Expression Of Tryptophanyl-tRNA Synthetase Influences Cell Proliferation Of BALB/c Mouse Lymphoma Animal Model

ObjectiveLymphoma is the malignent tumor of immune system originating in lymph node or extronodal lymphoid tissue.Each year the incidence and mortality of non-Hodgkin lymphoma (NHL)have increased. Recently,it has been the consensus that the imbalance of cell-mediated immune responses or immunotolerance participates in pathogenesis of malignent tumor.Recent studies have shown that,trytophan is the unique amino acid associated with the disorder of cell-mediated immune responses.indoleamine 2,3-dioxygenase(IDO) catalyzes the initial and rate-limiting step of tryptophan degeneration along the kynurenine pathway to create a low tryptophan microenvironment, and exerts the suppressive activity of cell proliferation.In contrast,tryptophanyl-tRNA synthetase(TTS) is the ubiquitous enzyme responsible for the association of tryptophan to its cognate tRNA, with the result of Trp-tRNA complex used for protein synthesis, and resists IDO-induced trytophan depletion.Previous study has shown that IDO is overexpression in tumor cells of NHL,and acts as an important indicator of prognosis.Our experiment is performed to measure the differences of TTS expression among control group,lymphoma group and IDO inhibitor(1-MT)group, at the basis of hypodermic injection of A20 cells to construct BALB/c mouse B lymphoma animal model,and investigate the correlation of tryptophan metabolism and lymphoma cell proliferation to provide a new passway of clinical intervention treatment.Methods and materials 1.Construction of BALB/c mouse lymphoma animal model:4-8w BALB/c mouses were raised in non-pathogen environment, succeeded by hypodermic injection of 5 X 106 A20 cells to construct BALB/c mouse B lymphoma animal model.Observe the tumor growth three times a week,and execute the mouses after twenty days.2.Rabbit anti-mouse polyclonal antibody and immunohistochemistry streptavidin-peroxidase(SP)staining was used to detect the TTS expression in tumor tissue and lymph node.3.Western blot was used to analyze semiquantitatively the expression of TTS among control group,lymphoma group and IDO inhibitor(1-MT)group.4.All the statistical analyses were carried out using SPSS 17.0.The significance of different groups were evaluated by means of ANOVA,and P<0.05 was considered statistically signigicant.Results1.7 days after the inoculation of A20 cells, subcutaneous tumor mass could be touched in the right axillary and dorsal region of the mouses, with a diameter of about 0.3-0.5cm. Tumor formation rate was above 95%. After execution(20 days after inoculation), the maximum diameter was found to be about 3-4cm. The tumor showed a round or elliptical shape under gross anatomy, the surface of which was nodular or lobulated without contact connective tissue envelope. The nearby skin, chest walls and other tissues were infiltrated. The lymph nodes beneath right axilla were enlarged and merged. It could be seen under optical microscope that the tumor cellls were uniform in size and had a pervading distribution. Their nuclei were trachychromatic and their cytoplasm was rich. The normal structure of tumor metastatic lymph nodes were destroyed and focal filtration cell mass could be seen.2.TTS is experessed in cytoplasm. The relatively large tumor cells with local filtration could be found in the tumor metastatic lymph nodes and TTS positive cells mainly centralized in the filtration region. The positive cells pervaded in tumor tissue, had rich cytoplasm and were dyed tan or tawny. Compared with normal lymph nodes, the TTS expression in tumor tissue and tumor metastatic lymph nodes rose with significant difference (P＜0.05). 3.Only a small quantity of TTS is expressed in normal lymph nodes of mouses. Compared with normal control, TTS had a significant higher expression in tumor tissue in both the tumor group and treatment group (P＜0.001,P=0.043). Compared with tumor group, TTS expression significantly decreased in treatment group (P=0.01). No significant change was found in TTS expression of tumor metastatic lymph nodes (P=0.695).ConclusionsThe method of hypodermic injection of 5 X 106 A20 cells is easy to operate, the rate of tumor information is about 95%,metastasis to lymph node and spleen is definite,and the mortality of mouses is very low,in our study,this method is effective in construction of BALB/c mouse lymphoma animal model.IDO and TTS involved in the disorder of tryptophan metabolism may relate to the abnormal metabolism of tumor cells and the induction of immunotolerance.Overpression of TTS may resist IDO-induced trytophan depletion,and response for the proliferative status of tumor cells.