家蚕血细胞系BmHc对脂多糖(LPS)和20-羟蜕皮酮(20E)的敏感性

Sensitivity of BmHc Cell Line From Bombyx mori to Lipopolysaccharide and 20-hydroxyecdysone

为研究家蚕的细胞信号转导及外源基因在家蚕血细胞中的表达,用TC-199培养基建立起一个血细胞系BmHc。BmHc细胞系由原血球、小球细胞、颗粒细胞和浆细胞等血细胞组成,细胞倍增时间约30 h。脂多糖(LPS)刺激血细胞合成抗菌蛋白和多肽,启动细胞吸收Ca2+参与细胞的增殖。用Flu-3 AM处理细胞30 min并结合TCS-SP2激光共聚焦扫描显微镜技术对Ca2+的分布、定位进行研究的结果表明,Ca2+主要分布在原血球和颗粒细胞的膜表面,但有部分荧光分布深入到原血细胞核,显示细胞核参与了钙信号的转导途径。同等条件下,用20羟蜕皮酮(20E)处理仅可见轻微的荧光反应。LPS激发并打开了细胞膜上的Ca2+通道,引起细胞内Ca2+浓度的变化。在培养基内加入20E引起的Ca2+浓度变化相对较小,因此,20E不是细胞膜上Ca2+通道的有效激发剂。

To study signal transduction path way in intracellular cells of insect and express foreign gene in cultured hemocytes, a BmHc hemocytes cell line from B. mori was established in TC-199 medium. The BmHc cell line consists of all types of hemocytes including prohemocytes, spherulocytes, granulocytes and plasmatocytes, etc, Doubling time of the cell line is about 30 h, LPS elicited hemocytes to synthesize antibacterial proteins and peptides and prompted cells to uptake more Ca^2 ＋ to take part in active cell proliferation. Flu-3 AM study with TCS-SP2 laser scanning confocal microscope showed that Ca^2 ＋ distributed on the membrane of prohemocytes and granulocytes, but some of them were on the signal path way to the prohemocytes nuclei in 30 min after LPS induction; while under the same condition, the membrane of the cells was only slightly stained with Flu-3 AM after treatment with 20E. LPS elicited and opened Ca^2 ＋ channel and caused the change of [ Ca^2＋]. A relatively stable change of [ Ca^2＋] in the cells when 20E was added to the culture medium that indicated 20E is not an effective enhancer for sensitive Ca^2＋ channel on the cell membrane.