蝎毒对Hela细胞的体外药理学研究

以东亚钳蝎毒素和梁氏异蝎毒素来研究蝎毒对Hela细胞增殖的影响,MTT法试验结果:20,40,60μg/m L的2种蝎毒对Hela细胞均有抑制增殖的作用,具有浓度和时间的依赖关系,随着浓度升高和时间的延长,Hela细胞增殖的抑制作用增强。淋巴细胞转化试验结果:20,40,60μg/m L的2种蝎毒均能促进淋巴细胞转化,并且梁氏异蝎毒素的作用更强。乳酸脱氢酶(LDH)释放试验结果:2种毒素均能促使LDH的释放,随着蝎毒浓度的增加和时间的延长,Hela细胞溶液上清液中LDH释放量逐渐增加。由此证明,这2种蝎毒均可抑制Hela细胞增殖,有望成为一种抗肿瘤药物。     

p21基因在牛卵成熟中的表达检测及其真核表达载体构建

本研究旨在检测p21基因在牛卵成熟过程中的表达,并构建其真核表达载体。首先利用RT-PCR和免疫荧光染色对不同成熟阶段牛卵内p21的mRNA和蛋白表达进行检测。然后从牛成纤维细胞中克隆了p21基因并构建pVenus-P21真核表达载体。经脂质体2000介导重组质粒pVenus-P21转染Hela细胞,通过荧光显微镜观察、RT-PCR检测,确认重组质粒在Hela细胞内的表达及定位。最后应用体外转录试剂盒将p21-venus体外转录为cRNA,并注射牛卵母细胞,荧光显微镜下观察其表达及定位。结果显示,在牛卵体外成熟过程中,各时期均存在p21基因mRNA及蛋白的表达。构建的真核表达载体pVenus-P21能够在Hela细胞中正确表达及定位。p21-venus cRNA显微注射牛卵后其融合蛋白也能实现准确定位,为研究P21在卵母细胞成熟以及胚胎发育过程中的作用奠定了基础。     

孕酮对奶牛子宫内膜上皮细胞凋亡和容受性的影响

为研究孕酮对奶牛早期胚胎附植的影响,本试验使用不同质量浓度孕酮处理奶牛子宫内膜上皮细胞,CCK8检测细胞活力,流式细胞术分析细胞凋亡,实时荧光定量RT-PCR和Western blot检测凋亡相关基因(p53、Cyto-c、Bax、Bcl-2、Caspase-9、Caspase-8和Caspase-3)和容受性相关基因(TLR-4、NF-κB、IL-6、VEGF、LIF和EGF)的mRNA和蛋白表达变化。结果显示：10,25,50,100μg/L孕酮对细胞活力未见影响;10,100μg/L孕酮对细胞凋亡未见影响,对细胞p53、Cyto-c、Bax、Bcl-2、Caspase-9、Caspase-8和Caspase-3的mRNA及蛋白的表达未见影响,但显著下调了TLR-4 mRNA和蛋白表达水平,显著上调了LIF和VEGF mRNA和蛋白表达水平。结果表明孕酮不影响奶牛子宫内膜上皮细胞的凋亡,但能提高子宫容受性,降低免疫反应。     

马立克病病毒致瘤蛋白Meq转录调节鸡p21基因的表达

为研究马立克病病毒(Marek's disease virus,MDV)致瘤蛋白Meq对细胞周期蛋白依赖性激酶抑制蛋白p21表达的调节作用,采用RTPCR、Western blot方法检测Meq蛋白表达在鸡胚成纤维细胞(Chicken embryo fibroblasts,CEF)中对p21 mRNA和蛋白水平的影响。然后,构建p21启动子的荧光素酶报告载体,检测Meq蛋白对p21的启动子活性的影响。最后,分别用MDV Md5强毒株和CVI988/Rispens疫苗毒株感染CEF细胞,利用Western blot检测MDV感染后p21蛋白水平的变化。结果:过表达Meq上调p21的mRNA和蛋白表达水平,Meq能够激活p21基因启动子活性,MDV感染细胞中p21蛋白的表达水平也有明显升高。这些发现为了解Meq在MDV感染中的作用机理奠定了基础。     

大蒜新素减少Rac1/Cdc42、MMP2表达抑制Hela细胞转移能力的研究

为探讨大蒜新素对Hela细胞转移能力的抑制作用,采用100μmol/L的大蒜新素处理Hela细胞24h(以等量的PBS为对照),Western blot检测Rac1和Cdc42蛋白表达,半定量RT-PCR法检测MMP2mRNA的表达。结果显示,大蒜新素处理组Hela细胞Rac1和Cdc42蛋白表达明显降低(P0.05),MMP2mRNA表达明显下降(P0.01)。说明大蒜新素抑制Hela细胞的转移能力可能与减少Rac1/Cdc42、MMP2的表达密切相关。     

硒增强二十二碳六烯酸在脂多糖诱导巨噬细胞炎性反应中的抗炎作用

为了探究硒是否影响二十二碳六烯酸(DHA)在脂多糖(LPS)诱导巨噬细胞炎性反应中发挥的抗炎作用。小鼠巨噬细胞系RAW264.7细胞分别经10μg/m L DHA、10μg/m L DHA+0.05μmol/L亚硒酸钠、1μg/m L LPS、10μg/m L DHA+1μg/m L LPS、10μg/m L DHA+1μg/m L LPS+0.05μmol/L亚硒酸钠诱导24 h,同时设置无添加的正常组。采用半定量反转录PCR检测细胞肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和白细胞介素10(IL-10)mRNA表达量,酶联免疫吸附测定(ELISA)法测定培养液上清液中TNF-α、IL-1β、IL-6和IL-10含量。结果显示,添加亚硒酸钠(0.05μmol/L)不仅显著或极显著增强了DHA(10μg/m L)对LPS(1μg/m L)诱导RAW 264.7细胞中促炎细胞因子TNF-αmRNA表达(P0.05)和IL-1β生成(P0.01)的抑制作用,还极显著增强了DHA(10μg/m L)对LPS(1μg/m L)诱导RAW264.7细胞中抗炎细胞因子IL-10 mRNA表达的促进作用(P0.01)。由此提示,硒可增强DHA在LPS诱导巨噬细胞炎症反应中的抗炎作用。     

不同生物素水平对3T3-L1脂肪细胞脂肪分解及相关基因表达的影响

为研究不同生物素水平对3T3-L1脂肪细胞脂肪分解及相关基因表达的影响,本试验利用三联诱导法将3T3-L1细胞经诱导分化为3T3-L1脂肪细胞。当3T3-L1脂肪细胞密集后分别采用0(对照组)、0.2、0.5、1.0μmol/L生物素处理,分别在12、24与48h时检测细胞上清液中甘油释放量、脂肪甘油三酯水解酶(ATGL)、激素敏感性脂肪酶(HSL)、脂滴包被蛋白A(perilipin A)及脂肪分化相关蛋白(ADRP)相对表达量。结果显示,在12h时,1.0μmol/L组甘油释放量、ATGL基因mRNA相对表达量均极显著低于对照组(P0.01),0.5、1.0μmol/L组HSL基因mRNA相对表达量显著低于对照组(P0.05),1.0μmol/L组perilipin A、ADRP基因mRNA相对表达量均极显著高于对照组与0.2μmol/L组(P0.01);在24h时,各试验组甘油释放量、1.0μmol/L组ATGL基因mRNA相对表达量均极显著低于对照组(P0.01),0.5、1.0μmol/L组perilipin A基因mRNA相对表达量极显著高于对照组与0.2μmol/L组(P0.01);1.0μmol/L组ADRP基因mRNA相对表达量极显著高于其他组(P0.01);在48h时,1.0μmol/L组甘油释放量、ATGL基因mRNA相对表达量均极显著或显著低于对照组(P0.01;P0.05),1.0μmol/L组perilipin A基因mRNA相对表达量显著高于对照组与0.2μmol/L组(P0.05),0.5、1.0μmol/L组ADRP基因mRNA相对表达量极显著高于对照组(P0.01)。综上所述,生物素可通过促进perilipin A与ADRP表达,同时抑制ATGL与HSL表达,达到抑制脂肪分解的效果,且浓度为1.0μmol/L时效果最佳。     

防御素基因在乌金猪与约大乌猪组织中的表达及东亚飞蝗抗菌活性物质对防御素基因表达的调节作用

本试验旨在检测β防御素-1(p BD-1)、β防御素-2(p BD-2)和β防御素-3(p BD-3)基因在乌金猪和具有乌金猪血缘的约大乌猪不同组织中的表达,并探讨东亚飞蝗抗菌活性物质(LAAS)对猪胎儿皮肤成纤维细胞(PFSF)免疫应激参数和防御素(p BDs)基因表达的调节作用。采用实时荧光定量PCR方法检测2月龄乌金猪与约大乌猪心脏、肝脏、肺脏、肾脏、空肠、回肠、十二指肠、皮肤、肌肉、胰腺、睾丸或卵巢这12种组织中p BD-1、p BD-2和p BD-3基因的表达差异;采用培养至第4~5代的PFSF,通过脂多糖(LPS)诱导建立免疫应激模型,在DMEM/F12培养液中添加不同浓度(0、75、150、300、600和1 200μg/m L)的LAAS,考察LAAS对PFSF免疫应激参数和p BD-1、p BD-2、p BD-3基因表达的影响。结果表明:1)乌金猪血清溶菌酶(LSZ)活性、免疫球蛋白G(Ig G)含量显著高于约大乌猪(P0.05),血清一氧化氮(NO)含量极显著低于约大乌猪(P0.01)。2)约大乌猪大多数组织中p BD-1、p BD-2和p BD-3基因的表达量高于乌金猪;2个品种猪的p BD-1和p BD-3基因均在皮肤和卵巢中表达量较高,p BD-2基因在肝脏和肾脏中表达量较高。3)300μg/m L LAAS极显著降低了培养液中乳酸脱氢酶(LDH)活性(P0.01);LAAS极显著增加了培养液中NO含量(除75μg/m L组正常细胞外)(P0.01),正常细胞和应激细胞均在LAAS浓度为600μg/m L时达到最高;LAAS浓度为0、75和150μg/m L时应激细胞一氧化氮合酶(NOS)活性显著或极显著高于正常细胞(P0.05或P0.01);添加1 200μg/m L LAAS可极显著降低培养液中的LSZ活性(P0.01),LAAS浓度为150、300、600和1 200μg/m L时应激细胞与正常细胞中LSZ活性差异显著或极显著(P0.05或P0.01)。4)应激细胞p BD-1、p BD-2和p BD-3基因的表达量均高于正常细胞;当LAAS浓度为150μg/m L时,p BD-1、p BD-2和p BD-3基因的表达量最高。结果提示,乌金猪和约大乌猪防御素基因的表达存在明显的品种差异性和组织特异性,适宜浓度的LAAS能够降低PFSF中LDH活性,提高NO含量及NOS、LSZ活性,上调应激细胞与正常细胞防御素的表达量。     

漏芦通过TLR4/NF-κB通路抑制脂多糖诱导的小鼠乳腺上皮细胞炎症反应

为探讨漏芦醇提物（RUEE）对脂多糖（LPS）诱导的小鼠乳腺上皮细胞（HC11细胞）的抗炎作用及机制,试验利用RUEE(80μg/mL)、LPS(1μg/mL)单独处理以及RUEE(80μg/mL)+LPS(1μg/mL)共处理HC11细胞,采用荧光定量PCR检测炎性细胞因子及Toll样受体4(TLR4)mRNA表达水平,采用Western blotting检测核转录因子κB(NF-κB)通路关键因子的蛋白表达量。结果表明：LPS诱导可明显提高HC11细胞肿瘤坏死因子-α(TNF-α)、环氧合酶-2(COX-2)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)的mRNA表达水平（P<0.05）;明显上调TLR4 mRNA表达及NF-κB p65和NF-κB抑制蛋白α(IκBα)的磷酸化水平（P<0.05）。RUEE预处理可显著降低LPS诱导的HC11细胞TNF-α、COX-2、IL-6、IL-1β的mRNA表达（P<0.05）;显著下调TLR4 mRNA表达及NF-κB p65和IκBα的磷酸化水平（P<0.05）。由此可知漏芦醇提物可通过抑制TLR...     

伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒(PRV)在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞(3D4/21)p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.