An Experimental Study On The Effect Of Tumor Specific CTL Combined With Thymosin α1 Against Tumor In Vivo

Purpose: To study the influence of thymosin alpha 1 (Tα1) on the killing effect of the malignant melanoma in mice led by tumor pecific cytotoxic T lymphocytes (CTLs) in vivo.Methods:Mononuelear cells of the mouse spleen are isolated by adhesion method. Dendritic cells are separated from mononuelear cells of the mouse spleen through adherent ways and are cultivated and amplified with mouse recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) 10ng/ml, recombinant mouse IL--4 (rmIL-4) 20ng / ml for 5 days, then tumor necrosis factor-α(TNF-α) 10ng/ml is added to induce DCs to maturation on the fifth day of cultivation .Onthe eighth day of cultivation, mouse spleen derived DCs are collected to detect the changes in phenotype and morphology by flow cytometry detection and scanning electron microscopy. Malignant melanoma cell line (B16) are repeatedly frozen and thawed to extract the supernatant as the whole-cell antigen of B16, which is loaded to DCs on the fifth day of cultivation. Meanwhile, TNF-αis added to stimulate DCs maturation. Mouse spleen derived T cells are separated and purified by villus column then are co-cultured with the B16 cells antigen-loaded DCs to induce B16 cells antigen-specific CTL. The density of B16 cells is adjusted to 5×106/ml and the model of C57BL/6 mouse malignant melanoma is established by subcutaneous injection of 0.2ml B16 cells. On the 9th tumor-bearing day, the mice are randomly divided into 3 groups, 6 mice in each group, as following:①Group A (NS control group);②Group B (CTL transfusion group);③Group C (CTL Combined with Tα1 transfusion group). The way of treatment: CTL transfusion through the tail vein of mice, Tα1 through subcutaneous injection of a number of locations around tumor. After each group is given to the 1st treatment on the 9th tumor-bearing day, CTL transfusion once every six days, each time 5×106/0.2ml, a total of 3 times; Tα1 (400μg/kg) subcutaneous injection once every 3 days, each 0.2ml, a total of 6 times. A, B, C of each group are randomly divided into two sub-groups, each sub-group of six mice.ⅠSub-group: to observe tumor growth and survival of mice: 1 times /3 days. The tumor volume is calculated and the tumor growth curve is drawn in accordance with V=1/2ab2, where"a"denotes the maximal diameter and"b"denotes minimal diameter. These Measurements about tumor volume stopped until any group of mortality got 50%.At the same time the survival rate of mice is calculated by recording the living time of mice. II Sub-Group: to measure the tumor weight and volume: On the 21th tumor-bearing day, all the 18 mice in threeⅡsub-groups are killed by the cervical dislocation method. Then tumor is removed and weighted. According to the formula, the volume and weight inhibition rate are calculated. The post-treated pathological changes in there II Sub-Groups of mice are discovered by observing tumor tissue with HE staining. And the apoptotic situation of the post-treated tumor cells is detected through transmission electron microscopy.Results: On the 18th tumor-bearing day, the average tumor volume of three groups are separately: (3.08±0.30cm3)in group A, (2.25±0.29cm3) in group B, (1.41±0.25cm3) in group C . The Comparison among three groups of tumor volume shows statistically significant (F=53.107, p<0.001). Group C of average volume is smaller than group A and group B (p<0.01).On the 21th tumor-bearing day, the volume of tumor of 2 mice in group C with good therapeutic effect further reduces. To the 32th tumor-bearing day the tumor in one of the 2 mice almost disappeared and it still remained alive to the end of the experiment. By observing the changes of different times in tumor volume curve, the curve of group C rose gently and the curve of group B rose gently at the beginning of the experiment but it significantly increases the upward trend after the 18th tumor-bearing day. On the 21th tumor-bearing day, theⅡsub-group of mice is anatomized to measure the average tumor volume and weight. Each group of the average tumor volume are separately (5.05±0.65cm3) in group A, (3.49±0.60cm3) in group B, (1.51±1.07cm3) in group C and the average tumor weight are separately (4.58±0.47g) in group A, (3.20±0.41g) in group B, (1.34±0.99g) in group C. The volume inhibition rate are 30.89% in group B, 70.10% in group C and the weight inhibition rate are 30.13% in group B , 70.74% in group C, (p <0.01).⑵the survival observation of mice in the I sub-group: the survival time and survival rate of group C both are significantly higher than group A and B. The survival curves of group C decline slowly. All of the mice in group A die to the 32th tumor-bearing day. And group B and group C maintaine 83.3% of the survival rate from the 9th tumor-bearing day to 45th tumor-bearing day but the B group of mice all die to the 55th tumor-bearing day, while 83.3% of the mice in group C are still alive. The median survival time of group C is 60±3.67 days, which is significantly higher than group A 27±1.84 days and group B 49±1.84 days (p <0.01).⑶After HE staining, it is found that tissures in group B and C reduce much more in the infiltrative density of tumor cell and increase more apparently in the hemorrhage and necrosis than in group A under a Electron Microscope.Conclusions:⑴We succee in establishing this model that tumor-specific CTL is carried out transfusion therapy through the mouse tail vein after it is induced in vivo. And this model is supposed to be the foundation for further research.⑵Tα1 can extend anti-tumor functional time of CTL and improve the capacity of its killing tumor cell in vivo.⑶The treatment with tumor-specific CTL Combined with Tα1 produces better effect than only with tumor-specific CTL to malignant melanoma in mice. And Tα1 has a higher value in inhibiting tumor growth, prolonging tumor-bearing mice survival time and improving the survival rates.⑷Tumor immunotherapy with immune adjuvant can effectively enhance the efficiency of killing tumor.