Objective The whole process of vaccine preparation is time-consuming and technically challenging. Here the hGM-CSF-engineered K-562 cell line was constructed to simplify tumor vaccine preparation process. Methods The eukaryocyte expressing plasmid pcDNA3.1/GM-CSF was first constructed and its accuracy was verified through sequencing. The pcDNA3.1/GM-CSF was transfected into COS-7 cells to verify GM-CSF expression and cytokine activity using TF-1 cell line. Then the plasmid was transfected into K-562 cell line using liposome method, and was selected under G-418 and sub-cloned by limiting dilution. GM-CSF product from the monoclone GM-CSF-K-562 cell lines was quantified using ELISA method. Results We successfully constructed the hGM-CSF eukaryocyte expressing plasmid and hGM-CSF expressing K-562 cell line. Conclusion The construction of K-562/GM-CSF line will simplify the preparation of tumor vaccine, thus facilitating the application of tumor vaccination therapy in clinical application.

Objective To explore the active immunotherapeutic effects of whole-cell leukemia vaccine combined with 1-methyl-tryptophan (1-MT, inhibitor of idoleamine 2,3-dioxygenase, IDO) treatment on leukemia. Methods The tumor-bearing mice model was made by hypodermic injection of FBL-3 cells. Then these mice were divided into 5 groups, normal group, PBS control group, CTX chemotherapy group, vaccine treated group and vaccine combined with 1-MT treated group (1-MT group), respectively. The main outcome measures including general condition, response rate, tumor size, metastasis and survival time were investigated. Results The mice of PBS control group were slow to move and much heavier (including tumor nodules) than the other groups. No obvious difference was observed in activity, eating behavior and weight between normal group, vaccine treated group and 1-MT treated group. The mice of CTX chemotherapy group were observed epilation, arched body and worn, and those weights decreased significantly compared with other group. The treatment-related mortality of vaccine-treated group and 1-MT group was lower than that of CTX chemotherapy group significantly (0, 0 vs 40 %). There were no significant difference in complete remission rates between vaccine treated group and 1-MT group (61.1 % vs 70.0 %, χ2 = 0.221, P >0.05). But the recurrence rate of 1-MT group was lower than vaccine treated group (0 vs 36.36 %). The tumor nodules growth of recurrent mice could be inhibited by 1-MT. The mean survival time of vaccine treated group and 1-MT group were longer than that in CTX chemotherapy group and PBS control group (χ2 = 52.13, P <0.01). Conclusion Whole-cell leukemia vaccine can inhibit tumor growth and prolong tumor-bearing mice survival time with remarkable curative effects and few side effects. Vaccine combined with 1-MT treatment can significantly reduce tumor recurrence rate, and 1-MT was still effective in inhibiting recurrence of tumor nodules growth after vaccine treatment.

Objective To construct Fas-targeting siRNA-expressing plasmid, and explore the significance of Fas inhibition in the treatment of acute aplastic anemia. Methods U6 promoter cassette and siFas sequence were obtained by PCR method, and cloned into modified pcDNA3.1 to produce plasmid pU6-siFas, which was transfected into P815 cells with limpofectin 2000, and then Fas expression was detected by immunohistochemistry. Results The plasmid pU6-siFas could efficiently reduce the expression of Fas and confer G-418 resistance in P815 cells. Conclusion The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique, and lay foundation for further studies of the therapeutic effect of Fas inhibition on acute aplastic anemia.