Effect And Mechanism Of PBMC Dealed With Aloe Vera Polysaccharide On Hepatoma Carcinoma Cell

Based on comparative and general document analysis on Aloe vera polysaccharide pharmacology and primary hepatic carcinoma therapy,this project mainly explored the effect and mechanism of peripheral blood mononuclear cell(PBMC)dealed with Aloe vera polysaccharide(AP)on hepatoma carcinoma cell.Literature investigation indicated that AP extracted from Aloe barbadensis Miller had pharmacological effects on immunity modulation,anti-carcinoma,antiradiation,liver protection,and so on.The anti-carcinoma effect was one of the hot points in our country research fields.However,most studies mainly used the animal as experimental model,and the research results were needed more clinical confirmation.In order to further explore the anti-carcinoma effect and mechanism of AP,this project observed the effect of PBMC dealed with AP on hepatoma carcinoma cell.Objective:To explore the effect of PBMC dealed with AP on hepatoma carcinoma cell and discuss the mechanism of AP on anti-carcinoma.Methods:1.The crude polysaccharide part was precipitated from the alcoholic liqueor after its solution subsequent standing at 4℃overnight,then the precipitated repeatedly washed sequentially with possibly less amounts of ethanol,acetone and ether,respectively,protein was deleted by trichloracetic acid.The refined crude polysaccharide(AP1)was dissolved in distilled water followed by filtration,and the supernatant was then precipitated with 100K molecular mass and 30K molecular mass to obtain the polysaccharide-enriched fraction(AP11,AP12,AP13).Content of each polysaccharide were measured by anthrone-concentrated oil of vitriol assay.2.Human lymphocyte separating medium was used to isolate PBMC through Ficoll Hypaque density gradient separation.Effects of AP11,AP12 and AP13 on PBMC in different concentration were observed by using the method of Alamar Blue assay,and effects on HepG2 were observed by MTT assay.3.Effect of AP11 alone or combined with Anti-CD3McAb and rhlL-2 on PBMC was measured by Alamar Blue assay.Effect of PBMC and its supernatant on the growth of HepG2 was measured by MTT assay.Effect of AP11 alone or combined with Anti-CD3McAb and rhIL-2 on the level of IFN-γand TNF-αsecreted by PBMC was measured by ELISA assay,and the FasL express on interfered PBMC was measured by flow cytometry assay.Effect of interfered PBMC on the level of IFN-γ,TNF-αand AKP secreted by HepG2 was measured respectively by ELISA assay and detection kit,moreover the cell cycle,apoptosis rate and Fas express of HepG2 were observed by flow cytometry assay.4.Used hepatoma tissue from hepatic excision as sample,primary culture hepatoma carcinoma cell was isolated by using the method of pancreatic enzyme transient digestion and simultaneous blowing,and its morphology was observed by inverted microscope.Then added AP,PBMC and PBMC dealed with AP to primary culture hepatoma carcinoma cell after two days cultivation,and observed the effect on carcinoma cell morphology. Otherwise the effect on the carcinoma cell growth was measured by MTT assay.Results:1.The proportion of aloe vera polysaccharide in AP1,AP11,AP12,AP13 were respectively 58%,79%,84%and 85%,and the concentration of protein in these polysaccharides were respectively 15.61μg/mL,13.33μg/mL,21.03μg/mL and 10.70μg/mL.2.PBMC was successfully isolated from peripheral blood samples,and its survival rate is more than 95%.Aloe vera polysaccharide had promotion on PBMC in vitro,compared with control group,AP11(200μg/mL)could promote the growth of PBMC significantly(P＜0.01).Compared with control group,AP11,AP12 and AP13 in different concentration had no obvious effect on the growth of HepG2(P＞0.05).3.Compared with the PBMC control group,AP11 alone or combined with Anti-CD3McAb and rhIL-2 could improve the growth of PBMC in different degree. Compared with the HepG2 control group,supernatant of each interfered PBMC groups had no obvious effect on HepG2(P＞0.05),but PBMC and interfered PBMC could inhibit the growth of HepG2 in different degree.Under inverted microscope,the control group cell mainly present polygon or anomal-epithelioid,Cellular nucleus was atypia obviously,nucleolus was clear and big, compared with the control group,cells in BC+H group,AP+H group,CI+H group and CIAP+H group were lower,and became fusiform,tadpole shape,some cells and some became shrinking,dark and round.Effect of AP11 on the level of IFN-γin supernatant of PBMC:The result indicated that, compared with BC group,the level of IFN-γin CI group and CIAP group remarkably increased(P＜0.01).Compared with AP group,the level of IFN-γin CI group and CIAP group remarkably increased(P＜0.01),and AP group and BC group had significant difference(P＜0.01).Effect of AP11 on the level of TNF-αin supernatant of PBMC:The result indicated that, the level of TNF-αin every group had no obvious difference(P＞0.05).Effect of effector cell(interfered PBMC)on the level of IFN-γin supernatant of target cell(HepG2):The result indicated that,compared with H group,the level of IFN-γin BC+H group,AP+H group,CI+H group and CIAP+H group remarkably increased(P＜0.01). Compared with BC+H group,the level of IFN-γin AP+H group,CI+H group and CIAP+H group remarkably increased(P＜0.01).Effect of interfered PBMC on the level of TNF-αin supematant of target cell(HepG2): The result indicated that,compared with H group,the level of TNF-αin BC+H group had increasing tendency,the level in AP+H group,CI+H group and CIAP+H group remarkably increased(P＜0.01).Compared with BC+H group,the level of TNF-αin AP+H group obviously increased(P＜0.05),in CI+H group and CIAP+H group remarkably increased(P＜0.01).Flow cytometry detection indicated that,cells of the control group had no hypodiploid apoptotic peak before diploid cell DNA peak(G1 peak),the apoptotic rate was lower(5.13%),mostly in S stage(43.33%),less in G1 stage(52.27%).Compared with the control group,cells of CIAP+H group displayed obvious apoptotic peak before G1 peak, the apoptotic rate remarkably increased(30.37%,P＜0.01),the S stage proportion decrease(24.37%,P＜0.05),and mostly in G1 stage(75.63%,P＜0.05).Cells in the other interfered groups displayed apoptotic peak,the apoptotic rate and proportion of S stage decreased in different degree,and proportion of G1 stage increased in different degree.Effect of AP11 on FasL express on PBMC:flow cytometry detection indicated that, compared with control group,FasL express on PBMC was enhanced in AP group,CI group and CIAP group.Effect of interfered PBMC on Fas express on HepG2:flow cytometry detection indicated that,compared with H group,Fas express on HepG2 was remarkably enhanced in BC+H group,AP+H group,CI+H group and CIAP+H group(P＜0.01).4.Primary culture hepatoma carcinoma cell was successfully isolated from hepatoma tissue by using the method of pancreatic enzyme transient digestion,simultaneous blowing and monocellular suspension.After 6 hours cultivation,the cell adhered to the plate.48 hours later,each group had cells from 20 to 100,some of which spread out completely. Under inverted microscope,living cell mainly present polygon or anomal-epithelioid,few fusiform cell could be found.Cellular nucleus was atypia obviously,nucleolus was clear and big,cytoplasm was little,particles and cavity were founded in intracytoplasm. Compared with the control group,AP group had no obvious effect on primary culture hepatoma carcinoma cell,but the PBMC and interfered PBMC group had significantly effect on primary culture hepatoma carcinoma cell.Some cell became fusiform from polygon,and some became shrinking,dark and round.MTT assay indicated that,compared with the control group,AP11 had no obvious effect on primary culture hepatoma carcinoma cell(P＞0.05),but PBMC and interfered PBMC significantly inhibited the hepatoma cell growth(P＜0.01).Conlusions:1.Provided high pure crude AP11,AP12,AP13 on base of traditional extraction technology.2.PBMC could successfully be isolated from peripheral blood samples by using Ficoll Hypaque density gradient separation.The activation of PBMC was related to blood donor health and the growth condition.The result indicated that AP could promote the growth of PBMC in vitro.Compared with control group,AP11(200μg/mL)could promote the growth of PBMC significantly(P＜0.01).Compared with control group,AP11,AP12 and AP13 in different concentration had no obvious effect on the growth of HepG2(P＞0.05). 3.AP11 alone or combined with Anti-CD3McAb and rhlL-2 could significantly improved the growth of PBMC,increased the level of IFN-γ,and enhanced FasL express, which indicated that AP11 could improve PBMC proliferation,and enhanced IFN-γsecretion.Maybe its antitumor effect was related to immunologic enhancement and inducing apoptosis.PBMC dealed with AP11 could induced HepG2 apoptosis and changed its cell cycle, could increase the level of IFN-γand TNF-α,decreased the level of AKP and enhanced Fas express on target cell.These result indicated that the mechanism of AP on anti-carcinoma was relative with decreasing carcinoma cell activation,changing its cell cycle,increasing the level of IFN-γand TNF-α,and inducing apoptosis through Fas/FasL pathway.4.Primary culture hepatoma carcinoma cell sucessfully isolated,which activity was related to the carcinoma phase and the growth condition in vitro.The result indicated that AP had no obvious effect on primary culture hepatic carcinoma.PBMC and interfered PBMC could significantly inhibit autoallergic hepatoma carcinoma cell growth by MTT assay and inverted microscope observation.