灵长类动物中TRIMCyp融合基因模式及对逆转录病毒复制的限制作用

TRIM5-CypA融合基因(TRIMCyp)是一种独特的TRIM5基因形式。迄今已发现新大陆猴中包括鹰猴在内的夜猴属所有代表种,以及在北平顶猴、巽他平顶猴、食蟹猴、印度恒河猴和熊猴等旧大陆猴中均存在这种基因融合现象,但在新大陆猴与旧大陆猴中的TRIMCyp融合基因的基因融合模式和表达剪接方式不同。新大陆猴TRIMCyp融合基因是由CypA假基因的cDNA序列通过LINE-1逆转座子介导的逆转座方式插入至TRIM5α基因的第7和第8外显子之间的内含子中形成,而旧大陆猴TRIMCyp融合基因则是由CypA假基因的cDNA序列以相似的逆转座方式插入至TRIM5基因的3’非翻译区(untranslatedregions,UTR)形成。TRIMCyp融合基因在不同灵长类动物中的存在比例、基因型、TRIMCyp融合蛋白的表达以及对逆转录病毒的限制活性均有所差异。鹰猴和平顶猴的TRIMCyp融合基因研究较多,鹰猴TRIMCyp融合蛋白可能以与TRIM5α相似机制限制HIV-1的感染,而平顶猴TRIMCyp融合蛋白则丧失了限制HIV-1的作用。这两个功能截然不同的融合基因为TRIM5α作用机制研究提供了难得的实验材料,也为建立HIV-1感染的新型灵长类动物艾滋病模型奠定了科学依据。该文综述了TRIMCyp融合基因在灵长类动物中的分布、存在形式及其限制逆转录病毒复制的作用机制等方面的研究情况。     

熊猴存在TRIM5/TRIMCyp杂合子基因型

缺乏合适的动物模犁是制约艾滋病研究取得重大突破的关键瓶颈之一.细胞内的抗病毒蛋白被称为限制因子.研究不同灵长类动物抗HIV-1宿主限制因子的存在形式及作用机制对建立合适AIDS灵长类动物模型有十分重要的意义.TRIM5α是哺乳动物细胞中一种重要和关键的限制因子,它以物种依赖的方式限制包括HIV-1在内的逆转录病毒的感染.TRIM5-CypA融合基因是存在于新大陆猴与旧大陆猴中的一种独特的TRIM5基因形式.为了研究不同灵长类动物TRIM5基因的存在方式,该文对熊猴、藏婀猴、红面猴及中闰恒河猴4个物种共110只灵长类动物进行了TRIM5-CypA融合模式的研究.首次发现熊猴也存在TRIM5-CypA基因融合现象.熊猴TRIMCyp融合基因形成模式类似于北平顺猴TRIMCyp融合基因模式,即CypA假基因的cDNA序列通过逆转座方式插入到TRIM5基凶的3'-UTR区域.基因序列分析表明,该基因与北平顶猴相应基因序列高度相似;并且其TRIM5内含子6的3'-剪接位点也相应存存G-to-T突变现象(G/T).这提示熊猴也极有可能像北平顶猴一样表达TRIM5-CypA融合蛋白,从而导致熊猴可能跟北平顶猴一样可能被HIV-1感染.因此,熊猴极有希望成为一种新的HIV/AIDS灵长类动物模型.     

灵长目动物TRIM5基因的进化及其抗逆转录病毒的种属差异性

灵长目动物TRIM5基因能编码TRIM5α蛋白,它在逆转录病毒侵染宿主细胞的早期阶段抑制病毒复制。同时科学家也在灵长目动物中发现了TRIM5基因的另外一种融合蛋白基因型——TRIMCyp,由于发生的剪接机制不同,新、旧大陆猴分别形成了不同的融合蛋白,从而导致它们抗病毒活性的广泛差异。TRIM5基因在灵长目动物进化过程中产生了很多突变位点,这些突变位点也使灵长目动物抗逆转录病毒的活性存在种属差异。本文简述了TRIM5基因及编码蛋白的结构,从自然选择的角度阐述了灵长目动物TRIM5基因的进化,揭示了由TRIM5基因多样性所造成的抗病毒种属差异,从而为研究灵长目动物的进化以及人猴共患疾病模式生物的构建提供一定的指导。     

打靶恒河猴CD4~+ T细胞的TRIM5α基因影响其感染HIV的能力

目的:探讨打靶恒河猴CD4~+ T细胞的TRIM5α基因对其感染HIV-1能力的影响。方法:从恒河猴的外周血中通过磁珠分选获得CD4~+ T细胞,并采用流式检测阳性率。构建打靶TRIM5α基因的TALEN质粒,通过电转导入CD4~+ T细胞,流式分选出转染TALEN质粒的细胞,提取基因组T7E1酶切检测打靶效率。HIV-1病毒感染打靶TRIM5α的CD4~+ T细胞,并通过ELASA检测病毒感染的情况。结果:成功地从恒河猴的外周血中分选出了CD4~+ T细胞,流式检测阳性率为99.5%。打靶TRIM5α基因的TALEN质粒转染CD4~+ T细胞的转染效率约为24.8%,并可成功打靶TRIM5α,打靶效率约为40%。ELASA检测结果表明打靶TRIM5α的恒河猴CD4~+ T细胞对HIV病毒的感染能力增强。结论打靶恒河猴CD4~+ T细胞的TRIM5α基因可使其易感HIV病毒,为进一步建立恒河猴HIV-1感染动物模型奠定基础。     

发现平顶猴易感HIV-1重要分子机制

在国家自然科学基金和“973”项目的支持下，中国科学院昆明动物研究所研究员郑永唐组和宿兵组合作，发现在旧大陆猴平顶猴对HIV-1易感的重要分子机制，并首次发现新型天然免疫分子mnTRIMCyp。此项研究进一步阐明了天然免疫分子TRIM5α限制HIV-1感染和复制的作用机制，也为国际HIV／AIDS研究广泛关注的HIV／AIDS非人灵长类动物模型建立提供了重要思路。     

食蟹猴微卫星DNA标记遗传监测及多态性分析

目的研究国内食蟹猴种群的遗传背景特性,建立食蟹猴种群遗传质量监测方法。方法采用微卫星DNA遗传标记技术对50只食蟹猴种群个体进行遗传质量监测及DNA多态性分析。结果从100个微卫星DNA位点中筛选出20个多态性高的位点,其食蟹猴种群个体的等位基因数目为5-10条,个体间均呈现高度的多态性;其观察等位基因数（Na）为5.0～10.0,有效等位基因数（Ne）为4.6118～8.3404,基因多样性（H）为0.7832～0.8801和香隆信息指数（I）为1.5651～2.1592。结论本实验有效地分析了食蟹猴种群的遗传多态性,为今后筛选特异性微卫星位点来建立食蟹猴种群遗传质量监测方法提供了理论依据。     

24种食蟹猴MHC B座位等位基因序列特征与进化分析

食蟹猴(Macaca fascicularis,Mafa)是重要的医学研究动物模型,被大量用于药物及移植医学试验。为探讨本地食蟹猴MHC I类基因B座位的多态性,本研究经大量PCR,克隆与测序,从10只食蟹猴中分离到24种Mafa-B等位基因的全长序列,其中4种为新序列,已递交NCBI数据库,并给予系统命名。在每个个体中均获得6至11条Mafa-B等位基因,显示食蟹猴的MHC-B座位出现扩增,食蟹猴MHC-B为重复基因座的特征与恒河猴类似。氨基酸序列比对分析得出,MHC-B基因在抗原肽结合区高度多态。     

食蟹猴MHC I类A型新等位基因序列分析

采用自行设计的MHC I类A型基因的引物从10只食蟹猴扩增得到11个3 000bp左右MHC等位基因。通过在Blast比对之后,证实有7个Mafa-A等位基因,4个Mafa-AG等位基因,新的等位基因序列已递交GenBank。由于每个个体可以检测出多个不同的Mafa-A等位基因,这说明食蟹猴Mafa-A基因经过多次复制,并且推测Mafa-AG是Mafa-A衍生物。同时,研究了等位基因在个体中的分布和系统进化树分析。     

五个鸡群中催乳素基因密码子使用频率与产蛋量的关系分析

通过鸡催乳素基因编码区基因扫描,发现3个SNP位点,分别是外显子2的C1607T、外显子5的C5749T和T5821C,3个SNP均没有改变氨基酸的编码.同时发现不同的单倍型间存在不同的密码子使用频率.对5个鸡群共370只鸡进行SSCP的基因型检测,共发现7种单倍型,结合44周龄产蛋量分析,发现不同单倍型的平均产蛋量存在显著性差异.结合密码子使用频率分析,发现使用高频密码子的单倍型个体产蛋量相对高.通过酶联免疫方法检测催乳素表达量,结果显示,使用高频密码子的个体,激素水平较高,其中使用2个高频密码子的单倍型个体和使用1.5个密码子的单倍型个体之间存在极显著差异.研究结果表明,密码子使用频率与产蛋量在一定的范围内呈现正相关趋势.     

食蟹猴腹腔注射低剂量百草枯致胰腺α-突触核蛋白的病理变化

本研究旨在通过食蟹猴经腹腔注射低剂量百草枯来探讨胰腺α-突触核蛋白(α-synuclein,α-syn)的病理变化。由广西南宁灵康赛诺科生物科技有限公司提供的12只(8~10岁)健康雄性食蟹猴为实验动物,随机分为实验组A、B和正常对照组C,每组4只,实验组分别于给药百草枯(paraquat,PQ)5个月,2个月后安乐死(对照组每阶段2只)。利用免疫组织化学方法和免疫蛋白印记方法检测各组食蟹猴胰岛α-syn的表达特点和表达量。结果显示,α-syn免疫活性物质主要见于胰岛细胞胞浆,并且α-syn在各组食蟹猴胰岛存在差异表达,其中给药百草枯5个月后的食蟹猴胰岛α-syn表达量最高,对照组表达量最低。我们的研究表明食蟹猴腹腔注射低剂量百草枯可致胰岛α-syn表达上调,百草枯是造成胰岛腺α-syn差异表达的关键因素之一,其结论将为相关疾病的模型制作、早期诊断及干预提供新的思路。     

Scurvy in capybaras bred in captivity in Argentine

In order to determine if the absence of vitamin C in the diet of capybaras (Hydrochoerus hydrochaeris) causes scurvy, a group of seven young individuals were fed food pellets without ascorbic acid, while another group of eight individuals received the same food with 1 g of ascorbic acid per animal per day. Animals in the first group developed signs of scurvy-like gingivitis, breaking of the incisors and death of one animal. Clinical signs appeared between 25 and 104 days from the beginning of the trial in all individuals. Growth rates of individuals deprived of vitamin C was considerably less than those observed in the control group. Deficiency of ascorbic acid had a severe effect on reproduction of another population of captive capybaras. We found that the decrease in ascorbic acid content in the diet affected pregnancy, especially during the first stages. The results obtained suggest that it is necessary to supply a suitable quantity of vitamin C in the diet of this species in captivity.     

Changes in lactate dehydrogenase activity in chicken tissues in hyperthyreosis in ontogenesis

The lactate dehydrogenase activity in reactions of lactate oxidation and synthesis was studied in subfractions of the chicken brain, heart and liver at the embryonal, early postembryonal and adult stages of development after thyroxine administration. It has been shown that during embryogenesis thyroxine predominantly enhanced the rate of lactate oxidation in the mitochondrial tissues. A marked increase in the lactate synthesis was found in cytoplasm of the adult chicken tissues. Specificity of enzyme activity alterations was detected in the chicken brain during ontogenesis after thyroxine administration.     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。