Extracting Proteins That Can Induce The Apoptosis Of HepG₂ In Vitro Culture From Guppy Embryos By Response Surface Method

Malignant tumor, an obstinate illness threatening health and life, is mainly cured by the chemical treatment, although researcher has made great progress in treating it. Because present chemical medicines always cause significant secondary effects, new antitumor drugs are greatly necessary to be developed.In 1970, Pierceetal has pointed that tumor has great relation with biological development. Therefore, studying apoptosis of tumor cells from the view of development attracts more attention.In this thesis, we adopted embryonic extracts、total embryonic proteins、small embryonic proteins、big embryonic proteins of guppy to induce the apoptosis of HepG2 in vitro culture. The result detected by optical microscope、electron microscope and flow cytometry showed that all of them can induce the apoptosis of HepG2 except small embryonic proteins.To further study the optional extraction conditions of protein which can induce apoptosis of HepG2, we use ammonium sulfate with small temperature coefficient and solubility, and dose not easily lead to protein denaturation to extract protein from guppy embryo. Extracting proteins with salting-out extraction is mainly affected by salt concentration, buffer pH, protein concentration, and the influence of the temperature. The BBD of Response Surface Method (RSM) was employed in this experiment to study the impact of biological process factors and their interaction evaluation. Selecting three factors (Ammonium sulfate saturation (A) and ratio of raw materials to PBS(B) extraction pH (C)) with significant effect according to the single factor experiment and then adopting three factors 3 level of to RSM to analyze the value of apoptosis. After the analysis of the significance of single factors and interaction, the optimum extraction conditions and the experimental value is 67.87% which were well matched with predicted value 68.8836% by the model.