The Effect Of Th17 Cells On Colitis Associated Colorectal Cancer Regulated By TL1A

As the incidence rate of inflammatory bowel disease is continously rising in our country, the researches on its mechanism and on the prevention and treatment of its complications have been raising concern. The colitis associated colorectal cancer(CAC) is closely related to IBD. With regard to the long-term complication that people most worry about, its occurrence is related to the disease extent, inflammation degree, and age of onset, morbidity time, family history of colorectal cancer, and other factors. The study on its pathogenesis can provide powerful help for effectively preventing and treating the colorectal cancer clinically.CAC refers that the intestinal tract goes through continuous hyperplasia based on the repeated inflammation stimulation, and finally it is transformed to cancer from the atypical hyperplasia.As inflammation is the initial factor for cancer, interleukin-17(IL-17), TNF-α and interleukin-6(IL-6), which is the significant proinflammatory factors inside human bodies, are involved in the development of many inflammatory disease and autoimmune diseases. They also play very important roles in the development and progression of tumors. The research on the mechanism for the cancer molecular activation induced by colitis shows that abnormal hyperthyroidism occurs to NF-κB(nuclear factor kappa B, NF-κB) and Wnt signaling path and their target genes at the different stages of the conversion of inflammation to caner. The latest studies reveal that Wnt signaling path, which becomes abnormally activated in sporadic colorectal cancer cases, could synergize the cancerization factors, such as TNF-α、IL-17 and IL-6, in regulating and controlling the morbidity of colitis associated colorectal cancer.TNF-like ligand 1 aberrance(TL1A) is a newly found member of the superfamily of tumor necrosis factors. Both in vivo and in vitro studies have proved that TL1 A is able to prompt the phenotypic activation of T-cells and APCs, enhance the activation of Th17 produced by mononuclear cells, result in up-regulate the expression of IL-17, TNF-α and IL-6, promote the occurrence of intestinal mucosa inflammation and participate in the onset of IBD. Will it, as the immune enhancer of T cell, influence the occurrence of colitis associated colorectal cancer by advancing the inflammatory reactions? However, the relevant literature is still found scarce. Under such circumstances, the LCK-CD2-TL1A-GFP-transgenic(L-Tg) mice and wild type(WT) C57BL/6 mice are used to establish the model of experimental colitis-associated colorectal cancer and research the functions of TL1 A for the purpose of finding out the answers.Objective: To establish a colitis associated cancer mouse model and investigate the role of TL1 A in the development of colitis associated colorectal cancer.Methods: ①LCK-CD2-TL1A-GFP Tg mice were identified by real-time Q-PCR.②The model of experimental colitis associated colorectal cancer was established through the co-induction of AOM and DSS. The L-Tg mice and C57BL/6 WT mice(8-12 weeks) were randomly divided into 8 groups respectively, namely, Control group, AOM group, DSS group and AOM+DSS group with 10 mice in each group. Normal diet was provided to Control group; normal diet was provided to AOM group after the mice in this group received the intraperitoneal injection of 10 mg/Kg AOM single dosage; in DSS group, the 2.5%DSS was provided for 7 days and distilled water provided for the following 14 days. This cycle should be repeated for 4 times; in AOM+DSS group, the mice received the intraperitoneal injection of AOM 10 mg/Kg single dosage and periodically drunk 4 cycles of 2.5% DSS. ③ The assessment of the degree of intestinal mucosa inflammation included the body weight(BW) change, disease activity index(DAI), the colon morphologic changes, histopathological score and myeloperoxidase(MPO); ④The assessment of tumor formation situation included the calculation of the number, diameter and formation rate of tumor and the degree of dysplasia were histopathological evaluated; ⑤Western blot technology used to detect the quantitative expression of IL-17 A, TNF-α and IL-6 protein. ⑥make correlation analysis between the MPO, the expression of IL-17 A, TNF-α, IL-6 and the tumor number, tumor size and dysplasia scores.Results: ① The TL1 A transgenic mice were identified by PCR. The TL1 A DNA located at 192 bp in Tg mice, while it did not express at 192 bp in WT mice;②Compared with the mice in Control group and AOM group, the body weight of the L-Tg mice and WT mice from the DSS group and AOM+DSS group reduced but DAI grade significantly increased. The DAI score increase is more obvious in AOM+DSS/Tg group compared with AOM+DSS/WT(2.3±0.76 vs 1.28±0.47, P<0.05); mice in DSS group suffered from hyperaemia and edema in their colon wall while the mice in AOM+DSS group had thickened colon wall with narrowed and distorted enteric cavity; under the light microscope, abundant inflammatory cells were found infiltrated in the colon tissues with ulcers in the injured epithelium mucosae and obvious increase in the pathological score. The mice in AOM+DSS group were plagued by distorted and deformed gland, not uniform cell sizes, variable forms, deep-dyed big nucleolus and atypia proliferation; in WT and Tg mice, the MPO activity is no defference between Contrl group and AOM group. When compared with the Control group and AOM group, MPO in DSS group and AOM+DSS group significantly increased, and it was more obvious in Tg group than in WT group;③ The number, size and formation rate of tumor were calculated. Compared with the WT mice, the mice in AOM+DSS/Tg group had more number of tumor bodies(5.62±1.30 vs 3.5±1.19, P<0.05), larger diameter(2.98±0.79 vs 2.08±0.37, P<0.05) and obviously higher tumor formation rate(88.9% vs 66.7%), the hyperplasia score is also increased statistically compared with that in WT mie(2.75±0.5 vs 1.7±0.8, P<0.05);④Western blot was utilized to detect the expression of IL-17 A, TNF-α and IL-6, showing that compared with the Control group and AOM group, the expression level of IL-17 A, TNF-α and IL-6 in AOM+DSS group significantly increased, and it was more obvious in AOM+DSS/Tg group than in AOM+DSS/WT group(P<0.05). ⑤ MPO was positive correlated with tumor number and size(r=0.6, r=0.55, P<0.05); the expression of IL-17 A, TNF-α and IL-6 were positive correlated with tumor number and dysplasia score(P<0.05), as well as IL-17 A, TNF-α were positive correlated with tumor size(P<0.05).Conclusion: The high expression of TL1 A will advance the course of colitis associated colorectal cancer, whose mechanism might be related to the secreation of inflammatory factors induced by TL1 A.