芦荟多糖干预的PBMC对肝癌细胞的作用及其机制研究
|
摘要
本课题在较全面综述近年来芦荟研究进展及肝癌中西医综合治疗研究的基础上,重点开展了芦荟多糖干预的外周血单个核细胞(peripheral blood mononuclear cell,PBMC)对肝癌细胞生长的作用及其机制的实验研究。文献研究表明,芦荟多糖具有调节免疫、抗肿瘤、抗辐射、保肝、降血糖、促进创伤和溃疡愈合及抗炎、抗病毒等多方面的药理活性。其中,芦荟多糖的抗肿瘤作用是我国近年芦荟研究的热点之一,但已有的研究多为动物实验研究,研究成果多数尚待临床研究证实。本课题通过研究芦荟多糖AP11干预的PBMC对肝癌细胞生长的作用,从细胞水平探讨了芦荟多糖的抗肿瘤作用机制。
目的:
通过研究芦荟多糖AP11干预的PBMC对肝癌细胞生长的作用,探讨芦荟多糖的抗肿瘤作用机制。
方法:
1、采用水提醇沉法提取芦荟粗多糖,无水乙醇、丙酮、乙醚洗涤,三氯乙酸除蛋白,得精制芦荟粗多糖AP1,选用10万分子量与3万分子量的超滤膜截留不同分子量芦荟多糖AP11(分子量＞10万)、AP12(3万＜分子量＜10万)、AP13(分子量＜3万),蒽酮-浓硫酸法测定各多糖含量,考马斯亮蓝染色法测定蛋白含量。
2、采用人淋巴细胞分离液、Ficoll Hypaque密度梯度离心法分离PBMC。AlamarBlue法测定不同浓度芦荟多糖AP11、AP12、AP13对PBMC生长的影响,MTT法测定不同浓度芦荟多糖AP11、AP12、AP13对HepG2生长的影响。
3、Alamar Blue法测定芦荟多糖AP11单独及联合Anti-CD3McAb、rhIL-2对PBMC生长的影响。MTT法测定多糖AP11干预后PBMC上清及PBMC细胞悬液对HepG2生长的影响。酶联免疫吸附试验(ELISA)测定多糖AP11对PBMC各组上清中IFN-γ、TNF-α水平的影响,流式细胞仪检测其FasL的表达。ELISA法测定干预后各组PBMC对HepG2上清中IFN-γ、TNF-α水平的影响,AKP检测试剂盒测定对AKP水平的影响,流式细胞仪检测干预后HepG2细胞周期、凋亡率及其Fas的表达。
4、以手术切除肝癌组织为标本,胰酶短暂消化加吹打分离、单细胞悬液培养法原代培养肝癌细胞,通过倒置显微镜观察其形态学特征。并于分离培养的第2天,加芦荟多糖AP11、芦荟多糖AP11干预及未干预的PBMC于癌细胞中,倒置显微镜观察其对癌细胞形态的影响。MTT法测定其对癌细胞生长的影响。
结果:
1、从库拉索芦荟凝胶冻干粉中提取的芦荟多糖AP1、AP11、AP12、AP13中多糖所占比例分别为58%、79%、84%和85%;蛋白含量分别为15.61μg/mL、13.33μg/mL、21.03μg/mL和10.70μg/mL。
2、人淋巴细胞分离液、密度梯度离心法可成功分离PBMC,细胞的存活率可达95%以上。不同分子量芦荟多糖对PBMC的生长有一定的促进作用。与PBMC空白对照组相比,AP11(200μg/mL)组可显著促进PBMC生长(P＜0.01)。与空白对照HepG2组相比,各浓度AP11、AP12、AP13组对HepG2生长的影响没有明显差异(P＞0.05)。
3、与空白对照PBMC组(BC组)相比,芦荟多糖AP11及其联合Anti-CD3McAb、rhIL-2对PBMC的生长有不同程度地促进作用。与空白对照HepG2组(H组)相比,干预后各组PBMC效应细胞的上清液对HepG2生长的影响无明显差异(P＞0.05),干预后各组PBMC效应细胞可不同程度抑制HepG2的生长。
光镜下观察可见,H组细胞以多边形细胞为主,细胞核异型性明显,核仁清晰,核大,胞浆透明。与H组相比,BC+H组、AP+H组、CI+H组CIAP+H组细胞镜下生长视野减少,均出现不同程度的形态改变,大多细胞呈梭形、蝌蚪形,部分细胞皱缩,变黑、变圆。
芦荟多糖AP11对PBMC上清中IFN-γ水平的影响:与BC组相比,CI组和CIAP组的IFN-γ水平显著提高(P＜0.01);与AP组比,CI组和CIAP组的IFN-γ水平显著提高(P＜0.01),与BC组相比差异显著(P＜0.01)。
芦荟多糖AP11对PBMC上清中TNF-α水平的影响:BC组、AP组、CI组和CIAP组之间的TNF-α水平无明显变化(P＞0.05)。
效应细胞对靶细胞HepG2上清中IFN-γ水平的影响:与H组相比,BC+H组、AP+H组、CI+H组与CIAP+H组IFN-γ水平显著提高(P＜0.01);与BC+H组相比,AP+H组、CI+H组与CIAP+H组IFN-γ水平显著提高(P＜0.01)。
效应细胞对靶细胞HepG2上清中TNF-α水平的影响:与H组相比,BC+H组有增高的趋势,AP+H组、CI+H组与CIAP+H组TNF-α水平显著提高(P＜0.01);与BC+H组比,AP+H组TNF-α水平有明显提高(P＜0.05),CI+H组与CIAP+H组TNF-α水平显著提高(P＜0.01)。
流式细胞仪检测结果显示,H组细胞在正常二倍体细胞DNA峰(G1峰)前无明显的亚二倍体凋亡峰,细胞凋亡率很低(5.13%),更多处于S期(43.33%),较少处于G1期(52.27%);与H组相比,CIAP+H组中靶细胞在G1峰前出现明显的凋亡峰,凋亡率显著增高(30.37%,P＜0.01),S期比例降低(24.37%,P＜0.05),较多处于G1期(75.63%,P＜0.05)。与H组相比,其余各组靶细胞都有凋亡峰出现,其凋亡率、S期比例都有不同程度降低,G1期比例有不同程度增加。
芦荟多糖AP11对PBMC FasL表达的影响:与BC组相比,AP组、CI组和CIAP组的FasL荧光表达率升高。
效应细胞对HepG2 Fas表达的影响:与H组相比,BC+H组、AP+H组、CI+H组与CIAP+H组的Fas荧光表达率升高。
4、胰酶短暂消化加吹打分离、单细胞悬液培养的方法,可成功获取原代肝癌细胞。培养6h后即可在倒置显微镜下观察到贴壁细胞,48h可观察到细胞呈单个或团块状贴壁生长,每个团块约有20～100个不等的细胞,有的细胞已经完全贴壁展开。倒置显微镜下,活细胞以多边形或不规则上皮样细胞为主,有少量梭形细胞。细胞核异型性明显,核仁清晰,核大,胞质少,胞浆内可见丰富颗粒及空泡。与未加多糖干预的空白对照组相比,癌细胞在数量及形态上没有明显的变化。与未加PBMC干预的癌细胞相比,芦荟多糖AP11干预及未干预的PBMC作用于癌细胞后,癌细胞形态发生明显变化,细胞混合后6h,一部分细胞形态由多边形变为梭形,一部分细胞皱缩,变黑、变圆。MTT实验结果表明,与癌细胞空白对照组比,芦荟多糖AP11对原代肝癌细胞生长的影响没有明显差异(P＞0.05);芦荟多糖AP11干预及未干预PBMC组可明显抑制肝癌细胞的生长(P＜0.01)。
结论:
1、在传统多糖提取工艺的基础上,得到纯度较高的不同分子量芦荟多糖AP11、AP12、AP13,为本研究提供了理想的实验用药。
2、成功建立PBMC分离方法。PBMC体外分离后第1天,其数量有下降趋势,第4～6天比较稳定,我们选用处于稳定期的细胞作为实验用细胞模型。PBMC的活性与供血者体质及体外培养条件有关。芦荟多糖AP11(200μg/mL)组可显著促进PBMC生长。不同分子量的芦荟多糖对HepG2的生长没有明显抑制作用。
3、芦荟多糖AP11单独及其联合Anti-CD3McAb与rhIL-2应用可显著促进体外PBMC的增值,同时可增加PBMC IFN-γ的分泌,增强FasL的表达。表明其可以促进PBMC的增殖,并能增强其分泌杀伤肿瘤活性细胞因子IFN-γ的分泌,提示其抗肿瘤作用与增强机体免疫,诱导细胞凋亡有关。
芦荟多糖AP11干预的PBMC效应细胞上清液对HepG2的生长没有明显抑制,而PBMC效应细胞可以显著抑制HepG2的生长,并能诱导HepG2靶细胞的凋亡、改变其细胞周期。此外,还能增加混合培养后细胞上清中IFN-γ、TNF-α的水平,降低AKP的水平及增强靶细胞Fas蛋白的表达。提示芦荟多糖AP11的抗肿瘤作用与降低肿瘤细胞活性、改变细胞周期、提高IFN-γ和TNF-α水平,通过IFN-γ与TNF-α的协同作用及激活Fas/FasL途径、诱导凋亡有关。
4、胰酶短暂消化加吹打分离、单细胞悬液培养的方法,可成功获取原代肝癌细胞。其活性与肝癌患者发病阶段及体外培养条件有关。芦荟多糖对原代培养肝癌细胞的生长及形态没有明显作用。芦荟多糖干预及未干预的自体PBMC对原代肝癌细胞有明显的抑制作用。
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.
目的:
通过研究芦荟多糖AP11干预的PBMC对肝癌细胞生长的作用,探讨芦荟多糖的抗肿瘤作用机制。
方法:
1、采用水提醇沉法提取芦荟粗多糖,无水乙醇、丙酮、乙醚洗涤,三氯乙酸除蛋白,得精制芦荟粗多糖AP1,选用10万分子量与3万分子量的超滤膜截留不同分子量芦荟多糖AP11(分子量＞10万)、AP12(3万＜分子量＜10万)、AP13(分子量＜3万),蒽酮-浓硫酸法测定各多糖含量,考马斯亮蓝染色法测定蛋白含量。
2、采用人淋巴细胞分离液、Ficoll Hypaque密度梯度离心法分离PBMC。AlamarBlue法测定不同浓度芦荟多糖AP11、AP12、AP13对PBMC生长的影响,MTT法测定不同浓度芦荟多糖AP11、AP12、AP13对HepG2生长的影响。
3、Alamar Blue法测定芦荟多糖AP11单独及联合Anti-CD3McAb、rhIL-2对PBMC生长的影响。MTT法测定多糖AP11干预后PBMC上清及PBMC细胞悬液对HepG2生长的影响。酶联免疫吸附试验(ELISA)测定多糖AP11对PBMC各组上清中IFN-γ、TNF-α水平的影响,流式细胞仪检测其FasL的表达。ELISA法测定干预后各组PBMC对HepG2上清中IFN-γ、TNF-α水平的影响,AKP检测试剂盒测定对AKP水平的影响,流式细胞仪检测干预后HepG2细胞周期、凋亡率及其Fas的表达。
4、以手术切除肝癌组织为标本,胰酶短暂消化加吹打分离、单细胞悬液培养法原代培养肝癌细胞,通过倒置显微镜观察其形态学特征。并于分离培养的第2天,加芦荟多糖AP11、芦荟多糖AP11干预及未干预的PBMC于癌细胞中,倒置显微镜观察其对癌细胞形态的影响。MTT法测定其对癌细胞生长的影响。
结果:
1、从库拉索芦荟凝胶冻干粉中提取的芦荟多糖AP1、AP11、AP12、AP13中多糖所占比例分别为58%、79%、84%和85%;蛋白含量分别为15.61μg/mL、13.33μg/mL、21.03μg/mL和10.70μg/mL。
2、人淋巴细胞分离液、密度梯度离心法可成功分离PBMC,细胞的存活率可达95%以上。不同分子量芦荟多糖对PBMC的生长有一定的促进作用。与PBMC空白对照组相比,AP11(200μg/mL)组可显著促进PBMC生长(P＜0.01)。与空白对照HepG2组相比,各浓度AP11、AP12、AP13组对HepG2生长的影响没有明显差异(P＞0.05)。
3、与空白对照PBMC组(BC组)相比,芦荟多糖AP11及其联合Anti-CD3McAb、rhIL-2对PBMC的生长有不同程度地促进作用。与空白对照HepG2组(H组)相比,干预后各组PBMC效应细胞的上清液对HepG2生长的影响无明显差异(P＞0.05),干预后各组PBMC效应细胞可不同程度抑制HepG2的生长。
光镜下观察可见,H组细胞以多边形细胞为主,细胞核异型性明显,核仁清晰,核大,胞浆透明。与H组相比,BC+H组、AP+H组、CI+H组CIAP+H组细胞镜下生长视野减少,均出现不同程度的形态改变,大多细胞呈梭形、蝌蚪形,部分细胞皱缩,变黑、变圆。
芦荟多糖AP11对PBMC上清中IFN-γ水平的影响:与BC组相比,CI组和CIAP组的IFN-γ水平显著提高(P＜0.01);与AP组比,CI组和CIAP组的IFN-γ水平显著提高(P＜0.01),与BC组相比差异显著(P＜0.01)。
芦荟多糖AP11对PBMC上清中TNF-α水平的影响:BC组、AP组、CI组和CIAP组之间的TNF-α水平无明显变化(P＞0.05)。
效应细胞对靶细胞HepG2上清中IFN-γ水平的影响:与H组相比,BC+H组、AP+H组、CI+H组与CIAP+H组IFN-γ水平显著提高(P＜0.01);与BC+H组相比,AP+H组、CI+H组与CIAP+H组IFN-γ水平显著提高(P＜0.01)。
效应细胞对靶细胞HepG2上清中TNF-α水平的影响:与H组相比,BC+H组有增高的趋势,AP+H组、CI+H组与CIAP+H组TNF-α水平显著提高(P＜0.01);与BC+H组比,AP+H组TNF-α水平有明显提高(P＜0.05),CI+H组与CIAP+H组TNF-α水平显著提高(P＜0.01)。
流式细胞仪检测结果显示,H组细胞在正常二倍体细胞DNA峰(G1峰)前无明显的亚二倍体凋亡峰,细胞凋亡率很低(5.13%),更多处于S期(43.33%),较少处于G1期(52.27%);与H组相比,CIAP+H组中靶细胞在G1峰前出现明显的凋亡峰,凋亡率显著增高(30.37%,P＜0.01),S期比例降低(24.37%,P＜0.05),较多处于G1期(75.63%,P＜0.05)。与H组相比,其余各组靶细胞都有凋亡峰出现,其凋亡率、S期比例都有不同程度降低,G1期比例有不同程度增加。
芦荟多糖AP11对PBMC FasL表达的影响:与BC组相比,AP组、CI组和CIAP组的FasL荧光表达率升高。
效应细胞对HepG2 Fas表达的影响:与H组相比,BC+H组、AP+H组、CI+H组与CIAP+H组的Fas荧光表达率升高。
4、胰酶短暂消化加吹打分离、单细胞悬液培养的方法,可成功获取原代肝癌细胞。培养6h后即可在倒置显微镜下观察到贴壁细胞,48h可观察到细胞呈单个或团块状贴壁生长,每个团块约有20～100个不等的细胞,有的细胞已经完全贴壁展开。倒置显微镜下,活细胞以多边形或不规则上皮样细胞为主,有少量梭形细胞。细胞核异型性明显,核仁清晰,核大,胞质少,胞浆内可见丰富颗粒及空泡。与未加多糖干预的空白对照组相比,癌细胞在数量及形态上没有明显的变化。与未加PBMC干预的癌细胞相比,芦荟多糖AP11干预及未干预的PBMC作用于癌细胞后,癌细胞形态发生明显变化,细胞混合后6h,一部分细胞形态由多边形变为梭形,一部分细胞皱缩,变黑、变圆。MTT实验结果表明,与癌细胞空白对照组比,芦荟多糖AP11对原代肝癌细胞生长的影响没有明显差异(P＞0.05);芦荟多糖AP11干预及未干预PBMC组可明显抑制肝癌细胞的生长(P＜0.01)。
结论:
1、在传统多糖提取工艺的基础上,得到纯度较高的不同分子量芦荟多糖AP11、AP12、AP13,为本研究提供了理想的实验用药。
2、成功建立PBMC分离方法。PBMC体外分离后第1天,其数量有下降趋势,第4～6天比较稳定,我们选用处于稳定期的细胞作为实验用细胞模型。PBMC的活性与供血者体质及体外培养条件有关。芦荟多糖AP11(200μg/mL)组可显著促进PBMC生长。不同分子量的芦荟多糖对HepG2的生长没有明显抑制作用。
3、芦荟多糖AP11单独及其联合Anti-CD3McAb与rhIL-2应用可显著促进体外PBMC的增值,同时可增加PBMC IFN-γ的分泌,增强FasL的表达。表明其可以促进PBMC的增殖,并能增强其分泌杀伤肿瘤活性细胞因子IFN-γ的分泌,提示其抗肿瘤作用与增强机体免疫,诱导细胞凋亡有关。
芦荟多糖AP11干预的PBMC效应细胞上清液对HepG2的生长没有明显抑制,而PBMC效应细胞可以显著抑制HepG2的生长,并能诱导HepG2靶细胞的凋亡、改变其细胞周期。此外,还能增加混合培养后细胞上清中IFN-γ、TNF-α的水平,降低AKP的水平及增强靶细胞Fas蛋白的表达。提示芦荟多糖AP11的抗肿瘤作用与降低肿瘤细胞活性、改变细胞周期、提高IFN-γ和TNF-α水平,通过IFN-γ与TNF-α的协同作用及激活Fas/FasL途径、诱导凋亡有关。
4、胰酶短暂消化加吹打分离、单细胞悬液培养的方法,可成功获取原代肝癌细胞。其活性与肝癌患者发病阶段及体外培养条件有关。芦荟多糖对原代培养肝癌细胞的生长及形态没有明显作用。芦荟多糖干预及未干预的自体PBMC对原代肝癌细胞有明显的抑制作用。
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.
引文
[1]皮文霞,丁辉,程爱斌,等.芦荟多糖的纯化工艺与体外抗肿瘤活性[J].中国天然药物,2007,5(6):425-427.
[2]邹翔,汲晨锋,高世勇,等.3种芦荟多糖抗肿瘤作用的比较分析[J].哈尔滨商业大学学报,2004,20(1):14-17.
[3]苗艳艳,禄保平.芦荟多糖对肝癌细胞及外周血单个核细胞作用的研究[J].河南中医学院学报,2007,22(3):17-18.
[4]邹翔,王洪亮,孙宇.3种芦荟多糖体外抗肿瘤作用的比较研究[J].哈尔滨商业大学学报,2004,20(5):512-518.
[5]邓小云,丁登峰,戴美红,等.植物多糖药理作用研究进展[J].中医药导报,2006,12(9):86-88
[6]Leung YK,Liu C,Koon JCM,et al.Polysaccharide biological response modifiers[J].Immunology Letters,2006,105:101-114.
[7]王莉,包旭,陈淑杰,等.巴巴多斯芦荟多糖提取物对小鼠免疫功能的影响[J].华西药学杂志,1999,14(4):23.
[8]芦荟产业专业委员会.http://www.aisc.com.cn/allfile/2003-1-1/news2006210110622.asp
[9]Womble D,Helderman JH.The impact of acemannan on the generation and function of cytotoxic T-lymphocytes[J].Immunopharmacol.Immunotoxicol,1992,14(1-2):63-77.
[10]叶子,师建国,张希国.CD3AK细胞的研究进展[J].现代肿瘤医学,2006,14(8):1030-1033.
[11]秦建国,韩奉立,韩民,等.CD3AK细胞过继治疗对肝癌患者T淋巴细胞亚群及功能活性的影响[J].中国普外基础与临床杂志,2002,9(3):183-185.
[12]Womble D,Helderman JH.The impact of acemannan on the generation and function of cytotoxic T-lymphocytes[J].Immunopharmacol Immunotoxicol,1992,14(1-2):63-77.
[13]Shah MA,Schwartz GK.Cell cycle-mediated drug resistance:an emerging concept in cancer therapy[J].Clin Cancer Res,2001,7(8):2168-2181.
[14]龚非力.医学免疫学[M].北京:科学出版社,2000:360.
[15]Dobrzanski MJ,Reome JB,Dutton RW.Immunopotentiating role of IFN-gamma in early and late stages of type 1 CD8 effector cell-mediated tumor rejection[J].Clin Immunol,2001,98(1):70-84.
[16]Trubiani O,Bosco D,Di Primio R.Interferon-gamma(IFN-gamma)induces programmed cell death in differentiated human leukemic B cell lines[J].Exp Cell Res,1994,215(1):23-7.
[17]Ahn EY,Pan G,Vickers SM,et al.IFN-gammaupregulates apoptosis-related molecules and enhances Fas-mediated apoptosis in human cholangiocarcinoma[J].Int J Cancer,2002,100(4):445-51.
[18]关燕清,杨鑫华,何丽梅,等.TNF与IFN协同诱导癌细胞凋亡机制的研究进展[J].华南师范大学学报(自然科学版),2006,(3):122-126.
[19]古翠萍,张沂平.TNF-α抗肿瘤作用机制新进展[J].中国肿瘤,2007,16(2):102-105.
[20]巫协宁.临床肝胆系病学[M].上海:上海科学技术文献出版社,2002:113.
[21]Abrahams VM,Kamsteeg M,Mor G.The Fas/Fas ligand system and cancer:immune privilege and apoptosis[J].Mol Biotechnol,2003,25:19-30.
[22]Griffith TS,Ferguson TA.The role of FasL-induced apoptosis in immune privilege[J].Immunol Today,1997,18:240-244.
[23]Sartorius U,Schmitz I,Krammer PH.Molecular mechanisms of death-receptor-mediated apoptosis[J].Chembiochem,2001,2(1):20-29.
[24]Nagata S,Golstein P.The Fas death factor[J].Science,1995,267(5203):1449-1456.
[25]刘军,王占民,刘博,等.人肝细胞癌细胞系HCC-9903的建立及其生物学特性研究[J].中华外科杂志,2001,39(11):872.
[26]孙晓东,钟雪云.原发性肝癌细胞原代培养制备过程中细胞分离方法的改良[J].中国病理生理杂志,1998,14(6):767-768.
[2]邹翔,汲晨锋,高世勇,等.3种芦荟多糖抗肿瘤作用的比较分析[J].哈尔滨商业大学学报,2004,20(1):14-17.
[3]苗艳艳,禄保平.芦荟多糖对肝癌细胞及外周血单个核细胞作用的研究[J].河南中医学院学报,2007,22(3):17-18.
[4]邹翔,王洪亮,孙宇.3种芦荟多糖体外抗肿瘤作用的比较研究[J].哈尔滨商业大学学报,2004,20(5):512-518.
[5]邓小云,丁登峰,戴美红,等.植物多糖药理作用研究进展[J].中医药导报,2006,12(9):86-88
[6]Leung YK,Liu C,Koon JCM,et al.Polysaccharide biological response modifiers[J].Immunology Letters,2006,105:101-114.
[7]王莉,包旭,陈淑杰,等.巴巴多斯芦荟多糖提取物对小鼠免疫功能的影响[J].华西药学杂志,1999,14(4):23.
[8]芦荟产业专业委员会.http://www.aisc.com.cn/allfile/2003-1-1/news2006210110622.asp
[9]Womble D,Helderman JH.The impact of acemannan on the generation and function of cytotoxic T-lymphocytes[J].Immunopharmacol.Immunotoxicol,1992,14(1-2):63-77.
[10]叶子,师建国,张希国.CD3AK细胞的研究进展[J].现代肿瘤医学,2006,14(8):1030-1033.
[11]秦建国,韩奉立,韩民,等.CD3AK细胞过继治疗对肝癌患者T淋巴细胞亚群及功能活性的影响[J].中国普外基础与临床杂志,2002,9(3):183-185.
[12]Womble D,Helderman JH.The impact of acemannan on the generation and function of cytotoxic T-lymphocytes[J].Immunopharmacol Immunotoxicol,1992,14(1-2):63-77.
[13]Shah MA,Schwartz GK.Cell cycle-mediated drug resistance:an emerging concept in cancer therapy[J].Clin Cancer Res,2001,7(8):2168-2181.
[14]龚非力.医学免疫学[M].北京:科学出版社,2000:360.
[15]Dobrzanski MJ,Reome JB,Dutton RW.Immunopotentiating role of IFN-gamma in early and late stages of type 1 CD8 effector cell-mediated tumor rejection[J].Clin Immunol,2001,98(1):70-84.
[16]Trubiani O,Bosco D,Di Primio R.Interferon-gamma(IFN-gamma)induces programmed cell death in differentiated human leukemic B cell lines[J].Exp Cell Res,1994,215(1):23-7.
[17]Ahn EY,Pan G,Vickers SM,et al.IFN-gammaupregulates apoptosis-related molecules and enhances Fas-mediated apoptosis in human cholangiocarcinoma[J].Int J Cancer,2002,100(4):445-51.
[18]关燕清,杨鑫华,何丽梅,等.TNF与IFN协同诱导癌细胞凋亡机制的研究进展[J].华南师范大学学报(自然科学版),2006,(3):122-126.
[19]古翠萍,张沂平.TNF-α抗肿瘤作用机制新进展[J].中国肿瘤,2007,16(2):102-105.
[20]巫协宁.临床肝胆系病学[M].上海:上海科学技术文献出版社,2002:113.
[21]Abrahams VM,Kamsteeg M,Mor G.The Fas/Fas ligand system and cancer:immune privilege and apoptosis[J].Mol Biotechnol,2003,25:19-30.
[22]Griffith TS,Ferguson TA.The role of FasL-induced apoptosis in immune privilege[J].Immunol Today,1997,18:240-244.
[23]Sartorius U,Schmitz I,Krammer PH.Molecular mechanisms of death-receptor-mediated apoptosis[J].Chembiochem,2001,2(1):20-29.
[24]Nagata S,Golstein P.The Fas death factor[J].Science,1995,267(5203):1449-1456.
[25]刘军,王占民,刘博,等.人肝细胞癌细胞系HCC-9903的建立及其生物学特性研究[J].中华外科杂志,2001,39(11):872.
[26]孙晓东,钟雪云.原发性肝癌细胞原代培养制备过程中细胞分离方法的改良[J].中国病理生理杂志,1998,14(6):767-768.