Esearch Of Immunomodulatory Effect And Anti-tumor Activity In Vitro Of Glycosaminoglycan From Crassostrea Rivularis

Aims:This paper has investigated the physicochemical properties, immune regulation andanti-tumor activities of glycosaminoglycan from Crassostrea Rivularis by enzymatic lysis.Methods:Use DEAE-52and Sephadex G-200to get the pure glycosaminoglycan. Charge the purityand the relative molecular weight by HPLC. Use IR to determine the characteristicabsorption peaks. Use chemical methods to determine the content of components. UseCTX to establish immunosuppression animal model. Weigh the thymus and spleen thencalculate the immune organs index. The swallow ability of the macrophage cell wasdetected by the neutral red and the carbon text. Use MTT method to check the ability of theNK cells. Check the TNF-α, IFN-γ, IL-2by ELISA. Check the NO, serum hemolysincontent, antibody generating cell number by colorimetric method. Use MTT method tojudge the anti-tumor activity in vitro.Results:(1) The the removal of protein of Glycosaminoglycan from Crassostrea Rivularis wascompared by different pH values. The membrane filtration and desalination was used topurify the glycosaminoglycan. Then the obtained CG was purified by DEAE-52columnchromatography and Sephadex G-200chromatography. Two ingredients CGIa and CGIIawere obtained. The chemical composition of CGIa and CGIIa were examed. The rate ofglucosamine to uronic acid of CGIa was1:1.38, and the content of sulfate was20.62%, therelative molecular weight was5.17×105Da. The rate of glucosamine to uronic acid ofCGIIa was1:1.58, and the content of sulfate was18.49%, the relative molecular weightwas2.64×105Da. IR showed CGIa and CGIIa both had the characteristics absorption ofglycosaminoglycan. CGIIa had the α-glycosidic bond.2. The effect of glycosaminoglycan from Crassostrea Rivularis on immune modulation ofmice:(1)Immune organs index: CG(50mg/kg，100mg/kg) could enhance the index of spleen andthymus, had significant difference of the control group（P﹤0.05， P﹤0.01, had theability to fix the injury of CTX(P﹤0.05，P﹤0.01).(2) The increase of the spleen cell numbers: CG, CGIa and CGIIa (10μg/mL,25μg/mL, 50μg/mL,100μg/mL) all could increase the spleen cell number with ConA. The highestindex is1.44by CGIa(25μg/mL). The effect of CG、CGIa and CGIIa combined with ConAshowed the same trend of work alone, and had a strong relation.(3)The effect of mononuclear phagocyte of mice: CGIa and CGIIa(10μg/mL,25μg/mL,50μg/mL,100μg/mL) all had the ablity to swallow the neutral ted (P﹤0.05，P﹤0.01),CG(25mg/kg,50mg/kg) could enhance the carbon index(P﹤0.05).(4) The text of anti-body number text and the serum hemolysin: About the anti-body text,the mice which ate CG(25mg/kg,50mg/kg,100mg/kg) had a significant difference of thecontrol group (P﹤0.01), and the CTX group（P﹤0.001）. About the serum hemolysincontent, the groups(50μg/mL and100μg/mL) had a significant difference of the controlgroup and the CTX group(P﹤0.05, P﹤0.01).(5) NK cell text: CG, CGIa, CGIIa (10μg/mL,25μg/mL,50μg/mL,100μg/mL) all couldincrease the killing activity of NK cell（sP﹤0.05, P﹤0.01）, and had a strong trend of dose.The highest result is39.09%.(6) The cell factors: CGIa and CGIIa (25μg/mL,50μg/mL,100μg/mL) all could enhancedthe increase of TNF-α and NO(P﹤0.05,P﹤0.01), and had a strong trend of dose. CGIaand CGIIa (25μg/mL,50μg/mL,100μg/mL) could enhance the increase of IFN-γ (P﹤0.01)and IL-2(P﹤0.05, P﹤0.01). The maximum quantity of TNF-α, IFN-γ and IL-2are912.941pg/mg,651.579pg/mg,2.821pg/mg.3. The anti-tumor activity in vitro: CG, CGIa and CGIIa (10μg/mL,25μg/mL,50μg/mL,100μg/mL,200μg/mL) could inhibit the growth of K562, CNE-2Z and Hela cells. Workedwith5-Fu, the inhibition rate has increased.Conclusion:(1) The molecular weight of CGIa and CGIIa were5.17×105Da and2.64×105Da, and theresults of IR and chemical analysis showed they were glycosaminoglycan.(2)The results of immune organs index, spleen cell numbers, mononuclear phagocyte,anti-body number text, the serum hemolysin, NK cell text were all positive, showed CGhad the ability to increase the immune. May be the immune function takes effect byenhancing the production of cell factors.(3) CG, CGIa and CGIIa had the ablity to inhibit the growth of tumor cells, such as K562,CNE-2Z and Hela cells. They also could bombined with5-Fu to enhance the inhibition oftumor cells. In the basis of the anti-tumor effect and the results of3.4.2.1, we found thatCG had the anti-tumor ability and caused no injury of the normal cells.