热休克凋亡食管癌细胞负载自体树突状细胞制备及其表型特征的分析

Preparation and characterize of autologous dendritic cells loaded by heat shocked apoptotic esophagus cancer cells

目的:建立自体热休克凋亡食管癌细胞抗原制备、树突状细胞(DC)体外诱导以及抗原负载方法,为DC肿瘤疫苗的临床应用提供技术基础。方法:采用酶消化法从手术切除的食管癌新鲜组织获得单细胞悬液,热休克后用桦脂酸诱导其凋亡制备成细胞抗原;采用人工肝素抗凝采集外周静脉血,分离单个核细胞(PBMC),贴壁法获得单核细胞,经GM-CSF与IL-4体外诱导成未成熟树突状细胞(imDC);负载细胞抗原后制备成DC肿瘤疫苗,并对DC疫苗的形态、数量及表型特征进行分析。结果:肿瘤细胞抗原每克组织得率(9.89±2.46)×106,平均凋亡率(67.60±3.23)%。imDC活率>95%,imDC表型分析为CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+和CD11c+CD86+,表达率分别为(88.30±3.59)%(、1.25±0.55)%、(6.13±1.79)%和(72.90±5.01)%。DC疫苗活率>95%,DC疫苗表型为CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+和CD11c+CD86+,表达率分别为(89.60±3.91)%、(34.20±3.85)%、(64.90±8.05)%和(86.10±6.22)%。负载自体抗原DC诱导CTL杀伤自体肿瘤细胞的杀伤率为(65.90±6.25)%,明显高于杀伤食管癌肿瘤细胞株的杀伤率〔(32.56±2.68)%〕,P<0.01。结论:本方法稳定、安全及可靠,可制备出在体外诱导特异性CTL的DC肿瘤疫苗。

OBJECTIVE：To establish a method for the preparation of heat shocked esophagus cancer cells antigen,and autologous dendritic cell（DC） induced in vitro including the antigen loading.METHODS： Single cell suspension was obtained from the surgical fresh tissues of esophagus cancer by enzyme digestion,and then the cell antigen was prepared through apoptosis induced by betulinic acid after heat shock.The peripheral venous blood was collected under anticoagulation adopting artificial heparin,and the separated peripheral blood mononuclear cells（PBMC） by adherent methods were induced into immature DC（imDC） by GM-CSF and interleukin IL-4 in vitro.DC tumor vaccine was prepared following cell antigen loading,and then the DC tumor vaccine＇s configuration,quantity,and phenotype character were analyzed.RESULTS：Tumor cell antigen gaining-rate was（9.89±2.46）×106 per gram tissue and average apoptotic rate was（67.60±3.23）%.imDC＇s viability was more than 95%.The imDC phenotype character analysis showed that the expression rate of CD11c＋ HLA-DR＋,CD11c＋ CD80＋,CD11c＋ CD83＋ and CD11c＋ CD86＋ were（88.30±3.59）%,（1.25±0.55）%,（6.13±1.79）%,（72.90±5.01）%,respectively.DC vaccine＇s viability was more than 95%.The DC phenotype character analysis showed that the expression rates of CD11c＋ HLA-DR＋,CD11c＋ CD80＋,CD11c＋ CD83＋,and CD11c＋ CD86＋ were（89.60±3.91）%,（34.20±3.85）%,（64.90±8.05）% and（86.10±6.22）%,respectively.The CTLs induced by autologous antigen loaded DC was significantly higher against autologous tumor cells（（65.90±6.25）%） than that against tumor cell lines（（32.56±2.68）%,P0.01）.CONCLUSIONS： The method for autologous dendritic cells loaded by heat shocked apoptotic esophagus cancer cells is of stabilization,security and credibility.The mature DC tumor vaccines can be prepared for the potential clinical application.