摘要
目的：探讨从胃癌干细胞中提取伴侣分子？抗原肽的方法及其免疫功能。方法低温超声裂解筛选胃癌干细胞和胃癌细胞，经盐析粗提蛋白及透析后，采用十二烷基硫酸钠？聚丙烯酰胺凝胶电泳（ SDS？PAGE）分析伴侣分子？抗原肽表达量，采用溴化氰活化琼脂糖凝胶4B亲和层析分离、纯化伴侣分子？抗原肽，采用反向高压液相色谱、SDS？PAGE和Western blot法进行蛋白纯度分析，采用淋巴细胞增殖实验和细胞毒活性实验检测伴侣分子？抗原肽的免疫活性。结果成功制备并鉴定胃癌干细胞的伴侣分子？抗原肽，主要组分为HSP60？抗原肽、HSP70？抗原肽、HSP90？抗原肽和HSP110？抗原肽。0．75μg HSP70？抗原肽、1．00μg HSP70？抗原肽和1．00μg HSP90？抗原肽对淋巴细胞有明显的增殖作用，其A490值分别为0．26±0．03、0．45±0．05和0．32±0．04，而相应剂量的 HSP60？抗原肽和HSP110？抗原肽不能激活淋巴细胞。1．00μg HSP70？抗原肽组和1．00μg HSP70组的细胞杀伤率分别为（45．0±2．0）％和（16．0±2．0）％，差异有统计学意义（P＝0．012）。1．00μg HSP90？抗原肽组和1．00μg HSP90组的细胞杀伤率分别为（36．0±5．0）％和（13．0±4．0）％，差异有统计学意义（P＝0．048）。结论胃癌细胞中伴侣分子？抗原肽含量极低，而胃癌干细胞中伴侣分子？抗原肽含量明显增多，经提纯后可制备纯度较高的伴侣分子？抗原肽。提取的伴侣分子？抗原肽具有较强的免疫原性，可在体外制作瘤苗，可能在胃癌靶向治疗上有较好的应用价值。
Objective To explore the method of extracting chaperone antigen peptide complexes from gastric cancer stem cells and its immune function. Methods Gastric cancer stem cells and gastric cancer cells were screened by low temperature ultrasonic lysis. After salting out and dialysis, the lysate supernatant was processed with SDS？PAGE to analyze the expression of chaperone antigen peptide complexes, and then was separated and purified with CNBr？activated SepharoseTM 4B. Reverse high pressure liquid chromatography （ HPLC） , SDS？PAGE and Western blotting were used to analyze the purity and nature of the acquired albumen. Lymphocyte proliferation assay and lymphocytotoxicity assay were used to ditermine the immunological activity of the chaperone？antigen peptide complexes. Results The chaperone antigen peptide complexes of gastric cancer stem cells were prepared and identified successfully, of which the main components were the antigen peptides of HSP60, HSP70, HSP90 and HSP110. 0.75 μg and 1.00 μg HSP70？antigen peptide and 1. 00 μg HSP90？antigen peptide activated lymphocytes significantly. Their A490 values were 0.26±0.03, 0.45±0.05 and 0.32±0.04, respectively, while the corresponding doses of HSP60？antigen peptide and HSP110？antigen peptide did not activate lymphocytes. The killing rates of 1. 00 μg HSP70？antigen peptide and 1.00μg HSP70 were （45.0±2.0）% and （16.0±2.0）%, respectively, showing a significant difference （P=0.012）. Similarly, the killing rates of 1.00 μg HSP90？antigen peptide and 1.00 μg HSP90 were （36.0±5.0）% and （13.0±4.0）%, respectively, also showing a significant difference （P=0.048 ） . Conclusions The amount of chaperone antigen peptide complexes in gastric cancer cells is extremely low, but it is obviously increased in gastric cancer stem cells. After purification, the chaperone antigen peptide complexes with high purity can be prepared. The extracted chaperone antigen peptide complexes have stronger immunogenicity, and can be used to make tumor vaccine in vitro, which may have a good application value in the targeted therapy of gastric cancer.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2017年第2期109-114,共6页
Chinese Journal of Oncology
基金
杭州市萧山区科技局课题（2013301）
关键词
胃肿瘤
肿瘤干细胞
伴侣分子-抗原肽
亲和层析
瘤苗
Stomach neoplasms
Tumor stem cells
Chaperone antigen peptide
Affinity chromatography
Tumor vaccine
