The Study Of The Immune Therapy Of Specific CTL On Glioma Carcinoma In Portability Mice

Objective：Dendritic cells (DCs) is the strongest functional antigen processingcell (APC) in the animal body. After caughting tumor cells, DC migrates to the lymphnode and presents tumor antigen to T lymphocytes, inducing it to a tumor peculiarcytotoxic T lymphocytes (cytotoxic T lymphocyte, CTL). CTLs can kill cancer cellsspecifically. We cultured mice spleen T lymphocytes in vitro. The T lymphocyteswere activated by the DC, which had loaded glioma antigen.Then the T lymphocytesbecame an anti-tumor of specific CTL. Then non-specific CTL and specific CTL wereinjected into the mice bearing glioma tumor. This research revealed the nCTL andsCTL groups all inhibited proliferation of cells in the mice bearing glioma carcinoma.Methods：The cells isolated from the mice’s bone marrow was cultivated byadding rhGM-CSF, rhIL-4and TNF-α. We had observated the change of cellsregularly.We collected the part of cells by flow cytometry technique analysis (flowcytometry, FCM) and determined the DC cells can be used. After stimulated byglioma cells which freezed and thawed, DC cells was changed to be the DC vaccine.We isolated T cells of mice spleen, cultured with DC vaccine together.Thus specificCTL was induced successfully in vitro. At the same time, the nCTL and sCTL cellswere injected to the mice bearing C6cells. After two weeks the mice were divided tothree groups randomly. Six mice were in one group.The mice were injected by i.v.every five days:(1) The control group (each mice is injected only physiological saline0.2mL).(2) sCTL group (each mouse is injected antigen sCTL5×106/mL0.2mL).(3) nCTL group (each mouse is injected nCTL5×106/mL0.2mL). We measuredtumor long diameter (a), short diameter (b) every5days. We calculated tumor’s sizeand tumor’s suppressionl ratio by the formula-the volume (V)=πab2/6, analyzed thetumor’s growth inhibition. We killed the mice to obtain the tumor organization30days later. The tumor tissue was fixed in formalin solution, dehydrated, and imbeddedin paraffin, then dyeing HE conventionally.We observed tumor cells’ form by themicroscope. Results:1. By induced, some DC cells was observed under the microscope aftersecond day.The shape of cells from circular form become irregular, pleomorphism,visible thorn shape of cytoplasm swelled. Before induced, DC processed specificitysurface markers of CD80、CD86.The percentage of it was1.25±0.09and1.74±0.11.After DC was cultivated with rhGM-CSF, rhIL-4, and induced by rm TNF-α, the levelof marker of DC was higher significantly. The percentage of it was99.02±5.34and99.47±6.28respectively (P <0.01).2. Before the T lymphocytes from mice bonemarrow were induced in vitro, the percentage of CD3+, CD4+, CD8+cells were63.84±3.21,26.32±1.42and30.29±3.42. After T lymphocytes induced by the DC andtumor antigen, in the stimulation of cell factor rmIL-2, the percentage of CD3+,CD4+, CD8+cells of specific CTL group were72.37±2.54,27.59±1.91and68.35±2.74. After T lymphocytes induced by the DC without tumor antigen, thepercentage of CD3+, CD4+, CD8+cells of nonspecific CTL group were70.28±2.34,25.29±2.14and63.59±3.24. T lymphocytes with DC induction compared with theminducted, the percentage of CD3+and CD8+T cells changed significantly (P <0.01).The percentage of CD4+T cells not changed significantly (P>0.05).3. The rate ofthe model mice bearing glioma was100%.4. Every treated group could inhibite thegrowing of the implanted carcinoma. sCTL group was the most significant inhibitionof the three group,which the average gross tumor volume post-treated15days was70±2.94mm3. sCTL group was higher than nCTL group (93±2.43mm3) and controlgroup (122±5.25mm3)，the result was different significancely (P=0.000). The tumorinhibition rate of sCTL group(72.85±4.38)％was significantly higher than nCTLgroup (47.14±2.96)％，the result was different significancely（P<0.05）.5. The tumortissue in treated group had a large number of lymphocyte infiltration by HE assay.6.We had observed that the apoptotic glioma carcinoma cells act a phenomenon of longtail by comet experimenta obviously.Conclusions: The nCTL and sCTL cell inhibited the glioma carcinomaproliferation of the mice bearing C6cells in vivo. This result demonstrated that CTLscould carry out the clinical application of glioma carcinoma.