| BackgroudClassical Hodgkin lymphoma(cHL) is a very special form tumor, whose tumor cells are H/RS cell that accounted for one percentage of parenchymal cells. Up to ten years ago, Kuppers confirmed that H/RS derived form B lymphocyte with single cell analysis and IgH gene rearrangement. There are lots of immune cells (including T lymphocytes, B lymphocytes, Plasm cells, Neutrophils, Eosinophils and Mast cells, etc.) and cytokines, which is the basic conditions for survival of H/RS cells. It is apparent that we must notice not only H/RS cell but also the microenvironment of immune cells and cytokines on the research of Hodgkin lymphoma.The microenvironment surrounding H/RS cells is necessary for survival of H/RS cells. Recent years tested that the survival of H/RS cells depends on the signal of microenvironment surrounding H/RS cells, that is to say, if the background cells die, H/RS cells must die, so H/RS cell of primary tumor is extremely difficult to culture in vitro. Because of Hodgkin lymphoma onset in the lymph nodes depending on lymph node status and survival of tumor environment, it'll be an ideal cell line of co-exist of HRS cells and background cells. At present there is a few H/RS cell lines were derived from the peripheral blood, bone marrow or ascites, not came from lymph nodes of HL patient's, including L428, L540, L591, DEV, HD-LM2 and KM-H2, Although these cells cannot copy the state of HL nodes, H/RS cells can live in other organs except nodes during the late phase of HL. There is few H/RS cells in the peripheral blood of early HL patient's. There are still lots of inflammatory cells surrounding H/RS cells, which is similar to lesion of HL nodes. H/RS cells cannot exist in normal mice for immune rejection. H/RS cells can exist in severe combined immunodeficiency mice to form tumor without background of nonneoplastic inflammatory cells, there is not HL animal models that have the characteristics of HL at present.At the same time chemokines may not only be involved in the attraction of other cells into the lymphoma microenvironment but also have direct effects on H/RS cells survival and proliferation. Our results suggested that the defferent chemokines exist in cHL and Diffuse Large B cell Lymphoma(DLBCL) by Bioinformatics analysis, including RANTES, CCL17 and CCL22 etc of HL. RANTES is in a relatively central location Complex cytokine network diagram. Studies have shown that H/RS cells attract CD4+ background cells dependenting the RANTES chemokine. Foreign and our previous experimental results tested that the most number of cells in HL microenvironment cells is CD4+T cells. CD4+ CD25+regulatory T cells can hold Cytotoxic T lymphocytes back, which play a very important role in formation of cHL too.Foreign studies have shown that the activity abnormal increasing of NF-κB is related to the occurrence and development of cHL. our previous results show that the H/RS cells with low CD99 gene expression and sustained activation of NF-κB signaling. Pre-constructed by our group mCD99L2-A20 cell line, low expression of mouse CD99 gene, identified its does have a H/RS cell phenotype, but How mCD99L2 A20 cell RANTES expression and activity of NF-κB is? Whether is the cell similar to H/RS?In order to research cHL clearly, the best way is to establish cHL animal model similar to cHL patient's, which may meet the research about cHL, a variety of background cells and cytokines supporting H/RS cells survival, proliferation and apoptosis suppression. There is no tumor formation by intravenous injection mCD99L2-A20 cells during our previous research. therefore, construction of animal models of HL falled into a plight. In view of HL belongs to the immune system disease, immune defects are closely related. Diabetes can suppress the body's immune system, research has shown that type 1 diabetes complicated by higher risk of lymphom than normal people. Therefore, If diabetic BALB/c mice is inoculated by lymphoma cell line of similar mice. Not only avoid exclusion of population, but also avoid immune rejection of health BALB/c mice. Also provides inflammatory cells in a state of immune suppression. It benefits to form tumor and useful to raise the background reaction in tumor tissue.In this research, Fluorescent quantitative PCR(QPCR), confocal microscopy, Immunocytochemistry(ICC), immunohistochemistry(IHC), enzyme linked immunosorbent assay(ELISA), Co-culture experiments, Western blotting, Flow cytometry and formation tumor in vivo were used. At first, the expression of RANTES in HL and DLBCL, analysed the role of RANTES to HL formation. Detect the expression of RANTES in mCD99L2-A20 cell, inoculated by intravenous injection of diabetic BALB/c mice to tumor, similar to cHL mouse model, and analysed the role of RANTES in this model.Objective1. Explore the expression of RANTES in Hodgkin lymphoma and diffuse large B cell lymphoma cells and tissue, and analyse the role of RANTES to form Hodgkin lymphoma.2. Detect the expression of RANTES in L428, L428-CD99, A20 and mCD99L2-A20 cells. Analyse the relation RANTES expression and CD99 gene.3. Construct type 1 diabetic BALB/c mice and detect the expression of RANTES chemokine in these mice peripheral blood.4. Compare the tumor of mCD99L2-A20 cells in BALB/c mice and type 1 diabetes BALB/c mice, and analyse the expression of RANTES chemokine in these mice peripheral blood.Method1. Detecting the expression of RANTES in Hodgkin lymphoma and diffuse large B cell lymphomaHL cell line L428 and diffuse large B cell lymphoma cell lines Lyl, Ly8, Ly10 cells were cultured. Observing these cell morphology, extracting RNA, analysing RANTES mRNA by RT-PCR and real time RT-PCR, detected RANTES protein by confocal Microscope. Detecting the RANTES expression in cells and tissue of HL and DLBCL by Immunocytochemistry and Immunohistochemistry, analysing the optical density value data of RANTES protein. detecting the secreted RANTES protein by enzyme linked immunosorbent(ELISA), detecting the chemotaxis of CD4+ cell by co-culture experiments with L428 cell.2. Analysing the relation of RANTES and CD99 and NF-κB signal pathwayL428 and L428-CD99, A20 and mCD99L2-A20 lymphoma cell lines were cultured. Observing these cell morphology, RNA extraction, analysing RANTES mRNA by RT-PCR and real time RT-PCR, detecting RANTES protein by confocal Microscope. detecting the RANTES expression in cells and tissue of HL and DLBCL by Immunocytochemistry and Immunohistochemistry, analysing the optical density value data of RANTES protein. detecting the secreted RANTES protein by enzyme linked immunosorbent(ELISA), detecting the expression of CD99 and p-iκBa protein by Western blotting.3. Constructing BALB/c mouse model of type 1 diabetes and detecting RANTES60 male BALB/c mice were randomly divided into A, B, C, D four groups, n= 15, A group were administered intraperitoneally with 150 mg/kg STZ, B group was with the A group by intraperitoneal injection of equal sterile citrate buffer, C group were 200 mg/kg STZ intraperitoneally 5 times, D group and the C group with the same amount of sterile citrate buffer 5 times by intraperitoneal injection, monitoring changes in fasting blood glucose at different time points, A, B, C, D four groups were free diet. CD4+CD25+Treg cells were detected by flow cytometry in peripheral blood of diabetic mice and normal mice. RANTES protein secretion of peripheral blood of diabetic mice and normal mice was detected by ELISA, respectively.4.Constructing type 1 diabetes in BALB/c mice with cHL-like modelmCD99L2-A20 cells that are silimar to H/RS cells were inoculated intravenously with type 1 diabetes BALB/c mice and normal BALB/c mice were each 20 to observe the animals, the time of tumor, tumor rate, subcutaneous tumor growth, take HE staining tumor tissues pathology, the expression of RANTES is detected by immunohistochemistry, tumor tissues were cultured, DNA-PCR detection of vector integration, CD4+ CD25+ Treg cells were detected by flow cytometry in peripheral blood of diabetic mice and normal mice. RANTES protein secretion of peripheral blood of diabetic mice with tumor or no tumor was detected by ELISA. Result1. the expression of RANTES in Hodgkin lymphoma and diffuse large B cell lymphoma(1) OCI-Lyl as a control, the expression of RANTES mRNA in L428, OCI-Ly8 and OCI-Ly10 cells are 8.154±0.809,0.990±0.062 and 1.008±0.0565 folds.The mRNA expression of RANTES in L428 cell is higher than that of OCI-Lyl or OCI-Ly8 or OCI-Ly10 cell, the two is statistically significant (P=0.000), respectively. OCI-Lyl and OCI-Ly8 (P=0.959), OCI-Lyl and OCI-Ly10 (P=0.968), OCI-Ly8 and OCI-Ly10 (P=0.927), compared with no statistical differences.(2) The RANTES protein expression of L428 cells is higher than that of OCI-Lyl, OCI-Ly8 or OCI-Ly10 cells, the expression RANTES protein of cHL tissue is higher than that of DLBCL tissue, the expression RANTES protein of cHL tissue expression is higher than that of L428 cells,the expression RANTES protein of DLBCL tissue is higher than that of OCI-Lyl, OCI-Ly8 or OCI-Ly10 Cells, all have a statistical comparison of the two School differences (P=0.000). OCI-Lyl and. OCI-Ly8 (P=0.811), OCI-Lyl and OCI-Ly10 (P=0.279), OCI-Ly8 and OCI-Ly10 (P=0.397), have no significant difference.(3) The RANTES secretion of L428 cells than that of OCI-Ly1, OCI-Ly8 or OCI-Ly10 cells, comparison of the two were statistically significant (P=0.000). OCI-Lyl and OCI-Ly8 (P= 0.885), OCI-Lyl and OCI-Ly10 (P=0.979), OCI-Ly8 and OCI-Ly10 (P=0.906), compared with no significant difference.(4) when co-cultured with L428 chemotaxis of CD4+T cells (340.556±15.852), higher than the co-culture with OCI-Ly1 can chemotaxis of CD4+T cells (79.333±10.782), comparing the two was statistically significant (P=0.000); the same time if the medium to the L428 plus CCL5, can lead to chemotaxis of CD4+T cells increased (571.111±14.049), if added to the medium anti-CCL5 antibody, can lead to chemotaxis of CD4+T cells Reduction (149.222±16.092), both statistically significant compared (P=0.000).2. the relation of RANTES and CD99 and NF-κB signaling pathway(1) during passage mCD99L2-A20 cells found in the cytoplasm of large cells rich nuclei significantly increased, there are more dual-core, multi-core cell; L428-CD99 cells in the large cells cells cells significantly reduced cell More consistency.(2) RT-PCR and quantitative PCR test:the mCD99 mRNA of mCD99L2-A20 cells was 0.519±0.004 times that of A20 cells, the mRANTES mRNA of mCD99L2-A20 cells was 15.224±0.984 folds that of A20 cells, the hCD99 mRNA of L428-CD99 cells was 24.503±0.681 times that of L428 cells, the two-phase Statistically significant compared (P=0.000), the hu-RANTES mRNA of L428-CD99 cells decreased 80.932±2.062 folds than that of L428 cells, the two compared statistically significant (P=0.000).(3) fluorescence detection:the RANTES protein expression of mCD99L2 A20 cells than that of A20 cells, the RANTES protein expression of L428 cells than that of L428-CD99 cells.(4) Western blotting detection:mCD99L2-A20 cells in mCD99 protein was weaker than A20 cells, p-iκBa protein stronger than A20 cells, mCD99L2-A20 cells by NF-kB signaling pathway inhibitors BAY had no effect on mCD99 protein, but p-iκBa protein expression was significantly decreased, A20 cells by NF-kB signaling pathway activator, LPS had no effect on mCD99 protein, but the p-iκBa protein expression was significantly increased. L428 cells in hCD99 protein was weaker than L428-CD99 cells, L428 cells by NF-kB signaling pathway inhibitors BAY had no effect on hCD99 protein, but the p-iκBa expression decreased, L428-CD99 cells by NF-kB signaling pathway activator LPS treatment had no effect on the hCD99 protein, but the p-iκBa protein was enhanced.(5) the amount of RANTES secretion in mCD99L2-A20 cells was 432.722±10.735, higher than the amount of RANTES secretion in A20 cells 39.343±1.819, both are significant differences (P=0.000).and mCD99L2-A20 cells were NF-kB signaling pathway inhibitor BAY treatment capacity of the secretion of RANTES 116.391±6.241, significantly reduced compared with before treatment, both statistically significant compared (P=0.000), A20 cells by NF-kB signaling pathway activator, LPS RANTES secretion after the amount of 246.838±7.163, significantly increased compared with before treatment, compared to statistical significance between the two (P=0.000). L428-CD99 cells to produce RANTES in the amount of 44.612±3.228, lower than the amount of L428 cells to secrete RANTES 839.388±2.605, both statistically significant compared (P=0.000), and L428 cells were treated with inhibitors of NF-kB signaling pathway after the secretion of RANTES BAY amount 109.107±5.100, significantly reduced compared with before treatment, both statistically significant compared (P=0.000), L428-CD99 cells by NF-kB signaling pathway activator secreted RANTES after LPS volume of 443.618±12.514, significantly increased compared with before treatment, both statistically significant compared (P=0.000).3. Construct BALB/c mouse model of type 1 diabetes(1)It was found that 86.67% and 93.33% male mice were developed diabetes mellitus in group A and group C, respectively. they have no significant difference (P=0.559).(2) CD4+ CD25+ Treg were 4.059%±0.384% in peripheral blood of diabetic mice and 8.576%±0.536% in peripheral blood of normal mice, compared the two groups was statistically significant (P=0.000).(3)RANTES was 124.551±3.683 pg/ml in peripheral blood of diabetic mice and 87.222±6.707 pg/ml in peripheral blood of normal mice, compared the two groups was statistically significant (P=0.000).4.Construction of type 1 diabetes in BALB/c mice with MCD99L2-A20cells into the tumor model(1) mCD99L2-A20 cells into the tumor cases:there are 6 of 20 type 1 diabetic BALB/c mice (30%) formation tumor, tumor in the armpit or groin, the tumor formation time of 34.67±2.16 days. after three months by tail vein injecting 20 normal BALB/c mice, killing mice, with no tumor formation.(2) mCD99L2-A20 cells into tumors of diabetic BALB/c mice grew more slowly than A20 cells tumor of normal BALB/c mouse (preliminary data by our group), there was significant difference between the two groups (P=0.000).(3) pathology:HE section showed that mCD99L2-A20 cells scattered in the lymphocytesbackground, the number of CD3 positive cells in the background was more than the number of CD20 positive cells by immunohistochemical detection.(4) tumor tissue of diabetic BALB/c mice:DNA-PCR detection of mCD99L2-A20 interference vector integrated into the cell genome, RT-PCR gene mRNA levels showed mCD99L2 lower than A20 cells.(5) CD4+CD25+ Treg in peripheral blood of diabetic tumor mice was 31.293%±1.573%, higher than the no tumor diabeticmice 4.493%±0.238%, there was significant between the two groups (P=0.000).(6) RANTES in peripheral blood of diabetic tumor mice was 257.293±5.696, higher than the no tumor diabeticmice 124.342±3.882, there was significant between the two groups (P=0.000).Conclusion1. L428 cells with high secretion of RANTES is conducive to more CD4+T cells. 2. Gene expression and secretion of RANTES of mCD99L2 A20 cell are controlled by NF-kB signaling pathway.3. mCD99L2-A20 cell transplanted tumor model is partly like the characteristics of early cHL.4. RANTES levels are increased in peripheral blood of cHL-like model.New point1. This study compared Hodgkin's lymphoma with diffuse large B cell lymphoma, detected RANTES expression of cell lines and tissues differences in the two lymphoma, analysed the high secretion of RANTES can chemotaxis more CD4+ T cell, which is conducive to the formation of Hodgkin's lymphoma.2. This study demonstrated high RANTES expression of mCD99L2-A20cells and the cell was similar to the characteristics of H/RS cells, type 1 diabetes BALB/c mice with immune suppression and high expression of RANTES characteristics was used to HL-like mice model. mCD99L2-A20 cells were injected into by the tail vein inoculation of diabetic mice at first time.3. The cHL-like mice model is the lymph nodes tumor, which is characteristic of T lymphocytes surrounding big cell, and high expression of RANTES. for the further regulating RANTES expression to construct the typical animal models of cHL, which lay a theoretical and experimental basis.
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