| Objective:Malignant tumor has already become a serious threat topeople’s health and life with an increasing incidence. With the constantimprovement of the deepening of the molecular biology research and treatment,the level of the diagnosis and treatment of tumor has been greatly improved.Biotherapy is the fourth largest cancer treatment after surgery, chemotherapyand radiotherapy, and it is one the hot research of cancer treatment. Cytokineinduced killers cells are a cluster of heterogeneous cells. They are preparedfrom human peripheral blood mononuclear cells, bone marrow mononuclearcells or cord blood mononuclear cells with the co-stimulation of some kinds ofcytokines (IL-2、IL-1、IFN-γ、CD3McAb). They are immune effector cells. Thecharacteristics of non-MHC-restricted tumoricidal CIK cells have antitumoractivity of T cells and NK cells. CIK cells are characterized by rapidproliferation, high and broad anti-tumor activities, mild toxicities, enhancesthe anto-immune of function in patient and clean up the remaining tummor.CIK cells have widespread using in adoptive immune tharepy. So far, thereport is much more in basis study of lymphoma cell line and lymphomaclinical application treated by CIK, but it is still at primary stage of nude micein vivo which bearing lymphoma. First, established the model of nude micebearing Jukat cells tumor and grouped, One group is the control group whichused physiological saline, the other group is injected CIK cells. Theexperiment is via cheak the nude mice’s change of the tumor volume. Thedifference of nude mice tissue vascular VEGF (endothelial growth factor) andMVD(microvessel density), investigate and look into the CIK cells, have thedepressant effect to Jukat nude mice and discuss the CIK cells mechanism.And Provide the basis for the clinical application of CIK cella in theChlidren’s lymphoma. Methods:1Cell culture of Jukat: Jukat was cultured in RPMI1640medium,supplemented with10%heated-inactivated fetal bovine serum, at37℃ina humidified of5%CO2in-air atmosphere. The cells was passaged everytwo days.2Prepare CIK cells and amplificate them in vitro: Used the lymphocyteseparation medium and the methods of density gradient centrifugationseparated human peripheral blood mononuclear cells(PBMC) fromhealthy person periphery blood, induced to become CIK cells by multicytokine(IFN-γ, CD3McAb, IL-2) in vitro induction. These cells werecultured, passaged and renewed the cytokine.3Construct human Jukat cell xenografts in nude mice and grouped: Thenude mice model was established by injecting Jurkat cells1.2×10~8/ml,0.2ml/one mouse to the mice’s oxter. When the tumor was grown well,divided into2groups randomly, experimental group and control group,each group were5nude mice. Experimental group injected CIK cellswhich had already cultivated in vitro for two weeks. They injected dosewere3×10~7/ml,0.2ml/one mouse. The other group was the control groupwhich used physiological saline. Each group were added every secondday, totally three times.4To killed the nude mice and looked into the VEGF and MVD in tumortissue from the two group mice were detected by immunohistochemistry:At the thirty-fifth days all mice of each group were killed to take thetomor tissue. Used the sliding caliper to measure the tumor’s size, figuredout the volume, calculated the tumor’s growth inhibition ratio. Lookedinto the VEGF and MVD in tumor tissue from the two group mice weredetected by immunohistochemistry.All measurement data used SPSS13.0statistical software for statisticalanalysis, and showed by means±standard deviation ((X|-)±S), the means datebetween groups tested by two-samples t-test. The relation between VEGF andMVD tested by linear regression. There was a statistical difference if P value less than0.05.Results:1The incidence of the transplanted tumor: Nude mice that were inoculatedJukat cells were17in total,among which10mice have grown apparenttumors after five days, tumor growth rate was58.8%.2The tumor growth of nude mice bearing Jurkat cells tumor: The micetumor of the treatment group grew more slowly than the control group (atthe fifth week132.74±6.04mm~3vs164.82±6.19mm~3, p＜0.001), theinhibited tumor’s rate of the treatment group was19.46%.3Under the microscrope: Cell density of tumors in the control group,and alarge, irregular, visible mitosis. The experimental group microscopyvisible to a small number of tumor cells, and a large deeply stained,irregular, mitotic rate is significantly less than the control group.4The expression of VEGF in tumor tissue from the two group mice: Theintractrellular VEGF immunostaining was assessed using asemiquantitative scale. The positive cell of VEGF expression in the CIKgroup was lower than the control group (p=0.018). CIK cells could inhibitVEGF protein experssion in lymphoma.5The MVD in tumor tissue from the two group mice: Yellow vWF antigenlabeled vascular endothelial cells, tumor microvessels irregular branchdisorders, most showed cluster-like arrangement. Two group within thetumor vascular structures often lack integrity, only wall arranged in alayer of endothelial cells, lack of smooth muscle, basement membranethinning or absent, compared with the control group, the experimentalgroup tumor neovascularization less vascular take shape relative rule,with less anastomosis. After the administration, MVD in the CIK groupwas lower than the control group, p＜0.001. CIK cells’ depression toJurkat cells in vivo were related to inhibited the expression level of themicrovessel density.6The relationship of endothelial growth factor (VEGF) and microvesseldensity (MVD): The relationship between vascular endothelial growth factor and microvessel density is positively correlated. The linearregression regression equation is Y=3.27+0.329x， P=0.005. Thecorrelation coefficient is r=0.807, the coefficient of determination isR2=0.652.Conclusion:1CIK cells could significantly inhibite the proliferation of Jurkat cells invivo.2CIK cells’ depression to Jurkat cells in vivo were related to inhibited theexpression level of the endothelial growth factor(VEGF) and microvesseldensity(MVD).3The relationship between vascular endothelial growth factor andmicrovessel density is positively correlated. Endothelial growth factor(VEGF) is the important factor in promoting tumor angiogenesis. |
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