Experiment On The Anti-Gastric Cancer Effects Of Dendritic Cells Tumor Vaccine Loaded With Gp96-polypeptide Complex

Objective: Gastric carcinoma is one of the most common malignant tumors in the world. Current strategies for cancer therapy include surgery, chemotherapy, radiotherapy and so on. But they all have many defects such as obvious side effects, nonspecificity and low efficiency, which limit the foreground of their application. Dendritic cells (DCs) are the most potential professional antigen presenting cells, which act as an important role in stimulating T lymphocyte proliferation and anti-tumor immune response. Anti-tumor specific immune response of dendritic cells vaccine from peripheral blood mononuclear cell has been manifested. Nowadays, the immune response which was primed by commonly applicable dendritic cells vaccine has not acquired satisfactory effect because of their many disadvantages. The heat shock protein gp96 is a stress-inducible glycoprotein which is known to be constitutively and ubiquitously expressed in the endoplasmic reticulum of mammalian cells. It has low level expression in normal tissue and obviously overexpression in some tumor tissues, and has a closely relationship with tumors' differentiation and stages. As a molecular chaperone, gp96 has been implicated in the activation of dendritic cells for the initiation of adaptive immunity. Through the receptors on the surface of DCs, gp96 entrances to DCs directly and induces DCs maturation, up-regulates the expression of its surface molecules, leads to MHC class I restricted representation of gp96-associated peptides and CTL activation. Many experiments have shown that good effects in immunological reaction of anti-tumour immuned with gp96-polypeptide complex from tumour tissues. Because of lower quantities and function defect of DCs in tumour patients, gp96-polypeptide complex vaccine activated a lower immune reaspone, and so, DCs vaccine loaded with gp96-polypeptide complex in vitro might gain better effect. Therefore, the expression of gp96 in gastric cancer tissues and the relation with human gastric cancer are firstly investigated. From the gastric cancer tissues of high expression gp96, we purify the gp96-polypeptide complex and co-culture with DCs in order to study whether autologous gp96 polypeptide complex/dendritic cells vaccine could induce peptide specific cytotoxic T lymphocyte (CTL) response. Methods: SABC immunohistochemistry and in situ hybridization were used to evaluate the expression of gp96 at the mRNA and protein level in gastric cancer tissues and normal control from clinical samples and the relationship between gp96 expression and clinical pathological feature were investigated. The gp96-polypeptide complex from gastric cancer tissues of high expression gp96 were purificated utilizing con A sepharose affinity chromatography and mono Q ion exchange chromatography ,and then loaded DCs with it . Fluorescence-activated cell sorter (FACS) was used to detect surface molecules expression of dendritic cells and 3H-TDR was used to detect the capacity of DCs to activate T lymphocyte proliferation. 51Cr release test was performed to evaluate the gp96-peptide specific CTL response induced by DCs in two target tumor cells including the primary cultured tumor cells and SGC7901 cells. Enzyme-linked immunosorbent assay(ELISA) method were used to detect IL-12 ,IL-10 and interferon-γ (IFN-γ) level of supernatant secreted by activated dendritic cells and T lymphocytes .Results: 1. Heat shock protein gp96 was expressed both in human gastric cancer tissues and normal gastric tissues at the mRNA and protein level by in situ hybridization and immunohistochemistry. There was an obviously over expression in gastric cancer tissues both at the mRNA and protein level compared to normal gastric tissues and had a closely relationship with differentiation, stage and metastasis, but showed no correlation with other clinical pathological features. 2. From high expression heat shock protein gp96 gastric cancer tissues, the gp96-polypeptide complex could be purified utilizing conA sepharose and monoQ chromatography. Approximately 30μg g...