研究內容
本研究對棉鈴蟲幼蟲神經細胞原培養基作了大量改進，增補了昆蟲細胞生長所必需的成分，提高了神經細胞貼壁率。並成功用於甜菜夜蛾幼蟲神經細胞的離體培養，在此基礎上，鑑定了甜菜夜蛾幼蟲神經細胞所表達的電壓門控性離子通道類型，即TTX-敏感型鈉通道、L-型鈣通道、瞬時鉀通道和延遲整流鉀通道，這和棉鈴蟲幼蟲神經細胞所表達的電壓門控性離子通道類型是相同的。
首次利用膜片鉗技術研究了苦皮藤素Ⅳ對棉鈴蟲幼蟲神經細胞鈉通道的影響，結果表明它對棉鈴蟲幼蟲神經細胞鈉通道具有濃度依賴性阻滯作用，而且影響鈉通道動力學特徵，使鈉通道激活電壓和峰電壓向正電位方向發生移動。並與臨床上常用的局部麻醉劑利多卡因進行了比較，進一步探討了苦皮藤素Ⅳ的麻醉機理。而且，研究也表明苦皮藤素Ⅳ對甜菜夜蛾幼蟲神經細胞鈉通道同樣具有阻滯作用，但甜菜夜蛾與棉鈴蟲的敏感性有差異。
鑑定了棉鈴蟲表達的高電壓激活鈣通道類型，比較研究了胞外液中分別以〓或〓做載流子，所記錄鈣電流的異同。在此基礎上，初步研究了苦皮藤素Ⅳ對棉鈴蟲幼蟲神經細胞鈣通道的影響。 首次研究了我國常用的生物鹼植物殺蟲劑-苦參鹼對棉鈴蟲幼蟲神經細胞鈉通道的影響。結果顯示苦參鹼對棉鈴蟲幼蟲神經細胞鈉通道具有濃度依賴性阻滯作用，但不改變其激活電壓、峰電壓和I-V曲線形狀。
套用透射電鏡技術比較研究了雙醯肼(RH-5992)和雙硫醯肼類殺蟲劑(W200013)對棉鈴蟲皮細胞和新表皮形成的影響。W200013在中毒症狀、生測和超微結構水平表現出和RH-5992相同的作用，都可使棉鈴蟲產生早熟、異常的致死蛻皮。因此，雙硫醯肼與雙醯肼類殺蟲劑相同，都是通過作用於表皮的蛻皮激素受體而發揮其殺蟲作用的。
總之，本研究初步闡述了苦皮藤素Ⅳ的麻醉作用機理和苦參鹼對鈉通道的影響，揭示了新型雙硫醯肼類蛻皮激素殺蟲劑誘導異常蛻皮的作用機制。
外文摘要
Studies on the action mechanisms of new type of biorational insecticides
ABSTRACT
Studies on the action mechanisms of new type of biorational insecticides
ABSTRACT
Botancical insecticides and nonsteroidal ecdysone agonists of insect growthregulators (IGRs) are two kinds of important biorational insecticides with novel modesof action, which are less harmful to nontarget organism than conventional chemicalinsecticides. This paper emphasized on the effects of Celangulin Ⅳ and matrine on ionchannel currents in cultured nerve cell of insect by using whole cell of patch clamptechnique. Furthermore, the effects induced by new type nonsteroidal ecdysoneagonist on cuticle structure and moulting process of Helicoverpa armigera werecarried out.This research developed a modified culture medium to improve the attachment ofnerve cells. Furthermore, supplemented with essential ingredients that promote thenerve cell growth. All these improved the number of attachment nerve cells .Theculture medium also adapt to the neuron cells of Spodopteta exigua to grow in vitro.The voltage gated ion channels in central neurons of S. exigua had been characterizedby whole-cell patch champ recording. It exhibits tetrodotoxin-sensitive sodium current,L-type high-voltage activated calcium current, delayed rectifier potassium current (〓)and 〓 sensitive to 4-aminoloyridine. The types of voltage gated ion channels incentral neurons of S. exigua are same as H. armigera.Celangulin Ⅳ is the active ingredient which has narcotic action against someinsects. The effects of Celangulin Ⅳ on inward sodium channel current (〓) incultured nerve cell of cotton bollwom (H. armigera) were studied by patch champtechnique. The recording results showed that Celangulin Ⅳ reduced sodium inwardcurrent in a concentration dependant manner, affected the kinetics of sodium channeland shifted the active and peak current potential to the positive direction. Lidocaine isa clinical local anesthetic. The effects of lidocaine on 〓 in cultured nerve cell ofcotton bollworn were studied comparatively. The results indicated that lidocaine alsohad effects on 〓, but 〓 could be inhibited with more potency of Celangulin Ⅳ thanthat of lidocaine. At same time, the patch clamp recording showed that Celangulin Ⅳinhibited the sodium current in cultured nerve cell of S. exigua. The effects are similarto H. armigera on the whole, but S. exigua has different sensitivity to Celangulin Ⅳcompared with H. armigera.That high-voltage activated calcium current was identified by nifedipine is L-type〓 channel. Moreover, we compare with the calcium current in the external solutionsof 〓 and 〓 as carrier. 〓 displaced 〓 as divalent cation carrier, the peakamplitude of the inward current was bigger, and there was a slower decline of thecurrent during the voltage step. In addition, the effects of Celangulin Ⅳ on inwardcalcium channel current (〓) in cultured nerve cell of H. Armigera were studied.Matrine is another kind of conventional alkaloids from plants against insect pests.Its effects on inward sodium channel current (〓) in cultured nerve cell of H.armigera were studied. The recording results indicated that Matrine inhibited thesodium current in a dose dependent manner and without changing the active potential,peak potential and shape of current-voltage relation curve.The action mode of sulphur-sulphur dibenzoylhydrazine-type nonsteroidalecdysone agonists was studied on the larva of cotton bollworn, H. Armigera(Lepidoptera) by transmission electron microscope. The results revealed thatW200013 was same as dibenzoylhydrazine-type insecticide RH-5992 on the toxicsymptoms, bioassay results and ultrasturctural changes of integument. They allinitiated a precocious and lethal larval moult. It proved that W200013 exhibits itsnematicidal action by bind to ecdysteroid receptors as RH-5992. In conclusion, the research illustrated the narcotic action mechanisms ofCelangulin Ⅳ and effects of Matrine on sodium channel current. Furthermore, theabnormal moult mode induced by new sulphur-sulphur dibenzoylhydrazine-typenonsteroidal ecdysone agonist was explained.
