荧光素标记检测乙型肝炎病毒表面抗原特异性杀伤性T淋巴细胞方法的探讨

用绿色荧光前体化合物Calcein AM标记靶细胞,经过与杀伤性T淋巴细胞(CTL)效应细胞数小时的孵育,通过分析培养上清中的荧光强度检测CTL效应。通过对一系列标记条件的研究：不同浓度Calcein AM标记靶细胞、不同洗涤缓冲液、不同标记细胞浓度、不同洗涤次数、不同裂解液配方,确定Calcein AM荧光素标记法的最佳条件。用该方法检测乙型肝炎病毒表面抗原的DNA疫苗免疫Balb／c小鼠对P815S细胞的CTL效应,E／T比为10时,杀伤效率接近饱和,达到65％。通过荧光显微镜直接观察杀伤后的P815S细胞,细胞破裂程度与计算出的杀伤效率有一定相关性。     

从HFRS患者的PBMC建立BLCL及其意义

体外研究汉滩病毒(Hantaan virus,HTNV)S基因及其5'端、3'端在BLCL(EBV-transformed Blymphoblastoidcellline,BLCL)内稳定表达的意义,为核蛋白T细胞表位的研究奠定基础.本研究从肾综合征出血热(HFRS)患者的外周血单个核细胞(PBMC)建立与HTNV特异性CTL同源的患者自身的EB病毒转化的B淋巴母细胞系,将含有不同HTNV S基因片段的重组真核表达载体转染入BLCL,获得长期稳定表达,作为靶细胞系,为下一步进行CTL杀伤试验提供靶细胞系.设计4条引物,用PCR方法从PBV220-S22原核质粒中扩增出S基因全读码框(37-1326bp)及S基因5'端(37-501bp),S基因3'端(502-1326bp),用TA克隆将其克隆入pcDNA3.1/V5-His-TOPO载体中,成功构建pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C真核表达载体,并通过脂质体转染至BLCL细胞中,进行了稳定表达.间接免疫荧光成功检测到pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C在BLCL细胞中的表达.pcDNA3.1-S及pcDNA3.1-S-N、pcDNA3.1-S-C真核表达载体有较高的转染效率,目的基因能在宿主细胞中长期稳定表达,有利于研究HTNV-S基因在T细胞表位研究中的意义.建立的特异性CTL克隆对表达完整NP、NP羧基和氨基端肽段的靶细胞均有比较明显的杀伤效应,平均杀伤率分别为50.2%、39.0%和25.4%.HTNV-NP优势T细胞表位可能主要位于病毒核蛋白的羧基端.     

HIV—1CN嵌合基因与IL—2基因共表达产物诱导产生CTL的实验研究

经脂质体介导共表达中国流行株HIV-1gag-gp120基因与IL-2基因的核酸疫苗质粒pGPIL-2转染BHK-21细胞,以间接免疫荧光法鉴定其表达.取pGPIL-2免疫鼠脾细胞,检测pGPIL-2诱导的细胞毒性T细胞杀伤活性.杀伤实验结果证明,经基因免疫获得的CTL效应细胞,可杀伤HIV-1\{CN\}嵌合基因转染的靶细胞.提示pGPIL-2可有效诱导CTL的产生,该研究结果为进一步设计中国流行株HIV-1核酸疫苗提供了重要实验依据.     

雷帕霉素对CAR-T细胞功能的影响

为探索雷帕霉素对CAR-T细胞体外杀伤靶细胞、杀伤后的扩增及细胞因子分泌能力的影响,该研究在体外使用靶向CD19的CAR-T细胞与CD19⁺CD22⁺Raji靶细胞的T细胞特异性杀伤模型,在不同效靶比、不同时相点、不同雷帕霉素浓度下,通过流式细胞绝对计数检测细胞杀伤率发现雷帕霉素不影响CAR-T细胞对靶细胞的杀伤能力。通过羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)检测CAR-T细胞扩增能力,发现雷帕霉素通过抑制CAR-T细胞增殖而非促进凋亡的方式降低其活化后的扩增数量。通过流式细胞术微球阵列法(CBA)检测细胞因子分泌情况,发现雷帕霉素可显著性降低CAR-T细胞被靶细胞活化后的细胞因子分泌水平。综上所述,雷帕霉素可在不影响CAR-T细胞杀伤功能的前提下,降低其炎性因子分泌水平。     

多表位肽抗原诱导特异性CTL抗慢性髓性白血病细胞的体外实验研究

目的：在应用基因工程技术人工表达获得多表位BCR-ABL融合蛋白的基础上,对该融合抗原在体外诱导对自血病细胞的特异性杀伤效应进行检测,探索慢性髓性自血病（CML）免疫治疗的新途径。方法：从外周血单个核细胞培养树突细胞（DC）,以BCR-ABL融合抗原脉冲刺激DC,诱导特异性细胞毒T淋巴细胞（CTL）产生;MTT法检测CTL对白血病靶细胞的特异性杀伤活性。结果：以BCR-ABL融合抗原刺激产生的CTL能特异性抑制b3a2＋的靶细胞生长,包括K562细胞（P〈0．01）和HIJA-A2＋／b3a2＋的CML原代细胞（P〈0．05）,而对HIA-A2-或b2a2＋靶细胞无明显抑制作用。结论：设计表达的多表位BCR-ABL融合抗原能在体外诱导特异性抗CML免疫反应,抑制b3a2＋自血病细胞生长,有望为进一步的体内实验奠定基础。     

非复制重组痘苗病毒表达人免疫缺陷病毒Bostwana C亚型Nef蛋白及免疫效果的观察

从含人类免疫缺陷病毒Ⅰ型(HIV-1)Bostwana C亚型全基因的质粒pJM4-HIV中克隆了nef基因,并利用非复制型痘苗病毒载体构建表达Nef蛋白的重组病毒NTVJ1175nef,经PCR和Southern blot鉴定,nef基因正确整合到痘苗病毒基因组的J片段上;感染人源细胞后,经Western blot和免疫荧光检测表明,重组病毒能很好地表达Nef蛋白,并定位于细胞质中.NTVJ1175nef免疫BALB/c小鼠后,经Pep-IFN-'γ-Assay法检测,可诱导产生针对表位肽P1特异的可分泌IFN-'γ的C 8 T细胞(占脾细胞总数0.20%);经乳酸脱氮酶(LDH)法检测证实,诱导的细胞毒性T细胞(CTL)可特异性地杀伤表位肽P1特异P815靶细胞.这些结果表明,NTVJ1175nef具有良好的细胞免疫原性,为下一步构建表达包含HIV早期抗原的多组分重组痘苗病毒候选疫苗奠定了免疫学基础.     

Epstein-Barr病毒特异性T细胞对重组痘苗病毒表达LMP和EBNA2的靶细胞的识别

本文用EB病毒转化自体淋巴细胞所建立的类淋巴母细胞系(LCL),以及用EB病毒潜伏感染膜蛋白(LMP)基因和核蛋白-2(EBNA2)基因与痘苗病毒重组的重组病毒(Vac-LMP和Vac-EBNA2)感染的自身纤维母细胞,同时作为刺激细胞和靶细胞,以~(51)Cr释放法检测5例血清中EB病毒VCA—IgA抗体阳性者及1例阴性健康者外周血单个核细胞(PBMC)的特异性T细胞杀伤效应。结果表明,用自身LCL激活的EB病毒特异性T细胞杀伤效应高峰出现在第14～28天;参与杀伤性细胞免疫反应的T细胞亚群主要是T3、T8阳性的细胞毒性T细胞,其对靶细胞的识别及杀伤受HLA-I的限制。用重组牛痘病毒感染的纤维母细胞作靶细胞或刺激细胞,有1例供者可接受LMP,另1例可接受EBNA2的刺激,并对相应的靶细胞产生特异性T细胞杀伤反应,表明EB病毒-LMP和EBNA2可能既是EB病毒特异性T细胞的刺激抗原,又是其识别的靶抗原。     

自然杀伤T细胞抗胞内菌感染的研究进展

自然杀伤T细胞(NKT细胞)是免疫细胞中一类具有特定标记的T细胞亚群,能特异性识别南抗原呈递细胞表面CD1d分子所呈递的糖脂类抗原.活化后的NKT细胞能分泌多种细胞因子,参与机体的天然免疫和获得性免疫反应,还能直接杀伤靶细胞,具有效应细胞的功能.研究发现,NKT细胞在抗胞内菌感染中发挥着重要作用.     

口蹄疫重组鸡痘病毒诱导猪特异性细胞毒性T淋巴细胞(CTL)杀伤活性检测

目的:评价口蹄疫重组鸡痘病毒vUTAL3CP1诱导猪产生特异性细胞毒性T淋巴细胞(CTL)杀伤活性的能力。方法:用PCR方法亚克隆O型FMDVVP1基因C末端部分片段(第130～213AA)。将其插入真核表达载体pDisplay中,构建质粒pDisplay-mVP1。将pDisplay-mVP1转染PK15细胞,经3次G418加压筛选,并用RT-PCR和IFA鉴定,证明获得表达目的基因的PK15/pDisplay-mVP1细胞。最后,利用该细胞作为靶细胞,用LDH法检测口蹄疫重组鸡痘病毒vUTAL3CP1免疫猪的特异性CTL杀伤活性。结果:vUTAL3CP1免疫组在效靶比25∶1和50∶1时,CTL裂解活性分别达到42.84%±32.1%和61.94%±42.8%,显著高于灭活疫苗组与其它对照组(p<0.01)。结论:vUTAL3CP1可以诱导猪产生高水平的特异性细胞毒性T淋巴细胞(CTL)杀伤活性,为进一步的FMDV基因工程疫苗的研究奠定了基础。     

Fas介导细胞凋亡及相关免疫调节作用

Fas/CD95作为肿瘤坏死因子(TNF)/神经生长因子(NGF)分子受体超家族成员,在细胞凋亡及体内免疫调节方面发挥重要的作用。细胞在接收到经Fas传递的凋亡信号后,主要经胞浆caspase和线粒体两种途径引发细胞程序性死亡。Fas介导T/B淋巴细胞的凋亡,在这些细胞的早期分化发育和维持机体免疫平衡中起主要作用。CTL和NK等杀伤细胞也通过Fas-FasL介导的细胞凋亡而发挥对靶细胞的杀伤效应。因此,Fas-FasL系统在肿瘤、自身免疫性疾病、艾滋病等疾病过程中发挥重要的作用。     

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.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

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.