体外转录的自我复制型mRNA疫苗研究进展*

自我复制型mRNA是一种灵活的疫苗平台,该平台的开发基于甲病毒表达载体,其中复制必需基因得以完整保留,而结构蛋白基因则被来自病原的抗原基因替换。由于避免了病原培养、毒力返强和现存免疫的干扰,使其成为疫苗快速设计的理想平台。大量研究数据显示,此类疫苗可应用在人、小鼠、兔、猪、禽甚至鱼类体内诱导体液免疫和细胞免疫。过去,自我复制mRNA疫苗的研究采用重组单载体的模式,基因组骨架来源于辛德毕斯病毒、塞姆利基森林病毒和委内瑞拉马脑炎病毒。现在,反式复制型RNA和核酸修饰的反式复制型RNA作为下一代技术平台被寄予厚望。对基于甲病毒表达载体的mRNA疫苗技术的研究进展进行概述,重点介绍针对以流感病毒、新型冠状病毒和寨卡病毒等为代表的自我复制型mRNA疫苗研究现状,并探讨了该技术平台的未来发展方向。     

体外转录的自我复制型mRNA疫苗研究进展*

自我复制型mRNA是一种灵活的疫苗平台,该平台的开发基于甲病毒表达载体,其中复制必需基因得以完整保留,而结构蛋白基因则被来自病原的抗原基因替换。由于避免了病原培养、毒力返强和现存免疫的干扰,使其成为疫苗快速设计的理想平台。大量研究数据显示,此类疫苗可应用在人、小鼠、兔、猪、禽甚至鱼类体内诱导体液免疫和细胞免疫。过去,自我复制mRNA疫苗的研究采用重组单载体的模式,基因组骨架来源于辛德毕斯病毒、塞姆利基森林病毒和委内瑞拉马脑炎病毒。现在,反式复制型RNA和核酸修饰的反式复制型RNA作为下一代技术平台被寄予厚望。对基于甲病毒表达载体的mRNA疫苗技术的研究进展进行概述,重点介绍针对以流感病毒、新型冠状病毒和寨卡病毒等为代表的自我复制型mRNA疫苗研究现状,并探讨了该技术平台的未来发展方向。     

Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.     

血脑屏障体外模型研究进展

脑毛细血管上的特殊结构单元为大脑提供氧气和养分,与此同时形成一种限制性屏障,称为血脑屏障(BBB),该结构单元由单层脑微血管内皮细胞构成,内皮细胞外侧的周细胞、基膜以及星形胶质细胞的足突也参与了血脑屏障的形成.血脑屏障是一种选择性渗透屏障,大多数中枢神经系统候选药物在血脑屏障中的渗透性差,用实验动物进行药物筛选具有成本高、周期长、成功率低等缺点.此外,直接在人体中试验有违道德伦理,但建立可靠的体外血脑屏障模型可以简化实验过程、缩短试验周期、实验结果更易测定,因此建立体外BBB模型可以极大地加快中枢神经系统药物的研发.目前已研究的模型主要可以分为3类:单培养、共培养、三培养,这些模型由简单到复杂,与体内血脑屏障的相似性也越来越高.本文就目前现有的血脑屏障模型进行综述,以期未来在体外BBB模型设计中有新的思路.     

In Vitro Assays of Processive Myosin Motors

Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent single-molecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include density-dependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins.     

体外培养的神经元网络可塑性

神经元网络是大脑执行高级认知行为的结构基础,研究证明学习记忆及神经退行性疾病与神经元网络可塑性密切相关。因此,揭示调控和改变神经元网络可塑性的机制对理解神经系统信息交互以及疾病治疗具有重大意义。目前,基于微电极阵列（microelectrode array, MEA）培养的神经元网络是体外探究学习和记忆机制的理想模型,同时针对该模型的研究为预防和治疗神经退行性疾病提供了独特的视角。本文综述了基于MEA采集体外培养神经元网络的放电信号来构建功能网络的相关研究,分别从二维神经元网络和三维脑类器官发育,以及开环和闭环电刺激对神经元网络可塑性影响的角度,总结了体外培养神经元网络可塑性的相关研究,最后对该方向的应用前景进行了展望。     

体外遗传学方法及应用

体外遗传学是利用核酸分子本身一定的表型（如结合、催化等）在试管中分离筛选特定核酸分子序列进行研究的方法．由于体外遗传学方法改变了自然界缓慢的进化过程,使人为的进化得以简单地实现,同时也使许多核酸功能区的识别和确定由被动变为主动,为进一步探索基因的调控规律及主动地调节生化反应过程提供了有效的手段,近年来体外遗传学方法及在分子生物学方面的应用得到了很大的发展．     

Retroviral Integrase Proteins and HIV-1 DNA Integration

Retroviral integrases catalyze two reactions, 3′-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.     

In VitroCulture System for Iris-Pigmented Epithelial Cells for Molecular Analysis of Transdifferentiation

Dissociated cells of the iris-pigmented epithelium (IPE) from a 1-day-old chick grew in monolayer culture and stably maintained their differentiated state when cultured with standard culture medium. After replacement of the control medium by EdFPH medium, which is effective in inducing dedifferentiation of retinal pigmented epithelium (RPE) cells, all cells rapidly lost pigment granules, proliferated intensively, and dedifferentiated. By further addition of ascorbic acid, dedifferentiated cells accumulated and formed a large number of lentoids. This system provides a useful opportunity for analyzing cellular and molecular mechanism involved in each step of transdifferentiation. Furthermore, Northern blot data indicates that the up-regulation of pax-6 gene could be an important event during lens regeneration as well as during normal lens development.     

Apolipoprotein H Is Not Affected by In Vitro Glycosylation

Increased nonenzymatic glycosylation of all major classes of apolipoproteins has been demonstrated in diabetes. In this work we deal with the in vitro nonenzymatic glycosylation of apolipoprotein H, whose role in lipid metabolism is still poorly understood and whose levels increase in diabetes. Apolipoprotein H was isolated from human plasma and purified through a combination of affinity chromatography and continuous elution electrophoresis. The in vitro glycosylation was performed by incubating purified apolipoprotein H with high concentration of glucose. Our results indicate that the in vitro nonenzymatic glycosylation has no effect on the physical properties of apolipoprotein H, despite the fact that this apolipoprotein contains a high number of lysine residues. Since the in vitro concentration of glucose was far higher than the levels normally found in diabetic subjects, it is unlikely for apolipoprotein H to become glycosylated in diabetes.     

神经元凋亡的离体模型及其检测技术

近年来,随着细胞凋亡研究的深入,神经元凋亡与神经退变病的关系愈发引人注目,已建立多种神经元凋亡的离体模型.多种因素如营养剥夺、自由基、谷氨酸、低钙及β-淀粉样蛋白等均可诱发神经元凋亡.凋亡的检测,可先从酶或蛋白质的变化判断神经元的损伤情况,再结合形态学观察,最后通过DNA电泳等确证.     

Action of FMRFamide-like Peptides on Porcine Gastrointestinal Motility In Vitro

Decker, B., B. Vadokas, U. Kutschenreuter, K. Golenhofen, K. Voigt, G. P. Mcgregor and K. Mandrek. Action of FMRFamide-like peptides on porcine gastrointestinal motility in vitro. Peptides 18(10) 1531–1537, 1997.—Mechanical activity was recorded in circular and longitudinal smooth muscle preparations isolated from extensive regions of the porcine gastrointestinal tract in response to the FMRFamide-like neuropeptides F8Famide and A18Famide. In all preparations, the peptides were about equipotent in producing phasic contractions or enhancing spontaneous activity. The most prominent responses were observed in jejunal longitudinal strips which were on the average 91% (±4% SEM, n = 15; 10⁻⁶ M) of the histamine (10⁻⁵ M) responses. The peptide-induced phasic activity was completely abolished by nifedipine but was unaffected by tetrodotoxin, atropine, phentolamine, yohimbine, phenoxybenzamine, propranolol, methysergide, cimetidine, indomethacin, levallorphane or naloxone. Both peptides enhanced acetylcholine-induced contractions. However, bovine ileum and guinea-pig taenia coli was not affected by these peptides. The results indicate that F8F- and A18F-amide contract porcine gastrointestinal smooth muscle by acting directly via non-opioid receptors on L-type calcium channels. In addition an increase of the sensitivity to cholinergic stimulation occurs.     

维拉帕米对铜绿假单胞菌群体感应抑制作用的体外研究

考察维拉帕米对铜绿假单胞菌（Pseudomonas aeruginosa, PA）群体感应（Quorum sensing, QS）的抑制作用。测定维拉帕米最小抑菌浓度(Minimal inhibit concentration,MIC) ;构建培养环境,考察维拉帕米对PA生长的影响,绘制生长曲线;毒力因子表达实验中,分别考察维拉帕米对PA弹性蛋白酶表达、蛋白水解酶表达和绿脓毒素表达的影响。结果表明,维拉帕米MIC₅₀为128 μg/mL,MIC₉₀为512 μg/mL,最低抑菌质量浓度范围为128~512 μg/mL时,具有较好抑菌活性;生长曲线表明,维拉帕米质量浓度为16 μg/mL时,开始抑制PA的生长,随着浓度的增加,抑制作用逐渐增大;当维拉帕米质量浓度为512、256、128、64、32、16 μg/mL时,明显抑制弹性蛋白酶表达（P<0.01）,质量浓度为8 μg/mL时,对弹性蛋白酶有一定抑制作用（P<0.05）,维拉帕米质量浓度为512、256、128、64、32 μg/mL时,明显抑制蛋白水解酶的活性和绿脓毒素的表达（P<0.01）,当质量浓度为16 μg/mL时,对蛋白水解酶活性和绿脓毒素的表达有一定抑制作用（P<0.05）;维拉帕米对QS的抑制有浓度依赖。维拉帕米对PA的QS有明显抑制作用,体外可明显抑制PA生长,可作为抗菌增效的潜在开发药物。     

犬卵母细胞的体内成熟环境与体外成熟培养

犬(Canis familiaris)是生物医学研究的最重要模型动物之一.但由于生殖生理的特殊性,其卵母细胞的体外培养成熟率低,辅助生殖研究进展缓慢,严重制约了该动物在生物科学研究中的运用.在犬科动物体内,排卵前卵母细胞处于高浓度孕酮的卵泡环境中,在生发泡期排到输卵管内,并在此恢复和完成减数分裂.因此,犬卵母细胞体外成熟所需的条件不同于其他哺乳动物,目前主要采用以添加相关因子的M199作为培养液,但体外培养发育至MⅡ期的比率仅为15%～20%.所以,必须在了解犬卵母细胞体内成熟机制的基础上,建立一套类似于体内生理环境的体外成熟培养体系.本文在阐述犬卵母细胞体内成熟生理过程的基础上,对其体外成熟培养方法和影响因素的研究现状进行分析,为相关研究提供参考.     

Partial In Vitro Digestion of Active Gliadin-Related Peptides in Celiac Disease

Peptides corresponding to residues 75–86 (RPQQPYPQPQPQ) and 75–85 of the A-gliadin structure, which were shown to be active in an animal model of celiac disease, were digested in vitro with small intestinal mucosa from children with celiac disease in remission and with mucosa from normal children. The products of digestion were separated into two fractions by gel permeation chromatography. Undigested residues (M _r > 400 fraction) from both peptides contained mainly glutamine, proline, and tyrosine, while the digested materials (M _r < 400 fraction) contained mainly proline, glutamine and arginine. Much larger amounts of undigested peptides were obtained from digestion with celiac mucosa than from normal mucosa. The results with peptide 75–86 indicated that the octapeptide 77–84 (QQPYPQPQ) was the main residual component and this peptide was shown to be active in the assay. Peptide 77–84 was also obtained as a residue from digestion of peptide 75–85, together with heptapeptide 77–83. The results lend further support for a primary mucosal defect in celiac disease and indicate that residual peptides in the small intestine of patients with the disease still retain appreciable toxicity.     

In Vitro Angiogenic and Proteolytic Properties of Bovine Lymphatic Endothelial Cells

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-l, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.     

In Vitro DNA Tethering of HIV-1 Integrase by the Transcriptional Coactivator LEDGF/p75

Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.     

In VitroRearing ofTrichogramma minutumRiley (Hymenoptera: Trichogrammatidae) for Ten Generations, with Quality Assessment Comparisons ofin Vitroandin VivoReared Adults

Trichogramma minutumRiley were reared for 10 generations on an artificial diet containing a yeast extract, FreeAmine III, nonfat dry milk, chicken egg yolk, chicken embryo extract, andManduca sexta(L.) egg liquid. Quality control parameters, including adult longevity, sex ratio, pupation rate, percentage of pupae to emerge as adults, adult female body length, number ofHelicoverpa zea(Boddie) eggs parasitized by a female, and percentage of deformed females were assessed and compared to insects rearedin vivoon irradiatedH. zeaeggs. The development time was longer forin vitroreared insects, but there were more deformed females in thein vitroculture. The sex ratio, however, was generally not significantly different between thein vitroandin vivocultures. Thein vitroreared females generally were larger, lived longer, and parasitized moreH. zeaeggs. Emergence of adults was in excess of 75% in all but the firstin vitrogeneration and was generally not significantly different from adult emergence in thein vivoculture. These findings will be of value in the development of a practical system forin vitromass rearing ofTrichogrammafor use in biological control.     

我国体外诊断行业发展现状与对策建议*

体外诊断在疾病诊疗过程中扮演着非常重要的角色,素有“医生的眼睛”之称。新冠肺炎疫情让人们对生命健康的关注程度空前提高,在此背景下,体外诊断作为大健康产业的重要一环,迎来爆发式增长。概述全球和中国体外诊断行业发展现状,重点分析市场规模、竞争格局和国产化情况,分析现阶段我国体外诊断行业遇到的问题,并从突破原始性创新、加强资金保障、构建产业链生态、加大人才培养力度等方面提出建议和对策。