人血栓调节蛋白基因的表达及转基因小鼠的建立

用PCR方法从人的基因组DNA中扩增了人血栓调节蛋白（hTM)基因，将其克隆到带有人EF－1α启动子的pEF-neo哺乳动物表达载体上，得到表达质粒pEF-TM。在转染pEF-TM的COS－1细胞中，hTM得到了瞬时表达，并且正确地定位到细胞膜上。用pEF-TM转染猪内皮细胞（PEC），经G418筛先得到稳定转染的克隆。凝血活性测定结果显示，表达hTM的PEC凝血时间明显延长，表明其抗凝血能力增强。通过显微注射方法，得到了1只F0代hTM转基因小鼠。Southern杂交结果表明，该转基因小鼠整合了9个拷贝的pEF-TM DNA,并能将外源DNA遗传给后代。     

Research Progress and Countermeasures of Porcine Endogenous Retrovirus in Chinese Miniature

The paper is a brief introduction of porcine endogenous retrovirus (PERV) innovative research results in the world and also expounds the passing PERV existence situation on different varieties of miniature pig, analyzes the PERV-virus gene cloning and sequence, appraises method on cell level, create the platform of infection HEK293 cells research, study on pigs A3F inhibition of PERV, and reveal the innovative research results on the specific molecular genetics in the different strain of PERV, analyzes the advantages of Wuzhishan miniature pig inbred line such as low gene copy of PERV,and there is no passing PERV-C specificity and as well as looking forward to cultivate the methods for new strain of PERV negative pigs. It will provide a scientific counter measure and new perspective to solve the spread of disease risk of miniature pig PERV and product safety for human xenotransplantation and biomedical materials of research and development.     

中国小型猪内源性反转录病毒研究进展与对策

作者介绍了国内外猪内源性反转录病毒（PERV）研究进展,简述了中国不同品种小型猪PERV存在情况、PERV前病毒全基因克隆与序列分析、细胞水平鉴定方法、感染HEK293细胞研究平台的创建、猪A3F对PERV的抑制作用研究,以及从分子遗传学上揭示不同品系PERV特异性等创新性研究成果,分析了五指山小型猪近交系具有PERV基因拷贝少且没有PERV-C等特异性,以及展望培育中国PERV阴性猪新品系方法等研究,以期为人类异种器官移植和生物医用材料产品安全性研发,解决小型猪PERV疾病传播危险性提供科学对策和新的方向。     

豚鼠至大鼠原位肝移植肝下下腔静脉套管方法的改进

目的探讨豚鼠至大鼠异种原位肝移植中肝下下腔静脉套管的改进方法。方法豚鼠和SD大鼠各40只分别作为供、受体,随机分为实验组和对照组并随机配对。实验组采用改进的肝下下静脉套管方法进行肝下下腔静脉袖套管的安装及吻合,对照组采用常规方法进行肝下下腔静脉袖套管的安装及吻合。比较两组肝下下腔静脉袖套管安装时间、安装成功率、吻合时间、吻合成功率和手术成功率。结果实验组较对照组肝下下腔静脉袖套管安装时间、吻合时间和无肝期明显缩短,袖套安装成功率、吻合成功率和手术成功率明显提高,差异有统计学意义(P<0.05)。结论应用改进的肝下下腔静脉套管方法进行豚鼠至大鼠原位肝移植,手术成功率高,可用于豚鼠至大鼠原位肝移植实验研究模型。     

牛羊水间充质干细胞对小鼠酒精性肝病的影响

试验旨在分离培养牛羊水间充质干细胞（AF-MSCs），并研究其对酒精性肝病（ALD）小鼠治疗效果以及对肝组织中缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）及Toll样受体4（TLR4）mRNA表达的影响。从4~5月龄雌性胎牛羊水中获取AF-MSCs，进行RT-PCR、免疫荧光及诱导成脂鉴定。将48只ICR小鼠（雄性）随机分为4组：空白对照组（BC组）、模型对照组（MC组）、AF-MSCs组（MC+AF-MSCs组）和水飞蓟宾胶囊（SC）组（MC+SC组），每组12只。除BC组灌胃0.2 mL生理盐水，其余各组均灌胃56度红星二锅头（5 g· kg ^-1，2次· d ^-1），连续4周；第29天MC+AF-MSCs组进行肝外注射Dil标记的AF-MSCs（5×10⁶个·mL ^-1），MC+SC组分2次灌胃SC 2.4 mg（溶于56度红星二锅头）；试验结束后，测定小鼠血清谷丙转氨酶（ALT）、谷草转氨酶（AST）和甘油三酯（TG）活性。HE染色、免疫组化法观察造模情况，Dil示踪AF-MSCs在肝内定植分布；qRT-PCR法检测HIF-1α、VEGF及TLR4基因表达情况。RT-PCR、免疫荧光结果表明，分离得到牛AF-MSCs，且有诱导成脂分化能力。血清学结果显示，MC组ALT、AST和TG活性极显著高于BC组（P<0.01）。组织病理切片显示，MC组肝组织中出现脂肪性变、炎性细胞浸润及肝血窦充血等病理性变化，说明56度红星二锅头成功复制ALD小鼠模型；与MC+SC组相比，MC+AF-MSCs组ALT、AST活性显著降低（P<0.01），肝组织中脂肪性变减轻，肝血窦未见充血，说明AF-MSCs对肝功能的改善比SC效果显著；qRT-PCR结果显示，MC+AF-MSCs组下调HIF-1α、VEGF、TLR4基因的表达量较MC+SC组显著（P<0.01），说明AF-MSCs参与HIF-1α/VEGF信号通路的调控，抑制TLR4表达的效果优于SC；细胞示踪术证明，AF-MSCs定植在病变部位并趋化更多AF-MSCs向炎症部位迁移参与肝组织的修复。综上，本研究成功分离得到牛AF-MSCs，其能够参与调节氧化应激、血管生成及抑制炎性因子的释放，且改善ALD的效果优于SC。     

Histological differences in full-thickness vs. lamellar corneal pig-to-rabbit xenotransplantation

To evaluate the differences in graft survival and histopathological characteristics between full-thickness and lamellar orthotopic corneal xenotransplantation in a pig-to-rabbit model, we orthotopically transplanted a full-thickness or the anterior half of a pig's cornea onto the OD of 16 rabbits. As a result, the median survival were 16.83 and 29.07 days for the full-thickness and lamellar xenografts, respectively ( P  = 0.0005). Histologically, the full-thickness corneal xenografts had massive infiltration by eosinophils, whereas the lamellar xenografts showed predominantly mononuclear infiltrates ( P  < 0.05). Given these preliminary findings, lamellar corneal xenografts in rabbits survived longer than the full-thickness xenografts and each type of graft demonstrated different rejection mechanisms.     

卵巢异种移植在生殖生物学和动物保存中的研究进展

随着关于卵巢组织较有效的冷冻保存方法的发展,生殖系的保存和繁殖已变成了现实的目标。本文就卵巢冷冻保存和卵巢移植上的发展进步,异种移植在保存稀有和濒危物种及一些女性卵巢功能上的潜在价值,以及即将出现的异种移植技术进行综述。     

广西巴马小型猪SLA-DQB 基因 的克隆及序列分析

利用RT-PCR 的方法从广西巴马小型猪肝脏组织中扩增SLA-DQB 基因的编码区序列,旨在为下一步构 建SLA-DQB 转基因广西巴马小型猪奠定基础。结果表明,克隆的广西巴马小型猪SLA-DQB 基因编码区长687 bp,与 GenBank 参考序列（NM_001113694）相比,共有15 个氨基酸发生突变（位点为10、14、19、27、29、38、39、48、58、61、68、 72、76、78、183）。与普通猪、其他小型猪、奶牛、人类及小鼠的同源性分别为95.8%、95.8%、88.1%、86.2%和80.0%。     

Requirements for the welfare of baboons and pigs used in animal-to-animal xenotransplantation experiments

Objective   Review the welfare requirements of pigs and baboons used for xenotransplantation in research laboratories. Because of the requirements to maintain optimum health status, these animals are often kept in barren enclosures with little or no enrichment. They may also be exposed to procedures causing stress and discomfort. Although animal-to-human xenotransplantation is, at the present time, not approved in Australia, research is currently being performed to develop laboratory procedures, using the pig-to-baboon model.
Results and conclusion   We make recommendations for the husbandry of baboons and pigs used for xenotransplantation, to increase their welfare and minimise stress during experimental procedures, while attempting to preserve the health status required. It is proposed that novel standards should be devised and implemented for baboons, whereas existing pig welfare appraisal schemes could, with minor changes, be suitable for assessing the welfare of pigs used for xenotransplantation.     

Rat allogeneic mesenchymal stem cells did not prolong the survival of corneal xenograft in a pig-to-rat model

Objective  Recent reports have demonstrated that mesenchymal stem cells (MSCs) can modulate/suppress immunologic responses through interactions with different immune cells. We performed this study in order to investigate the immunomodulatory effects of MSCs in corneal xenotransplantation.
Animals studied  Pig and rat.
Procedures  We orthotopically transplanted pig corneas into rats and topically applied allogeneic rat MSCs to the corneas for 2 h immediately after transplantation. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. The rejected grafts were histologically examined using antibodies against CD4, CD8, CD161, and CD68. The expression of IL-2, IL-6, IL-10, and IFN-γ was also evaluated in the rejected grafts using an enzyme-linked immunosorbent assay.
Results  The survival of corneal xenografts was not significantly prolonged by MSC application (MST 10.5 days) compared with the controls (MST 9.67 days) ( P  = 0.4189). Histologically, the rejected grafts in both groups were massively infiltrated with neutrophils and macrophages. Some CD8⁺ T cells and rare NK cells were found in the rejected grafts. The levels of IL-6 and IL-10 were significantly increased in the rejected grafts from MSC-treated rats compared with the grafts from MSC-untreated rats. However, the levels of IL-2 and IFN-γ were not different between the two groups.
Conclusions  Topical application of allogeneic rat MSCs was ineffective in prolonging corneal xenograft survival in a pig-to-rat model.