| [Background]Burkitt lymphoma (Burkitt lymphoma, BL) is a highly aggressive,rapid growth of mature B cells non-hodgkin’s lymphoma, which is one of the most powerful proliferation and metastasis tumors.Now commonly treatments include surgery, radiation,chemotherapy,monoclonal antibody CD20treatment,hematopoietic stem cell transplantation.Due to its sensitive to chemotherapy, It can be suitable for children and strong tolerance adult patients in the short course strong chemotherapy. However the side effects of chemotherapy is very large,It is difficult for such patients like weak patients,the elder,HIV infecttive patients to tolerate high strong chemotherapy,besides they hardly benefit from the large dose of chemotherapy. So looking forward to a new drug or new treatment,which not only for specific tumor cell,at the same time, but also for how to transform the tumor cells more sensitive to the body’s immune system,reducding the impact to a minimum on bodys, that will be the research direction.Norcantharidin(NCTD)is synthetic derivatives of cantharidin demethylation, which has the similar medicinal value to cantharidin, including anti-virus, anti-tumor, anti-inflammation, anti-fibrosis, etc, but NCTD eliminates the side effects of urinary system irritation which cantharidin has, it retained anti-tumor effect and increase the number of white blood cells, meanwhile it is a natural silk/threonine phosphatase PP1and PP2A inhibitors. Current research shows that NCTD can inhibit the proliferation of many tumor cell lines and induce apoptosis and cell cycle arrest. NCTD do not reduce the number of white blood cells, and not inhibit body’s immune, when inhibit the tumor cell,that make it more advantage in anti-tumor.But relevant report about the effect of NCTD to Burkitt lymphoma Raji cell lines has not been seen.Therefore, we discussed whether to NCTD can inhibit the growth of Raji cells, provided the basis research of NCTD against aggressive malignant lymphoma.At present simple adoptive cellular immunotherapy(ACI)in most cancer patients did not obtain such high therapy effect as in vitro.Further studies have found that Burkitt lymphoma Raji cell lines can resist the NK cells kill,causing immune tolerance.The main reason is the cells surface high express HLA-Ⅰ molecules,and very low express NKG2D ligand including MICA/B and ULBP1-3. These ligands don’t express or lower express in the normal cells, which only express in the cancer cells that in stress status or the cells infeced pathogen.NKG2D matchs up with the ligands can directly activate NK cells causing a series of effects, meanwhile as a collaborative signal stimulation promote a P T cells, and γδ T cell activation and enhance its anti-tumor effect. Therefore,may be can enhance NKG2D and its ligands interaction, in order to remove tumor cells.It will be the focus to research how to improve NKG2D and its ligands expression,prompted Raji cells more sensitive to the immune killer cells.This study would be discussed that the biological characteristics of Burkitt lymphoma Raji cell lines influences by NCTD and the NCTD whether regulate Raji cells more sensitive to the immune killer cells and some preliminary mechanism of that.So as to provide the date of NCTD against malignant invasive Burkitt lymphoma in vitro research, find out NCTD whether can enhance the sensitivety of Raji cells to killer cells, which can improve adoption cellular immune therapy, and establish a new immune treatment road.Part I The biological characteristics of Burkitt lymphoma Raji cell lines influence by NCTD[Objective] To investigate the effects of NCTD on human Burkitt lymphoma Raji cells on proliferation and cell cycle arrested in vitro.[Methods] Applied Trypan blue assay to detect the inhibiting rate on Raji cells and peripheral blood mononuclear cells (PBMC), in which concentration of NCTD (0.15265、0.3125、0.625、1.25、2.5ug/ml) were different. The self-renewal and proliferating potential were examined with methylcellblose conlony-forming units(CFU) assay,through Raji cells treat by NCTD (0,0.25,0.5,1ug/ml) directly group and NCTD pretreatment48hours a group.The cell cycle of Raji cells after48h treatment by NCTD(0.5ug/ml)was detected by flow cytometry(FCM).The analysis was performed using SPSS13.0software package.Thg data was represented as the mean±standard deviatiion（x±s).Comparisons of means among groups of the inhibiting rate on Raji cells and PBMCs,the amount of Raji cells colonies were performed using factorial design analysis of vriance.The cell cycle was performed using independent-samples t-text,P<0.05were considered to be statistically significant.[Results] By using Trypan blue assay, we found that NCTD can inhibit the proliferation of Raji cells significantly with dose-and time-dependant manners, whereas compared with Raji cells to PBMC in different consentration at the same time,with statistically significant(F=1005.664,P=0.000).The values of IC50are0.88 ug/ml,0.47ug/ml,0.28ug/ml at24hours,48hours,72hours.Besides NCTD neither inhibit the proliferation of PBMC also non-toxic effect, with statistically unsignificant in all concentration(F=0.866, P=0.517).The CFU assay shows that NCTD (0,0.25,0.5,1ug/ml) direct treament group and the group of NCTD pretreatment48hours significantly inhibit Raji cells cloning forming ability (F=530.933, P=0.000),With the increase of NCTD concentration. It means that Raji cells has cloning forming ability in vitro, and NCTD can inhibit the ability of Raji cells. In lug/mlNCTD direct treatment, Raji cells cloning forming ability obviously inhibit at14d and21d.The number of cloning reduced at (1.00±1.00) and (2.67±1.15)/hole. In a word The number and size of cloning in NCTD direct treatment group obviously less than NCTD pretreatment group (F=48.945, P=0.000).Raji cells were induced cell cycle arrest by the NCTD (0.5ug/ml) treatment after48hours.The proportion of cell cycle at G1/G0and S phase were reduced by6.35%,13.23%(t=0.711, P=0.516)(t=1.708, P=0.163), but G2M phase increase19.58%(t=7.227, P=0.002),to show the cell cycle arrest at G2/M phase.[Conclusion]1、In different concentrations of NCTD can inhibit Raji cells proliferation with dose-and time-dependant manners.But NCTD neither inhibit the proliferation of PBMC also non-toxic effect, with statistically unsignificant in all concentration.2、NCTD significantly inhibit the cloning formation ability of Raji cells,espacially for which cells wich fast self-renewal and high potential proliferation,and continuously and steadily drug concentration enhance the inhibition effect.3、NCTD inhibits Raji cells proliferation may associate with the arrestion of cell cycle at G2/M phase.Part II Norcantharidin enhances the cytolytic sensitivities of Burkitt lymphoma Raji cells to peripheral blood mononuclear cells [Objective] To explore the cytolytic sensitivities and preliminary mechanism of untreated PBMC and the PBMC activated by IL-2,IL-15on Raji cells from Burkitt lymphoma before and after treated by NCTD.[Methods] LDH texted untreated PBMC and the PBMC stimulated by IL-2,IL-15against K562cells and Raji cells which before or not treated by NCTD (0.5ug/ml) were analyzed by LDH releasing assay at different effector-to-targat cell ratios(E:T).The expression of NKG2D on the surface of PBMC activated by IL-2,IL-15was assayed by FCM.The expressions of NKG2D ligands(MICA、 MICB、ULBP1、 ULBP2、ULBP3) was assayed by FCM before and after treated by NCTD (0.5ug/ml) for48h.The analysis was performed using SPSS13.0software package.The data was represented as the mean±standard deviatiion (x±s).Comparisons of means among groups of the cytotoxic rates at K562cells and Raji cells were performed using factorial design analysis of vriance, the express rates of NKG2D and NKG2D ligands was performed using independent-samples t-text,P<0.05were considered to be statistically significant.[Results] The cytotoxic rates of unactivated PBMC in effect-target rate10:1and20:1to K562cells were (15.82±5.12)%and (27.67±3.66)%, in the same condition, the cytotoxic rates of IL-2,IL-15activated PBMC to K562cells were (52.42±3.89)%and (79.55±9.22)%,increased by36.60%,51.88%(F=168.076, P=0.000). It means that IL-2, IL-15induced PBMC cytotoxic effect is obviously superior to the PBMC without induction, with highly cytotoxic activity without rely on the MHC I molecules of tumor cells.Raji cells natural resist to untreatment PBMC.In E:T10:1,20:1the cytolytic rates of untreat PBMC to Raji cells were only (5.04±1.25)%and (8.59±2.19)%, but the rates in Raji cells after treat by NCTD (0.5ug/ml) for48h were (23.63±6.20)%and (41.80±4.09) Respectively,increasing by18.59%,33.21%(F=25.905, P=0.007; F=153.492, P =0.000). Under the same conditions,the cytolytic rates of PBMC activated by IL-2,IL-15to Raji cells were (13.19±3.67)%and (19.89±4.15)%, and the rates in Raji cells after treat by NCTD were (38.97±2.76)%and (63.09+7.30)%, respectively increasing by18.59%,33.21%(F=94.368, P=0.001; F=79.519, P=0.001). NCTD can obviously increase cytolytic rates in PBMC of untreatment and IL-2, IL-15induction to Raji cells (F=285.832, P=0.000), in addition the cytolytic rates of activation PBMC obviously increase than the untreated PBMC (F=61.593, P=0.000). the NKG2D expression rate rise19.63%after IL-2, IL-15activation PBMC a with statistical significant difference (t=3.518, P=0.024). molecule ULBP2expression on the surface of Raji cells rise significantly11.66%(t=5.036, P=0.007), after NCTD (0.5ug/ml) treatment for48hours,and the expression of orther ligands change nosignificant(P>0.05).[Conclusion]1、NCTD can obviously inhances the cytolytic sensitivities of Burkitt lymphoma Raji cells to before and after IL-2, IL-15activation PBMC.2、 NCTD raised the ULBP2expression on Raji cells surface, but the expression of MICA/B,ULBP1and ULBP3are no statistical significance.3、IL-2, IL-15stimulated PBMC obviously more cytolytic than the PBMC without induction, mechanisms may increased the NKG2D expression on IL-2, IL-15PBMC surface. |
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