The Study Of Function Of The Cytochrome C On Apoptosis In Spodoptera Litura Sl-1 Cells

Apoptosis is the programmed cell death process which caused by a variety of factors mediated in multicellular organisms. Since the early 90s of last century, there are widespread and thorough studies on apoptosis from mammals, nematode and Drosophila, the molecular mechanisms of apoptosis has also made enormous progress. As we known, the pathway in apoptosis include two ways, and the main pathway is Mitochondrial apoptotic pathways. Although the molecular mechanisms of apoptosis is highly conserved in evolution process, the studies which in the endogenous mitochondria-mediated apoptosis indicated the pathway is different:the apoptosis is induced by mitochondrial release of cytochrome C into the cytoplasm in mammalian cell, different from Drosophila and nematode is different. The study of apoptosis in Lepidoptera is similar to mammals. The mammalian cytochrome C has been widely used in the study of apoptosis in lepidopteran insects so far, and in order to clarify the cells apoptosis' molecular mechanisms and function of cytochrome C in lepidopteran insect,we construct a prokaryotic expression plasmid (pET-28a-Cyt C) for protein expression and purification, the purified proteins of endogenous Cyt C is used for the study of cell-free apoptosis system; on the other hand, we also construct the eukaryotic expression vector of Cyt C, and then transfect it into SL-1 cells with the ultimate ways of morphology, cellfluorescence quantitative method and streaming technology to detect the role of endogenous Cyt C in SL-1 cell apoptosis.At first we induce the prokaryotic expression plasmid to express the target protein, at 24?,0.4mM IPTG for eight hours, the expression product was verified by His antibody, we found the MW of the protein is 15KD, extract over a nickel affinity chromatography column, be eluted by 350mM imidazole, then get off imidazole by centrifugal filter to obtain a purified endogenous cytochrome C protein. The purified protein was subjected to SDS-PAGE electrophoresis and gel cutting rabbits immunized four times to obtain sl-cytc polyclonal antibody. The purified protein for SL-1 is studied in cell-free system, then we found that endogenous Cyt C protein induces apoptosis in cell nuclei occurs without apoptosis SL-1 system; while the existence of cytochrome C antibodies and purified protein can inhibit apoptosis.The study also successfully constructed three eukaryotic expression vectors of Spodoptera Cyt C, PIEl-Cytc-EGFP, PIE1-EGFP-Cytc and PIE1-EGFP-C1. We use the vectors to transfect SL-1 cells for dectecting fusion protein by western blot; while we use mitochondrial fluorescent dyes and GFP to observe the localization of cytochrome C in cells, then after transfecting successfully we can observe the apoptosis of nuclei by DAPI staining, measure the activity of caspase-3 by fluorescence quantitative method, and flow cytometer together to verify the role of endogenous cytochrome C in the SL-1 cell apoptosis, we not only found that endogenous Cyt C protein can induce cell apoptosis, but also found that the different insert locations of cytochrome C can lead to the different apoptosis rates.In summary, we found that endogenous Cyt C is involved in the process of apoptosis, and also we proved the existence of mitochondrial Cyt C-mediated apoptosis in the SL-1.