| The SODs are a family of enzymes that very efficiently catalyze the dismutation of the superoxide radical anion(O2·-). There are mainly three categories of SOD according to their distributions in cells: SOD1 (Cu/Zn SOD, mainly located in eukaryotic cytoplasm), SOD2 (Mn/Fe SOD, mainly located in eukaryotic mitochondria) and SOD3 (EC-SOD, mainly located in the extracellular matrix). All the SODs have the ability to catalyze superoxide anion to have dismutation reaction and thus become an important scavenger for oxygen free radicals. Among them, SOD1 was the first discovered and studied widely.The oxygen free radicals(OFR) converted from oxygen molecules directly or indirectly. There are uncoupled electrons in their outer orbit, including superoxide anion free radical, hydroxyl radical, hydrogen peroxide. With reactive chemical properties , they gain or loss electrons easily and thus initiate free radical chain reaction. The free radicals in vivo are produced by the stimulus such as high temperature, radiation or just a by-product of normal metabolism process. Produced in proper amount, superoxide is a normal and useful metabolite, serving important roles as a signaling molecule in processes such as cell division, and even serving to act as a terminator of lipid peroxidation. When flagrantly overproduced, however, the radical can initiate lipid peroxidation, protein oxidation, and DNA damage, leading to cell dysfunction and death by apoptosis or necrosis. The radical and/or the SODs have been implicated in a broad range of disease states including inflammatory diseases, diseases of ischemia and reperfusion injury, neurodegenerative diseases, diabetes, cancer, and many others. It's helpful to those diseases when redundant free radicals cleaned out.Two main reactions involved in the elimination of free radicals. The first is to transform superoxide anion to oxygen molecule and hydrogen peroxide by SOD, the second is to transform hydrogen peroxide into oxygen molecule and water. SOD plays an essential role in such process. If the major SOD genes were disrupted in E. coli, the organism could no longer grow in oxygen on minimal medium. Considerably increased SOD could decrease the amount of free radicals and ameliorate liposome peroxidation, inflammatory activity and aging. So it would be possible to regulate the amount of free radicals and thus control physical and pathological conditions by manipulating the SOD content.The mechanism of wrinkle generating involves exogenous factors such as light aging, injury and infection and endogenous factors such as genetic regulation according to aging and injury caused by endogenous free radicals. The free radicals produced by the ultraviolet ray ( UV ) of sun light will cause skin aging. SOD and CAT genes also are damaged by UV, as a result, the production of SOD and CAT decrease and free radicals accumulate and the skin be damaged. So, the free radicals play a very important role in the wrinkle generating. If the amount of SOD could increase moderately, the skin would be protected and wrinkles diminished.Despite the long time of the SOD study, SOD-based antioxidant therapy has not made a greater and more rapid impact on clinical medicine: the problem of oxidant-antioxidant balance. As a protein with large molecular weight, SOD is expensive, short-life, besides, it can not overcome the barrier of cell membrane to work inside the cells. The immunogenicity and dose problems are hard to solve too. Methods to overcome such problems includ drug vectors transporting the drugs into cells, SOD mimics and up-regulating the endogenous SOD production. In this study, a system was designed to screen out the polypeptide drugs which could up-regulate the endogenous SOD production.Reporter genes are a kind of genes whose products could be detected easily and could be distinguished from background proteins. The transcription elements of certain gene cloned into reporter gene vector, the expression level of the gene can be detected directly by the expression of reporter gene. Red fluorescent protein gene encodes a red fluorescent protein that has been optimized for high expression in mammalian cells (excitation maximum = 558 nm; emission maxmum = 583 nnm) without any co-factor or substrate, thus it could detect the gene expression of living tissue and cells without cell disruption.In this study, a human SOD1 promoter was cloned into the pDsRED1-1 vector containing red fluorescence protein reporter gene and transfected into NIH 3T3 cell line, subsequently a permanent cell line was screened out. The red fluorescence of this cell line remarkably increased after stimulated by PMA, and the value of fluorescence increased in a linear manner. The cell line provided a convenient and sensitive tool to screen the drugs which could up-regulate the expression of endogenous SOD1.In order to avoid the immunogenicity problem by protein, a peptide library was constructed for drug screening, which contains 12 random amino with a theoretical capacity of 2012 to guarantee the capability and diversity of the library. Since most of the large molecules could not entry the cell membrane, HIV-Tat PTD was chosen as the drug vector to transport the fusion protein into the cells. HIV-Tat can penetrate the cell membrane in a energy independent, receptor independent and classical internalization independent way. It can carry various cargoes including nucleotide, polypeptide and protein into the cells and is an ideal transporter for polypeptide. Besides, EGFP in the fusion protein can be a taq to tell the location of the protein in the cells.An effective work-flow has been established to purify the polypeptide drug candidates and to screen out the potential drugs. The histidine taq was selected to purify the proteins and then the purified proteins were incubated with the certain premanent cells for a certain time, and then the value of fluorescence inside the cells was detected to judge the effect of the peptide on human SOD1 promoter.To sum up, this study used the gene technology and cell line screening technology to obtain a permanent cell line that can be a tool to detect the expression level of human SOD1 promoter, and used genetic recombination and mutagenesis technology to construct a random polypeptide library containing 12 continuous random polypeptides, transmembrane transporting carrier and EGFP. Based on the cell line and random polypeptide library, a work-flow was established to screen out the polypeptide drug which could up-regulate the endogenous SOD1 expression, thereby provided a system for the following drug screening work. |
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