Ⅰ型禽腺病毒AV208株在鸡肝癌细胞中增殖规律的研究

【目的】摸索鸡肝癌细胞系(LMH)培养条件,并采用LMH细胞系对Ⅰ型禽腺病毒AV208株(FAVⅠ-AV208)进行增殖规律的研究。【方法】细胞以不同血清浓度培养并以不同接种比例传代。观察最佳感染复数下的细胞病变和病毒增殖情况,并研究病毒接种物浓度与蚀斑形成数量之间的关系。【结果】LMH细胞系的最适培养条件为,以10%FBS的培养基以1:5比例传代培养。FAVⅠ-AV208毒株可以在LMH细胞中良好增殖并达到较高的滴度(107.5TCID50/0.1 mL)。在最佳感染复数(MOI)为0.01时,72 h细胞即出现明显的细胞病变(CPE),特征为细胞变大变圆,集聚成不规则的葡萄串状。病毒接种物浓度与蚀斑形成数量间呈线性相关。【结论】LMH细胞是FAVⅠ较合适的病毒培养系统,为研制禽腺病毒重组疫苗提供了有利工具。     

八种昆虫离体细胞系对灭多威农药的敏感性研究

采用ＭＴＴ法,研究了灭多威农药对八昆虫离体细胞系敏感性的差异。结果显示,同一浓度处理下,不同细胞系细胞病变的程度不同,敏感性差异很大。ＬＣ５０值在１０＾２－１０＾５μｇ／ｍｌ范围内变化,其中,白纹伊蚊细胞系是最敏感的,而小菜蛾细胞系几乎不敏感。     

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822)

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28 °C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.     

Progress in the study of virus detection methods: The possibility of alternative methods to validate virus inactivation

Virus inactivation validation studies have been widely applied in the risk assessment of biogenic material-based medical products, such as biological products, animal tissue-derived biomaterials, and allogeneic biomaterials, to decrease the risk of virus transmission. Traditional virus detection methods in an inactivation validation study utilize cell culture as a tool to quantify the infectious virus by observing cytopathic effects (CPEs) after virus inactivation. However, this is susceptible to subjective factors because CPEs must be observed by experts under a microscope during virus titration. In addition, this method is costly and time- and labor-consuming. Molecular biological technologies such as quantitative polymerase chain reaction (qPCR) have been widely used for virus detection but cannot distinguish infectious and noninfectious viruses. Therefore, qPCR cannot be directly applied to virus inactivation validation studies. In this paper, methods to detect viruses and progress in the challenge of differentiating infectious and noninfectious viruses with the combination of pretreatment and qPCR techniques such as the integrated cell culture-qPCR (ICC-qPCR) method are reviewed. In addition, the advantages and disadvantages of each new method, as well as its prospect in virus inactivation validation studies, are discussed.     

四种动物病毒的细胞培养及血凝检测的比较研究

四种动物病毒的细胞培养及血凝检测的比较研究李天宪,赵林,罗怡珊,冯锋（中国科学院武汉病毒研究所,武汉４３００７１）关键词细小病毒,细胞培养,细胞病变,血凝试验云豹肠炎病毒（ＬＰＶ）、水貂肠炎病毒（ＭＥＶ）、犬肠炎病毒（ＣＰＶ）和猫泛白细胞减少症病毒（...     

Mos 3' UTR regulatory differences underlie species-specific temporal patterns of Mos mRNA cytoplasmic polyadenylation and translational recruitment during oocyte maturation

The Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal Mos mRNA. However, the precise temporal requirements for Mos protein function differ between oocytes of model mammalian species and oocytes of the frog Xenopus laevis. Despite the advances in model organisms, it is not known if the translation of the human Mos mRNA is also regulated by cytoplasmic polyadenylation or what regulatory elements may be involved. We report that the human Mos 3' untranslated region (3' UTR) contains a functional cytoplasmic polyadenylation element (CPE) and demonstrate that the endogenous Mos mRNA undergoes maturation-dependent cytoplasmic polyadenylation in human oocytes. The human Mos 3' UTR interacts with the human CPE-binding protein and exerts translational control on a reporter mRNA in the heterologous Xenopus oocyte system. Unlike the Xenopus Mos mRNA, which is translationally activated by an early acting Musashi/polyadenylation response element (PRE)-directed control mechanism, the translational activation of the human Mos 3' UTR is dependent on a late acting CPE-dependent process. Taken together, our findings suggest a fundamental difference in the 3' UTR regulatory mechanisms controlling the temporal induction of maternal Mos mRNA polyadenylation and translational activation during Xenopus and mammalian oocyte maturation.     

Detailed dosimetry calculation for in-vitro experiments and its impact on clinical BNCT

PurposeBoron Neutron Capture Therapy (BNCT) is a form of hadrontherapy based on the selective damage caused by the products of neutron capture in ¹⁰B to tumour cells. BNCT dosimetry strongly depends on the parameters of the dose calculation models derived from radiobiological experiments. This works aims at determining an adequate dosimetry for in-vitro experiments involving irradiation of monolayer-cultured cells with photons and BNCT and assessing its impact on clinical settings.M&MDose calculations for rat osteosarcoma UMR-106 and human metastatic melanoma Mel-J cell survival experiments were performed using MCNP, transporting uncharged particles for KERMA determinations, and secondary particles (electrons, protons, ¹⁴C, ⁴He and ⁷Li) to compute absorbed dose in cultures. Dose-survival curves were modified according to the dose correction factors determined from computational studies. New radiobiological parameters of the photon isoeffective dose models for osteosarcoma and metastatic melanoma tumours were obtained. Dosimetry implications considering cutaneous melanoma patients treated in Argentina with BNCT were assessed and discussed.ResultsKERMA values for the monolayer-cultured cells overestimate absorbed doses of radiation components of interest in BNCT. Detailed dose calculations for the osteosarcoma irradiation increased the relative biological effectiveness factor RBE_1% of the neutron component in more than 30%. The analysis based on melanoma cases reveals that the use of survival curves based on KERMA leads to an underestimation of the tumour doses delivered to patients.ConclusionsConsidering detailed dose calculation for in-vitro experiments significantly impact on the prediction of the tumor control in patients. Therefore, proposed methods are clinically relevant.     

Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro

Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.