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关于果蝇翅成虫盘的长期体外培养

Towards long term cultivation of Drosophila wing imaginal discs in vitro.

作者信息

Handke Björn, Szabad János, Lidsky Peter V, Hafen Ernst, Lehner Christian F

机构信息

Institute of Molecular Life Sciences (IMLS), University of Zurich, Zurich, Switzerland.

Department of Biology, Faculty of Medicine, University of Szeged, Szeged, Hungary.

出版信息

PLoS One. 2014 Sep 9;9(9):e107333. doi: 10.1371/journal.pone.0107333. eCollection 2014.

Abstract

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.

摘要

黑腹果蝇的翅成虫盘是研究细胞生长、增殖和死亡控制以及器官发生过程中模式形成和形态发生的重要实验系统。适用于该系统的精确遗传方法促进了发育生物学领域具有根本重要性的概念进展。如果能在体外研究翅成虫盘的长期发育,其实验可及性和多功能性将进一步提高。例如,培养系统将允许以最大的时间和空间分辨率进行实时成像。然而,正如本文清楚表明的那样,标准培养方法会导致解剖后的翅成虫盘在培养数小时内迅速停止细胞增殖。使用已建立的S期和M期细胞标记以及新型报告转基因RGB细胞周期追踪器进行分析,结果显示体外培养会干扰整个间期的细胞周期进程,而不仅仅是在G1期。此外,对可诱导转基因的EGFP表达进行定量分析发现,盘培养对细胞周期进程之外的基本细胞功能有快速的不利影响。盘移植实验证实,这些有害后果并非反映分离过程中翅成虫盘的致命损伤,这清楚地表明是培养基不足。对替代培养基进行了评估,包括在原位生长过程中包围翅成虫盘的血淋巴。但发现分离的幼虫血淋巴甚至比目前的培养基更不合适,这可能是由于血淋巴分离或盘培养过程中的转化过程所致。分析了翅成虫盘培养过程中重要的生长调节途径的意义,以及胰岛素和翅成虫盘与幼虫组织共培养作为内分泌因子潜在来源的影响。基于我们的分析,我们制定了一种培养方案,可延长培养盘中的细胞增殖。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16c1/4159298/616fc970c34a/pone.0107333.g001.jpg

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