| BackgroundMultiple myeloma (MM), the most common plasma cell bone malignancy, accounts for 1% of all cancers and affects as many as 10% patients with haematological malignancy patients. With the development of modern induction therapy, hematopoietic stem cell transplantation and the application of thalidomide, the efficacy of MM are rising. However, most patients will relapse or resistance to the drugs. Minimal residual disease (MRD) is the main obstacles of improving the cure rate and survival rate, and is also the root of drug resistance and relapse. MM is thought to be an incurable disease. Therefore, the clinician need to find a more effective method of treatment. At present, bio-immune therapy for MM is the focus of tumor research. We hope the chemotherapy combining immune therapy can completely control and clear the MRD, so as to achieve the purpose of curing MM.Immune therapy is a new broad prospects of the new model of cancer treatment.Studies have shown that lymphocytes play an important role in tumor immune therapy. As a member of lymphocytes, NK cells has been a research focus.NK cells are a class of lymphocytes, existing in the peripheral blood as well as bone marrow and other tissues and organs.They can damage tumor cells and some cells with pathogenic infection, while the host's own tissue is not normally affected. NK cells damage tumor cells and cells with pathogen infection, through pattern recognition regulated by their various NK cell receptors (NK receptors, NKRs) interaction with their respective ligands. NKRs can be divided into inhibitory receptors and activating of receptors, according to their function. At present, we have found two receptors that can activate of NK cell's killing effect, mainly including natural cytotoxicity receptors (NCRs) and NKG2D receptors. NCRs include 3 kinds of hypotypes, NKp46, NKp44 and NNKp30.Recent studies have shown that primary malignant melanoma, breast cancer, ovarian cancer, gastric cancer and colon cancer tumor cells can be killed by alloreactive natural killer (Allo-NK) cell. However, the cytotoxic activity of Allo-NK cells against MM cells is lower.ObjectiveBased on the biological behavior of RPMI 8226 cells and the function of NK cells, we will investigate whether the cytokines could enhance the cytotoxicity of allo-NK cells against RPMI 8226 cells, and explored the correlated molecular mechanisms. On expounding these problems, we can find a new method for biological treatment of MM, and provide experimental and theoretical basis for establishment of a new potential therapeutic way for MM patients.Methods1. Observation of the biological characteristics of human myeloma cells RPMI 8226 In this study, we observed the morphology and growth characteristics of RPMI 8226 cells under light microscope; analyse the cell cycle of RPMI 8226 cells by flow cytometry; and analyse the resistance spectrum of RPMI 8226 cells by MTT.2. The collection, isolation and culture of NK cells The human PBMC were been washed by PBS and divided into groups A-F. The group A is the control group, and group B-F were stimulated with added cytokine IL-2; IL-7; IL-21; IL-2+IL-7; or IL-2+IL-7+IL-21.3. The determination of NK cell's number and surface receptors After the cells of groups A-F were incubated for 5d, we analyse the proportion of NK cells by flow cytometry; analyse the expression of activated receptors (NKG2D,NKp4,NNKp30) by flow cytometry; analyse the expression of NKG2D and NKp46 genes by Real-time quantitative PCR.4. The cytotoxicity of allo-NK cells against RPMI 8226 cells After the cells of groups A-F were incubated for 5d, and then the cytotoxic effect of allo-NK cells against RPMI 8226 cells was analyzed with CFSE/PI double staining.Results1. Human myeloma RPMI 8226 cells were round, larger, single or spherical suspended growth. The cell population doubling time is 37.11h.The cell cycle is: G0/G1 phase cells accounted for 26.5%, S phase cells accounted for 57.6%, G2/ M phase cells accounted for 16.0%.2. Flow cytometry showed:the proportion of NK cells in group A (control group) and group B (IL-2), group C (IL-7), group D (IL-21), group E (IL-2+IL-7) and group F (IL-2+IL-7+IL-21) were (7.23±1.86)%, (13.47±1.68)%, (5.33±0.97)%, (11.47±0.81)%, (12.73±2.97)%, (12.60±2.66)% respectively. In addition to group C, the proportion of NK cells in the other groups increased compared with the control group. The expression of NKG2D, NKP44, NKP30 in cell surface is (37.97±1.93)%,(7.03±0.83)%,(4.30±1.65)%; (64.00±6.34)%,(8.93±0.89)%,(7.30±1.65)%; (52.73±10.39)%,(1.90±0.60)%,(7.37±2.50)%; (64.40±12.52)%,(6.03±0.49)%,(6.40±1.06)%; (70.37±13.16)%,(6.80±0.90)%,(5.23±3.81)%; (74.43±11.63)%,(8.00±1.25)%, (6.38±5.99)%.In addition to the group of IL-7, other groups could increase the expression of NKG2D in the cell surface.3. RT-PCR results showed that the gene expression of NKG2D and NKP46 in group B-F showed no significant difference in compare with group A (control group).4. T ratio of 20:1, the NK cell killing of RPMI 8226 cells in group A-F were (2.13+0.42)%,(58.73+1.80)%,(1.90+0.60)%,(14.43+1.22)%,(34.47+2.35)%, (37.4 7+0.60)%. In addition to group C (IL-7), the cell-killing rate on the RPMI 8226 cells in group B (IL-2), group D (IL-21), group E (IL-2+IL-7) and groups F (IL-2+IL-7+IL-21) were significantly increased than group A (control group), and also were statistically significant differences compared with the group A. The cytotoxic effect of the group added IL-2 is the strongest among the groups.Conclusions1. We can enhance the cytotoxic effect of NK cells against RPMI 8226 cells with the single factor IL-2, IL-21, two-factor IL-2+IL-7 and multi-factor IL-2+IL-7+ IL-21. And, single factor IL-2 can strengthen the cytotoxic effect.2. That the cytokines could enhance the NK cell quantity and the expression of cell surface activation acceptor (NKG2D) is the possible mechanism. |
PDF Full Text Request