用PCR-SSP方法检测中国恒河猴Mamu-DRB*W101和Mamu-DRB*W201基因

利用序列特异引物聚合酶链反应（polymerase chain reactionsequence-specific primers，PCR-SSP）方法扩增中国恒河猴的主要组织相容性复合体II类基因Mamu-DRB*W101、- DRB*W201，初步了解中国恒河猴中Mamu-DRB*W101、- DRB*W201基因的阳性率。采集中国恒河猴静脉血，用DNA提取试剂盒提取全血DNA，分别用Mamu-DRB*W101、- DRB*W201特异引物PCR扩增Mamu-DRB*W101、- DRB*W201基因的第二外显子区域，并对扩增出的阳性条带进行测序，与已知序列对比验证序列是否正确。共检测了来自136只中国恒河猴的样本，PCR检测出Mamu-DRB*W101阳性个体10只，Mamu-DRB*W201阳性个体也是10只，其阳性个体所占比率均为7.35%。测序结果表明，PCR扩增产物的核苷酸序列与基因库中的序列完全一致。本研究表明中国恒河猴中存在Mamu-DRB*W101、- DRB*W201基因阳性个体，为中国恒河猴在AIDS研究中的应用及进一步分析中国恒河猴MHC II类基因提供了基础。     

云南地区恒河猴MHC部分基因频率及多态性的分析

目的不同种群恒河猴（Macaca mulatta）主要组织相容性复合体（major histocompatibility complex,MHC）基因的差异性,很大程度上影响了用其作为动物模型的实验研究结果的稳定性、可靠性和可重复性。清晰了解云南地区恒河猴种群免疫遗传学背景对于运用该种群开展各项研究非常重要。恒河猴基因Mamu（Macaca mulatta）-A＊01就被公认与猴免疫缺陷病毒（SIV）的感染与延缓病程相联系。Mamu-Ⅱ类等位基因Mamu-DRB1＊0306、0309有研究表明也与SIV感染后的病程发展有关。方法针对目前研究热点的Mamu-A＊01基因及Ⅱ类Mamu-DRB1＊0306、0309基因,采用序列特异性引物-聚合酶链式反应方法（PCR-SSP）,对云南地区的140只恒河猴（其中92只来源于云南景东野外自然繁殖群、48只来源于本灵长类中心自繁群）进行检测。并对阳性样本用部分测序结合单链构象多态性分析（SSCP）的方法加以验证。结果Mamu-A＊01检测结果为阴性。Mamu-DRB1＊0306、0309基因频率分别为32%及45%。结论初步显示云南地区恒河猴种群Mamu-A＊01基因可能缺失或基因频率很低,但存在Mamu-DRB1＊0306、0309基因。初步证明SSCP方法代替大规模测序是可行的,为云南地区恒河猴在AIDS研究中的应用提供了部分依据,一定程度上填补了该种群的遗传背景资料。     

中国恒河猴Mamu-A*01基因筛查及其对抗原肽p11C的特异性CTL反应

目的 筛查中国恒河猴Mamu-A*01基因,比较中国恒河猴和印度恒河猴的Mamu-A*01基因序列和功能是否相同.方法 PCR方法检测128只中国恒河猴,用特异性引物扩增Mamu-A*01基因,将PCR扩增后的产物克隆测序后与印度恒河猴的Mamu-A*01基因进行同源比对;酶联免疫斑点检测 (ELISPOT) 方法分别检测5只Mamu-A*01基因阳性和5只阴性恒河猴针对SIV、SHIV抗原肽p11C的特异性CTL反应.结果 共筛查出5 只Mamu-A*01基因阳性恒河猴 (3.91%),经测序分析后与印度恒河猴的同源性可达99.1 %.这5只均为SIV/SHIV感染恒河猴,其中四只SIV感染的猴的ELISPOT结果显示针对p11C的高频CTL反应,斑点数在500-1400/106 PBMCs之间,而另1只SHIV感染的恒河猴及5只阴性猴没有斑点出现.结论 中国恒河猴含有Mamu-A*01基因,基因频率有区域性差异,中国恒河猴的Mamu-A*01可提呈特异性抗原肽p11C.     

ELISPOT方法检测SIVmac239感染猴特异性细胞免疫水平

目的为了完善现有的SIV／恒河猴模型,掌握恒河猴被SIV感染后体内细胞免疫应答状态,为评价HIV疫苗提供方法和数据上的参考,我们测定了SIV感染猴体内病毒特异性的细胞免疫水平。方法实验前选出4只无SIV、sTLV、SRV／D和B病毒感染的恒河猴,用SIVmac239病毒液静脉感染实验猴,使用RT-PCR、流氏细胞术和ELISPOT等方法,监测SIVmac239病毒在恒河猴体内复制情况、感染猴的外周免疫损伤情况和细胞免疫情况,持续测定一年。结果实验结果显示IFN-γ ELISPOT方法能有效的评估实验猴的细胞免疫情况,IFN—YELISPOT结果和CD4＋T细胞数无相关性,与血浆病毒载量稍有相关。结论本实验明确了SIVmac239感染中国恒河猴体内CTL的基本趋势和范围,了解了外周血病毒载量、外周免疫损伤与细胞免疫状况之间的联系,完善了SIV／SAIDS模型评价指标,为使用此模型评价抗病毒药物或疫苗提供了基础条件。     

用ELISA和Dot-ELISA方法检测植物病原的比较研究

比较了ELISA和Dot-ELISA方法检测番茄巨芽类菌原体(Tomato Big Bud Mycoplatma-Like Organism,TBB-MLO)、番茄斑萎病毒(Tomato Spotted Wilt Virus,TSWV)和青枯假单胞菌(Pseudomonas solanacearum)的差异,结果表明,两种方法对番茄斑萎病毒无明显差异,对番茄巨芽类菌原体,用Dot-ELISA比用ELISA的检测灵敏度高50—100倍,对青桔假单胞菌,用Dot-ELISA比用ELISA检泓抗原     

用改进的MVR-PCR方法检测河北地区汉族人群D7S21基因座多态性

为研究D7S21基因座在河北汉族人群分布的多态性,应用MVR-PCR ( Minisatellite Variant Repeat-Polymerase Chain Reaction)方法和聚丙烯酰胺梯度凝胶电泳银染法对124名河北汉人无关个体D7S21基因座进行了快速检测,并进行数字编码。每一个体平均得到36个数字编码,未发现任何两个无关个体所有编码相同,两无关个体36个编码相同的概率为3.48×10-18。三种重复单位a型、t型和0型出现的概率分别为：48.5 ％、49.4％和 2.1％。该基因座杂合度为0.9876,非父排除率为0.9746,多态性信息含量为0.9872。研究表明,D7S21基因座在河北汉族人群中具有高度的多态性,聚丙烯酰胺梯度凝胶电泳银染法简便、快速,具有一定的实用价值。 Abstract:To study the polymorphism at D7S21 locus in Hebei Han population,124 unrelated individuals were detected rapidly by Minisatellite Variant Repeat-Polymerase Chain Reaction (MVR-PCR) and polyacrylamide gradient gel electrophoresis followed by silver staining,and digital codes were obtained.About 36 digital codes were obtained from each individual.No two unrelated individuals shared the same codes.The probability of identity in 36 digital codes was 3.48×10-18.The percentage of three repeat units,a-type,t-type and 0-type was 48.5%,49.4% and 2.1% respectively.The heterozygosity (H),excluding probability of paternity（EPP）and polymorphism information content（PIC）were 0.9876,0.9746and 0.9872 respectively.The results suggested that D7S21 locus has highly polymorphism in Hebei Han population.The method-polyacrylamide gradient gel electrophoresis followed by silver staining was simple,rapid and practical.     

抗人B淋巴细胞单克隆抗体清除中国恒河猴体内B淋巴细胞效果观察

目的用抗人B淋巴细胞单克隆抗体（利妥昔单抗注射液,Rituximab）通过静脉滴注的方法敲除中国恒河猴体内B淋巴细胞,并观察其敲除效果,为建立B淋巴细胞缺失的恒河猴动物模型提供基础的实验数据。方法选取健康的中国恒河猴两只,静脉滴注抗人B淋巴细胞单克隆抗体,定期采集外周血、腹股沟淋巴结和十二指肠黏膜组织,制备淋巴细胞悬液,应用流式细胞术的方法系统性测定B淋巴细胞及T淋巴细胞亚群的变化。结果静脉滴注利妥昔单抗注射液后24 h,中国恒河猴外周血中B淋巴细胞的缺失即能达到100%,持续约14 d;腹股沟淋巴结中B淋巴细胞在静注后7 d缺失100%;十二指肠黏膜组织中B淋巴细胞在静脉滴注后7 d缺失达到90%,维持28 d。并且在成功敲除B淋巴细胞的情况下,CD4＋T及CD8＋T淋巴细胞无明显波动,维持在一个稳定的水平。结论利妥昔单抗注射液能有效去除中国恒河猴体内B淋巴细胞,为建立B淋巴细胞缺失动物模型奠定基础。     

Frequency of the major histocompatibility complex Mamu-A*01 allele in a closed breeding colony of rhesus monkey (Macaca mulatta) from Brazil

Background  Rhesus monkeys are relevant models for human diseases. The simian immunodeficiency virus (SIV) infection is an useful macaque model for assessing human immunodeficiency virus (HIV) vaccine strategies. Susceptibility and resistance to viruses have been associated with particular major histocompatibility complex (MHC) molecules. Several epitopes in the HIV structural and non-structural protein restricted by distinct MHC class I haplotypes are important targets for human cytotoxic T lymphocytes, which mediate protection against SIVmac infection. Mamu-A*01 , for example, is a MHC class I molecule of rhesus monkeys that presents a peptide from SIV gag protein.
Methods  Our study determined the frequency of Mamu-A*01 in a closed colony of rhesus monkeys from Brazil by polymerase chain reaction.
Results  A high frequency of the allele was found in the study colony.
Conclusion  This colony provides a significant source of A*01 -positive animals to investigators.     

Adoption by captive parturient rhesus macaques: Biological vs. adopted infants and the cost of being a “twin” and rearing “twins”

Four rhesus macaque (Macaca mulatta) mothers each spontaneously adopted and reared an abandoned, unrelated neonate in addition to their own neonate. Data on relative time spent in maternal contact and who maintained proximity were collected for the biological and adopted “twins” and singleton control infants using focal animal sampling. Infant weight gain and the subsequent conception history for each mother were obtained for the following year. Biological infants spent more time in maternal contact than their adopted “twin” siblings. When in contact with their mothers, biological “twins” spent more time in the ventro-ventral position and more ventral time alone than adoptees. Mothers initiated more contacts with their biological infants than their adopted infants, suggesting these differences may be due to differential maternal behavior. “Twins” gained weight at a slower rate than singletons, and mothers rearing “twins” produced significantly fewer offspring the following season. Am. J. Primatol. 43:259–264, 1997. © 1997 Wiley-Liss, Inc.     

Body fat,rank, and nutritional status in a captive group of Rhesus Macaques

Using weight and skinfold thickness to calculate relative body fat, the fat content of 21 captive female rhesus macaques was estimated. Although age and. pregnancy had no effect on fat, rank was significant, especially during winter, with high-rank females having the highest fat scores. Rank therefore has a significant effect on the health of captive animals and may also affect the health of individuals in feral groups.     

恒河猴感染SARS-CoV的病毒学、血清学检测

目的对感染SARS-CoV的8只恒河猴进行病毒学、血清学指标检测。方法SARS-CoV经鼻腔接种8只恒河猴,在感染的第1天开始到5、7、10、15、20、30和60天分别安乐处死时,不同时间取咽拭子、血液和脏器,进行病毒分离,RT-PCR检测和抗体测定。结果RT-PCR证实感染病毒检出时间为5~16d,8只猴中的5只分离到了病毒,感染15d后可检测到抗体。结论感染SARS-CoV的恒河猴不仅出现与SARS患者类似的临床和病理学改变,也在一定时期内排毒,出现特异免疫反应,这些指标均可作为药物筛选、疫苗评价等方面的重要参数。     

The influence of birth timing upon infant growth and survival in Captive Rhesus Macaques (Macaca mulatto)

Weights, growth rates, and mortality data of 815 captive-born Macaca mulattainfants were studied to determine if date of birth influences infant growth and survival. The six groups studied displayed a unimodal spring-summer birth season that has become systematically more restricted since 1977. Males exhibited higher rates of stillbirth and neonatal death and were more frequently born outside the normal birth season, when infant mortality was more common. Within the normal birth season, infant weight increased linearly with birth date, and infant growth rate declined linearly with birth date. Female infants with weights and growth rates near the developmental norm, especially those born in the middle of the birth season, have the greatest probability of survival. Males are more likely to survive if their weights and growth rates exceed the developmental norm, and thus male infants might be initially more costly to produce than female infants. These results are inconsistent with the hypothesis that offspring of high-ranking males, which conceived predominantly in the first third of the breeding season, enjoy a selective advantage.     

气管插管法接种H5N1病毒感染恒河猴及系统组织病毒检测

目的测试气管插管法接种高致病性禽流感病毒H5N1感染恒河猴的优势效果及疾病分析,为有效感染恒河猴、制备H5N1疾病模型提供实验依据。方法使用人源H5N1病毒液经气管插管滴入恒河猴上呼吸道进行感染,观察感染恒河猴的临床表现,每天采集咽拭子、鼻灌洗液,在感染前2d感染后第3、5、7天采血,感染后第3和7天分别解剖1只恒河猴,取支气管淋巴结、肠淋巴结、鼻甲、心、肝、脾、肺、肾、肠、气管、脑及血液进行病毒分离、核酸载量检测和血常规测定。结果感染后第2天恒河猴出现食欲下降,活动减少,并伴有一过性体温升高,白细胞数和淋巴细胞数下降。咽拭子、鼻灌洗液、肺、心、气管、脑、肝、肾、肠和血液中都能分离到H5N1病毒。结论气管插管法接种H5N1病毒能有效感染恒河猴,并在猴体内多组织中分离、检测到病毒,为制备完善的H5N1模型和检测指标确定、进一步研究H5N1病毒的致病机制等奠定了基础。     

Bax/Bcl-2 mRNA在恒河猴药物流产胎盘中的表达

目的：通过检测重要的细胞凋亡调节因子Bax和Bcl-2的表达,探讨恒河猴胎盘细胞凋亡与药物流产的关系。方法：利用RT-PCR和原位杂交的方法检测恒河猴对照组和药物流产组(RU-486与AG诱导)胎盘中Bax和Bcl-2mRNA的表达。结果：Bax mRNA在流产胎盘中的表达量明显高于对照组胎盘。在胎盘绒毛滋养层细胞可见明显表达,基底层蜕膜细胞也有表达。Bcl-2mRNA在流产胎盘中的表达量明显低于对照组胎盘,表达部位与Bax类似。结论：RU-486和AG可能通过上调Bax mRNA和下调Bcl-2mRNA的表达,导致胎盘组织凋亡细胞的数量明显增加,从而影响胎盘的正常结构和功能,增加了流产的危险性。