Y Q Jiang1, Q M Guo1, X P Xu1, J C Liang1, Y Y He1, S H An1, F Su1, C Y Li2, C X Huang2. 1. Department of Radiotherapy, the First People's Hospital of Xiaoshan, Hangzhou 311201, China. 2. Department of Oncology, the Affiliated Hospital of Hangzhou Normal University, Hangzhou 310015, China.
Abstract
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 A(490) 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 1.00 μg 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.
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 A(490) 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 1.00 μg 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.