浙江眼镜蛇(Naja naja atra)蛇毒镇痛多肽的分离纯化及其性质的研究

研究浙江眼镜蛇 (Najanajaatra)蛇毒的镇痛活性组分 ,为寻找镇痛效果好 ,而无成瘾性的新型镇痛药奠定基础。采用CM Sephadex离子交换色谱和SephadexG 5 0凝胶过滤色谱对浙江产眼镜蛇蛇毒中的镇痛活性组分进行分离纯化。采用PAGE IEF及HPLC等实验方法对产物纯度进行鉴定。采用SDS PAGE法和HPLC法测定产物的分子质量 ,用IEF法测定产物的等电点 ,用小鼠热板法与醋酸扭体法考察产物的镇痛活性。结果表明眼镜蛇毒经 3次柱色谱后 ,产物AAP经鉴定为单一组分。AAP经SDS PAGE法和HPLC法测定的分子质量分别是 7 2 8Mr 和 7 2 5Mr,等电点是 9 5 8。AAP的痛阈提高百分率和扭体抑制率分别为 91 3% ,77 2 %。眼镜蛇毒经

中华眼镜蛇(Naja Naja Atra)毒神经生长因子对PC_(12)细胞κ阿片受体mRNA表达的影响

目的研究眼镜蛇毒神经生长因子(nNGF)对PC12细胞κ阿片受体mRNA表达的影响,探讨nNGF诱导PC12细胞分化的分子机制。方法用RT-PCR方法半定量地考察nNGF及κ阿片受体的一些拮抗剂和激动剂对PC12细胞κ阿片受体mRNA表达的影响。结果(1)在25,50和100 ng/mL 3个nNGF剂量组中,50和100 ng/mL nNGF显著下调PC12细胞κ阿片受体mRNA的表达。(2)50 ng/mL nNGF分别处理PC12细胞1,3,5,7,10 d后,结果显示κ阿片受体mRNA表达随时间有降低的趋势,7 d时显著下调(P<0.05),10 d时特别显著(P<0.01)。(3)10μmol/L吗啡上调PC12细胞κ阿片受体mRNA表达,并能逆转nNGF对该κ阿片受体mRNA表达的下调效应。10μmol/L纳络酮对PC12细胞κ阿片受体的表达无显著影响,也能逆转nNGF对该κ阿片受体mRNA表达的下调效应。但吗啡和纳络酮共存时不能逆转NGF对该κ阿片受体mRNA表达的下调效应。结论nNGF诱导PC12细胞分化时,能下调其κ阿片受体mRNA表达,这种下调能分别被吗啡或纳络酮逆转,但不能被吗啡和纳络酮共存时逆转。

中华眼镜蛇（Naja naja atra）毒细胞毒素对动物急性及长期毒性研究

目的探讨中华眼镜蛇毒中分离纯化的细胞毒素（ＣｈｉｎｅｓｅＣｏｂｒｏＶｅｎｏｍ－Ｃｙｔｏｔｏｘｉｎ，ＣＶ－ＣＴＸ）对动物急性及长期毒性的作用。方法急性毒性：小鼠５０只，尾静脉一次注入不同剂量的ＣＶ－ＣＴＸ注射液，观察７ｄ内死亡率。犬５只，戊巴比妥钠静脉麻醉后，分别静脉恒速注入不同剂量的ＣＶ－ＣＴＸ，观察５ｈ内血压、心电图及呼吸的变化。长期毒性：大鼠１６０只分别腹腔注射生理盐水２０ｍｌ、ＣＶ－ＣＴＸ２５０，５００，１０００μｇ／ｋｇ·ｄ－１，连续４０ｄ。结果（１）小鼠急性ＬＤ５０及其９５％可信限为１１５８．７（１０７０～１２５４．６）μｇ／ｋｇ，中毒症状呈匍伏、毛松、懒动直到心跳停止，解剖未见重要脏器（心、肺、肝、脾、肾）明显变化。（２）犬静脉恒速注入１００μｇ／ｋｇ间隔１ｈ，共５次，呼吸、血压、心电图未见明显变化，一次超过５００μｇ／ｋｇ，可使心律不齐而停搏，呼吸停止。（３）大鼠长期毒性实验，３个剂量组，连续用药４０ｄ，无一死亡，肝、肾功能及血液生化均无明显影响。病理检查高剂量组部分动物肝脏中度浊肿及水样变性和散在点状坏死，肺有散在性出血；中剂量组肝脏轻度浊肿，偶伴有水样变性，个别动物有小点状坏死，肺有点状出?

眼镜蛇(Naja naja atra)毒膜毒素MT-Ⅰ的生物学效应研究

从眼镜蛇（Ｎａｊａｎａｊａａｔｒａ）毒中纯化出膜毒素ＭＴ－Ⅰ，该毒素与磷脂酶Ａ２产生协同溶血效应，使小鸡颈二腹肌等标本产生进行性挛缩，对体外Ｈｕｔ－７８细胞有杀伤作用。

福建连江产眼镜蛇(Naja naja atra)核型及等臂染色体研究

本文较详细地分析了福建连江产眼镜蛇的核型,绘出其染色体组型模式图。它与浙江产眼镜蛇的核型相比,有一对大染色体着丝点位置差异显著。文中首次报道了眼镜蛇的等臂染色体,并初步探讨了用双线期染色体进行蛇类染色体组型分析的方法。

Therapeutic Potential of Naja Naja Atra Venom in A Rat Model of Diabetic Nephropathy

Objective To study the protective effects of naja naja atra venom （NNAV） in a rat model of diabetic nephropathy （DN）. Methods The rat diabetes model was induced by intraperitoneal injection of streptozotocin （STZ）. Thirty-two model rats were randomly divided into one DN group （n=8） and three treatment groups （n=8 each） that received NNAV at doses of 30, 90, or 270 I^g/（ks.day） via oral gavage, another eight rats as normal controls. After 12 weeks, all rats were sacrificed and the changes in serum and urine biological index levels were determined by colorimetric assay. Microalbumin （mALB）, N-acetyl-13- glucosaminidase （NAG） and cystatin C （CysC） concentrations were measured by ELISA. Renal tissues were sliced for pathological and immunohistochemical observations. Results Comparied with the DN group, serum glucose was decreased by 31.04%, total cholesterol 21.96%, triglyceride 23.78%, serum creatinine 19.83%, blood urea nitrogen 31.28%, urinary protein excretion 45.42%, mALB 10.42%, NAG 20.65%, CysC 19.57%, whereas albumin increased by 5.55%, high-density lipoprotein-cholesterol 59.09%, creatinine clearance 19.05% in the treatment group by NNAV administration at dose of 90 μg/（kg-day）. NNAV also reduced the levels of malondialdehyde in serum （22.56%） and kidney tissue （9.79%）, and increased superoxide dismutase concentration in serum （15%） and decreased it in renal tissue （8.85%）. In addition, under light microscopy kidney structure was improved and glomerular hypertrophy decreased by 8.29%. As shown by immunohistochemistry, NNAV inhibited transforming growth factorl by 6.70% and nuclear actor-KB by 5.15%.

Age-related Variation in Snake Venom: Evidence from Two Snakes (Naja atra and Deinagkistrodon acutus) in Southeastern China

In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-related variation in electrophoretic profiles was found in both species and proteolytic and fibrinogenolytic activity was higher in neonate than adult venoms. Neonate D. acutus venom had higher 5＇ nucleotidase, PLA2, hyaluronidase and gelatinolytie activity, but lower esterolytic activity, than adult venom. Neonate and adult D. acutus venoms showed identical phosphomonoesterase, LAO and fibrinolytic activities. Neonate N. atra venom had higher phosphomonoesterase and LAO activity, but lower 5＇ nucleotidase, PLA2, hyaluronidase and Ache activities than adult venom. Neonate and adult N. atra venoms showed similar gelatinolytic activity. Further, age-dependent immunoreactivity was found in both species, and cross-reactions between homologous venoms and antiserums were closely related to venom composition. We speculate that age-related variation in venom characteristics is possibly driven by evolutionary forces associated with ontogenetic shifts in dietary habits, competition and predation pressure.

Identifying Intraspecific Variation in Venom Yield of Chinese Cobra(Naja atra) from Ten Populations in China's Mainland

Detailed information on venom yield is helpful in preparing antivenoms and treating snakebites, but such information is lacking for many species of venomous snakes. The Chinese cobra(Naja atra) is a large sized, venomous snake commonly found in southeastern China, where it causes a heavy burden of snakebites. To examine the effects of various factors(morphology, sex, age, season, and geographical origin) on the venom yield in this snake, we collected venom samples of 446 individuals(426 adults and 20 neonates) from 10 populations of N. atra over an eightyear period. We used two variables, lyophilized venom mass(venom yield) and solid content of venom(% solids), to quantify the venom yield. We used linear regression analysis to check if venom yield was related to morphological factors, one-way ANOVA and one-way ANCOVA to detect the sexual, ontogenetic, and geographic variation in venom yield, and repeated-measures ANOVA to examine seasonal shifts in venom yield. Our results indicate that venom yield of N. atra is positively related to the morphological traits examined, with male snakes expelling more venom than females. Venom yield in N. atra was age-related, with elder snakes always expelling more venom than younger ones. Geographic variation in venom yield was also observed, while seasonal variation was not. The solid content of venom was lower in males than in females, but this was not related to morphology, season, age, or geography. Our findings suggest that venom yield in N. atra is influenced by multiple factors, as well as by the interactions among these factors.

Unhatched and Hatched Eggshells of the Chinese Cobra Naja atra

Changes in structure and composition of the eggshell resulting from embryonic mobilization of minerals from the eggshell are found in all oviparous reptiles studied thus far. In this study, we measured samples of unhatched and hatched eggshells of the Chinese cobra Naja atra to determine the percentage of ash and the phase composition of calcium carbonate. The mean percentage of ash was significantly higher in unhatched eggshells （24.6%） than in hatched eggshells （22.3%）. The dominant phase in unhatched eggshells was the calcite form of calcium carbonate. In addition to the peaks of calcite, a few small peaks were found to be caused by the aragonite and vaterite phases of calcium carbonate, implying that there are small amounts of aragonite and vaterite in the eggshell. The concentration of the various phases calculated from the intensity of the X-ray diffraction spectra allowed the estimation that percentages of calcite, aragonite and vaterite were about 92%, 4% and 4%, respectively. Hatched eggshells produced similar spectral characteristics as unhatched eggshells, with one exception. The dominant phase composition in the hatched eggshell was also calcite, but the amount of the aragonite phase had a marked increase. Our study adds evidence that embryonic mobilization of minerals from the eggshell may result in changes in structure of the eggshell.

Lineage Diversification and Niche Evolution in the Chinese Cobra Naja atra (Elapidae)

Theroleof nicheevolution(niche conservatism or niche divergence)in lineage diversification is a poorly studied area.The Chinese cobra Naja atra(Elapidae)has diverged into three lineages:Lineage E in eastern China,Lineage S in southern China and Vietnam,and Lineage W in western China.However,whether the ecological niche is conserved or divergent among these three lineages is unknown.In the present study,we used ecological niche models in geographical space to study the ecological differences among lineages.We compared the niche overlap in environmental space to test niche conservatism and niche divergence.Our results showed that the three lineages of N.atra shared an ecological niche space between Lineages E and S/W,with the climatic niches of Lineages S and W representing a specialized fraction of the climatic niche of Lineage E.We speculated that the niche divergence between Lineages S and W was a consequence of geographical barriers limitinggeneflow.Ourstudyprovides evidence for lineage diversification associated with both geographical isolation and climatic niche evolution,suggesting that early niche divergence between Lineages S and W,followed by niche conservatism,causes niche divergence among lineages.

Characterization of multidrug-resistant Klebsiella pneumoniae isolated from the Chinese cobra Naja atra in a Beijing suburb

The emergence and spread of antibiotic resistance genes among Bacteria are a serious threat to global health.Their occurrence in animals which are in contact with humans is also important.The Chinese cobra(Naja atra,Elapidae),though a highly venomous species,is appreciated as food and as a source of materials used in traditional Chinese medicine.We are here reporting the isolation of multidrug-resistant Klebsiella pneumoniae(Enterobacteriaceae)from the lung of Naja atra,obtained from a snake farm in a Beijing suburb.Our study analyzed,using gene sequencing,the occurrence of antibiotic resistance genes(ARGs)in three K.pneumoniae isolates from two snakes.In addition,bacterial clones were identified by biochemical tests and phylogenetic analysis.Tests of antimicrobial susceptibility showed that all K.pneumoniae isolates were resistant to a host of antibiotics(piperacillin,cefazolin,gentamicin,tetracycline,doxycyclin,ciprofloxacin,levofloxacin,lomefloxacin,ofloxacin,norfloxacin,nalidixic acid,chloramphenicol,nitrofurantoin,sulfamethoxazole,and sulfamethoxazole/trimethoprim)but were susceptible to cefotaxime,cefixime,aztreonam,bramycin,amikacin,kanamycin,netilmicin,and streptomycin.Eighteen ARGs were detected in total DNA extracted from the isolates.Results showed three quinolone resistance genes(oqxA,oqxB,qnrB),the gyrA gene that confers resistance to beta-lactam antibiotics,and the emerging aac(3)-II gene that confers resistance to aminoglycosides.K.pneumoniae is an important opportunistic human pathogen and the emergence of multidrug-resistant K.pneumoniae in N.atra suggests the increasing risk of pathogen transmission between humans,livestock,and wildlife.Given the close association between foodborne pathogenic microorganisms and humans,it is key factor to identify these antibiotic resistance genes profile thereby minimize the risk of K.pneumoniae transmission.

中华眼镜蛇咬伤的治疗进展

中华眼镜蛇咬伤多见于中国东南部,主要表现为局部肿胀、疼痛和组织坏死。尽管传统内科治疗如使用抗蛇毒血清中和毒素,但对局部损伤恢复作用有限,往往需要外科干预治疗。院前处理包括快速识别、肢体制动、清洁伤口和迅速就医。院内治疗有清创术、局部负压封闭引流术(VSD)、植皮术和皮瓣移植术等治疗方法,以减少感染,促进创面修复和愈合。围手术期的对症支持、中医和功能康复治疗,对毒蛇咬伤患者的恢复有积极作用,可减少致残率和改善患者预后。

中华眼镜蛇细胞毒素致小鼠局部皮肤溃疡模型的构建研究

目的:探讨不同部位皮下注射中华眼镜蛇细胞毒素(CTX)致小鼠局部皮肤溃疡模型构建。方法:将57只小鼠随机分为5组:CTX组(n=18)、粗毒组(n=18)、CTX前肢组(n=7)、CTX后肢组(n=7)和CTX背部组(n=7),饲养21天观察皮肤溃疡坏死面积及病理变化。结果:CTX组造模成功率100%,粗毒组造模成功率仅5.5%。选择四肢皮肤注射CTX造模较背部成功率更高,不同注射部位溃疡伤口均可见典型炎症坏死表现。结论:CTX皮下注射小鼠后肢可构建成功率高,稳定,典型的中华眼镜蛇毒素中毒局部皮肤溃疡模型。

眼镜蛇(Naja naja atra)神经毒素与心脏毒素分离纯化研究进展

眼镜蛇神经毒素与心脏毒素分别具有显著的镇痛活性和抗肿瘤活性。这两种毒素同源性高,等电点,分子质量以及蛋白结构都很相似,给分离纯化造成一定难度。获得单一成分的神经毒素和心脏毒素是理化性质分析、药理活性研究和临床应用的前提,文章综述了多年来舟山眼镜蛇蛇毒中神经毒素与心脏毒素的分离纯化研究进展。

富血小板血浆与贫血小板血浆治疗中华眼镜蛇细胞毒素致小鼠皮肤溃疡的效果比较

目的 比较富血小板血浆(PRP)与贫血小板血浆(PPP)治疗中华眼镜蛇细胞毒素致小鼠皮肤溃疡的效果。方法 将中华眼镜蛇细胞毒素造模成功的30只中小鼠随机分为对照组(n=10)、PRP组(n=10)、PPP组(n=10),造模后第9天各组分别给予单纯生理盐水、PRP、PPP治疗,比较3组小鼠皮肤溃疡面积、溃疡创面愈合率、创面面积差绝对值与HE染色的病理改变。结果 低速组的PRP、PPP符合工作浓度标准。PRP凝胶、PPP凝胶治疗小鼠溃疡愈合时间可缩短3 d。PRP组与PPP组创面愈合率及3 d内面积差绝对值均大于对照组(均P<0.05)。镜下见PRP凝胶、PPP凝胶可减少炎症细胞浸润,促进小鼠上皮细胞再上皮化等病理改变。结论 PRP凝胶、PPP凝胶均可促进中华眼镜蛇伤溃疡皮肤修复,缩短愈合时间。

中华眼镜蛇神经毒素相转化水凝胶微针的制备及其初步镇痛作用研究

目的以中华眼镜蛇神经毒素为模型药物制备相转化水凝胶微针,并对其质量以及镇痛作用进行评价。方法采用两步法制备神经毒素相转化水凝胶微针。利用光学显微镜和扫描电子显微镜对微针形貌进行考察,筛选最佳处方,在最佳处方下对微针的载药量、机械性能、体外溶胀、体外透皮等进行综合评价。最后通过热板实验考察神经毒素相转化水凝胶微针的镇痛作用。结果针尖部位最佳处方为聚乙烯醇/葡萄糖水溶液(84/16,W/W),背衬层最佳处方为聚乙烯醇水溶液(25%,W/W)。光学显微镜和扫描电镜下微针针体垂直,排列整齐,矩阵清晰;HE染色结果表明微针可以刺入皮肤表层为药物递送提供通道。180 min后溶胀率达到210%;载药量可达到23.4%;12 h后体外释放率接近85%;6 h后微针的体外透皮率达到80%,而中华眼镜蛇神经毒素水溶液几乎没有穿过皮肤。药效学实验表明,神经毒素相转化水凝胶微针具有镇痛作用。结论本实验制备的微针可以刺破皮肤,在体内可以达到镇痛的疗效,为蛋白类药物剂型的开发提供了新的思路和策略。

应用仿生亲和层析从广东产舟山眼镜蛇(Naja naja atra)蛇毒中分离纯化镇痛多肽

研究眼镜蛇毒镇痛组分的分离纯化方法,为寻找镇痛效果好,而无成瘾性的新型镇痛药物,在合成出可用于亲和吸附眼镜蛇毒心脏毒素层析介质的基础上,结合阳离子交换UND SphereTMS,凝胶过滤Sehpadex G-50,疏水层析Phenyl SepharoseTM CL-4B,从广东产舟山眼镜蛇毒中分离出镇痛多肽。此外,将凝胶过滤Sehpadex G-50应用于仿生亲和层析之前检测分离效果。得出在一步仿生亲和层析后,用疏水层析的方法分离效果更佳,仿生亲和层析适于第一步分离。

蛇毒神经生长因子的分离纯化及鉴定

采用ＳｅｐｈａｄｅｘＧ５０、ＣＭ－Ｃｅｌｕｌｏｓｅ３２柱层析，从中华眼镜蛇中分离出神经生长因子（ＮｅｒｖｅＧｒｏｗｔｈＦａｃｔｅｒ，ＮＧＦ）。经ＳＤＳ－聚丙烯酰胺凝胶电泳及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ法证明所得到的ＮＧＦ为单一组分，相对分子质量约为１．３×１０４．经凝胶等电聚焦电泳测得ＮＧＦ等电点ＰＩ约为７．０左右．经ＨＰＬＣ及电泳图像分析系统测得纯度为９８％．此ＮＧＦ经８ｄ鸡胚背根神经节体外培养证明，具有促进神经纤维生长的活性．将所纯化的ＮＧＦ免疫大白兔获得抗血清。

舟山眼镜蛇毒细胞毒素的分离纯化及其体外抗肿瘤活性

目的从眼镜蛇毒中分离纯化细胞毒素 F(CTX F)并鉴定其活性。方法应用凝胶过滤、离子交换柱色谱及疏水柱色谱等方法从舟山眼镜蛇毒中分离纯化CTX F ,以SRB法观察CTX F对体外培养癌细胞的杀伤作用。结果眼镜蛇毒粗毒经凝胶过滤获得 4个蛋白峰 ,将CTX所在第Ⅳ峰用阳离子交换柱色谱获A、B、C、D、E、F和G等7个组分 ,其中E、F和G具CTX活性 ,将F组分再经凝胶过滤和疏水色谱进一步纯化得不含磷酯酶A2 (PLA2 )的CTX纯品 ,暂定名为CTX F ,它对多种癌细胞株有杀伤作用。结论应用凝胶过滤、离子交换和疏水色谱等方法可从眼镜蛇毒中获得不含PLA2 的CTX 。

分离纯化眼镜蛇蛇毒因子的一种经济方法

将较大批量的眼镜蛇粗毒依次通过ＳｅｐｈａｄｅｘＧ７５，ＤＥＡＥ－ｃｅｌｌｕｌｏｓｅＤＥ５２和Ｂｉｏ－ＧｅｌＰ２００柱层析可高效地分离纯化眼镜蛇蛇毒因子（ＣＶＦ），５０ｇ粗毒可制备电泳纯ＣＶＦ９６０ｍｇ，产率大大高于既往文献报道，整个制备过程仅需时数日。通过ＳＤＳ聚内烯酰胺凝胶电泳测得其分子量为２２６０００，其溶血活性从抗补体活性均与文献报道相近。所以，本实为一种经济，高效的实验室大量制备ＣＶＦ的方法。