The Anti-tumor Effective Of Co-culture DC-LAK Cells And The Distribution Of Them After Different Pathway Infusion In Tumor-bearing Mice

Objective: Dendritic cells were generated from bone marrow cell of the mouse and stimulate them with tumor freeze-melt antigen, co-cultured with Lymphokine activated killer cells from mouse splenic lymphocyte.Observe the anti-tumor effective of co-culture DC-LAK cells and the distribution of them after different pathway infusion in tumor-bearing mice, and in order to find out their anti-tumor mechanisms and provide preclinical rationale experimental theory.Methods: 1 Mouse lung cancer cell line Lewis lung carcinoma (LLC) was cultivated, and it was collected in exponential phase of growth. Monoplast suspension which was of 1×107/ml density 0.2ml was injected in right-fore-limb armpit of C57BL/6 mice. 2 Mice tumor tissue was freezed thawing to prepare tumor antigen and determine the proteinum content. 3 Preparation of Dendritic cells and LAK cells. Bone marrow cells which came from femoral bone and shin bone of C57BL/6 mice were cultured in RPMI1640 medium supplemented with recombinant mouse rmGM-CSF 50ng/ml and rmIL-4 25ng/ml for 7 days. The DCs were stimulated with lysates LLC antigen in the 7th day. After 48 hours, FACS was used to explore the immunphenotype(CD80,CD86)of Dendritic cells. Spleen cell suspensions were prepared in asepsis, and splenic lymphocytes were isolated by Ficoll-Hypaque.These cells were further cultured in RPMI1640 medium supplemented with recombinant mouse IL-2 1000iu/ml for 7 days.4 DCs and LAK cells were further cultured in RPMI1640 medium supplemented with rmGM-CSF 25ng/ml and rmIL-4 12.5ng/ml for 2 days. The rate of DC:LAK was 1:5. 5 The in vitro killing rate of co-culture DC-LAK cells. Effector cell is co-culture DC-LAK cells; target cell is LLC cell, Killing activity was examined by MTT. The rate of E:T was 20:1 and 40:1.6 Transfusion of exogenous killer cells.48 tumor-bearing mice were randomly divided into para-tumor subcutaneous injection group and intraperitoneal injection group. Each group was randomly divided into four groups(1h,4h,24h,72h)with 3 mice in it. DC-LAK cells or LAK cells 3×107/0.2ml labeled by CFSE had been infusioned in the mouse had been infusioned LLC cell ten days.7 Detection of the exogenous killer cells in vivo. Blood,liver,spleen,lung and tumor of each mice which had been infusioned exogenous killer cells were harvested in the time of 1h,4h,24h,72h. The simplex cell suspension was made and detect with Laser scanning confocal microscope (LSM).Results: 1 DCs can be purified from bone marrow cells. The cells showed branching-like and pseudopod-like.DCs stimulated by tumor freeze-melt antigen highly express CD80, CD86(CD80 42.4%,CD86 75.31%,CD80&CD86 41.36%).2 the DC-LAK cells group can be observed when DC and LAK cells co-culture for 24 hours.3 The Killing ability of effector cells on LLC cell line. When E:T is 40:1,the killing ability of LAK cells on LLC cell line is 63.45±2.46%,the DC-LAK group is 81.02±2.18%, There are significant difference between two groups(p<0.01).When E:T is 20:1,the Killing ability of LAK cells on LLC cell line is 57.92±2.11%,the DC-LAK cells group is 75.10±3.68%, There are significant difference between two groups(p<0.01).4 Detection of the exogenous killer cells in vivo.(1)After exogenous killer cells by the way of peritoneal injection transfusion to the tumor-bearing mice, in one hour, the distribution of the labeled DC-LAK cells in lung achieved to the peak value; In four hours, the distribution of them in liver and spleen achieved to the peak value; in twenty-four hours, the distribution of them in the tumor achieved too, at this time, the distribution of them was obviously higher than in other organs(p<0.05).In 72 hours, the distribution of them in spleen was the highest(p<0.05).During the hole sight surveying, the distribution of peripheral blood was low. Compared with LAK cells group, in 1hour,4 hours,24 hours, the distribution of DC-LAK cells group in tumor was higher (p<0.05).In 72 hours, the two groups did not exist statistics difference.(2)After exogenous killer cells by the way of tumor-side hypodermic injection transfusion to the tumor-bearing mice, in 1 hour, the distribution of the labeled DC-LAK cells in lung achieved to the peak value; in 4 hours, the distribution of them in liver and achieved to the peak value; in 24 hours, the distribution of them in the tumor and spleen achieved too, at this time, the distribution of them was obviously higher than in other organs(p<0.05).In 72 hours, the distribution of them in tumor and spleen was the highest(p<0.05).During the hole sight surveying, the distribution of peripheral blood was low. Compared with LAK cells group, in 1 hour,4hours,24 hours, the distribution of DC-LAK group in tumor was higher (p<0.05).In 72 hours, the two groups did not show exist statistics difference.(3)Compared of the two ways of transfusion. DC-LAK cells mainly distributed in liver and spleen by the way of intraperitoneal injection; DC-LAK cells mainly distributed in tumor by the way of tumor-side hypodermic injection. The distribution of them in tumor was higher the tumor-side hypodermic injection group than the intraperitoneal injection group.Conclusion: 1 A large number of high purity cells which have DCs characteristic of morphology and function, can generated by GM-CSF and IL-4 from mouse bone marrow.2 DCs stimulated by tumor freeze-melt antigen can induce more efficient and specific killing activity of LAK cells against LLC in vitro.3 the DC-LAK cells after intraperitoneal injection in tumor-bearing mice are major accumulated in liver and spleen. After para-tumor subcutaneous injection in tumor-bearing mice, DC-LAK cells are major accumulate in tumor tissue. Both tow pathway, DC co-culture with LAK cells can raise the accumulation of LAK cells in tumor tissue. The distribution of DC-LAK cells of para-tumor subcutaneous injection group is more than intraperitoneal injection group all the time.