| Objective Yam is the rhizome of Dioscorea oppsita Thunb,which has been used as both food and medicinal since ancient times.Dioscorea oppsita Extract(DOE)is rich in vitamins,proteins,amino acids,starches and trace elements,as well as polysaccharides,saponins and polyphenols,which can enhance immunity,antioxidant,regulate intestinal flora,lowering blood sugar and blood lipids.Among them,yam polysaccharide is one of the main active ingredients and is also a research hotspot in recent years.In this paper,yam extract was used as the main research object,and the polysaccharide content in the extract was determined by phenol-sulfuric acid method.The effects of DOE addition concentration on CHO cell growth,oxidative stress and antibody expression and the effect of DOE addition time on CHO cell growth and antibody expression were explored through cell experiments.Methods1.Firstly,the phenol-sulfuric acid method was used to determine the polysaccharide content of the active ingredient in the yam extract.2.In the experiment exploring the effects of DOE addition concentration on CHO cell growth,oxidative stress and antibody expression,DOE was completely dissolved with distilled water to prepare a 5 mg/m L DOE stock solution,and then sterilized with 0.22μm microporous membrane filter before adding to the cell culture medium,so that the concentrations of DOE in the final medium of each treatment ware 1,5,8,10 and 15 mg/m L,respectively,and no DOE was added as the control.Cell was cultured with fedbatch culture.Cell density,cell viability,intracellular malondialdehyde(MDA)content and superoxide dismutase(SOD)activity,antibody expression in cell supernatant,monoclonal antibody content was measured by flow culture,meanwhile the antibody charge variants were detected and glycosyl composition,relative content and protein binding activity of the antibody were analyzed.3.In the experiment exploring the time effect of adding DOE on CHO cell growth and antibody expression,DOE stock solution was added on the 0、3、5、7、9、11 and 13 day of cell culture to make the DOE concentration in the final culture medium of each treatment was 5 mg/m L,and DOE was added on the 0th day as the control.Cell density,cell viability,antibody expression and monoclonal antibody content in cell supernatant were measured respectively.Meanwhile antibody charge variants were detected,and glycosyl composition,relative content and protein relative binding activity of antibody were analyzed.Results1.The polysaccharide content in DOE was(48.67±0.86)%.2.In the experiment on the effect of DOE addition concentration on CHO cell growth,oxidative stress,and antibody expression,5 mg/m L DOE can significantly promote the growth of CHO cells,improve the living cell density,cell viability and SOD activity,and reduce the content of MDA,as well as increase the intracellular antibody yield and monomer content.Adding DOE can change the content of some glycolforms of antibody to a certain extent.With the increase of DOE concentration,the relative content of G0 F and Man5 decreased first and then increased slowly,the content of G1 F gradually increases,and the content of G0 is relatively stable.After adding DOE,there were no significant difference in the contents of acid charge variants,main peak and basic charge variants(P>0.05),and the relative binding activity of the antibody remained between 102% and 135% of the reference sample,and DOE did not affect the binding activity of the antibody.3.In the experiment on the time effect of adding DOE on CHO cell growth and antibody expression,with the time delay of adding DOE,the living cell density,cell viability,antibody yield and monomer content of cells would all decrease.In the N-glycosylation modification of antibodies,different times of adding DOE can affect the modification effect of cells on some certain glycoforms to a certain extent.Compared with T0,T3 cells had less effect on the content of four glycoforms and T5 cells on the content of Man5.With the delay of DOE adding time,the content of G0 F and Man5 glycoforms gradually increased,the content of G1 F glycoform gradually decreased and the content of G0 glycoform was stable.The results also showed that there was no significant difference in charge variant content and reduced fragments with different addition times of DOE(P>0.05).The data showed that the relative binding activity of the antibody in the DOE treatment group at different addition times remained between about 106% and 126% of the reference sample at different adding times,and the DOE treatment at different adding times did not affect the binding activity of the antibody.ConclusionThe active ingredient polysaccharide content in DOE was(48.67±0.86)%;The concentration and time of addition of DOE in the process of cell culture played an important role in cell growth and antibody expression.Adding 5 mg/m L DOE on the 0th day of cell culture has the best promoting effect on cell growth and antibody expression,which provides a reference for future studies on the effects of Chinese extracts on CHO cell growth and antibody expression. |
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