响应面法优化提取虎耳草总酚及其抑制五步蛇毒PLA2酶活性的相关性

为了探讨虎耳草总酚（TP）与抑制五步蛇毒中磷脂酶A₂（PLA₂）之间的关系,本研究通过单因素试验及Box-Behnken试验对虎耳草TP的提取条件与抑制五步蛇毒中PLA₂活性进行优化,考察各试验因素对超声辅助提取虎耳草TP得率及其对五步蛇毒PLA₂活性抑制率（PIR）的作用,并对虎耳草TP含量与PIR的相关性进行分析。结果表明,响应面法提取虎耳草TP和PIR的最佳工艺条件为：粒径为60目,料液比为1∶50,超声功率为250 W,超声时间为1.8 h。此优化工艺条件下虎耳草TP得率为（8.69±0.46） mg·g^-1,PIR为（40.91±0.21）%;相关性分析结果显示虎耳草TP含量与PIR具有极显著正相关（P<0.01）,说明TP可能为虎耳草抑制五步蛇毒中PLA₂活性的物质基础。     

尖吻蝮人工养殖灌喂技术研究

目的探讨采用灌喂器人工灌喂尖吻蝮技术的可行性。方法采用湖南京湘源蛇类养殖有限公司自主研制的灌喂器人工灌喂尖吻蝮,观察记录随机抽取的30条尖吻蝮幼蛇及30条尖吻蝮成蛇在机器灌喂后每条幼蛇的体重数据,每3个月测量1次,观察12个月统计尖吻蝮的体重增长情况,并与常规灌喂方法饲养的尖吻蝮进行比较。结果灌喂器饲养的尖吻蝮幼蛇成活率为86.67%,平均体重达（482.39±40.19）g;灌喂器饲养的尖吻蝮成蛇成活率为96.67%,平均体重达（1346.13±117.51）g。而同期常规灌喂技术饲养的尖吻蝮幼蛇全部死亡,尖吻蝮成蛇成活率为76.67%,平均体重（878.56±82.39）g。结论人工灌喂器饲养的尖吻蝮幼蛇及成蛇的体重增长快速,成活率高,值得推广。     

尖吻蝮的人工繁育

In this article, we report our first experience of successful artificial propagation about the five paced pit viper ( Deinagkistrodon acutus ) and breeding to its second filial generation. In May, 1994, 18 adult snakes (8 males, 10 females) were captured in field, and were reared in man made environment. In July and August of the same year, 5 females laid 134 eggs in total (26 8±5 26), which were artificially incubated into 123 hatchlings. 100 hatchlings (average body weight 12 13±1 50 g)were selected to feed. Three years later, 58 snakes were alive (livability 58%). In September of 1997, April and May of 1998, some of the 58 snakes copulated. From July to August of 1998, 6 females laid 64 eggs in total, 58 of which were fertilized, and 54 were incubated into hatchlings in September of 1998. Therefore, we had successfully bred the second filial generation of the five paced pit viper in complete artificial environment.     

尖吻蝮自然越冬洞穴的结构观察

为了解决尖吻蝮在人工饲养条件下难以安全越冬的问题 ,在黄山地区对尖吻蝮的自然越冬进行了数年的调查 ,陆续观察了 2 3个越冬洞穴的结构。初步总结了尖吻蝮自然越冬的生态 ,并用数据和图示方式较详细地报道了尖吻蝮自然越冬洞穴的构造 ,介绍了人工饲养过程中保证尖吻蝮安全越冬的管理方法。     

墨旱莲对4种蝮蛇毒引起的炎症和出血的影响

目的探讨墨旱莲提取液对短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒及尖吻蝮蛇毒所致的炎症和出血的影响。方法应用短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒及尖吻蝮蛇毒所致大鼠足跖肿胀的致炎模型,观察墨旱莲提取液对蛇毒所致大鼠足跖肿胀的影响。墨旱莲提取液分别与不同蛇毒混合,给小鼠腹部皮下注射,观察其对蛇毒引起的小鼠皮下出血的影响。结果墨旱莲提取液15g／kg连续2次灌胃给药,对短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒或尖吻蝮蛇毒所致大鼠足跖肿胀的急性炎症造模和短尾蝮蛇毒棉球肉芽肿的慢性炎症造模(20g／kg)均有明显的抑制作用,对这些蛇毒引起的小鼠皮下出血也能明显抑制。结论墨旱莲提取液对短尾蝮蛇毒、蛇岛蝮蛇毒、白眉蝮蛇毒及尖吻蝮蛇毒引起的炎症和出血均有明显的抑制作用。     

尖吻蝮仔蛇的人工模拟生态养殖试验

目的通过建立一个适应于尖吻蝮仔蛇生长发育的稳定的多功能生态系统的人工模拟生态养殖蛇场,以最大限度地降低仔蛇的饲料成本,从而达到降低人工养殖尖吻蝮蛇的成本。方法选取一个室外围墙式养蛇场作为试验基地。设计为适应于蛇、蛙、昆虫、蚯蚓、小杂鱼等动物生长繁衍的人工模拟生态环境,建立一个稳定的多功能蛇场生态系统,以尖吻蝮蛇100条,在试验以前随机抽取10条测量其体重和体长,并以活体动物饲料喂养法和科学饲养管理法进行试验养殖,并在进入第一、第二次冬眠前均随机抽取10条蛇仔测其体重和体长。结果本次试验与本蛇场室内人工饲养尖吻蝮仔蛇相比,不仅在体重、体长和肥满度方面均相差不大,而且仔蛇的存活率还有所提高。结论表明该人工模拟生态养殖蛇场能够满足尖吻蝮仔蛇生长发育的需要,达到了预期的目的。     

Purification and Immunoprotection Evaluation of AaHIV from Complex Venom Metalloproteinases of Deinagkistrodon acutus

The aim of this study was to investigate the immunoprotective effects of AaHIV in mice. After purification, a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) was performed. Bicinchoninic acid was used to determine the molecular weight and concentration of AaHIV. AaHIV, venom complex (VC), and phosphate buffered saline (PBS) were subsequently used to immunize the mice three times, and the blood was sampled 1 week after the third immunization to determine the serum immunoglobulin G (IgG) antibody titer. A skin‐bleeding inhibition assay and toxin‐eliminating assay were performed on the immunized mice. The purity and concentration of AaHIV were 86.6% and 1.20 mg/mL, respectively. The AaHIV group exhibited higher antibody titers than the VC group. The survival rate of the AaHIV group (7/10) was significantly higher than that of the PBS group (0/10) (P = 0.0031). The high titer of antibodies induced by AaHIV partially neutralized the bleeding activity of the Deinagkistrodon acutus venom complex.     

Molecular phylogeography of endangered sharp-snouted pitviper (Deinagkistrodon acutus; Reptilia, Viperidae) in Mainland China

Using phylogenetic and population genetic approaches, the present study reports the phylogeographic structure of the sharp-snouted pitviper (Deinagkistrodon acutus), a threatened snake species with commercial and medicinal importance in China. The entire mitochondrial ND2 gene (NADH dehydrogenase subunit 2) sequences of 86 individuals of D. acutus from 14 localities across its range in China were determined. Based on the results of phylogenetic analyses, distribution of diagnostic sites, haplotype network, and AMOVA hierarchical analysis, an east-west division of the whole D. acutus population could be observed. Geographically, a line formed by a lake, river, and mountain chain (the Poyang Lake, Gan River to the southern end of the Wuyi Mountains), results in vicariance and approximately vertically splits the range into two and the whole population into two main lineages (western and eastern). The bifurcating tree suggested generally west to east dispersal trend. The data fit the isolation by distance (IBD) model well. Star-like clusters in haplotype network, significantly negative values of Fs statistics, and unimodal mismatch distributions all suggest recent demographic expansions in four areas. The results show that isolation, dispersal, bottleneck, and expansion jointly constitute the history of D. acutus. In a haplotype network, the excessive predominance of central haplotypes, few medium-frequency haplotypes, predominance (73.1%) of the singletons among the derived haplotypes, most of which are connected to the central haplotype by only one mutational step, unsymmetrical campanulate unimodal curve of mismatch distributions and leftwards shift of the peaks, all suggest that the whole D. acutus population is a young population with low genetic diversity. Based on the data, the first priority for conservation action should be given to the Huangshan unit.     

不同地理单元尖吻蝮蛇毒蛋白组分比较研究

目的探讨不同地理单元尖吻蝮蛇毒蛋白组分有无差异性。方法采用非变性聚丙烯酰胺凝胶电泳(NativePAGE)方法,比较采自安徽黄山(黄山单元)和贵州梵净山(西部单元)尖吻蝮蛇毒组分。结果梵净山尖吻蝮的蛇毒蛋白表达量和条带数目均高于黄山尖吻蝮。结论黄山单元和西部单元的尖吻蝮蛇毒蛋白组分具有差异性。     

尖吻蝮（Deinagkistrodon acutus）生长过程不同时期排毒量及蛇毒组分的变化

对人工培育的4个年龄段尖吻蝮蛇毒和野生成体尖吻蝮蛇毒进行了聚丙烯酰胺凝胶电泳分析。结果显示：3龄以前尖吻蝮蛇毒蛋白电泳图谱,无论是在分带、泳动率及相应组分的量等方面,均呈现一定的差异,但随着尖吻蝮蛇龄的增长,蛇毒蛋白电泳图谱与野生成蛇蛇毒蛋白电泳图谱越来越接近。3龄尖吻蝮生长发育达到性成熟,其蛇毒蛋白电泳图谱与野生成蛇趋于一致。蛇毒组分的变化,提示了尖吻蝮的生长发育进程。