狂犬病病毒糖蛋白转基因小鼠对该病毒形成免疫耐受

对带有绵羊ＭＴ启动子－狂犬病病毒糖蛋白基因的（ＭＴ－Ｒｇｐ）的ＴｇＮ（ｏＭＴ－Ｒｇｐ）２Ｌｇｅ、ＴｇＮ（ｏＭＴ－Ｒｇｐ）４Ｌｇｅ和ＴｇＮ（ｏＭＴ－Ｒｇｐ）６Ｌｇｅ３株转基因小鼠系（子代）进行了表达检测。ＥＬＩＳＡ结果表明，小鼠肝脏、肾脏中均有糖蛋白表达产物，以肝脏的表达量较高；对肝脏的免疫组化检测结果显示，糖蛋白分布在肝脏实质细胞，主要位于细胞膜上和胞浆内。对８只ＴｇＮ（ｏＭＴ－Ｒｇｐ）２Ｌｇｅ子代鼠一次接种灭活的狂犬病病毒８２０２株，接种前和接种后３周的血清抗体水平变化在转基因鼠和非转基因鼠之间有明显区别，非转基因鼠抗狂犬病病毒抗体水平明显升高，而转基因鼠抗体水平基本保持不变。经统计学分析，转基因鼠的抗体水平变化相差不显著，表明转基因小鼠对该病毒形成了部分免疫耐受性。     

Uncoupling proteins and energy expenditure in mice divergently selected for heat loss

The objective of this study was to determine whether variation in energy expenditure created by selection on heat loss is mediated by uncoupling protein-1 (UCP1) in brown adipose tissue. Divergent selection for heat loss developed lines of mice with high (MH) and low (ML) maintenance energy expenditure. Concentration of UCP1 mRNA in brown adipose tissue (BAT) was 93% greater in ML than in MH mice (P < 0.02). Two new lines of mice, KH and KL, were bred by backcrossing a UCP1 knockout gene into the MH and ML lines, respectively; KH and KL with both knock-out (-/-) and wild type (+/+) UCP1 genotypes were generated. At 13 wk of age, KH mice exhibited greater heat loss (166 kcal x kg(0.75) x d(-1)) than KL mice (126.4 kcalkg(0.75) x d(-1)) regardless of the UCP1 knockout (P < 0.0001). Concentration of UCP2 mRNA in BAT was 74% greater in UCP1 knockout mice (-/-) than in wild type (+/+; P = 0.0001). We conclude that response to selection for increased energy expenditure was not mediated by increased expression or function of UCP1.     

Performance of mouse lines divergently selected for heat loss when exposed to different environmental temperatures. II. Feed intake, growth, fatness, and body organs

Mouse populations differing in metabolic rate have been developed through selection for high (MH) and low (ML) heat loss, along with the unselected controls (MC). Objectives of the study were to compare the MH, ML, and MC lines for feed intake, growth, body fatness, and organ weights when reared at 12, 22, and 31 degrees C, and investigate potential line x environment interactions. Feed intake was recorded weekly from 3 to 9 wk of age, and BW at 3, 6, and 9 wk of age. Body fat percent and organ weights were measured at 9 wk of age. No line x environment interactions were detected for any of the traits measured. The MH mice consumed more feed than ML mice from 5 to 9 wk of age. Between 8 and 9 wk of age, MH mice consumed 13% more feed than the ML mice, but they were relatively leaner (14.45 vs. 16.32% body fat); MC mice were intermediate for both traits. Mice in the cold environment consumed the greatest amount of feed, and those in the hot environment consumed the least. Males consumed more feed than females, and the difference was greater in the cold than in the hot environment. No differences in BW were found between the lines. Mice in the 22 degrees C environment were heavier than their age-matched counterparts in the other two environments, and males were heavier than females at all ages. Relative to BW, the three lines had similar tail length, body length, and liver weight. Mice in the cold environment had heavier spleens and livers than those in the hot environment but relatively shorter bodies and tails; the normal environment was intermediate for these traits. Results from this study indicate that selection to decrease maintenance requirements did not produce mice with any less ability to grow and perform under an array of environmental temperatures.     

鼠源重组UBC13蛋白对脂多糖诱导的小鼠急性炎症的影响

试验旨在探讨鼠源重组UBC13蛋白对脂多糖(lipopolysaccharide,LPS)诱导的小鼠急性炎症的影响。将24只SPF雌性小鼠随机分成4组:PBS组,LPS模型组,重组UBC13蛋白高、低剂量组(分别为100和25μg/只),每组6只。LPS模型组与各蛋白剂量组腹腔注射20 mg/kg LPS,PBS组腹腔注射等体积PBS;注射结束1 h后,各蛋白组按相应剂量背部皮下多点注射重组UBC13蛋白,PBS组与LPS模型组注射等体积PBS。给予蛋白24 h后处死小鼠。收集小鼠肺脏、脾脏、胸腺及肝脏组织,计算脏器指数,HE染色观察组织病理学变化,实时荧光定量PCR检测肺脏、脾脏、胸腺和肝脏中IL-1β、TNF-α、IL-6 mRNA的相对表达量,以及肺脏中iNOS mRNA的相对表达量,综合评价鼠源重组UBC13蛋白对LPS诱导小鼠急性炎症的影响。结果显示,与PBS组相比,LPS模型组小鼠肺脏、脾脏及肝脏指数均显著或极显著升高(P<0.05;P<0.01),且肺脏、脾脏和肝脏组织均出现病理变化。实时荧光定量PCR结果显示,与PBS组相比,LPS模型组肺脏、脾脏、胸腺和肝脏中IL-1β、TNF-α、IL-6 mRNA相对表达量均极显著升高(P<0.01),肺脏中iNOS mRNA相对表达量也极显著升高(P<0.01);与LPS模型组相比,UBC13蛋白高剂量组肺脏、脾脏和肝脏中病理变化明显改善,肺脏、肝脏、脾脏中IL-1β、TNF-α、IL-6及肺脏中iNOS mRNA表达量均极显著降低(P<0.01);胸腺中TNF-αmRNA表达量显著降低(P<0.05),IL-6 mRNA和IL-1β表达量极显著降低(P<0.01)。表明鼠源重组UBC13蛋白可下调炎性因子的表达,从而改善LPS诱导的小鼠急性炎症反应。     

高脂饲粮和高碳水化合物饲粮对大鼠脂肪代谢的影响

本试验旨在以相同代谢能水平为基础,研究高脂饲粮和高碳水化合物饲粮对大鼠脂肪代谢的影响。选取48只8周龄雄性SD大鼠,随机分为3组,每组8个重复,每个重复2只。3个组分别饲喂高脂饲粮(HF组)、高碳水化合物饲粮(HC组)和对照饲粮(CON组),试验期9周。结果表明:1)与CON组相比,HF组、HC组大鼠的初重、末重、日增重均无显著差异(P0.05),日采食量极显著降低(P0.01),饲料转化效率极显著升高(P0.01)。2)各组大鼠的Lee’s指数、肾脏指数、胃指数、脾脏指数、肝脏指数无显著差异(P0.05)。3)与CON组和HF组相比,HC组大鼠血清甘油三酯、总胆固醇、高密度脂蛋白胆固醇含量显著或极显著升高(P0.05或P0.01);与HC组和CON组相比,HF组大鼠血清葡萄糖含量极显著增加(P0.01),血清尿素氮含量极显著降低(P0.01)。与CON组相比,HF组和HC组大鼠肝脏甘油三酯含量极显著升高(P0.01),HF组大鼠肝脏总胆固醇含量极显著升高(P0.01)。4)与CON组相比,HF组和HC组大鼠肝脏磷酸烯醇式丙酮酸激酶(PEPCK)mRNA表达量极显著增加(P0.01);与CON组和HC组相比,HF组大鼠肝脏胆固醇调节元件结合蛋白1(SREBP1)mRNA表达量极显著增加(P0.01)。综上所述,相同代谢能水平下,HF组和HC组大鼠体重无显著增加。HC组大鼠血脂升高;HF组大鼠血清葡萄糖、肝脏总胆固醇含量升高,血清尿素氮含量下降。HF组和HC组大鼠肝脏甘油三酯含量升高,且通过肝脏PEPCK和SREBP1基因表达调控途径影响脂肪代谢     

Identification of Equine Herpesvirus 5 in Horses with Lymphoma

Equine multinodular pulmonary fibrosis, equine herpesvirus 5 (EHV-5), and multicentric lymphoma were discovered in one patient. Review of gamma herpesvirus activity in humans revealed a propensity for lymphoproliferative disorders associated with infection. The objective was to determine the frequency of EHV-5 in lymphoma tissues and compare with the frequency found in the lymph nodes of clinically normal horses. Case control investigation of lymphoma-positive tissues and analysis via polymerase chain reaction (PCR) for EHV-5 was performed on 12 horses. Prospective collection and PCR analysis of lymph nodes (mesenteric or submandibular) for EHV-5 was performed on 21 control horses. Thirteen samples of lymphoma-positive tissues and fluid were submitted for PCR analysis for EHV-5. Of these, 67% was positive. In the control horse population, 14% was positive for EHV-5 (P = .004). Neoplastic samples positive for EHV-5 were classified as T-cell rich B-cell lymphoma (three), T-cell lymphoma (one), one was nondifferentiated, and two were not stained. Gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as Kaposi sarcoma and Burkitt lymphoma. This study reveals an increased frequency of EHV-5 (gamma herpesvirus) in horses diagnosed with lymphoma compared with healthy control horses. Although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia is unknown, EHV-5 may be an etiologic agent associated with the development of some types of equine lymphoma.     

Performance of mouse lines divergently selected for heat loss when exposed to different environmental temperatures. I. Reproductive performance, pup survival, and metabolic hormones

Mouse populations differing in metabolic rate have been developed through selection for high (MH) and low (ML) heat loss, along with the unselected controls (MC). Objectives of the study were to compare the MH, ML, and MC lines for reproductive performance, pup survival, and metabolic hormones when reared at 12, 22, and 31 degrees C, and to search for line x environment interactions. Conception and litter size were recorded on the parent generation mice introduced to the environments at 11 wk of age and bred after a 3-wk acclimatization period. Survival of pups (preweaning to 3 wk; postweaning from 3 to 9 wk of age) was measured with continuous exposure in the designated environment from birth to the time of measurement. Corticosterone, triiodothyronine (T3), and thyroxine (T4) serum concentrations were measured on the parent generation after producing litters and on the pup generation at 9 wk. No line x environment interaction was detected for conception rate, preweaning mortality, postweaning survival, pup weaning weight, or body temperature. There were no differences in conception rate among lines and environments. Environments affected survival of pups, but there were no line differences. Rectal body temperatures were greater for MH than ML mice, and MC mice were intermediate; body temperature of mice did not differ among the environments. Lines differed significantly in litter size only in the 22 degrees C environment. No significant line differences were found for serum corticosterone or serum T3 or T4. Line x environment interaction was detected only for litter size and for serum corticosterone concentration in dams. Contrary to the other two lines, ML dam performance relative to MH and MC was not affected negatively by either of the thermal environments. Results from this study do not raise concern that selection to decrease maintenance requirements will produce livestock with any greater liability to cope and perform under an array of environmental temperatures.     

Aberrant development of mammary glands, but precocious expression of beta-casein in transgenic females ubiquitously expressing whey acidic protein transgene

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.     

Interpretation of laboratory tests for canine Cushing's syndrome

Hypercortisolism (HC) is a common disease in dogs. This article will review the laboratory tests that are available for diagnosis of HC and laboratory tests for differentiating between causes of HC. An emphasis will be made on the clinical process that leads to the decision to perform those tests and common misconceptions and issues that arise when performing them. To choose between the adrenocorticotropic hormone (ACTH)-stimulation test and the low-dose dexamethasone suppression test (LDDST), the advantages and disadvantages of both tests should be considered, as well as the clinical presentation. If the index of suspicion of HC is high and other diseases have been appropriately ruled out, the specificity of the ACTH stimulation test is reasonably high with an expected high positive predictive value. Because of the low sensitivity, a negative result in the ACTH stimulation test should not be used to rule out the diagnosis of HC. The LDDST is more sensitive but also less specific and affected more by stress. A positive result on the urine cortisol:creatinine ratio does not help to differentiate HC from other diseases. A negative result on the urine cortisol:creatinine ratio indicates that the diagnosis of HC is very unlikely. The LDDST is useful in differentiating pituitary-dependent HC from an adrenal tumor in about two thirds of all dogs with HC. Differentiation of HC from diabetes mellitus, liver diseases, and hypothyroidism cannot be based solely on endocrine tests. Clinical signs, imaging studies, histopathology, and response to treatment should all be considered.     

In vivo alternative testing with zebrafish in ecotoxicology

Although rodents have previously been used in ecotoxicological studies, they are expensive, time-consuming, and are limited by strict legal restrictions. The present study used a zebrafish (Danio rerio) model and generated data that was useful for extrapolating toxicant effects in this system to that of humans. Here we treated embryos of the naive-type as well as a transiently transfected zebrafish liver cell line carrying a plasmid (phAhRE-EGFP), for comparing toxicity levels with the well-known aryl hydrocarbon receptor (AhR)-binding toxicants: 3,3'',4,4'',5-pentachlorobiphenyl (PCB126), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene. These toxicants induced a concentration-dependent increase in morphological disruption, indicating toxicity at early life-stages. The transient transgenic zebrafish liver cell line was sensitive enough to these toxicants to express the CYP1A1 regulated enhanced green fluorescent protein. The findings of this study demonstrated that the zebrafish in vivo model might allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development. We have also shown that the transient transgenic zebrafish liver cell line can be used for research on AhR mechanism studies.     

猪瘟病毒及其猪瘟疫苗研究进展

猪瘟（ＨＣ）是猪的一种高度传染性疾病。它遍布于世界各国,对经济造成巨大的损失。中国猪瘟兔化弱毒疫苗在防治猪瘟中曾起到决定性作用,但近几年来出现免疫效果不理想,应用组织培养弱毒苗也发生免疫失败,究其原因比较多本文结合多年研究成果,对猪瘟病毒分子生物学特性和猪瘟疫苗研究进展进行了较为系统的综述,以期望寻找出彻底控制猪瘟的办法。     

Pathology of spontaneous tumour lesions in pullets and adult chickens in commercial farms - Short communication

Twenty pullets and adult chickens, aged 100 to 403 days, from several commercial chicken farms were examined by gross and histopathology. Grossly, all chickens had white-greyish masses in the visceral organs with or without enlargement of the peripheral nerves. Histopathological examination revealed Marek's disease (MD) lymphoma, lymphoid leukosis (LL) and myeloid leukosis (ML) in 14/20, 5/20 and 1/20 of the chickens, respectively. Lesions of the sciatic nerves in chickens diagnosed as having MD lymphoma were various. No neoplastic and/or inflammatory cells were noted in the peripheral nerves of chickens diagnosed as having LL and ML. These results indicated that MD lymphoma could also develop in older chickens; thus, microscopic examination is needed to identify MD in older chickens showing lymphocyte-derived tumours.     

Changes in hepatic enzyme activities in transgenic mice carrying human prototype c-Ha-ras gene treated with diethylnitrosamine

Activities of malate dehydrogenase (MDH) and aspartate aminotransferase in the malate-aspartate shuttle were significantly increased in the cytosolic fractions of livers with early neoplastic symptoms such as swelling and discoloration in transgenic mice carrying the prototype human c-Ha-ras gene, Tg-rasH2 mice, which were administered with diethylnitrosamine (DEN) as a carcinogenic chemical at a dose of 200 mg/kg body weight. Cytosolic MDH/LDH (lactate dehydrogenase) (ML) ratio increased significantly and was considered to be a useful marker to characterize the energy metabolism at early neoplastic stage in livers of the Tg-rasH2 mice.     

从疑似猪瘟病料中检出牛病毒性腹泻病毒

根据牛病毒性腹泻病毒（ＢＶＤＶ）ＮＡＤＬ株和猪瘟病毒（ＨＣＶ）Ａｌｆｏｒｔ株的核苷酸序列，设计合成了１对ＢＶＤＶ引物和３对ＨＣＶ引物。以从吉林、长春、哲盟３个地区经临床及病理学诊断为猪瘟的病料中提取的ＲＮＡ为模板，采用反转录－聚合酶链反应（ＲＴ－ＰＣＲ），分别以ＢＶＤＶ和ＨＣＶ引物进行扩增。结果，用ＢＶＤＶ引物从哲盟地区的疑似猪瘟病料中扩增出大小约４００ｂｐ的片段，而用３对ＨＣＶ引物在不同的条件下均未从该病料中扩增出相应大小的ＨＣＶ基因片段。此外，用ＨＣＶ的ＰＥ０／ＰＥ４引物从吉林、长春的病料中扩增出了ＨＣＶ的基因片段，但用ＢＶＤＶ引物从这两个地区的病料中均未扩增出ＢＶＤＶ的基因片段。从哲盟疑似猪瘟病料中扩增出的片段经克隆、序列测定及计算机分析证实，该片段的核苷酸序列和氨基酸序列与ＢＶＤＶ的同源性明显高于与ＨＣＶ的同源性。将哲盟病料接种于ＭＤＢＫ传代细胞，出现典型而规律的ＢＶＤＶ样细胞病变。由此证明，从哲盟疑似猪瘟病料中检测出的是ＢＶＤＶ而不是ＨＣＶ     

Natural infection of pigs with bovine viral diarrhea virus and its differential diagnosis from hog cholera.

Natural infection of pigs with bovine viral diarrhea virus (BVDV) through contact with infected cattle has caused problems in diagnosing hog cholera (HC). Low cross-reacting serum antibody titers against HC caused by BVDV infection were found in clinically normal pigs as well as those suspected of having HC. Bovine viral diarrhea virus was isolated from specimen tissues and initially identified as HC virus (HCV), using the fluorescent antibody cell culture technique. Additional cell cultures, as well as pig and calf trials, were necessary to identify it as BVDV. The isolate caused clinical signs of illness in the calves, whereas the pigs remained healthy. Bovine viral diarrhea virus may be detected in tissue sections or isolated in cell cultures and confirmed as HCV, using the HC fluorescent antibody conjugate. Laboratories performing the neutralization test for HC should use discretion when interpreting HC titers unless BVD titers are determined on the same serums.     

A novel phenotype for Lardon dwarfism in miniature Bos indicus cattle suggests that the expression of growth hormone receptor 1A in liver is required for normal growth

Mutations within the growth hormone receptor (GHR) gene that lead to an inactivated or truncated GHR protein cause abnormal growth and small adult size in a variety of species (Laron dwarfism). We studied a line of miniature Bos indicus cattle that have phenotypic (small mature size) and endocrine (increased blood growth hormone and decreased blood insulin-like growth factor-I concentrations) similarities to Laron dwarfs. Liver mRNA from miniature and control cattle was used to amplify a cDNA within the coding region of the GHR. The miniature cattle had GHR mRNA size (determined by Northern blot) and cDNA sequence that were similar to control cattle and, therefore, were unlike most Laron dwarf genotypes in which the GHR gene is mutated. Amounts of mRNA from liver as well as muscle (superficial neck and longissimus) were analyzed by ribonuclease protection assay for IGF-I, total GHR, GHR 1A (inducible, liver-specific GHR mRNA), and GHR 1B (constitutive GHR mRNA). Four control and five miniature bulls were tested. As expected, liver IGF-I mRNA was decreased in the miniature cattle (approximately 12% of control; P < 0.01). The amount of the total GHR as well as GHR 1A mRNA were also decreased in liver (17% and 19% of control, respectively; P < 0.01). Other GHR mRNA, including GHR 1B mRNA, were similar for miniature and control cattle. In muscle, there was a tendency (P < 0.10) for decreased IGF-I mRNA and increased GHR mRNA in miniature compared with control cattle. In summary, a novel phenotype for Laron dwarfism in Bos indicus cattle was associated with underexpression of GHR 1A mRNA, but not other GHR mRNA variants in liver. In addition to decreased GHR 1A mRNA, the miniature cattle had decreased liver IGF-I mRNA. Full expression of GHR 1A in liver, therefore, may be required for full liver IGF-I expression and normal growth.     

徐淮山羊H-FABP基因克隆、表达产物亚细胞定位的研究及转基因小鼠的制备

旨在克隆徐淮山羊心脏型脂肪酸结合蛋白(H-FABP)基因的cDNA,探讨其生物信息学功能。通过EGFP融合蛋白对该基因亚细胞水平的表达定位,探究该基因在异种生物体内表达情况,探索制备异种转基因动物的可能性。采用反转录PCR(RT-PCR)方法克隆徐淮山羊H-FABP基因cDNA,进行生物信息学分析,构建其融合表达载体pEGFP-H-FABP。脂质体(LTX)介导基因转染山羊成纤维细胞(GEF),48h后进行荧光检测,RT-PCR检测mRNA在细胞内的表达。利用睾丸注射将目的基因转入小鼠体内,在DNA和蛋白水平上检测目的基因的表达情况。结果,徐淮山羊H-FABP基因完整编码区(CDS)大小为402bp,编码133个氨基酸,GenBank登录号为AY466498.1。徐淮山羊H-FABP序列与人、鸡、褐家鼠、奶牛、野猪、驴及斑马鱼相应编码区同源性为89%、76%、85%、84%、93%、91%、70%,氨基酸序列同源性为90%、79%、88%、97%、95%、94%、72%。成功构建融合表达载体pEGFP-H-FABP,目的基因在mRNA水平上成功表达。目的蛋白定位于细胞质中,与在线预测结果相同(无信号肽,定位于细胞质中)。通过尾静脉注射和睾丸注射,该基因可以在小鼠体内实现暂态表达和持续性表达。本研究成功克隆了徐淮山羊H-FABP基因cDNA,而且H-FABP基因在进化过程中是保守的。该蛋白无信号肽,在单细胞水平上可表达于细胞质中,在小鼠体内也可以成功表达。