一种适于研究家蚕色素代谢的眠蚕体壁组织培养方法与黑色素合成抑制试验

A Tissue Culture Method Using Integument of Molting Larva as Material Suitable for Studying Pigment Metabolism in Bombyx mori and Inhibitory Experiment on Melanin Synthesis

利用家蚕幼虫眠时形成新表皮以及重新合成黑色素分布于新表皮上形成固有体色斑纹等特点,用4龄入眠后的家蚕幼虫体壁组织为材料,观察离体培养体壁的色素代谢变化,探索适宜的家蚕幼虫体壁培养基及培养温度条件,眠蚕体壁的取材方法及取材时间,色素代谢观察的外观标志性状等。实验结果表明:含10%优等胎牛血清的Grace培养基适于家蚕幼虫体壁组织培养,培养温度为26℃;取4龄期入眠11～12 h后剥去旧表皮的体壁组织更容易观察色素代谢的外观性状变化;体壁培养24 h后,刚毛再生和色素沉积是易于观察的色素代谢变化标志性状。通过对家蚕4龄眠蚕离体培养体壁组织的黑色素合成抑制试验,观察到体壁外观性状变化,证明酪氨酸羟化酶催化酪氨酸转化成多巴是家蚕黑色素合成代谢途径中的一个重要环节。

By utilizing the nature of molting Bombyx moil larva which forms new skin by initiating melanin synthesis again and distributing newly synthesized pigments in the new skin to form inherent body markings, we cultured the integument isolated from the molting 4th instar larvae and observed variation of pigment metabolism in the in vitro cultured integument to explore suitable culture medium and temperature, suitable sampling method and sampling time, and typical signs for observing pigment metabolism in integument of molting Bombyx mori larvae. The experimental results showed that： grace medium with 10% high grade fetal bovine serum was suitable for integument tissue culture of Bombyx mori larvae and the suitable culture temperature was 26 ℃; it was much easier to observe appearance change of pigment metabolism by sampling integument underneath the old skin at 11 -12 h of molting after culturing the integument for 24 h, setaregeneration and pigment deposition were typical signs of pigment metabolic changes easy to observe. The inhibitory experiment on melanin synthesis in the in vitro cultured integument of the molting 4th instar larvae displayed appearance change of the integument, demonstrating that the conversion of tyrosine into dopa catalyzed by tyrosine hydroxylase is an important link of melanin ana-bolic pathway in Bombyx mori.