昆虫血脑屏障对杀虫剂的阻滞和外排作用及其与棉铃虫抗药性相关机制研究

The vertebrate blood-brain barrier (BBB) is a specialized system of brain microvascular endothelial cells that shields the brain from harmful blood-derived substances, supplies brain tissues with nutrients. Transport across the BBB is strictly limited through both physical and metabolic barriers. It is estimated that approximately 100% of large-molecule neurotherapeutics and over 98% of small-molecule compounds never reach the market due to an inherent inability to cross the BBB. At the same time, the BBB-forming cells express a variety of transporters to pump harmful substances back into blood. Taken these properties together, the BBB is regarded as the bottleneck of the successful development of the central nervous system (CNS)-acting drugs. A structurally analogous diffusion barrier also exists in insects, but unlike the BBB, the endothelial lining of the blood vessels is often incomplete or leaky in insects, the barrier is established entirely by glial cells, it separates nervous system from the surrounding blood-like hemolymph, so the blood-brain barrier of insect is termed more accurately as hemolymph-brain barrier (HBB). The HBB shares central properties with BBB, both types of diffusion barriers prevent influx of metabolites from the circulation. The blocking effects of BBB on neurotherapeutic drugs prompt an idea that the HBB of Helicoverpa armigera may be the major obstacle for the penetration of insecticides, especially for the neurotoxic insecticides whose target sites are voltage-gated ion channels in membrane of neurons inside the central nerve cord or the brain. If it were true, how could the neurotoxic insecticides bind to the target ion channels and affect the functions of related ion channels and the signal transduction of nerve system? Are there any differences between the HBBs of sensitive and resistance insects? Is HBB a pivotal factor for insecticide resistant mechanism? Do the HBB-forming glia cells also express efflux transporters? Based on the information, the transmission electron microscope will be employed to compare the ultrastructure of HBB of sensitive and insecticide resistance insects. The penetrability of pyrethroids and other kinds of insecticides will be investigated using HPLC. The functional efflux transporters will be analyzed by molecular biological technique. And most importantly, an in vivo patch clamp recording set up will be built and used to analyze the blocking effects of HBB of Helicoverpa armigera on different kinds of neurotoxins. The function of neural signal transduction related to postsynaptic potential will also be recorded with the existence of pyrethroids. Finally, the types of voltage gated ion channels expressed in membrane of isolated sensory neurons will be explored and effects of pyrethroids on these channels will be compared using whole cell patch clamp technique. These studies are helpful for the elucidation of blocking effects of insect HBB on insecticides and scientific use of insecticides in field.

脊椎动物的血脑屏障（BBB）是维护中枢神经系统内环境稳定的重要屏障，100%的大分子和98%以上的小分子药物无法透过BBB，同时BBB的转运蛋白能将脑内毒素快速外排。昆虫血脑屏障（HBB）的生理功能类似于BBB。BBB对药物的阻滞作用提示HBB对杀虫剂的透过也有较强的抑制作用，那么以细胞膜离子通道为靶标的杀虫剂是如何到达膜通道或突触等作用部位的？杀虫剂能否透过HBB，HBB是否也表达外排转运蛋白？抗性和敏感昆虫的HBB有无差异？HBB是否为抗性形成的关键因素？本项目拟用透射电镜分析抗性和敏感棉铃虫HBB的超微结构及拟除虫菊酯等杀虫剂对HBB的影响；通过高效液相技术检测HBB对杀虫剂的透过率；分子生物学方法探寻HBB是否表达毒素外排转运蛋白；主要通过建立昆虫在体膜片钳记录方法，综合分析HBB对杀虫剂的阻滞作用及杀虫剂对神经信号传导的影响。研究有助于明确棉铃虫的抗药性机理，为科学施药提供线索。

通过透射电镜、高压气相-质谱联用、分子生物学和电生理等技术和方法检测了棉铃虫（H. armigera）和意蜂（Apis mellifera）P-gp在不同龄期及不同组织器官中相对表达量的差异；观察了棉铃虫血脑屏障结构，对比了拟除虫菊酯类杀虫剂对棉铃虫血脑屏障的影响；利用 Illumina MiSeq二代高通量测序技术对棉铃虫及意蜂肠道细菌的16S rDNA进行测序，分析了肠道细菌的多样性和丰富度；检测了杀虫剂对棉铃虫神经细胞离子通道的影响。结果发现，敏感和田间棉铃虫不同龄期幼虫及同一龄期幼虫不同组织器官的P-gp相对表达量有差异。敏感棉铃虫4龄幼虫的P-gp表达水平最高，田间棉铃虫1龄幼虫的表达水平最高。敏感棉铃虫中肠P-gp的相对表达量最高，田间种群棉铃虫腹神经索、体壁、马氏管相对表达量均明显高于敏感品系的。噻虫嗪处理不同时间，72h后棉铃虫P-gp的表达量最高，且中肠P-gp的表达量最高，是脂肪表达量的37倍。环溴虫酰胺作用后，棉铃虫腹神经索等的P-gp表达量上升。透射电镜结果表明棉铃虫高龄幼虫脑神经节的神经束膜和神经围膜的厚度之比大于稍低龄幼虫的，低龄幼虫脑神经细胞内的线粒体多为圆形，杆状线粒体数量较少，高龄幼虫脑神经元内杆状线粒体数量较多；气-质联用检测结果表明，七氟菊酯较难穿透棉铃虫腹神经索部血脑屏障，而溴氰菊酯可以穿透棉铃虫血脑屏障。高通量检测结果表明拟除虫菊酯类杀虫剂对棉铃虫肠道菌群的结构和代谢能力有明显影响，棉铃虫肠道有益菌的相对丰度下降，致病菌的相对丰度增加。Ⅰ型和Ⅱ型拟除虫菊酯类杀虫剂对棉铃虫肠道菌群结构和代谢功能的影响不同。膜片钳检测结果表明七氟菊酯、溴氰菊酯、环氧虫啶等杀虫剂对棉铃虫神经胞膜内向通道的峰值电流、I-V曲线、激活、失活等都有不同程度的影响。棉铃虫的电压门控钠、钾、钙、氯等通道为所检测杀虫剂的作用靶标。研究对杀虫剂作用机理分析和害虫抗药性治理具有比较重要的理论意义和实践价值。