Isolation and primary culture of stem cells from the midgut epithelium of the silkworm <i>Bombyx mori</i>
Isolation and primary culture of stem cells from the midgut epithelium of the silkworm <i>Bombyx mori</i>
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家蚕中肠上皮干细胞的分离及原代培养
DOI：
10.11416/jibs.89.3_055
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发表时间：
2020
影响因子：
--
通讯作者：
J. Kobayashi
中科院分区：
文献类型：
--
作者：
N. Fujiwara;M. Azuma;M. Itoh;J. Kobayashi
蜕皮过程对鳞翅目幼虫的发育和成虫的变态是必不可少的。然而，由于缺乏有效的体外分离和鉴定中肠干细胞的方法，限制了对细胞更新和再生的研究。在这里，我们展示了一种简单而安全的方案，用于从家蚕的蜕皮幼虫和蚕蛹中分离中肠干细胞。这种方法是基于干细胞的生理学，干细胞与上皮细胞的底部松散地联系在一起，在蜕皮或变态阶段繁殖。当它们延长并分化成成熟的形式时，它们最终会嵌入或取代现有的上皮细胞。使用大约10种昆虫，有可能获得500,000个高度同质性的细胞和80%的存活率。从四龄蜕皮幼虫和甲虫蛹中提取的干细胞呈球形，可在4℃下保存1~2周，无活性损失。然而，在Grace的昆虫培养液中培养一周后，活细胞数量呈稳步下降的趋势。补充剂如胎牛血清、富含脂肪的牛血清白蛋白或经热处理的天然家蚕血浆不支持干细胞增殖或抑制细胞死亡。尽管干细胞的连续培养仍然很困难，但所描述的中肠研究方法可能会被用于干细胞的生化研究和体外病理检测，这可能会为农业病虫害防治带来新的策略。
The process of molting is essential for the growth of lepidopteran larvae and their metamorphosis into adults. However, the investigation of cellular renewal and regeneration is limited by the lack of efficient methods for the isolation and characterisation of midgut stem cells in vitro. Here, we demonstrate an easy and secure protocol for midgut stem cell isolation from molting larvae and pharate pupae of the silkworm, Bombyx mori. This method is based on the physiology of stem cells, which lie loosely associated with the base of epithelial cells and multiply during the molting or metamorphosing phase. They finally intercalate or replace existing epithelial cells as they elongate and differentiate into mature forms. Using approximately 10 insects, it was possible to obtain> 500,000 cells with high homogeneity and> 80% viability. Stem cells prepared from the fourth instar molting larvae and pharate pupae were spherical and could be preserved at 4 C for 1-2 weeks with no viability loss. However, the number of living cells showed a steady decline when cultured in Grace’s insect culture medium for one week. Supplements such as fetal bovine serum, lipid-rich bovine serum albumin, or heat-treated native B. mori plasma did not support stem cell proliferation or suppress cell death. Although continuous stem cell culture is still difficult, the described method for midgut investigation could potentially utilized for biochemical studies of stem cells and pathological assays in vitro, which may lead to novel strategies for agricultural pest management.