Nesfatin-1调节脂肪代谢的研究进展

Nesfatin-1是一种新型饱腹因子,其前体蛋白NUCB2被激素原转化酶裂解为nesfatin-1、nesfatin-2和nesfatin-3等3个片段。Nesfatin-1广泛地分布在中枢神经系统和外周组织中,尤其是在下丘脑、垂体、肝和脂肪组织中大量表达。Nesfatin-1具有调节脂肪代谢、摄食、睡眠、生殖、胃功能、心血管和血糖等多种生物学功能。该文综述了nesfatin-1的发现、结构、组织分布和调节脂肪代谢的机制,以期为肥胖和脂肪肝的防控提供理论依据。     

糖尿病大鼠下丘脑Nesfatin-1对胃运动的影响及机制研究

目的:探讨下丘脑腹内侧核Nesfatin-1对正常大鼠及糖尿病大鼠胃运动的影响及其潜在机制。方法:正常大鼠随机分为0.08μg,0.8μg,8.0μg/0.5μL Nesfatin-1组;30μg/0.5μL astressin-B组;(0.8μg Nesfatin-1+30μg astressin-B)/0.5μL组;0.5μL生理盐水(NS)组;正常羊血清+假刺激(NR+SS)组;正常羊血清+电刺激(NR+ES)组;抗NUCB2/Nesfatin-1抗体+假刺激(anti-Nn-Ab+SS)组;抗NUCB2/Nesfatin-1抗体+电刺激(anti-Nn-Ab+ES)组。制作糖尿病大鼠模型,将糖尿病大鼠随机分为0.08μg/0.5μL Nesfatin-1组;0.8μg/0.5μLNesfatin-1组;8.0μg/0.5μL Nesfatin-1组;0.5μLNS组;NR+SS组;NR+ES组;anti-Nn-Ab+SS组;anti-Nn-Ab+ES组。大鼠胃部置入感应器后腹内侧核置管,记录清醒大鼠胃运动及电刺激海马CA1区后的胃运动。结果:与生理盐水组相比,下丘脑腹内侧核注射不同浓度Nesfatin-1,大鼠胃收缩幅度和频率显著降低,下丘脑腹内侧核注射0.5μL(0.8μg Nesfatin-1+30μg astressin-B)混合液后,相比单独给予0.8μg Nesfatin-1组,大鼠胃收缩幅度和频率显著升高。大鼠下丘脑腹内侧核注射0.5μL Nesfatin-1(0.8μg),大鼠胃收缩幅度和频率显著降低,下丘脑腹内侧核注射0.5μL(0.8μg Nesfatin-1+30μg astressin-B)混合液后,相比单独给予0.8μg Nesfatin-1组,大鼠胃收缩幅度和频率显著升高。下丘脑腹内侧核注射抗NUCB2/Nesfatin-1抗体后再电刺激海马CA1区,与正常羊血清+电刺激组相比,大鼠胃收缩幅度和频率进一步增强,下丘脑腹内侧核注射抗NUCB2/Nesfatin-1抗体后再电刺激海马CA1区,与单独注射抗NUCB2/Nesfatin-1抗体+假电刺激组相比,大鼠的胃收缩幅度和频率显著增高。下丘脑腹内侧核注射抗NUCB2/Nesfatin-1抗体后再给予电刺激海马CA1区,与正常羊血清+电刺激组相比,正常大鼠和糖尿病大鼠胃运动指数均显著增加,下丘脑腹内侧核注射抗NUCB2/Nesfatin-1抗体后再电刺激海马CA1区,与单独注射抗NUCB2/Nesfatin-1抗体+假电刺激组相比,正常和糖尿病大鼠的胃运动指数均显著增高。与正常大鼠相比,电刺激海马CA1区、下丘脑腹内侧核注射抗NUCB2/Nesfatin-1抗体后再给予电刺激海马CA1区,或下丘脑腹内侧核微量注射抗NUCB2/Nesfatin-1抗体,糖尿病大鼠胃运动指数均无显著差异。结论:海马-下丘脑Nesfatin-1信号通路参与胃传入信息和胃运动调控,该作用可能与CRF系统活动有关。     

下丘脑腹内侧核损毁对大鼠nesfatin-1/NUCB2表达的影响

目的:观察下丘脑腹内侧核(VMH)损毁对大鼠脂肪组织nesfatin-1/NUCB2表达的影响及其机制。方法:电损毁VMH,观察大鼠体重和脂肪组织变化,采用Western blot检测脂肪组织中nesfatin-1/NUCB2表达改变。腹腔注射6-羟多巴胺(50 mg/kg)以阻断交感神经;持续外周注射卡巴胆碱(180μg/kg)用以模拟VMH损毁,观察其对大鼠皮下脂肪nesfatin-1/NUCB2表达的影响。结果:与对照组和假手术组比较,VMH损毁后大鼠体重明显增加(P0.05),皮下脂肪(P0.05)和肠系膜脂肪(P0.05)也显著增多;Western blot分析结果显示,nesfatin-1/NUCB2在胰腺和肝脏中表达较多,但皮下、肠系膜脂肪和肩胛间棕色脂肪组织(i BAT)中表达较少,骨骼肌(腓肠肌)中鲜有表达;与对照组和假手术组比较,VMH损毁组大鼠nesfatin-1/NUCB2在肝脏、胰腺、骨骼肌和i BAT中表达无显著差异(P0.05),皮下脂肪(P0.05)和肠系膜脂肪(P0.05)nesfatin-1/NUCB2表达显著增多与对照组相比,6-羟多巴胺组nesfatin-1/NUCB2表达显著升高(t=3.43,P0.05),而卡巴胆碱组nesfatin-1/NUCB2表达无显著差异(t=0.37,P=0.72)。结论:VMH损毁后大鼠脂肪组织nesfatin-1/NUCB2表达改变可能通过抑制交感神经活动介导。     

茶树咖啡碱合成酶基因原核表达、及其抗体制备与鉴定

克隆出茶树咖啡碱合成酶基因,对其进行原核表达,并制备TCS1抗体,旨在从蛋白水平研究茶树体内TCS1的表达情况。根据Gen Bank登陆的TCS1基因的全长c DNA序列,找出其完整的ORF(开放阅读框),从茶树叶片c DNA中克隆了TCS1基因的开放阅读框,连接到p GEX-4T-2表达载体,经IPTG诱导表达重组蛋白p GEX-4T-2-TCS1。进行体外酶活检测后,亲和层析纯化重组蛋白,作为抗原免疫家兔,制备TCS1多克隆抗体。用ELISA方法检测抗体效价,Western blot检测抗体的特异性。通过优化诱导条件,得出重组蛋白的最佳表达条件为:30℃、4 h。诱导后的总蛋白、可溶性蛋白与包涵体蛋白均出现一条明显的外源蛋白条带。抗体经ELISA检测,效价为1∶2 000,Western blot检测表明抗体具有相对较好的特异性。构建了TCS1原核表达质粒,同时成功制备了抗TCS1的多克隆抗体。     

重组SCIRR39多克隆抗体的制备及检测

目的：在大肠杆菌中表达大鼠脊髓损伤与修复蛋白39（SCIRR39）的C端抗原表位,并制备其多克隆抗体。方法：从大鼠脊髓全横断损伤脊髓cDNA中扩增1386bp的Scirr39基因编码框,亚克隆该基因编码蛋白C端359～461位氨基酸残基的DNA片段,插入表达载体,转化大肠杆菌BL21（DE3）,IPTG诱导表达,SDS-PAGE分析表达情况,切胶纯化目的蛋白;利用多克隆抗体制备技术,制备重组SCIRR39蛋白的多克隆抗体;用ELISA方法检测抗体效价,Western印迹检测抗体的特异性。结果：SCIRR39蛋白C端抗原表位与GST的融合蛋白在大肠杆菌中以可溶形式高表达,相对分子质量为37．9×103;获得抗SCIRR39蛋白C端抗原表位的兔抗血清,其效价达到1：104;Western印迹显示多克隆抗体能特异识别重组SCIRR39蛋白的C端抗原表位。结论：在原核系统中表达纯化了重组SCIRR39抗原表位蛋白,制备的重组蛋白多克隆抗体将用于检测SCIRR39在脊髓损伤过程中的表达变化。     

小鼠子宫珠蛋白结合蛋白的生物信息学分析及其多克隆抗体制备

本文旨在制备小鼠子宫珠蛋白结合蛋白(mouse uteroglo binbinding protein,mUGBP)多克隆抗体,为后续研究工作奠定基础。通过生物信息学分析方法预测mUGBP蛋白的跨膜结构、理化特性、疏水性等因素,设计出两段多肽,分别与匙孔戚血蓝蛋白(keyhole limpet hemocyanin,KLH)交联后免疫新西兰兔,结果显示其中一个含13个氨基酸残基的多肽序列(221st～233rd)可用作抗原免疫动物,并成功获得高效价的抗mUGBP多克隆抗体。通过ELISA法检测其效价为1:108,亲和层析纯化后Westernblot检测抗体特异性,并将制备的抗体应用于免疫印迹及人和小鼠肺组织免疫组织化学和免疫荧光检测,结果显示本研究制备的抗体具有特异性,且用该抗体成功在人和小鼠气道上皮细胞和肺血管内皮细胞检测到UGBP蛋白表达。结果表明,本研究通过生物信息学方法成功预测了抗原表位,并据此预测成功制备了高效价、高特异性的抗mUGBP多克隆抗体。     

双峰驼血清IgG亚型的分离纯化及多克隆抗体的制备

双峰驼IgG亚型包含IgG1、IgG2和IgG3,其中IgG2和IgG3为重链抗体,在结构上与IgG1存在显著差异。为获取双峰驼血清中的IgG1、IgG2和IgG3,并分析其抗原特异性和抗体特异性,本文交替使用Protein A和Protein G亲和层析柱,对其分离纯化,并通过聚丙烯酰胺凝胶电泳进行鉴定;之后分别制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体,通过ELISA对制备的多克隆抗体的效价进行测定;最后应用Western blot评估这三个亚型多克隆抗体的特异性,进而对双峰驼血清中IgG1、IgG2和IgG3的抗原特异性进行分析。结果表明,应用Protein A和Protein G亲和层析柱成功分离纯化出双峰驼血清中的IgG1、IgG2和IgG3;并制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体效价均在1∶10000以上,并且所获得的多克隆抗体分别与IgG1、IgG2和IgG3之间均存在交叉反应,但兔抗双峰驼IgG1多克隆抗体较其它两个亚型多克隆抗体特异性低。结果证明,双峰驼IgG1、IgG2和IgG3均具有良好的免疫原性,三者结构虽存在显著差异,但其抗原特性类似。     

棉铃虫钙粘蛋白N端多肽多克隆抗体制备及对Bt抗性的初步检测

用重组表达的棉铃虫Helicoverpa armigera(Hübner)中肠钙粘蛋白N端多肽片段制备兔多克隆抗体,并利用其对Bt抗性进行鉴定。通过RT-PCR方法对棉铃虫中肠钙粘蛋白N端多肽的基因片段Cad285进行PCR扩增,将其克隆到pET-30a原核表达载体中,在大肠杆菌BL21(DE3)中经IPTG诱导表达,得到35ku的重组融和蛋白,融合表达的包涵体经过变性、Ni-NTA柱亲和纯化、复性等方法处理包涵体,获得可溶性纯化蛋白,用纯化后蛋白免疫新西兰兔制备多克隆抗体,ELISA检测其效价高于1∶16000;利用最终获得的多克隆抗体对室内纯合Bt抗/感品系的棉铃虫中肠钙粘蛋白进行Western blot分析,结果显示敏感和抗性品系之间有明显差异,表明其能够应用对Bt抗性进行初步检测。     

灰飞虱内生病毒HiPV外壳蛋白VP1多克隆抗体的制备及在病毒检测中的应用

【目的】前期发现水稻条纹病毒(rice stripe virus, RSV)可与介体灰飞虱Laodelphax striatellus体内的HiPV病毒(Himetobi P virus, HiPV)互作。本研究旨在制备HiPV外壳蛋白VP1的多克隆抗体,并评估其在HiPV病毒检测中的可用性,以为深入研究HiPV-RSV和HiPV-灰飞虱的互作机制提供技术支持。【方法】以RT-PCR方法从灰飞虱成虫体内扩增HiPV主要外壳蛋白基因VP1,然后将VP1基因亚克隆至原核表达载体pET-32a中,构建表达载体pET-VP1。将重组质粒转化大肠杆菌Escherichia coli BL21 (DE3),经IPTG诱导、Ni²⁺-NTA亲和层析纯化,获得重组蛋白,免疫新西兰大白兔,制备抗体。【结果】从灰飞虱体内克隆到774 bp的HiPV外壳蛋白基因VP1,经原核表达、纯化,获得分子量约47.5 kD的融合蛋白,免疫新西兰大白兔后获得VP1多克隆抗体。该抗体间接ELISA效价达1∶819 200,与HiPV外壳蛋白VP1有特异性反应,而与灰飞虱蛋白无交叉反应。利用该多克隆抗体建立了检测单头灰飞虱成虫体内HiPV的Western blot和免疫捕获RT-PCR方法,检测结果显示HiPV在携带和不携带RSV的灰飞虱高亲和性群体内均广泛存在。【结论】利用制备的HiPV的VP1多克隆抗体可特异性检测灰飞虱体内HiPV。本研究为HiPV病毒的快速检测以及HiPV-RSV互作、HiPV-灰飞虱互作研究提供了技术支持。     

人抗酶抑制因子-1的原核表达纯化及多克隆抗体的制备

原核表达纯化人抗酶抑制因子-1((antizyme inhibition factor-1,AZIN1),制备并鉴定抗AZIN1多克隆抗体。p ET-28a/AZIN1表达质粒转化大肠杆菌BL21(DE3)后,IPTG诱导蛋白表达,利用Ni-NTA树脂于变性条件下亲和层析纯化人AZIN1蛋白。将重组AZIN1蛋白用作抗原免疫BALB/c小鼠以制备多克隆抗体,ELISA检测抗AZIN1抗体效价,Western bloting、细胞免疫荧光、细胞免疫化学方法检测抗体的应用。结果显示,重组p ET-28a/AZIN1表达质粒经酶切及测序鉴定构建正确。细菌内重组AZIN1蛋白可被IPTG诱导表达并以包涵体的形式存在。用亲和层析法能有效纯化原核表达的AZIN1蛋白,该蛋白在小鼠体内能够诱导抗AZIN1特异性抗体产生,血清效价达到1640 000。制备抗体能够特异性识别和结合人及小鼠瘤细胞中表达的AZIN1蛋白,并可有效用于AZIN1的Western blotting、细胞免疫荧光和细胞免疫化学分析。成功原核表达和纯化了人AZIN1蛋白并制备了抗AZIN1多克隆抗体,为深入研究AZIN1在调控细胞增殖及在疾病防治中的作用提供了研究基础。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.