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.

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。

改进YOLO v4模型在版纳微型猪只行为识别中的研究

为了能够在猪只重叠、遮挡等复杂场景中实现版纳微型猪只行为的准确、高效识别,试验通过改进YOLO v4模型的方法来识别猪只行为,通过视频捕获的方式截取不同角度猪只行为图片,构建行为特征数据集;采用嵌入CBAM注意力机制的Res Net50残差网络结构作为改进YOLO v4模型的主干网络,并引入由深度可分离卷积、批标准化(BN)、Hard Swish激活函数组成的CH模块,代替主干网络中的传统卷积,提升模型检测精度的同时降低参数量;在PANet多尺度特征融合结构中引入双重3层1×1和3×3交替卷积运算替代上、下原采样方式,构成DPANet网络结构,增强对猪只行为图片中细节特征的提取,提高计算效率;基于参数共享理念与二阶段训练的迁移学习方法,优化训练过程以显著缩短训练时间,加速模型的收敛速度。结果表明:改进YOLO v4模型对猪只行为数据集的训练时间仅为6 h,而原模型训练时间则需要19 h;改进YOLO v4模型识别平均精度为93.97%,召回率为96.27%、参数量为0.26×10^(8),与Faster-RCNN、SSD、YOLO v4模型相比,平均精度与召回率分别提升8.88,15.36,8.68个百分点及16.09,41.34,30.40个百分点,参数量最大减少1.11×10^(8)。改进YOLO v4模型对识别爬栏探究、站立行走、进食、躺卧4种行为的准确率达到了98%、88%、92%、97%,与其他3种模型相比,站立行走、进食两种行为的识别效果远大于其他模型。说明改进YOLO v4模型在复杂场景下具有良好的准确性和有效性,能够精准识别猪只的不同行为。

北京某绿地蚊媒病毒的分离和鉴定

为了解北京某绿地蚊媒病毒的分布情况,本试验使用诱蚊灯捕蚊采集到蚊虫标本19000余只,利用蚊子细胞C6/36培养进行病毒分离,利用高通量测序和逆转录聚合酶链式反应(RT-PCR)鉴定分离株;RT-PCR测定分离株病毒的序列,并与国内外相应病毒序列进行比对分析。结果显示,样本经病毒分离获得2株可引起明显细胞病变的分离株,分别命名为BJ2021/01和BJ2021/02。高通量测序和RT-PCR检测结果显示,BJ2021/01分离株含有版纳病毒(BAV)、忍者病毒(SHTV)、沙捞越病毒(SWKV)和库蚊源类芜菁黄花叶病毒科病毒(CuTLV),为4种病毒的混合物;BJ2021/02为BAV。基因序列分析结果显示,BAV分离株BJ2021/02的12个片段与国内外BAV分离株的核苷酸同源性为58.1%~98.7%,编码氨基酸的同源性为54.4%~98.9%。系统进化分析结果显示,BJ2021/02分离株属基因A型。本试验结果丰富了我国BAV的基因组序列,为开展北京市BAV的流行病学研究提供了参考,为虫媒病毒病的预防和控制提供了病原学依据。

版纳微型猪1型糖尿病模型的构建方法初探

目的构建1型糖尿病微型猪模型,并探索有效延长模型猪生存周期的术后护理方案。方法选取7头版纳微型猪进行胰腺切除手术。术后3~5 d通过补充葡萄糖、维生素及抗生素等方法帮助其恢复。每日早晚两次检测血糖、尿糖水平,据此调整胰岛素补充量。每日巡视模型猪并记录其饮食欲、精神状态、虚弱程度、皮肤破损情况、粪尿量等数据,每周称量体重,直至动物死亡。根据模型猪状态及时给予葡萄糖注射液和乳酸林格氏液等以补充营养并纠正电解质失衡。结果7头版纳微型猪在胰腺切除术后均表现为随机血糖浓度大于11.1 mmol/L,远超正常猪的平均血糖值(6.0 mmol/L),尿糖阳性,且其体重呈渐进性下降等典型的糖尿病临床表征,提示1型糖尿病模型构建成功。此外,存活时间超过8周的1型糖尿病猪还出现了渐进性被毛脱落及皮肤破溃的情况;在模型猪无法站立乃至无法自主进食时进行安乐死,并采集其肝脏、肾脏及皮肤等受累及的器官组织进行病理组织切片及HE染色,病理组织切片也可见长期高血糖导致的肝脏淤血、肝糖原大量蓄积、肝细胞气球样变和渐进性肝脏纤维化,肾小球淤血、肾小管上皮细胞空泡变性和蛋白尿,真皮层出现淤血、血管壁变薄以及不同程度的角化不全和角化不良等肝脏、肾脏及皮肤组织病变。本研究构建的版纳微型猪糖尿病模型平均生存期为44 d,最长可生存121 d。结论通过胰腺切除术可成功建立版纳微型猪的1型糖尿病模型,进一步通过术后精心护理可获得并发症明显的长期1型糖尿病微型猪模型,这为1型糖尿病治疗策略研究提供了稳定的大动物模型。

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.

首次从新疆无名热病人分离到8株新环状病毒(BANNA)

从新疆境内的生产建设兵团124团采集到不明原因发烧病人血清98份,分离到8株病毒。感染2～4日龄乳小白鼠和三周龄小白鼠不发病。可引起C6/36细胞病变。电镜观察病毒为球形无囊膜颗粒,直径63～69nm,核衣壳直径为49～54um。超薄切片在感染细胞中常见到大量颗粒状包涵体,磷钨酸负染可见到排列整齐的壳粒结构,显示了典型的环状病毒形态特征。该病毒对酸、氯仿、胰蛋白酶及热敏感;1.1MMgCl_2在50℃作用15分钟可使其感染力提高,在-30℃作用1小时可使其感染力丧失;抵抗乙醚和5-碘脱氧尿苷。血清学鉴定,新分离病毒与披膜病毒科(Togaviridae)、黄病毒科(Flaviviridae)及布尼亚病毒科(Banyaviridae)的某些病毒的免疫腹水及免疫血清都不发生反应。与新环病毒(BANNA)的免疫腹水及免疫血清发生反应。用新环病毒酶标单克隆抗体(McAb)做酶标中和试验为阳性,中和指数为1023.3～18620.9。经初步鉴定,分离的8株病毒的抗原性与近年从我国南方分离的新环状病毒(BANNA)相一致。

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.

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。

版纳微型猪近交系用于镇痛类经皮吸收药物体外渗透性评价

【目的】通过比较版纳微型猪近交系皮肤(以下简称“猪皮肤”)和人皮肤的组织形态学以及镇痛类经皮吸收药物的体外渗透试验,验证猪皮肤在镇痛类经皮吸收药物评价方面的应用价值。【方法】通过HE染色、Masson染色、弹力纤维染色和透射电镜观察比较2种皮肤组织结构形态;选择水杨酸溶液、双氯酚酸二乙胺乳膏和利多卡因凝胶贴膏3种药物作为模型药物,通过Franz扩散池进行药物渗透试验,采用酶标仪和高效液相色谱仪检测透过皮肤的药物质量浓度。【结果】HE染色、Masson染色以及透射电镜结果显示:猪皮肤与人皮肤的分层和细胞构成相似,但是猪皮肤的表皮层极显著厚于人皮肤表皮层(P<0.01),人皮肤的真皮层极显著厚于猪皮肤真皮层(P<0.01)。版纳微型猪身体各部位皮肤从厚到薄依次是肩胛、臀部、背部和腹部。3种药物渗透的0~24 h内,大部分时间点3种药物透过猪皮肤和人皮肤的药物质量浓度均无显著差异(P>0.05)。【结论】猪皮肤和人皮肤组织结构相似,3种镇痛药物在2种皮肤中的渗透性相似,猪皮肤可用于镇痛类经皮吸收药物的体外渗透性评估。

东南亚十二节段RNA病毒荧光定量qRT-PCR和常规RT-PCR检测方法的建立

东南亚十二节段RNA病毒属为呼肠孤病毒科中的一个新属,该属包括版纳病毒(BAV)、芒市病毒(MSV)、卡皮罗病毒(KDV)和辽宁病毒(LNV)四种病毒。为建立上述四种病毒的群特异性核酸检测方法,本研究根据BAV、MSV、KDV和LNV毒株VP12基因序列的保守区,设计特异性引物和TaqMan探针,建立了荧光定量qRT-PCR和常规RT-PCR检测方法,并分别进行了特异性、灵敏性和重复性的检测。实验结果表明,两种检测方法分别对四种病毒均有特异性的扩增和荧光信号检出,而对流行性出血病病毒(EHDV)、蓝舌病病毒(BTV)、中山病病毒(CHUV)、广西环状病毒(GXOV)、阿卡斑病毒(AKAV)、云南环状病毒(YUOV)等病毒无扩增和无有效荧光信号检出,具有较好的特异性。灵敏性实验结果显示,荧光定量qRT-PCR检测四种病毒核酸的下限可达10 copies/μL,常规RT-PCR的检测下限为10^(2) copies/μL,荧光定量方法灵敏度是常规方法的10倍。四种病毒荧光定量qRT-PCR方法的组内和组间重复试验标准差均小于0.8,变异系数均小于3%,表明该方法均具有良好的稳定性。以上结果表明,本研究建立的BAV、MSV、KDV和LNV四种病毒的荧光定量qRT-PCR和常规RT-PCR方法具有快速、准确、稳定等优点,可为四种病毒的早期诊断、快速检测、流行病学调查和实验研究等提供有效的技术方法。

版纳微型猪近交系心脏的解剖观察

目的 观察中国版纳微型猪近交系心脏的大体解剖和组织学 ,为转基因猪心和异种移植提供形态学基础。 方法 应用灌注固定对 10头版纳微型猪近交系进行大体形态观察 ,测量心脏径线、心壁厚度、心脏的腔室、心脏大血管直径。使用苏木精 -伊红染色进行组织学观察。结果 版纳微型猪近交系心脏外形、各腔室及内部结构等与人心脏基本匹配 ,但是微型猪奇静脉直接开口于右心房 ;组织学观察微型猪心脏各部分结构组成及其细胞种类与人心脏相似 ,但是心肌闰盘较人的少 ,心室内膜下的蒲肯野氏纤维较人的粗大。结论 从解剖形态学及组织学观察 ,版纳微型猪近交系心脏与人心脏形态和结构相似 ,有成为异种移植供体的可能性。

版纳微型猪近交系133家系SLA-DQ cDNA的克隆和序列分析

目的阐明版纳微型猪近交系133家系(BMI-133)免疫相关基因及其可能在猪-人异种器官移植中的作用。方法提取外周血总RNA,通过反转录、cDNA合成及转化,最终获得了具有完整阅读框架的SLA-DQA和SLA-DQBcDNA克隆。结果序列分析表明,所获得的DQAcDNA长度为830bp,编码255个氨基酸残基;DQBcDNA长度为1103bp,编码262个氨基酸残基。结论和其他已报道的小型猪比较,本研究克隆的序列无论在核苷酸还是氨基酸水平上都与之存在差异,由此确定这是BMI-133的两个特有的新等位基因。另外,通过与人的相应序列对比发现,BMI-133与人类相应基因相比具有较高的同源性。随着研究的深入,BMI-133家系极有可能成为供异种器官移植的专用近交系猪群。

版纳微型猪近交系新生猪成纤维细胞的体外培养及其生物学特征研究

试验以版纳微型猪近交系新生猪耳部皮肤组织为材料,采用胰蛋白酶消化法培养成纤维细胞,并对其进行细胞形态观察、细胞冻存前和复苏后存活率测定、生长曲线绘制和染色体核型分析等生物学特征检测。结果表明:版纳微型猪近交系新生猪成纤维细胞呈典型的成纤维细胞形态;细胞冻存前和复苏后存活率分别为98.0%和94.07%;生长曲线呈"S"型,倍增时间为33 h;对所制备的染色体核型分析显示2n=38(XY),并在体外培养14代后仍能保持正常核型。本研究建立了稳定的版纳微型猪近交系新生猪成纤维细胞的培养方法,将为体细胞克隆、转基因和异种器官移植等相关的研究提供技术基础。

版纳微型猪近交系RNF4基因克隆、多组织qPCR表达及功能生物信息学分析

RNF4基因调节精细胞分化和增殖过程,文章以Gen Bank下载的猪及近缘物种RNF4 mRNA序列为参考序列,设计特异引物扩增版纳微型猪近交系(BMI)RNF4基因。应用qPCR分析15个重要组织mRNA表达谱,并对蛋白质序列作功能生物信息学分析,构建多物种系统进化树。研究获得BMI RNF4 573 bp编码区序列(Gen Bank登录号:KU705638,对应氨基酸登录号:AOC89056),编码190个氨基酸,蛋白质分子质量(Mw)为21.43 ku,等电点(pI)为6.95。多组织荧光定量表达分析表明RNF4基因在尿道球腺、睾丸、肝、肺中高水平表达;在其他11个组织中呈中低水平表达。功能生物信息学分析表明RNF4蛋白质存在1个保守结构域,无跨膜结构,无信号肽序列;N末端疏水,C末端疏水;有4类功能活性位点,位于细胞核概率为94.1%。系统进化分析表明,BMI与狗亲缘关系最近。结果为研究RNF4基因在猪精细胞分化和增殖方面作用奠定基础。

版纳微型猪近交系AQP3克隆、序列分析及组织表达

目的克隆版纳微型猪近交系aquaporin 3(AQP3)基因,并利用生物信息学方法分析其序列特征,研究其在猪各组织中的表达情况。方法从版纳微型猪近交系脾脏中提取总RNA,利用RT-PCR方法扩增猪AQP3编码区序列,将纯化的片段与pMD18-T载体连接,转化宿主菌DH 5α,筛选阳性克隆进行测序。并采用半定量RT-PCR方法分析版纳微型猪近交系不同组织中AQP3基因的表达情况。结果成功克隆出AQP3基因编码区全长873 bp的序列,GenBank登录号为HQ888860,其编码290个氨基酸。氨基酸序列同源性分析表明,与牛的AQP3基因同源性最大,达99%。多组织半定量RT-PCR研究表明AQP3 mRNA在BMI猪的脾脏、肾脏、肺、小肠中均有表达,其中脾脏中表达最高。结论成功克隆出了版纳微型猪近交系AQP3基因全长编码区,并对其编码的蛋白质功能进行了预测,为进一步的功能研究奠定了基础。