| Background and purpose:Pancreatic cancer is an insidious onset, characterized by fast growth and early Metastatic malignant tumors of the digestive system, is the one of the highest cancer mortality, there is currently no effective treatment measures, its incidence is an upward trend in the world. Due to the anatomical position of the pancreatic tissues, no obvious early symptoms and the lack of effective means of detection, most patients have advanced, poor prognosis, the5-year survival rate of less than5%. Early detection and early diagnosis is one of the effective measures to improve the prognosis of patients with pancreatic cancer. Therefore, looking for early diagnosis, with high sensitivity and specificity, cancer molecular markers to improve the prognosis of patients with pancreatic cancer has important clinical implications, will also provide the basis for new targeted therapy for pancreatic cancer technology.B7-H4(also called B7S1or B7x) is a newly-discovered members of the B7family, inhibits T-cell-mediated immune response through inhibiting T cell proliferation, cytokine production, and cell cycle[3-6]. B7-H4played an important role in tumor immune response. B7-H4mRNA was expressed in spleen, lung, thymus, placenta, liver, kidney, and other normal organization[3]. However, B7-H4protein was not expressed in normal tissues detected with immunohistochemistry (IHC)[3]. B7-H4was expressed in a high level in some tumor tissues, such as breast cancer, ovarian cancer, kidney cancer, prostate cancer and non-small cell lung cancer, and is closely related to tumor progress[5,7-11]. Given that there is no anti-B7-H4antibody which can be used for a variety of methods to detecting the expression of B7-H4, this research prepare the mouse monoclonal antibodies (mAbs) against B7-H4through preparing3T3-B7-H4cells as a immunogen which can stably express B7-H4. And the antibody’s biological properties were analyzed. And the expression and clinical significance of B7-H4in pancreatic cancer tissue was investigated.Methods:1. Balb/c mice were immunized with3T3-B7-H4cells which expressed extrinsic B7-H4. Then the spleen cells of the immunized mice were fused with Sp2/0myeloma cells by conventional hybridoma techniques. An indirect ELISA using lysate of3T3-B7-H4as antigen was established to screen antibody-producing hybridoma cell lines. Western blotting and Immunoprecipitation (IP) were applied to characterize the mAb.2. Immunohistochemical (IHC) staining was used to detect the expression of B7-H4in pancreatic cancer tissue. And the relationship between the expressions and pathology was evaluated.Results:1. We obtained a hybridoma cell line secreting mAb against B7-H4. The subclasse of this mAb was IgM, and light chain was Kappa. Western blotting and IP showed that the mAb specifically recognized B7-H4.2. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The B7-H4was diffuse expressed in cytoplasma and/or membrane of the pancreatic cancer tissue, which was much higher than that expressed in normal pancreatic tissue (4.00±1.44vs.1.12±0.78, P<0.01). The expression of B7-H4in the pancreatic cancer tissue was higher in patients with higher tumor grade (6.10±0.72vs.3.55±1.12, P<0.01) compared with lower tumor grade or lymph node metastatic carcinoma (6.14±0.66vs.3.70±1.25, P<0.01). The expression level of B7-H4was not related to the patients’ age and gender.Conclusions:mAb against B7-H4with high activity and specificity was prepared successfully. The expression of B7-H4in pancreatic cancer tissue was increased. The abnormal expression of B7-H4in pancreatic cancer is closely related with the tumor grade and lymph node metastasis. |
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