| BackgroundLymphoma is a kind of immune system malignant tumor, originated in the lymph nodes or other lymphoid tissues, its prominent and characteristic clinical manifestation, is painless and progressive lymphadenopathy and (or) the local mass. According to the histological classification standards of lymphoma, non-Hodgkin’s lymphoma, eventally B-cell lymphoma accounts for the highest proportion. The therapeutic programs of malignant lymphoma are usually multi-drugs combination chemotherapy, radiotherapy, hematopoietic stem cell transplantation. In recent years, with the advent of molecular targeted drug such as Rituximab, the continuous improvement of hematopoietic stem cell transplantation technology, the application of bio-immunological treatment, The treatment efficacy has been significantly ameliorated, while there are still a great many relapse, refractory and resistant lymphoma patients, so how to improve the efficacy of these patients, becomes the focus of many researchers.Thermotherapy is another important therpeutic method of the treatment of malignant tumours following surgery, chemotherapy, radiotherapy and biological immunotherapy. Hyperthermia can kill malignant cells, improve the cancer patients’ quality of life, extend the patients’survival time, and increase the sensitivity to radiotherapy and chemotherapy in patients with malignant tumour, generate complementary synergy with radiotherapy and chemotherapy, at the same time it can reduce the side effects of chemotherapy and radiotherapy, therefore be called "green therapy"by most medical professors. In addition, many sdudies have demanstrated that hyperthermia can not only directly kill tumor cells, much more important is that hyperthermia can also activiate the immune system, thus indirectly removal of the tumor cells.Natural killer cells (NKs), as one kind of lymphocytes, plays an important role in the antineoplastic immunity, and it can play cytotoxicity to kill tumor target cells, without being prior stimulated and (or) activated by tumor antigen. Therefore, recently, it has become a hot research topic of domestic and foreign scholars. NK cells express a variety kinds of receptors. According to the different biological functional effect, they are divided into inhibitory receptors and activating receptors. On the target cells surface, exist the corresponding ligands of the two differnt kinds of receptors. Through patterning recognition regulated by their various receptors, binding with their respective ligands, induce NK cells being activiated or inhibited. The activity level of NK cells damaging tumor cells is not only associated to the expression level of its inhibition and (or) activation receptors, but also closely related to the ligands’expression level on the target cells surface. Many studies have proved that a variety of different methods can induce NK cells expressing much more activated receptors, thereby enhance the cytotoxic ability of NK cells against target cells. Thus we assume that if we can use a certain method to improve the expression of the activiating recerptor ligands on the target cells surface, so as to enhance the sensitivity of target cells to NK cells.ObjectiveBased on the biological behavior of SU-DHL-6cells before and (or)after hyperthermia, we aim to investigate whether hyperthermia could change the sensitivity of SU-DHL-6cells to natural killer cells and explore the potential related molecular mechanisms, so as to provide a theoretical basis for hyperthermia being used to treatment lymphoma.Research MethodsChapter one the biological characteristics of human B lymphocytic cell lymphoma cell line SU-DHL-6In this study, the morphological and growth characteristics of SU-DHL-6cells were under the light microscope; The cell count method was used to draw the growth curvature and calculate the doubling time of cell proliferation; the immune phenotype and cell cycle distribution of SU-DHL-6were analysed by using flow cytometry; using MTT experimental method to detect the resistance spectrum of SU-DHL-6cells.Chapter two the inhibiting effect of thermotherapy on SU-DHL-6cells in vitro studyMTT method was used to analyze the growth inhibiting effect of different temperature level (40,42,43℃) hyperthemia to SU-DHL-6cells; After being treated by hyperthermia, observe the cells morphological changes under optical microscope; After the SU-DHL-6cells were treated by heat one hour, we analyze its cell cycle distribution and apoptosis rate by flow cytometry, and detect the level of heat shock protein70by using enzyme linked immunosorbent assay, ELISA.Chapter three before or after treated by hyperthermia, the sensitivity of SU-DHL-6cells to NK cells in vitro study.High purity of NK cells were isolated from three healthy volunteers’normal peripheral blood mononuclear cells, through immune magnetic beads sorting method as effector cells; The SU-DHL-6cells that treated by hyperthermia or not were cultured as target cells, measure the senstivity of them to NK cells by using the method that CFSE/PI double staining and flow cytometry when the efffector&target ratio was20:1. Chapter four preliminarily discuss the probable molecular mechanism of hyperthermia how to enhance the sensitivity of SU-DHL-6cells to NK cells.RT-PCR was used with the aim to observe the expression level of the gene of HSP70and NKG2D ligands in SU-DHL-6cells that were respectively cultured48hours long after being heat treated one hour under42℃hyperthermia.Results1. Human lymphoma SU-DHL-6cells were round, large, single or clouds suspended growth. The cell group doubling time is35.19hours. SU-DHL-6cells high expressed B lymphocytes surface marks. The cell cycle distribution:the G0/G1phase cells accounted for33.8%, the cells in S phase accounted for61.4%, G2/M phase cells accounted for4.85%. A variety of different chemotherapy drugs, the inhibition effect to SU-DHL-6cells growth were different and showed a concentration-dependent phonomenon.2. With the rise of temperature, the growth inhibitory effects of hyperthermia on the SU-DHL-6cells gradually increased; Under the optical microscopy, after being treated by hyperthermia, Intracellular pack slurry particles increased, the thin film membrane integrity, but the smoothness were not so good as untreated cells; The cell cycle distribution after hyperthermia:G0/G1phase cells accounted for25.1%, accounted for48.1%of cells in S phase, G2/M phase cells accounted for26.9%; The apoptosis rate after hyperthermia was34.4%. Hyperthermia induced HSP70expression,48h after, reached the highest level. With the incubation time going on, gradually returned to the normal status.3. T ratio of20:1, After heat treated, the SU-DHL-6cells become more sensitive to NK cells than before.4. After hyperthermia, the genes of HSP70and NKG2D ligands(except ULBP2) expression level were upregulated, after48h, beginning to go back to the normal status. Conclusions1. Hyperthermia could inhibit the growth and proliferation of SU-DHL-6cells, promote cells apoptosis and change the cell cycle distribution of different phases, the propotion of G0/G1and(or) S phase cells decreased, therefore the proportion of G2/M phase cells increased relatively.2. Hyperthermia could improve the sensitivity of SU-DHL-6cells to NK cells, one of the possible mechanisms may be that hyperthermia improve the amount of HSP70expression, thereby enhancing the immunogenicity of the SU-DHL-6cells, hyperthermia increasing the expression of SU-DHL-6cells surface activating receptor NKG2D ligands is another mechanism. |
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