一例猪繁殖与呼吸综合征的RT-PCR诊断与防制

2008年3月,新乡市某猪场发生了以一种经产母猪和初产母猪流产、死产、产木乃伊胎、死胎及弱仔为特征的疾病。采集流产胎儿和死胎的肺、肝、脾脏等病料,制悬液,取上清,进行细胞培养,观察细胞病变。另根据猪繁殖与呼吸综合征病毒美洲株VR-2332 ORF7基因序列设计一对特异引物,提取流产胎儿的肺、肝等组织中PRRSV的RNA,经RT-PCR扩增,检测猪繁殖与呼吸综合征病毒为阳性,结合病史和临床症状,诊断为猪繁殖与呼吸综合征。通过对该猪场采取紧急接种疫苗、彻底消毒等综合防制措施,控制了该病的流行与蔓延。     

Effect of Yiqi Huoxue Jiedu Fang on the growth,metastasis and angiogenesis of Lewis lung carcinomas

AIM: To study the effect of Yiqi Huoxue Jiedu Fang (YHJ), composed of ginsenoside, penex notogingseng and berberin, on tumor growth and metastasis and to explore its mechanism.METHODS: Murine Lewis lung carcinoma transplant model was established and mice were treated with YHJ by intraperitoneal injection. After 10 days, the inhibitory rate of tumor, pathology of tumor and PCNA of tumor cells were detected. After 20 days, numbers of metastatic foci on lung surface and microvessel density (MVD) were determined. Expression of VEGF in tumor and serum were also analyzed by immunohistochemical test and ELISA, respectively. RESULTS: YHJ reduced the weight of tumor and the amount of metastatic foci. The inhibitory rates of tumor at high and low dose of YHJ (24 mg·kg_^-1·d_^-1, 12 mg·kg_^-1·d_^-1) were 48.29% and 37.26%, and the number of metastatic foci was 1.67 and 3.50, while control was 6.44. Furthermore, PCNA of tumor cells, MVD of tumor and VEGF expression in serum and tumor were decreased in YHJ treatment goup as compared with control. CONCLUSION: YHJ remarkably inhibits Lewis lung carcinoma growth and metastasis in mice. Its mechanism may be related to inhibition of angiogenesis.     

西洋参和蜂花粉细粉及超微粉组织特征扫描电镜观察

将西洋参和蜂花粉分别进行常规粉碎和超微粉碎,利用扫描电镜对2种不同粉碎方式得到的粉末进行观察,比较2种中药细粉和超微粉形态学上的差异。结果表明：超微粉末颗粒变小,大小均匀,电镜下观察,绝大多数细胞壁破裂,基本无完整细胞存在。     

余甘子精粉的体外抗氧化活性分析

The antioxidant activities in vitro of refined powder of Phyllanthus emblica L. were detected by methods based on chemical measurement and cell modeling. The results of anti-peroxidation ability and iron reduction ability trials showed that the refined powder of P. emblica L. had strong ability of anti-peroxidation, and it increased with the increasing of concentration with a clear dose-response relationship. When the concentration reached 800 μg·mL^-1, the ability of anti-peroxidation was almost equal to the positive control VC with the same concentration. Meanwhile, it also had iron reduction ability that accompanied by significant dose-response relationship. By in vitro cell culture method, H₂O₂ was used to induce SH-SY5Y cells damages to establish the cell model of oxidative damage. The author detected the effect of different concentrations of powder on cell survival rate for the model cells by MTT assay. The results indicated that refined powder could show better protective effect to oxidative damaged SH-SY5Y cells in the range of 600 1 200 μg·mL^-1. When the concentration reached 1 200 μg·mL^-1, the cell survival rate was the highest and refined powder exhibited the best antioxidant activity. Both methods proved the strong antioxidant activities of refined powder of P. emblica L. in vitro.     

酵母富集微量元素铜的研究

酵母细胞具有富集某些微量元素的能力,它能将富集的无机态铜转化为有机态形式。采用富集培养方法,从22株酵母菌株中筛选出2株富集高含量铜的酵母菌,对其铜含量进行测定。结果表明：铜含量分别达到7．7mpdkg和7．9mpdkg,适宜的铜盐为氯化铜,其添加的离子浓度为90mpdkg。     

不同纤维来源饲粮和细胞壁降解酶对猪肠道微生物菌群多样性的影响

本试验旨在利用末端限制性片段长度多态性技术研究不同纤维来源饲粮和细胞壁降解酶对猪肠道微生物菌群多样性及其组成结构的影响。试验选用8头平均体重为(35.0±2.5)kg的"杜×长×大"三元杂交生长猪,统一安装回肠末端T型瘘管,随机分为4组,每组2个重复。试验分4期,每期每组猪按4×4拉丁方设计饲喂小麦麸、小麦麸加酶、大豆皮和大豆皮加酶4种试验饲粮之一。每期预试期15 d,正试期6 d。试验结果表明:1)大豆皮饲粮组猪回肠食糜微生物的多样性显著高于小麦麸饲粮组(P0.05)。2)小麦麸饲粮组显著提高猪回肠食糜中普氏菌属(Prevotella)和乳酸杆菌属(Lactobacillus)的丰度(P0.05),而大豆皮饲粮组则显著提高猪回肠食糜中瘤胃球菌属(Ruminococcus)及粪便中拟杆菌属(Bacteroides)、毛螺菌属(Lachnospira)的丰度(P0.05)。3)添加细胞壁降解酶显著提高各饲粮组猪回肠食糜中Lachnospira、真细菌属(Eubacterium)及粪便中Lachnospira的丰度(P0.05),但也同时降低了回肠食糜中Lactobacillus、Prevotella及粪便中Lactobacillus、Bacteroides和克雷伯氏菌属(Klebsiella)的丰度(P0.05)。综上所述,纤维饲粮可显著提高猪肠道内非淀粉多糖降解菌的丰度,而细胞壁降解酶则可选择性改变肠道微生物菌群的多样性及其组成。     

平邑甜茶延长根和吸收根抗凋亡基因的表达差异及其对2,4-D 的响应

 以3 年生平邑甜茶[Malus hupehensis（Pamp）Rehd. var. pinyiensis Jiang]盆栽树为材料,通过2,4-D 水溶液灌根处理,探讨了新根（延长根和吸收根）细胞死亡及其抗凋亡基因的表达特征。结果表明,在60 mg · L^-1 2,4-D 处理后的70 d 内,新根细胞死亡量和类caspase3/7（半胱天冬酶）活性先上升后下降,ATP 含量和根系活力的变化与之相反。平邑甜茶抗凋亡基因MhBAG、MhBI-1 与MhHSP70 在新根中均受2,4-D 诱导,随着2,4-D 处理时间的延长,MhBAG 基因的表达水平先下降后逐渐升高,MhBI-1 与MhHSP70 则先升高后下降。在2,4-D 处理下,吸收根细胞死亡量与根系活力的变化幅度明显高于延长根,吸收根MhBI-1 与MhHSP70 表达量的早期升高幅度大于延长根,表明吸收根细胞对2,4-D 的敏感性高于延长根;但在基因表达高峰时,延长根抗凋亡基因表达量的升高倍数明显高于吸收根,暗示延长根比吸收根有更强的抗凋亡能力。     

Establishment,characterization, virus susceptibility and transfection of cell lines from cobia,Rachycentron canadum (L.), brain and fin

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate‐buffered saline containing 0.25% trypsin at 25 °C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 °C in Leibovitz‐15 medium containing 10% foetal bovine serum. The cells have been sub‐cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.     

RNA干扰技术抑制新家坡石斑鱼虹彩病毒感染细胞多肽ICP46与绿色荧光蛋白融合基因的表达

新加坡石斑鱼虹彩病毒（singapore grouper iridovirus,SGIV）是一种严重的能引起全身性疾病的病原体,对石斑鱼养殖造成了重大的经济损失。将含有SGIV感染细胞多肽ICP46（infected cell polypeptides 46）基因的真核表达载体pEGFP-ICP46转染到胖头鲤细胞（fathead minnow cells,FHM）中进行融合表达,用荧光显微镜观察到ICP46-GFP融合蛋白主要分布于FHM细胞的细胞质中。根据SGIV-ICP46的序列,设计并体外化学合成了特异性干扰SGIV-ICP46的siRNA（siRNA-ICP46）,与pEGFP-ICP46共转染到FHM细胞中,通过荧光显微镜观察不同时间点荧光强度的变化。转染后24~48 h,实验细胞（共转染siRNA-ICP46和pEGFP-ICP46）和阳性对照细胞（共转染siRNA-GFP和pEGFP-ICP46）中的荧光微弱,发荧光的细胞数量较阴性对照（共转染siRNA-Negative和pEGFP-ICP46）少70%左右,但其后实验细胞和阳性对照细胞的荧光强度开始增强,在转染后72 h其与阴性对照组已差别不大。说明体外化学合成的siRNA-ICP46转染后24~48 h可有效抑制FHM细胞中外源导入SGIV-ICP46基因的表达。     

橘色双冠丽鱼黑色素细胞和黄色素细胞的分离、培养与鉴定

为探讨橘色双冠丽鱼(Amphilophus citrinellus)黑色素细胞和黄色素细胞的分离、培养与鉴定方法,观察鱼类色素细胞生长特征,本研究以橘色双冠丽鱼的尾鳍、鳞片、皮肤组织为材料,在25℃~28℃条件下,分别在胰蛋白酶溶液和胶原酶溶液中消化6 h和12 h,可有效分离出色素细胞团,细胞悬液经气孔尼龙网过滤后,于35%-45%-55% Percoll试剂中梯度分离和收集黄色素细胞和黑色素细胞。采用K-SFM培养基进行细胞原代培养和细胞纯化,抑制成纤维细胞和角朊细胞的生长,用十四烷酰佛波醇乙酸酯(TPA)、双抗、bFGF的DMEM培养基进行色素细胞传代培养;运用巴染色和透射电镜观察细胞形态,并用分子标记技术进行细胞鉴定。结果显示,细胞分离时,黑色素细胞位于Percoll试剂45%和35%浓度层之间,黄色素细胞位于Percoll试剂35%浓度层上,2种色素细胞均凝聚成絮状。透射电子显微镜观察发现,黑色素细胞内部含大量黑色素小体,而黄色素细胞内部含喋呤体和类胡萝卜素囊泡;黑色素细胞左旋多巴(L-DOPA)染色呈阳性;取第10代色素细胞进行体色相关基因黑皮素1受体(melanocortin receptor 1, MC1R)、酪氨酸酶(tyrosinase, TYR)和牛内皮素受体B(endothelin receptor B, EDNRB) PCR检测,显示基因扩增条带特异性强,表明获得的2种色素细胞具有良好的生物学活性。本研究建立了橘色双冠丽鱼黄色素细胞和黑色素细胞的分离培养与鉴定方法,为进一步开展鱼类体色细胞分化和体色形成的分子机理研究提供了细胞模型。