白血病骨髓基质细胞黏附培养对Jurkat细胞bcl-2家族成员表达的影响

Influence of the adhered-culture with bone marrow stromal cells on the expression of Jurkat cell and bcl-2 family

目的观察白血病骨髓基质细胞（BMSC）-Jurkat细胞共培养模型中Jurkat细胞的化疗敏感性，并探讨基质细胞黏附培养对Jurkat细胞bcl-2、bcl—xL及bax表达的影响。方法常规分离、培养BMSC，Jurkat细胞与mco照射的基质细胞层黏附培养，构建共培养模型，扫描电镜观察。以悬浮培养的Jutkat细胞为对照，MTT法测定Jurkat细胞DNR的‰，FITC—Annexin V／PI标记流式细胞仪定量Jurkat细胞的凋亡率，Westernblotting检测Jurkat细胞总蛋白中bcl-2、bcl—xL和bax的表达。结果白血病基质细胞黏附使Jurkat细胞对DNR的敏感性降低，IC50为2．30umol／L，而悬浮对照组为0．45umol／L，白血病基质细胞黏附组Jurkat细胞凋亡率分别为（6．05±0．54）％，与悬浮对照组（25．74±6．15）％比较差异有统计学意义（P〈0．01）。Western blotting检测结果显示，与白血病BMSC黏附培养4h即观察到bcl-2表达上调，24h和48h尤为明显，该家族另一凋亡抑制蛋白bcl—xL的表达未见明显变化，也未观察到促凋亡蛋白bax表达的明显变化。结论白血病BMSC黏附培养能介导白血病细胞耐药，其机制可能与基质细胞黏附使白血病细胞抗凋亡蛋白bcl-2表达上调有关。

Objective To investigate the chemotherapeutics sensitivity and expression of bcl-2, bcl-xL and bax of Jurkat cells co-cultured with bone marrow stromal cells （BMSC） isolated and cultured from leukemia patients. Methods BMSC were isolated and cultured from leukemia patients routinely. To construct the co-cultured model, Jurkat cells were co-cultured with of the irradiated layer BMSC by To and observed the model with scanning electron microscope. The Jurkat cells suspension-cultured were used as control. The apoptosis and IC50 were detected by the FACS can machine and M3T, respectively. The expression of bcl-2, bcl-xL and bax in Jurkat cells was detected by Western blotting. Results We found that the Jurkat cells in the model showed a decreased sensitivity to DNR, IC50 values for leukemic BMSC and nonadhered contol were of 2.30 umol/L and 0.45 umol/L, respectively. Moreover, Jurkat cells adhered to BMSC have a survival advantage over suspended cells following DNR exposure for 24 h, apoptosis percentages for leukemic BMSC group and nonadhered controls were of （6.05±0.54）% and （25.74±6.15）%, respectively. As compared with controls, leukemic BMSC group had significant difference in apoptosis percentages （P 〈0.01）. The expression of bcl-2 in Jurkat cells was up-regulated when adhered to BMSC for 4 h and the higher expression emerged after adhering for 24 h and 48 h. No marked change of bcl-xL and bax expressions were observed in the adhered Jurkat cells. Conclusion The adhered-culture with bone marrow stromal cells isolated from leukemia patients could make the leukemia cells acquire drug resistance, which was associated with the upregulated expression of bcl-2 in the leukemia cells.