Study On AChE Regulating Apopotosis Of Insect Cell

Sf9 cell line was isolated from the pupal ovarian tissue of Spodoptera frugiperda(Lepidoptera:Noctuidae).It is a host for most nuclear baculovirus and used extensively as a powerful tool in molecular biology.Bac-to-Bac Baculovirus Expression System was constructed based on AdMNPV-Sf cell line and is most widely used at present.Purification and recombination of baculovirus and production of recombinant proteins can be achieved by using Sf9 cell line.It is also a target cultivating cell in large scale.AChE(acetylcholinesterase)is a key enzyme in the biological nerve conduction,can rapidly hydrolyzed excitatory neurotransmitter acetylcholine and maintain normal neural synaptic transmission.In addition,this enzyme is involved in animal cell apoptosis.Previous studies revealed that this protein exhibits different expression level in apoptotic cells.Plant phytochemicals are one of the most important defense weapon of plants,which have a variety of effects on the animals and their cells,one of which is to induce apoptosis.In order to learn the relationship between insect cell AChE and apoptosis,the following works were performed and some valuable data were obtained.1.After treatment of Sf9 cells with different concentration and different time of xanthotoxin,tricin and camptothecin,cell apoptosis were detected by flow cytometry and Caspase 3 activity detection.Then AChE expression profiles were checked by using fluorescence quantitative PCR,Western blotting hybridization was used to detect protein content.More cells undergoing apoptosis in the group treated with camptothecin than that in control were observed under light microscope.Flow cytometry displayed that the sample treated with camptothecin showed more apoptosis than that in control.Caspase 3 had the highest enzyme activity in apoptotic cells treated with 1 ?mol/L of camptothecin for 24 h.The results from fluorescence quantitative PCR revealed the expression level of AChE increased 3.7-fold,6.2-fold and 2.5-fold in cells treated with 1 ?mol/L of camptothecin for 12 h,24 h and 36 h,than that in control,respctively.The data from the Western blotting hybridization revealed protein content incresed 15.83%,15.27%and 8.64%in apoptotic cells induced by camptothecin than that in control cells.2.A segment of AChE ORF(opern reading frame)was cloned into pGEM-T easy vector.The recombinant expression vector was transformed into E.coli cells and expressed to single-stranded RNA.Two inverted ssRNA molecules were mixed to form double-stranded RNA.Sf9 cell was transfected with the dsRNA by using lipofectamineTM 2000 reagent.The silencing of AChE was determined by fluorescent quantitative PCR and immunal blotting hybridization.Fluorescence quantitative detection revealed the expression level of AChE decreased 2.9-fold and 1.7-fold,respectively.Western blotting hybridization showed the expressed AChE proteins were decreased 7.08%and 3.32%than that in control.In this work,we studied Sf9 apoptosis induced by phytochemicals and the expression level of AChE in apoptotic cells.The data obtaine in this work will help in elucidation of the mechanism about how AChE regulates insect apoptosis,which laid a foundation for further research on the molecular interaction of insect and phytochemicals,and digging out novel insect-control strategies.