The Expressionand Clinical Significance Of PSGL-1 In Malignant Lymphoma

Background:Malignant lymphoma is a group of malignant tumor which originated in lymph nodes or outside of lymphoid tissues and it always had highly heterogeneous,anda high mortality rate.It ranked 10 in all of neoplastic disease.In recent years, with the gradually deepening understanding of malignant lymphoma on molecular mechanisms, cell genetics and other characteristics, some new immune therapy and molecular targeted therapy of malignant lymphoma make certain subtypes of better treatment and better prognosis. But there are still some lymphomas easy to relapse and transger in the course of treatment or after treatment because of their highly invasive characteristics. Therefore, it is important to discuss the invasion and metastasis of lymphoma. P-selectin glycoprotein ligand-1(PSGL-1, CD162) is a transmembrane sialic acid mucin in the leukocyte surface which has the homodimer structure. As the primary ligand of P-selectin, it can mediate cell adhesion, lymphocyte homing and regulating leukocyte rolling action on the surface of endothelial cells. In the inflammatory environment, PSGL-1 can promotes leukocyte and endothelial cell tight junction and cross the blood vessel walls to sites of inflammation.In many solid tumors, PSGL-1 is also important for tumor metastasis.The study found that tumor cell-derived PSGL-1 can promote tumor cell metastasis by binding the selectin from vascular endothelium, platelet and leukocyte.Some academics have already demonstrated that theexpression of PSGL-1 in human prostate and small cell lung cancer cells could had stronger invasion and metastasis than those who did not express. In 2007, Geert Raes and his colleagues proved in the gene animal models that, PSGL-1 on T cell hybridomas played an important role on hematogenous metastasis, that PSGL-1 could be expressed on T and B lymphocyte cell lines, and it could be contribute to hematogenous metastasis of lymphoma cells. But there was no relevant reports about PSGL-1 in hematogenous metastasis of human lymphoma.Therefore, in this study we would detect PSGL-1’s expression in lymphoma, and using cell experiments to understand the relevance of PSGL-1 with the biological behavior of malignant lymphoma cells,investigate its relationship with the expression and disease progression and blood metastasis of malignant lymphoma, try to explore the possible mechanism of action,providingsome theoretical basis for thediagnosis, treatmentandprognosis of malignant lymphoma. Objectives: 1. To learn PSGL-1‘s expression in lymphoma. 2. To explore the contactions with PSGL-1 and clinical datas of lymphoma patients. 3. To investigate the effect of PSGL-1’s expression on lymphomacells’.migration,proliferation and apoptosis. Methods: Part 1 Using immunohistochemical method to detect the expression of PSGL-1 in tissues of reactive lymphoid hyperplasia and lymphoma.Complete the collection of pathology organization of lymphoma and histopathology of reactive lymphoid hyperplasia,using immunohistochemical methods(adopted to non-biotinylated two-step test) to detect the PSGL-1’s expression of 30 cases of lymphoma and 10 cases of lymphadenitis.Complete the experiment in strict according to immunohistochemistry SP kit instructions. To complete statistical analysis, and then to learn the relationship between PSGL-1’s expression in lymphoma and the expression’s relevance tolymphoma type, differentiation and prognosis. Part 2 Constructing the targeted siRNA liposome of PSGL-1 gene and transfected into Raji Burkitt’s lymphoma cell line, using Western Blot to detected the silencing effect of PSGL-1 in Raji cellsPurchase Raji lymphoma cell lines and perform cell culture.Collected log phase growth over cells and divided into two groups. Constructed targeting siRNA liposome of PSGL-1 gene and transfected into Raji cells. Using low temperature ultracentrifugation method to extract cellular proteins. Protein concentrations were determined by the BCA method, To complete process In strict accordance with Western blot test kit instructions. Compare PSGL-1 expression in lymphoma before and after transfection, and prepare for the experiment of cell migration assay, cell proliferation and apoptosis. Part 3 Using Transwell method to contrast theRaji cell migration before and after the silencing PSGL-1 gene.Depending on the diameter of Raji cells, we choosed the pore size of 0.8μm transwell chamber. The cells were seeded on the interior with the medium added without fetal calf serum without antibiotics and lower medium was added withfetal calf serum and without antibiotics. Incubated at 37 ℃, 5% CO2 environment for 48 hours and couneted numbers of cells on the indoor. Comparaed results of the experimental and control groups, then analysised the impact on PSGL-1 protein for cell migration. Part 4 Using CCK 8, flow cytometry to contrast the proliferation and apoptosis of Raji cells before and after silencing PSGL-1 gene.According to CCK8 reagent operating instructions, added 10μl CCK8 into each control and experimental groups. The cells were placed in 37℃, 5% CO2 environment, cultured for 24 h, 48 h, 72 h, and then detected the absorbance values of 450 mM. Compare results between the two groups of cell proliferation at different time points. At the same time according to Annexin V-FITC / PI apoptosis detection kit instructions, to undergo apoptosis detection cells of each blank control group, negative control group, the experimental group. Understanding the impact of PSGL-1 protein on abilities of cell proliferation and apoptosis. Results: Part 1 PSGL-1’s distribution in lymphadenitis, indolent lymphoma, aggressive lymphoma tissues 1. PSGL-1 was highly expressed in malignant lymphoma and lower expression in reactivehyperplasia inflammation, the difference was statistically significant(P <0.05). Theresult suggested that PSGL-1’s expression were likely to indicate the occurrence ofmalignant lymphoma. 2. PSGL-1 was highly expressed in aggressive lymphoma and it had a lower expression ininert lymphomas, the difference was statistically significant(P <0.05). The resultssuggest that hightly expression of PSGL-1 is likely related to the degree of malignancyof lymphoma. 3. We analyzed the expressionsindifferent cell-sources lymphoma and found that theexpression of PSGL-1 was slightly higher in T cell-derived lymphomas than in Bcell-derived lymphomas,and there was no significant in T-cell or B-cell.This may bebecause of the too small sample volume, which will affect the statistical results. Part 2 The expression of PSGL-1 in human lymphoma tissue and the correlation between expression and clinical datas 1. In malignant lymphomas, the expression between PSGL-1 and Ki67 was positivelycorrelated, and the result was statistically significant(R2 = 0.539, P = 0.037). the resultshowed that PSGL-1 may be related to the degree of proliferation of malignantlymphoma,it seems that the higher degree of lymphoma cell proliferation, the higherexpression of PSGL-1. 2. Clinical,Statistical analysis showed that the expression of PSGL-1 may contribute tolymphoma extranodal metastasis, The results were statistically significant(P=0.026).The results showed that in patients with malignant metastases outside the organ,which was relatively highly expression of PSGL-1. Part 3Compared Raji cell migration before and after silencing gene of PSGL-1After transfection 48 h, counting numbers of Transwell small indoor cells by cell counting board.We found that, the number of the experimental group was significantly less than the number of the control group, the difference was statistically significant(P <0.01).Cell experiments showed that the situation of PSGL-1’s expression influenced the migration of Raji lymphoma cells, indicating that PSGL-1 helps lymphoma cells.to distant metastasis. Part 4Compared Raji cell proliferation and apoptosis before and after silencing gene of PSGL-1 1. After the transfection, siRNA group and NC group had no significant difference in cellproliferation, the difference was not statistically significant(P> 0.05).it suggestted thatthe expression of PSGL-1 may not participate in lymphoma cell proliferation. 2. Detected apoptosis situation of control group, negative control group and theexperimental groupby flow cytometry after the transfection.Experimental results showthat the apoptosis of negative control group and the experimental group had nodifference.It suggested thatPSGL-1 does not participate in lymphoma cell apoptosis. Conclusion: 1. PSGL 1-highly expressed in malignant lymphoma suggesting related lymphomamalignancy. 2. PSGL-1’sexpression correlated with the degree of lymphoma’s proliferation, extranodalmetastasis,prompting that expressing of PSGL-1 contributes to lymphoma bloodtransfer. 3. Expression of PSGL-1 does not participate in cell proliferation and apoptosis.