Induction Of T - Lymphoma Stem Cells And Their Resistance To Vincentine Extract

Background:Malignant lymphomas, a term independent disease, is primary in the lymphnodes and sites of immune lymphoid tissues and organs of the cancer cells. It can be found in any part of the body, such as lymph nodes, spleen and bone marrow. Lymphocyte tumor, that is characterized by weakness, belongs to Chinese medicine "evil nuclear", and often is caused by the loss of the internal organs.With the understanding of the origin and pathogenesis of malignant lymphoid tumors, the hypothesis of tumor stem cells derived from tumor formation is proposed. For the recognition of drug resistance, some scholars have pointed out that most of the traditional treatment methods, but can not effectively kill the stem cells in the tumor. The tumor stem cells that are not killed by treatment eventually become the source of tumor recurrence and metastasis, which is the main reason that are unable to cure the tumor at present. Herbal extract of vincristine is a first line chemotherapeutic drug for the treatment of acute leukemia, lymphoma, but its clinical effect is poor knowed, which can not completely cure patients.We first used CHIR99021 and PD173074 to treatment T lymphoma tumor stem cell for screening and culture, enrichment and identification of Hut-102 tumor cell spheres, and observed cancer stem cell characteristics of Hut-102 cell spheres whether vincristine herbal extract had stronger tolerance and explored the mechanism of the resistance to vincristine.Objective:In this study, we established a serum-free culture method for non-adherent microspheres of human T-cell lymphoma stem cells, and distinguished them. We observed tolerance of T-lymphocyte tumor stem cells induced by anti-tumor drug vincristine, and preliminary discussed the mechanism of tolerance.Method:Enrichment of sphere cells with stem-like properties from human T-cell lymphoma lines(Hut-102) were grown in a defined serum-free medium (SFM) with CHIR99021 and PD173074, two inhibitor of cell differention. The levels of Sox2, Bmil, C-Myc, OCT4, Nanog, Klf4 were analyzed by RT-PCR. By western blotting, we futher verified the OCT4 protein expression. The antigen expressions of CD34, CD44 were measured by Flow cytometry. The cell cycle of Hut-102 spheres was measured by PI and observed lymphocyte proliferation by CFSE. We enriched the Hut-102 spheres through the tail vein into transplanted nude mice, and determined the transplantation ability in vivo. We observed tolerance and preliminary discussed the mechanism. By CCK8 detection, we observed drug toxicity of Hut-102 sphere induced by anti-tumor drug include doxorubicin and Chinese medicine vincristine. By PCR method, we observed the drug resistance gene expression of Hut-102 sphere.Result:Using cell suspension culture, we successfully enriched Hut-102 sphere in serum-free medium with CHIR99021 and PD 173074 inhibitor.Hut-102 spheres have high expression of cancer stem cells characteristic gene, including Sox2, Bmil, C-Myc, OCT4, Nanog, Klf4, and the high expression of OCT4 protein, with overexpression of CD34, CD44 (74.20%,90.82%). By detection with the cell cycle, we found that most cells in the detection enriched spheres were in Gi/Go phase (64%), and low cells in S phase (30.56%) contrast to the Hut-102 cells. In the T lymphoma cell derived from caudal vein injection of Hut-102 spheres in athymic nude mice,67.74%±5.32% of cells showed Hut-102 cell surface markers (CD3).Hut-102 sphere had a strong resistance with the traditional Chinese medicine vincristine, which was 8 fold resistance than adriamycin. On the basis of the mechanism of drug resistance, we found that Hut-102 spheres were higher expression of ABCG2, ABCC10, ABCB4 with drug-resistant genes.Conclusion:For the first time in Hut-102 lymphoma cells, we enriched Hut-102 spheres in serum-free medium with CHIR99021 and PD173074 inhibitor. Hut-102 spheres had characteristic of cancer stem cells with transplantation ability properties in nude mice. Enrichment of Hut-102 spheres has strong tolerance properties with traditional Chinese medicine vincristine. By tolerance mechanism, we found high expression of resistance genes in Hut-102 sphere.