦肉率有极显著影响(P<0.01),对背膘厚和pH24有显著影响(P<0.05);MyoG基因1 655,1 688位点多态性对瘦肉率和pH24有显著影响(P<0.05),对背膘厚有极显著影响(P<0.01);结合前人的研究结果,可以初步推测MyoG基因是影响滇南小耳猪肉质性状的主效基因或是与影响滇南小耳猪胴体、肉质性状的QTL连锁的基因。实验结果可以为猪育种中提高瘦肉率等性状提供分子依据。

淀南地区古近系沙河街组层序地层模式

应用层序地层学原理,通过地震剖面、电测曲线、岩石类型组合等层序地层界面划分标志,将淀南地区古近系沙河街组划分为6个三级层序、17个体系域。除层序Ⅰ缺少低位域外,层序Ⅱ至层序Ⅵ均由低位域、湖侵域和高位域三个体系域构成。在层序地层单元划分和对比的基础上,建立了断层坡折与沉积坡折两种层序地层模式,研究了与岩性油藏分布的关系,认为坡折带附近是岩性圈闭成藏极为有利的部位,坡折带下倾方向是低位扇、地层超覆等油藏发育的有利部位,坡折带上倾方向是地层不整合、滩坝透镜体等油藏发育的有利部位。

云南省剑川县甸南镇白族传统制陶业调查

对云南省剑川县甸南镇海虹上登村白族传统制陶业进行了实地调查,记述了该项传统技艺的原料、工艺流程、产品特点、市场现状和相关的民俗事象.

利用76个微卫星标记分析滇南小耳猪的遗传多样性

滇南小耳猪是云南省优良地方猪种之一，其数量大，分布广。为了阐明其群体遗传变异情况，以对其进一步有效保护和合理利用提供科学依据，本研究采用分布在家猪19条染色体上的76个微卫星标记对该猪群体54个个体进行了群体遗传变异检测。共检测到338个等位基因，每个座位的等位基因数在2～8个之间，有效等位基因数在1．9560～5．71432：间，平均每个座位等位基因数（4．4474±1．2044）个，有效等位基因数（3．5091±0．9197）个，群体平均杂合度及平均多态信息含量分别为0．6917±0．09797和0．6388±0．1142。研究结果表明，滇南小耳猪群体遗传多样性较丰富。

滇南小耳猪电刺激采精及精子功能的检测

采用Lane PulsatorⅣ型直肠电刺激采精法对滇南小耳猪进行采精,采精仪电压参数设为4～5档,电刺激时间1～5 s,在麻醉情况下成功采精。据精液的色泽,明显分为3段:前段清亮(精液量28±12 mL;密度(2.5±1.2)×10~6个·mL^(-1);活力18.2±5.8)%,中段为胶冻絮状液体(26±14 mL;(4.7±3.9)×10~8个·mL^(-1);(69.3±15.4)%)和后段清亮(22±10 mL;(5.8±5.3)×10~6个·mL^(-1);(35.4±12.2)%)。与间隔3 d和14 d重复采精比较,间隔7 d采精的效果较好,中段精液的品质较好。中段精液对超教排卵母猪的人工授精实验中,卵子受精率为81.3%,囊胚率达到71.9%。直肠电刺激采精方法是适用于滇南小耳猪的安全有效的采精技术。

滇南中蜂的周年变化与气候的相关性分析

为研究滇南中蜂的周年状况及气候的相关性,探明各压力因素间与蜜蜂群势的联系,为滇南中蜂保种提供依据,于2018年1-12月每隔15 d用测子框测定90群蜜蜂群势,并以气候仪观察气象变化,分析蜜蜂的幼虫数、蛹数、工蜂量与蜜粉存储量的相关性,并探讨危及蜜蜂健康的各类潜在因素。结果表明:蜂群在冬、春季增殖激烈,全年中群势呈波澜起伏状;全年气候表现为冬春季降雨量少,温度波动较大,夏秋季节炎热湿润;主成分分析表明,温度是构成蜂群变化的最主要的成分(a=0.977);蜂群内部蜜粉存储量与工蜂量(r=0.580)及卵/幼虫量(r=0.386)呈显著的正相关性,与温度(r=-0.248)呈显著的负相关。在周年变化中,滇南中蜂群势受蜂群外部因素和蜂群内部因素影响,外部因素具有季节性,内部因素由蜂群管理方式决定。

滇南小耳猪血液生理生化部分指标测定

目的测定正常3～6月龄和8～12月龄封闭群滇南小耳猪血液生理与生化各项指标.方法常规方法检测滇南小耳猪18项血液生理指标、30项血液生化指标,统计各指标间的性别差异及两年龄段同类指标的对比分析.结果性别间绝大多数血液生理与生化指标差异不显著.3～6月龄血液生理与生化雌雄间比较,各指标差异均无统计学意义(P>0.05);8～12月龄血液生理雌、雄间HGB、RBC、HCT、MONO%和MONO有差异(P<0.05),雌雄间血液生化ALT、TBA、CHO、TG、LDL-C、APOB和CL有差异(P<0.05);两个年龄组雌雄合并相比,血液生理WBC、PLT、MPV、LY、MCHC、NEUT%和NEUT等7项指标有差异(P<0.05),生化指标ALB、UREA、HDL-C、GLU、APOA和FE等6项指标差异有统计学意义(P<0.05).结论滇南小耳猪两个年龄段之间以及雌、雄之间部分血液生理生化指标存在明显差异可为滇南小耳猪的标准化研究和相关实验研究提供基础资料和数据.

初情期前云南滇南小耳猪超数排卵和原核期胚胎体外发育的研究

目的对初情期前云南滇南小耳猪超数排卵和原核期胚胎体外发育进行研究.方法对8头初情期前云南滇南小耳猪注射1000IUPMSG和500IUhCG诱导超数排卵,以自然交配和人工授精技术、手术或离体冲卵技术回收原核期胚胎,并进行胚胎体外培养研究.结果超排的激素处理方法可成功诱导超生理数量的卵子发育和排卵及受精,最多可得到31枚受精卵.受精卵的囊胚率可达到87.5%.经过比较研究,人工授精可能优于自然交配的授精方式;雌激素可作为初情期前小母猪的超排成败的重要标志.但是,手术法冲卵的技术较离体冲卵的还很低.结论初情期云南滇南小耳猪可用于体内胚胎生产,其原核期胚胎体外发育具有比较高的的发育潜能.

猪新基因PFN3电子克隆、RT-PCR验证和组织表达分析

通过NCBI下载猪近缘物种的PFN3基因序列,在线BLAST比对获得猪EST序列,利用Lasergene软件电子克隆猪PFN3基因编码区序列及部分侧翼序列。针对编码区设计特异引物并以滇南小耳猪为研究材料进行实验验证,同时应用半定量RT-PCR技术对30个组织进行了表达分析。研究获得了猪PFN3 414 bp的编码区序列(GenBank登录号:JX457348,对应的氨基酸登录号:AFU74013)。生物信息学分析表明,该基因编码137个氨基酸,预测的蛋白质分子量(Mw)为14.73 ku,等电点(pI)为9.30。蛋白质结构分析显示,PFN3存在1个保守域,无信号肽;疏水性分析表明其N端和C端均具有亲水性;亚细胞定位显示,该蛋白位于细胞质的概率是94.1%。系统进化分析表明,猪与牛亲缘关系最近。组织表达分析表明,PFN3基因在30个被检组织中均有表达,在睾丸、肝、肾、结肠、盲肠、直肠及食管中高表达,在胸腺、甲状腺、附睾、淋巴结、十二指肠、空肠、回肠、胃、皮肤、大脑、小脑、垂体及下丘脑中中度表达,在颌下腺、肺、心、脾、肾上腺、肌肉、胰脏、脑干、舌下腺及脊髓中低表达。研究结果将为该基因在猪中的进一步功能研究奠定基础。