| Objective:Adoptive cellular immunotherapy (ACT) is the method of biotherapy to achieve the goal of cure cancer which transmit immune cells (specificity or non-specificity) of active anti-tumor. The immune cells can directly kill and wound the tumor or motivate the body immune reaction to kill and wound the cells. For the past few years, because of the reasonable application of complex treatment, a great part of non-Hodgkin lymphoma can cure or relief, but the patients suffer long term chemotherapy which lead the patient to have weak health condition, and the stem cells and periphery vessel could be damaged, patient's survivability is bad, need to seek new therapeutic tool. Now, cytokine-induced killer cells get into clinical trials, which have good curative effect to many malignant tumor reported in internal and abroad, it's a immunocyte remedy which have more potential. CIK is a kind of immune active cells, which have the characteristics of rapid proliferation great efficiency and broad spectrum in killing of tumor cells, and have a little effect on hematopoiesis. As to improve the sufferers'restore of immune, clear up the remain lesion, and decontaminate the marrow all have fine curative. So far, the report is much more in basis study of lymphoma cell line and lymphoma clinical application treated by CIK, but it still at primary stage of nude mice in vivo which bearing lymphoma. This experiment is via check the Burkitt lymphoma Raji cells vary of cells'cycle and apoptosis before and after use the CIK cells, and the nude mice's change of the tumor size. The difference of nude mice tissue bcl-2 and Ki-67, investigate and look into the CIK cells, have the depressant effect to Raji nude mice and discuss the CIK cells mechanism. So as to have a further application and provide the theory for CIK cells clinical utilization. Method:1 Subculture of the tumor cells: Set the Burkitt lymphoma Raji cells into 1640 medium which contains 10% FBS, on condition that 37℃, 5%CO2, saturated humidity, chose the cells that were in log phase into the experiment.2 Prepare CIK cells and amplificate them in vitro: Used the lymphocyte separation medium and the methods of density gradient centrifugation separated human peripheral blood mononuclear cells(PBMC) from healthy person periphery blood, induced to become CIK cells by multi cytokine(IFN-γ, CD3Mcab, IL-2) in vitro induction.These cells were cultured and passaged, subcultured every 3 days, took a small quantity of cell suspension, then used blood cell counting plate to take count of those cells, and maked the growth curve.3 Analyzed the cytotoxicity about CIK cells to Raji cells in vitro by MTT assays: set CIK cells and Raji cells into 96 pore plate by different Effect/Target rate(E:T are 10:1, 20:1, 40:1), each group had three double-pores, NS group in normally condition. Cultivating24, 48 hours later, we determined the OD by MTT, and figured out the antipersonnel activity.4 Established the model of nude mice bearing Raji cells tumor and grouped: The nude mice model was established by injecting Burkitt lymphoma Raji cells 1×107/ml, 0.2ml/one mouse to the mice's oxter. To survey the transplanted tumor's size every 3 days, record long diameter and short diameter to account the tumor volume using the formula and drew tumor growth curvature. When tumor grew big enough, randomly divided those mice into 3 groups (A, B, C). Each groups had 5 mice. Group A was the control group which used physiological saline, B, C injected CIK cells were the treatment groups which had already cultivated in vitro for 14 days. They injected dose were 2×107/0.2ml/one mouse, 4×107/0.2ml/one mouse for group B,C respectively. Each group were added every second day, totally three times.5 Killed the mice and took the tumor tissue: At the seventh day all mice of each group were killed to take and weight the tumor tissue. Used the sliding caliper to measure the tumor's size, figured out the volume, calculated the tumor's growth inhibition ratio and then turned the tumor tissue into monoplasts suspension by mechanical method. Using flow cytometry (FCM) to mensurate the cells'apoptosis rate and the expression level of the protein bcl-2 and Ki-67.Results:1 CIK cells had obvious lethal effect to Raji cells, at different Effect/Target rate(10:1,20:1,40:1), different time point (24h,48h) of CIK cells treated Raji cells, compared with control group, OD value of treatment group reduced in different degree. With the increasing of concentration and effect time of CIK cells, OD value reduced gradually, and the apoptotic rate increasing. At the time point of 24h, the apoptotic rate in different E/T is(15.7±1.4)%,(33.8±2.3)%,(49.2±2.1)%,at the time point of 48h, the apoptotic rate in different E/T is (27.9±1.7)%,(59.3±2.0)%,(78.3±1.6)%, the difference was significant at different Effect/Target rate in the same time point (P<0.05), the difference was also significant too at different Effect/Target rate same time point (P<0.05).2 The mice tumor of the treatment groups shrank than before, the inhibited tumor's rate of group B was (23.7±0.56)%, group C was (54.3±0.68)%, compared with control group, the difference was significant (P<0.05) , and between the treatment groups, there were significant statistics difference (P<0.05).3 The rate of tumor cells treated with different number CIK cells in G0/Gl stage was gradually higher (the percentage are 28.56±1.69,34.41±0.34, 53.43±1.87), the rate of S-stage and the rate of G2/M stage reduced gradually, the percentage of S-stage were (52.66±4.60)%,(49.62±1.22)%,(34.87±0.67)%, the percentage of G2/M stage were (17.87±0.49) %, (15.43±0.44)%, (11.97±1.23)%, and tumor cells'proliferation index which were (72.18±3.32)%,(64.40±1.31)%,(46.91±0.69)% reduced gradually too. Compared with control group, each stage and the PI difference were significant (P<0.05), and between the treatment groups, there were significant difference too(P<0.05).4 After treated with different concentration of CIK cells, there was no obvious apoptotic peak in group A, there were typical hypodiploid apoptotic peak in group B and C by FCM, With the increase of concentration of CIK cells, apoptotic crest value and the rate of apoptosis raised gradually. Tumor apoptosis rate of group B, C were (6.19±1.16)%, (13.17±0.17)% respectively, compared with (0.94±0.87)% of group A, the difference was significant (P<0.05).5 Analysed the bc1-2's FI and Ki-67's FI of tumor by FCM, after treated with different concentration of CIK cells, the bc1-2's FI were A: 1.00±0.01, B:0.96±0.01, C:0.87±0.02. The Ki-67's FI were A: 1.00±0.01, B:0.95±0.01, C:0.83±0.02, Corresponding the expression of bc1-2 and Ki-67 , group B,C were obviously lower than group A(P<0.05), the difference was significant (P<0.05), the difference between group B and C was significant too(P<0.05).Conclusions:1 CIK cells had obvious lethal effect to Raji cells. This lethal effect positive correlation with the increase of concentration and effect time of CIK cells, and it showed obvious relation between dose-dependence and time-dependence manners.2 The experimental in vivo result confirmed that CIK cells had a active suppressive effect to the tumor growth of nude mice bearing Raji cells tumor.3 CIK cells could change tumor cells'cycles'distribution of the transplant- ation inhibite of G0/Gl stage, prevented tumor cells to get into cleavage stage, rev down its generation.4 CIK cells'depression to Raji cells in vitro were related to inhibited the expression level of the protein bcl-2 and Ki-67.
|
PDF Full Text Request