Experimental Study Of Immunoregulation And Antitumor Function Of Gekko Gecko Peptides

OBJECTIVE In oder to isolate and purify protein polypeptide from Gekko gecko,and determine protein molecular weight and concentration of each component.METHODS Take fresh Gekko gecko executed, remove head and visceral,then cut into small tissue mass, weighing,use 0.3% NaCl solution diluted into 5ml/g,then use pulprefiner homogenate shattered to crush and homogenate, centrifuge with 4℃, 10000r/min after in liquid nitrogen repeated freezing and thawing 3 cycles, repeat 3 times and repeat the above operating 1 time. Get supernatant fluid filter by the filter paper, Remove the lipid of insoluble, centrifugal ultrafiltration use ultrafiltration centrifugal tube intercepting molecular mass for 50Kda after filtrate with 0.45 u m filter,remove macromolecular protein, the filtrate collected to be freezed and dry,then get brown solid powder through the Sephadex G-50 gel chromatography. To determine the protein purity and molecular weight through SDS-PAGE electrophoretic analysis, To determine concentration of each component through the ultraviolet spectrophotometer.RESULTS The Gekko gecko protein was separated to three main protein peaks by Sephadex G-50,through SDS-PAGE electrophoretic analysis,the molecular weight of protein componentsⅠwas between 20Kda-50Kda, the molecular weight of protein componentsⅡwas between 3Kda-20Kda,and the molecular weight of protein componentsⅢwas under 3Kda. Determination by the ultraviolet spectrophotometer, three groups of protein component concentration respectively was 0.32mg/ml,1.28 mg/ml,0.25 mg/ml,the ceollectin respectively was 140ml,161ml,147ml, content of protein respectively was 44.8mg,206.08mg,126.42mg,the total was 377.3mg,62.88% of freeze-dried protein powder of Gekko gecko,and three groups of protein componen of total respectively was 7.47%,34.34%,21.07%.CONCLUSIONS Applying the methods of ultrafiltration centrifugalization,freeze-dryingand gel chromatography to isolate and purify the Gekko gecko and get the protein with molecular weight between 50Kda-3Kda. OBJECTIVE To study the impact to the mice's immune function of Gekko gecko peptide in vitro,and screened the strongest components of immunity activity of Gekko gecko peptide.METHODS Preparation of different concentrations(0.1ug/ml,1ug/ml, 10ug/ml,100ug/ml) of Gekko gecko peptide solution and different concentrations(10U/ml, 100U/ml, 1000U/ml, 10000U/ml) of IL-2 solution. Studies of the influence of applications for immune cells of different concentrations and different components of Gekko gecko peptide in vitro alone or joint IL-2, determination of activity of NK cells and the tumoricidal activity of the macrophages of mice with MTS.RESULTS Gekko gecko peptide components II have three concentrations (1ug/ml,10ug/ml,100ug/ml) can significantly improve killer activity of NK cells (P<0.01), the activity of NK cells may reach up to (17.26±3.07)% when the concentration of lOug/ml, it improved 1.58 times respectively than normal control group;but the concentration of 0.1 ug/ml without ascending function.Gekko gecko peptide components I and III didn't improve the activity of NK cells when used alone. When lOug/ml of concentration of diefferent components Gekko gecko peptide mixed different concentrations(10U/ml,100U/ml,1000U/ml,10000U/ml)of IL-2, componentsⅡhas a synergistic effect (P<0.05) with IL-2;when different concentrations Gekko gecko peptide mixed 100U/ml of concentration of IL-2, 10ug/ml of Gekko gecko peptide componentsⅡcan strengthen the effect of activation NK cells of IL-2 (P<0.05).The three components of Gekko gecko peptide all can improve the tumoricidal activity of the macrophages, including components I andⅢhave significantly boosting effect (P<0.05) in concentration 1 ug/ml,10ug/ml,100ug/ml;four concentration of Gekko gecko peptideⅡcan significantly improve the tumoricidal activity of the macrophages, and the tumoricidal activity of the macrophages may raise to (31.99±6.97)% in the 1 Oug/ml,it improved 2.05 times respectively than normal control group,and had a remarkable enhancement effect (P<0.05). It can not strengthen the tumoricidal activity of the macrophages when in concentration of 0.1 ug/ml of Gekko gecko peptide componentsⅠandⅢCONCLUSIONS Of collected three main component of Gekko gecko peptides, Gekko gecko peptide componentsⅡcan improve the activity of NK cells use alone in vitro,and it had the strongest effect When the concentration of 10ug/ml; and when the concentration of 10ug/ml,it had a synergistic effect with IL-2 to activate NK cells. Gekko gecko peptide components I and III didn't improve the activity of NK cells when used alone. The three components of Gekko gecko peptide all can improve the tumoricidal activity of the macrophages in vitro, including components II in the concentration of 10ug/ml,it had a strongest effect;but it can not strengthen the tumoricidal activity of the macrophages when in concentration of 0.1 ug/ml of Gekko gecko peptide componentsⅠandⅢ. It prompt Gekko gecko peptide can improve the activity of immunocyte in vitro,thus it activate cellular immunity in body. OBJECTIVE To observe effects of Gekko gecko peptides to immunologic function of the mice immunosuppresive modal.METHODS Using the above experiment separation and selected Gekko gecko peptides componentsⅡin animal experiments,and its molecular weight was between 3Kda-20Kd.The immunosuppressed mouse model was established by cyclophosphamide cyclophosphamide intraperitoneal injection 40mg/kg/d,for 3 consecutive days, research the effect of Gekko gecko peptides irrigation stomach treatment on the mice immunosuppresive modal. Low immunity mice were respectively given Gekko gecko peptides componentsⅡ500,200mg/kg/d for 7 days through irrigation stomach treatment, by the way of MTS, determination of splenic lymphocyte proliferation rate,activity of NK cells and the tumoricidal activity of the macrophages. Low immunity mice were respectively given Gekko gecko peptides componentsⅡ500,200,100mg/kg/d for 7 days through irrigation stomach treatment, and the serum hemolysin formation was performed by HC50 assay to research the effect of Gekko gecko peptides to the serum hemolysin formation on the mice immunosuppresive modal.RESULTS The mice immunosuppresive modal were established by cyclophosphamide intraperitoneal injection,their various immune index all lower than normal control group,their difference was statistically significant (P<0.01).It can significantly enhance splenic lymphocyte stimulation index (P<0.05), the activity of NK cells(P<0.05) and the tumoricidal activity of the macrophages(P<0.05) of the immunosuppressed mouse model to give the mice Gekko gecko peptides componentsⅡwith with molecular weight between 3Kda-20Kd 500,200 mg·kg-1 by ig. for 7 days. It can significantly increase the production of serum hemolysin (P<0.01, P<0.01, P<0.05) of the immunosuppressed mouse model to give the mice Gekko gecko peptides componentsⅡ500,200,100mg·kg-1 by ig. for 7 days.CONCLUSIONS Gekko gecko peptides componentsⅡwith molecular weight between 3Kda-20Kda may enhance immune function of the immunosuppressed mouse model through regulate cellular immunity and humoral immunity. OBJECTIVE To study the immunoregulation of Gekko gecko peptides peptides in tumor-bearing mice and its growth inhibitory effects.METHODS Using the above experiment separation and selected Gekko gecko peptides components II in animal experiments,and its molecular weight was between 3Kda-20Kd. The mice with sarcoma-180 and Hepal-6 hepatic carcinoma as the animal models were used to study the therapeutic effect of Gekko gecko peptides and it united with CTX. And applied MTS to determine tumoricidal activity of peritoneal macrophage, the stimulation index of lymphocyte and natural killer cell activity. Then the tumor weight and the inhibiting tumor rate were calculated.RESULTS It can significantly enhance the tumoricidal activity of peritoeal Macrophages of the mice with sarcoma-180 and the phagocytosis of peritoneal Macrophage of the mice with Hepal-6 hepatic carcinoma to give the mice pure Gekko gecko peptides componentsⅡ400 mg/kg/d by ig. for 12 days(P<0.05),and it also can enhance lymphocyte proliferation capacity and netrual killer cells activity remarkably(P<0.05) after treatment with Gekko gecko peptides componentsⅡ.United with CTX, Gekko gecko peptides componentsⅡcan improve the above immune indexes which constrained by CTX and can enhance the tumor inhibition rate significantly(P<0.05).CONCLUSIONS Gekko gecko peptides componentsⅡwith molecular weight between 3Kda-20Kd have immunoregulation to the suppression of immune function which from tumour and chemotherapeutics,and it can achieve anti-tumor effect cooperate with chemotherapeutics this way.