Effect Of Bozhi Glycopeptide On Proliferation And Interleukin-10 Production In Gastric Cancer Patients' Peripheral Blood Mononuclear Cells

Purpose:1.To optimize isolation of peripheral blood mononuclear cells(PBMCs)from gastric cancer patients using density gradient centrifugation,so as to guarantee the quality of PBMCs isolated.2.With a mathematic model,to evaluate effects of three factors including centrifugal time,force and subjects(gastric cancer patients or not)on the proliferation of PBMCs,in order to clarify the possibility of being modulated of PBMCs by Bozhi Glycopeptide.3.To explore the effecting pattern of Bozhi Glycopeptide on the proliferation of PBMCs isolated from gastric cancer patients.4.To elucidate effects of Bozhi Glycopeptide on the production of interleukin-10(IL-10)in PBMCs from gastric cancer patients.Methods:1.A custom design was built with JMP 10.0 software,entering response with the thickness value of PBMCs,specifying centrifugal time and force as factors,defining the constraints of the factors,adding the two-factorinteractions and quadratic effects into model,then the design table was made.Data were collected including response values produced by Ficoll density gradient centrifugation of PBMCs in gastric cancer patients.The images of cell layers in test tubes were recorded using digital camera.The thickness values of isolated PBMCs were measured with Photoshop CS 6 software.Data were analyzed using standard least squares and maximizing desirability in JMP.To verify the effect of optimization,PBMCs were isolated with optimized centrifugal time and force,and then stained with trypan blue to assess cell viability.2.In custom design module in JMP,Bozhi Glycopeptide concentration(mg/L),cell culture time(h)and subjects(categorized as gastric cancer patients or not)were specified as factors,defining constraints of concentration and time.Values of optical density of PBMCs were set as respond.Experiments were performed according to the design table being made by JMP.Data were obtained by measuring the OD values by MTT method.The model was fitted using standard least squares method.3.Repeated measures experiments were made,in which PBMCs isolated from gastric cancer patients were grouped as follows: co-cultured with Bozhi Glycopeptide of0 mg/L,25 mg/L,50 mg/L,100 mg/L,150 mg/L,200 mg/L final concentration respectively,and each group was cultured for 4 h,12 h,24 h,48 h respectively.The OD values were measured using MTT method.Data were analyzed using JMP 10.0.4.PBMCs of four groups were co-cultured with Bozhi Glycopeptide of different final concentrations,at 16 hours,the IL-10 contents were measured using ELISA in each group.Results:1.F ratio from the fit model was 8.911,P<0.01.R2 was 0.88.The interaction between centrifugal time and force is not significant,P>0.05.Centrifugal time,force and their quadratic effects had significant effect on the thickness value of PBMCs,P<0.05.Surface plot by Surface Profiler showed complicated curve relationship betweenPBMCs thickness and the two factors.Optimized Centrifugal time = 10 minutes,and centrifugal force = 2307 r/min.The average cell viability ratio of PBMCs isolated under optimized factors was 95%.2.The interaction effect of drug concentration and culture time on PBMCs proliferation was significant,P < 0.05.The main effect of drug concentration was significant,P<0.01.P value of main effect of culture time was 0.055.The main effect of subject factor,and the interaction between time and concentration had no significant effects on PBMCs proliferation.Contour plot showed that time factor' role was little.The steepness of the prediction trace of concentration was much more obvious than that of time and subject.Maximize desirability profiling produced the optimized factors' values as follows: drug concentration = 196 mg/L,culture time = 4 h,subject = gastric cancer patients.Under the optimization condition,the OD = 0.354308,95% CI(0.30289,0.40573).3.Test for sphericity of the model showed that Mauchly criterion was 0.781,P<0.01.The adjusted interaction between concentration and culture time was significant,P<0.01.In the within-subject effect(Time),F = 286.077,P = 2.553E-43,both G-G(Greenhouse-Geisser)and H-F(Huynh-Feldt)adjustment showed significant P values.Between-subject(across-subject,concentration)effects were significant(F = 192.390,P= 1.894E-44).Compared with control group,Bozhi Glycopeptide of different concentration increased proliferation of PBMCs in different culture time respectively(P<0.05).4.IL-10 contents(x±s,pg/ml)in groups of 0 mg/L(control),50,100,200 mg/L(treatment)were(5.36±0.46),(6.73±0.25),(9.23±0.77),(12.35±0.86)respectively.Compared with control,IL-10 contents was significantly higher in treatment groups,Bozhi Glycopeptide with higher concentrations produced higher IL-10 contents thanthat in lower concentration groups.Conclusions:1.PBMCs Isolation in gastric cancer patients can be easily optimized using JMP software,the improved factors' values can be calculated by maximizing desirability in profiler,cell viability of PBMCs isolated by improved methods is high.2.The concentration of Bozhi Glycopeptide has significantly effect on the proliferation of PBMCs in the model containing three factors as follows: concentration,culture time and subject category.There are basis for Bozhi Glycopeptide to increase proliferation of PBMCs in gastric cancer patients,as a result of the similar proliferation effect in the non-gastric cancer patients.3.Bozhi Glycopeptide increase proliferation of PBMCs from gastric cancer patients in the time and concentration dependent way.4.Bozhi Glycopeptide significantly increases the production of PBMCs.