Preliminary Study On The Mechanism Of CCL5/RANTES In Diabetic Patients With Diffuse Large B Cell Lymphoma

The chemokine RANTES/CCL5is an inducible, secreted inflammatory cytokineof small molecular weight, and has functions of chemotaxis and activation of T cellsand mononuclear cell. The protein among them, containing68amino acid residuesand its molecular weight being8kd, is mainly formed by NK cells and T lymphocytes,and plays an important part in immunity to the tissue damage, infection and tumor.Objective:The purpose of this experiment is to investigate the mechanism of chemokineCCL5in diabetes mellitus with diffuse large B cell lymphoma (DLBCL), and toinvestigate the action of CCL5gene from molecular level, cell level and animal leveland partial molecular mechanism, in order to provide experimental data withreference value for the function mechanism of CCL5in diabetes with DLBCL.Method:Normal human B cells and DLBCL cells were cultured in vitro, and RT-PCRwas used to detect the expression of CCL5mRNA; human diffuse large B lymphomacell lines and rat diffuse large B cell lymphoma cell lines A20were cultured in twokinds of sugar concentration of5mmol/L and30mmol/L, RT-PCR was applied for thedetection of the expression of CCL5mRNA respectively; BALB/c mice wereintraperitoneal injected streptozotocin (STZ) of small dose to construct diabetic ratsmodel, and cell lines with a stable low expression of CCL5and high expression ofCCL5were established via lentiviral transfection technology. The three kinds of celllines of low expression of CCL5, high expression of CCL5and un-transfection wereinjected subcutaneously in the diabetic BALB/c mice and normal blood glucoseBALB/c mice, then were observed about the rate and the time of tumor formation andtumor size and texture; the expression of CCL5in each group was detected by tumortissue routine HE staining and immunohistochemistry; peripheral blood from mice in eachgroupwasextractedto detect theexpressionofCCL5inperipheralblood byELISA. Result：1. DLBCL cell lines of Ly1, Ly8, Ly10are cultured in vitro, the expression levelof CCL mRNAin each group is higher than the normal B cell(P<0.05).2. Glu30mmol/L cultivate human DLBCL cell strain Ly1, Ly8, Ly10and miceDLBCL cell strain A20, after culturing for2W, the CCL5mRNA are all higher thanthe one cultured by Glu5mmol/L（P<0.05）.3. Through injecting the diabetic mice by lower expression CCL5, highexpression CCL5, none transfection cell lines A20, the diabetic mice’s tumorformation rate is A1:93.3%; A2:60%; A3:66.6%, the tumor forming time arerespectively A1:7.0±0.85d; A2:9.5±2.8d; A3:9.0±1.8d. Higher than the normalblood sugar mice’s tumor forming rate B1：20%；B2:20%; B3:46.6%, the tumorforming time are respectively B1:12±1.3d; B2:14±2.5d; B3:12±4.2d.4. Using the immuno-histochemistry to detect the diabetic mice tumor formingtissue CCL5’s expression, and the result is higher than the normal blood sugar mice’stumor tissue.5.The result of ELISA detecting the diabetes mice’s CCL5expression is higherthan the mice with normal blood sugar in the peripheral blood.Conclusion:1. Human DLBCL cell strain’s expression of CCL5mRNA are higher than thenormal B cell;2. High concentrated glucose cultivate human DLBCL cell strain and miceDLBCL cell strain’s CCL5mRNA is higher than the low sugar cultivate one, thisshows in the high glucose environment, the DLBCL tumor cell can produce moreCCL5;3. The high expression CCL5’s cell strain is more easily to form tumor in thebody than the low expression CCL5’s cell strain, the tumor grow faster; and at thesame condition, the diabetes mice are more easily to form the tumor than the normalmice;4. The expression of CCL5in the tumor forming tissue of the diabetic mice ishigher than the tumor forming tissue of the normal mice; 5. The expression of CCL5in the serum of the diabetic mice is higher than thenormal mice.In conclusion, hyperglycemia can cause the increasing of the concentration ofthe CCL5, the high expression of CCL5may be one of the important factors to causeDLBCL in diabetes patients.