犬低温体外循环手术及其灌注管理的试验研究

试验应用现代体外循环技术，实施完全心肺转流的低温体外循环，首次在我国兽医临床建立了２８只犬低温体外循环实验模型。探索适用于小动物心脏直视手术的体外循环手术操作、心脏停搏与复苏、体外循环灌注管理的技术方法。研究表明，选择右侧第４肋间进胸，从右心房施行前后腔静脉插管及升主动脉根部置供血管是简便易行的手术通路。采用部分晶体液与全血作预冲液，体外转流维持中度血液稀释（平均２３６８±３２７％），最低温度控制２６℃左右（平均２６１４±０７７℃），血流复温至３６℃左右（平均３５９７±０６８）开放升主动脉，转流中灌注压维持５０～９０ｍｍＨｇ（平均７２±１３ｍｍＨｇ），灌注流量８０～１００ｍＬ·ｋｇ－１·ｍｉｎ范围，是低温体外循环下开展犬心内直视手术成功的基本保障。采用冷晶与冷温血停搏液不同灌注。结果显示，冷温血停搏液联合灌注组心脏自动复跳率最高，并且复跳迅速，心搏有力，正性肌力药应用剂量小，复跳后辅助循环时间明显低于冷晶停搏液灌注组（Ｐ＜００５）。     

快速固化水泥刨花板的工艺试验

以速生杉木加工剩余物为主要原料、Na2SiO3为快速固化添加剂制备水泥刨花板,研究了冷、热压两种压板方式、Na2SiO3添加量、热压温度、热压时间、水灰比、灰木比、水泥种类和刨花形态等因素对水泥刨花板主要性能的影响.结果表明,热压法可适用于水泥刨花板制造,最佳工艺条件为:Na2SiO3添加量10%、热压温度95℃、热压时间12 min、水灰比0.6、灰木比2.6,制备的水泥刨花板的静曲强度达到10 MPa;采用MgCl2后处理能显著改善板的性能.     

Expression of hypoxia-inducible factor 1 and neuroglobin in piglet cortex during deep hypothermic circulatory arrest

AIM: To observe the expression of hypoxia-inducible factor 1 (HIF-1) and neuroglobin (NGB) in piglet cortex during deep hypothermic circulatory arrest. METHODS: Wuzhishan piglets were randomly assigned to cardiopulmonary bypass group (CPB group), 40 min of circulatory arrest (CA) at 18 ℃ without cerebral perfusion (DHCA group) or with selective antegrade cerebral perfusion (SACP group). After 180 min of reperfusion, cortical tissue was harvested for determining HIF-1α and NGB expression by HE staining, Western blot and real-time PCR. RESULTS: Severer cerebral injury was observed in DHCA group than that in SACP group. After 180 min of reperfusion, HIF-1α protein and mRNA levels were significantly higher in DHCA group than those in CPB group (P<0.05). Accordingly, SACP animal had higher levels of HIF-1α protein and mRNA than those in DHCA group (P<0.05). Simultaneously, higher NGB protein and mRNA levels were found in DHCA group than those in CPB group after 180 min of reperfusion (P<0.05). The SACP animal had higher levels of NGB protein and mRNA than those in DHCA group (P<0.05). CONCLUSION: Up-regulation of HIF-1 and NGB are involved in the mechanism against cerebral injury resulting from DHCA in the cortex and possibly a part of cerebral protective effect of SACP.     

低温沼气功能菌群的选育与试验

通过低温定向培养,从高纬、高寒野外厌氧环境样品和冬季沼气池底泥样品中分别选育出野生低温功能菌群和沼气池低温菌群。在13℃和10℃低温条件下,野生低温菌群产气率达到0.14 m3/d.m3和0.10 m3/d.m3,但所产沼气的甲烷含量较低,只有51.0%和48.0%。同样温度下,沼气池低温菌群的产气率只有0.06 m3/d.m3和0.04 m3/d.m3,但所产沼气的甲烷含量较高,达到62%和56%。两类菌群混合发酵可显著改善产气性能,相同温度下,产气率可达到0.15 m3/d.m3和0.11 m3/d.m3,高于两类菌群单独发酵,是常温菌群的3倍以上;混合低温菌群所产沼气的甲烷含量为57.0%和52.0%,介于野生低温菌群和沼气池低温菌群之间,比常温菌群提高19.0%和16.0%。研究结果表明,混合低温菌群具有较好的低温产气性能和广阔的应用前景。     

Preservation of common carp germ cells under hypothermic conditions: Whole tissue vs isolated cells

The aim of this study was to optimize the conditions for hypothermic storage of spermatogonial stem cells (SSCs) and oogonial stem cells (OSCs) of common carp Cyprinus carpio. This was conducted by storing gonadal tissue or isolated cells for 24 hr under hypothermic conditions in the first experiment and by testing two different storage media (L‐15 or DMEM supplemented with 10% FBS and 25 mM HEPES) and regular medium change (every 4 days) during two weeks of hypothermic storage in the second experiment. During the first 24 hr, isolated cells showed no decrease in viability, while cells obtained from hypothermically stored tissues displayed significantly lower viability after only 6 hr (Tukey's HSD, p < 0.01) indicating that hypothermic storage of isolated cells is superior to storing tissue pieces. The 2‐week trial demonstrated that storage media have a profound influence, while regular medium exchange does not have a positive effect on cell viability. Viability of SSCs and OSCs after two weeks was approximately 40% and 25%, respectively; however, survival of ~70% was obtained after 10 days of storage for SSCs and 7 days for OSCs. Hypothermic storage developed in this study has many practical applications during the development of surrogate broodstock technologies for common carp, but also in carp hatcheries and for the conservation of genetic resources of closely related cyprinid species.     

赤松毛虫质型多角体病毒的引进和利用研究

从日本引进赤松毛虫CPV对我国松毛虫属(Dendrolimus)的七种松毛虫以及棉龄虫有较高的致死效果。根据 RNA 电泳图谱的研究,赤松毛虫 CPV 与在中国松毛虫属上的各复制株是不同的。目前,我国已发现有三种 RNA 类型,日本赤松毛虫CPV 属第一型。JADP-CPV、JYDPW-CPV、CYDPW-CPV 等属于第二型,CGDP-CPV 离心所得的上带多角体属于第三型。类型Ⅱ、Ⅲ属首次报道。和其他CPV一样,JDS-CPV 只侵染中肠上皮细胞。对第一代3龄和5龄虫的 LC_(50) 分别为1.6×10~5CPB/ml 和3.3×10~5CPB/ml,对同代3龄虫的 LD_(60)为2.1×10~3CPB/虫。JDS-CPV 不仅对幼虫防效好,而且也影响幼虫取食、体重以及下代幼虫存活率。此病毒与BT混用可以减少针叶被害72—80％。在油乳剂中于4℃下保存197天,在水悬液中保存 5年,对此病毒活性无明显影响。以林间松毛虫作宿主可进行大量增殖。一头6—7龄幼虫平均可产多角体2亿个。     

Expression and Protective Efficacy of Clostridium perfringens β toxin Derivative

This study was conducted to obtain the Clostridium perfringens β toxin (CPB) derivative and to evaluate its virulence and immunoprotection. Based on the known sequence, four amino acid mutations, R212E, L268G, Y266A and W275A, were introduced into the gene of Clostridium perfringens CPB. Meanwhile, genes of Th cell and N-terminal of flagellin were added to 5' of the CPB gene, respectively. Then the gene GTF_NCPB_m4 was optimized and synthesized, and was subsequently cloned into the prokaryotic expression vector pET-30a (＋) for expression and purification to get the recombinant protein, rTF_NCPB_m4. Reactivity of rTF_NCPB_m4with antiserum of Clostridium perfringens type C crude toxins was detected by Western blot. Meanwhile, the toxicity of rTF_NCPB_m4 to mice was evaluated. According to the method prescribed in Chinese Veterinary Pharmacopoeia (2015), rabbits were immunized with rTF_NCPB_m4 to prepare antiserum and detect the neutralizing titer against Clostridium perfringens type C crude toxins. Results showed that rTF_NCPB_m4 was presented predominantly in an insoluble form (inclusion bodies), and it could react with the antiserum of Clostridium perfringens type C crude toxins. rTF_NCPB_m4 with an injection volume of 50 μg was still not fatal to mice. Sera from rabbits immunized with rTF_NCPB_m4can neutralize 10-20 mouse minimum lethal doses (MLD) of Clostridium perfringens type C crude toxins after twice immunization. Moreover, rabbits immunized with rTF_NCPB_m4 can fully resist 1 rabbit MLD of Clostridium perfringens type C crude toxins challenge, whereas all of the rabbits died (4/4) in the control groups. These data suggest that rTF_NCPB_m4 is a potential vaccine candidate for the subunit vaccine of Clostridium perfringens type C.     

犬低温体外循环综合监测的试验研究

试验借助现代医学监测设备,首次在我国兽医临床对２８只低温体外循环的犬系统开展了生命体征综合监测的研究。结果表明,密切观察动态心电（ＥＣＧ）,结合平均动脉压（ＭＡＰ）、中心静脉压（ＣＶＰ）、心率（ＨＲ）的变化,是判断循环功能、冠状动脉灌注、心肌缺血改变、保证脑血流通畅以及辅助转流时间长短的主要参考指标。保持良好的血气监测水平,是达到满意组织灌注不可缺少的指标;进一步证实,体外循环期维持良好的血钾水平,并于主动脉开放后１０ｍｉｎ适时补钙,有利于心脏收缩与复苏,对减轻心肌再灌注损伤具有重要作用。研究提示为确保每只手术犬在低温体外循环的肝素标准化以及合理使用鱼精蛋白用量,激活全血凝固时间（ＡＣＴ）的监测必须个体化。     

PVAc添加量对热压法快速固化水泥刨花板性能的影响

以杨木刨花为原料,42.5#普通硅酸盐水泥为无机胶黏剂,Na2SiO3为快速固化添加剂,研究添加聚醋酸乙烯酯乳液胶对热压快固水泥刨花板性能的影响.结果表明:①添加聚醋酸乙烯酯乳液可显著改善板材的物理力学性能;②要保证热压法快速固化水泥刨花板达到较理想的物理力学性能,应将板材在卸压后的回弹率控制在≤10%的范围内.     

重组产气荚膜梭菌β毒素突变体的表达与免疫保护性分析

旨在获得产气荚膜梭菌β毒素（CPB）的重组突变体,并评价其毒力及免疫保护性。对已知的产气荚膜梭菌CPB编码基因进行优化设计,同时引入4个氨基酸突变位点,分别是第212位的精氨酸突变为谷氨酸,第268位的亮氨酸突变为甘氨酸,266位的酪氨酸和275位的色氨酸突变为丙氨酸。此外,在该基因5'端添加Th细胞表位（T）和鞭毛蛋白（flagellin）N末端的编码序列,经人工合成获得重组基因片段（GTF_NCPB_m4）。将GTF_NCPB_m4克隆至原核表达载体pET-30a（＋）中进行表达与纯化,获得重组蛋白rTF_NCPB_m4。利用Western blot方法检测rTF_NCPB_m4与C型产气荚膜梭菌毒素抗血清的反应性,并检测rTF_NCPB_m4对小鼠的毒力。随后,以rTF_NCPB_m4免疫家兔,按照《中华人民共和国兽药典》（2015年版）规定的方法检测家兔血清对C型产气荚膜梭菌毒素的中和抗体效价。结果表明,rTF_NCPB_m4主要以包涵体的形式表达且能与C型产气荚膜梭菌毒素抗血清反应。小鼠安全性试验显示,50 μg的rTF_NCPB_m4对小鼠仍无致死性;免疫rTF_NCPB_m4后,每毫升家兔二免抗血清可中和10~20个小鼠最小致死量（MLD）的C型产气荚膜梭菌毒素;1个家兔MLD的C型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了100%（4/4）的保护。以上结果说明,rTF_NCPB_m4在丧失毒力的同时保留了良好的免疫原性,从而为C型产气荚膜梭菌病基因工程疫苗的研制提供了重要的数据。