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背景:对白血病患儿在白血病细胞获得耐药、抗凋亡特性的过程中机制的研究目前甚少,多数研究集中在正常骨髓间充质干细胞和已建系的基质细胞,而未重视患儿骨髓间充质干细胞与白血病细胞之间的相互作用。目的:课题创新性提出白血病患儿骨髓间充质干细胞可能对白血病细胞株K562/AO2生长增殖及凋亡产生影响的理论假设。设计、时间及地点:细胞学体外实验,于2007-12/2008-08在广州医学院第一附属医院儿科实验室完成。材料:骨髓间充质干细胞来源于广州医学院第一附属医院住院的30例白血病患儿,其中急性淋巴细胞白血病患儿22例,急性粒细胞白血病8例,患儿家属对实验均签署知情同意书。K562/AO2细胞株由天津血液病研究所提供。方法:Ficoll密度梯度法分离培养白血病患儿骨髓间充质干细胞。设立2组:K562/AO2细胞组单独悬浮培养处于对数生长期的K562/AO2细胞;K562/AO2细胞+骨髓间充质干细胞共培养组在骨髓间充质干细胞贴壁呈融合状态时,加入1×108L-1处于对数生长期的K562/AO2细胞,24h后去除未黏附的K562/AO2细胞。主要观察指标:白血病患儿骨髓间充质干细胞对K562/AO2细胞生长的影响,AnnexinV-FITC法检测阿霉素对K562/AO2细胞凋亡的影响,流式细胞仪测定不同条件培养下的K562/AO2细胞周期,Taqman-MGB探针实时荧光定量PCR检测不同条件培养下耐药基因mdr1的表达。结果:与单独悬浮培养的K562/AO2细胞比较,K562/AO2细胞+骨髓间充质干细胞共培养组的K562/AO2细胞生长较为缓慢,无明显的对数生长期;早期凋亡细胞数明显减少(P<0.05);处于G0～G1期的K562/AO2细胞明显增多,S期细胞减少;K562/AO2细胞mdr1耐药基因的表达无明显差异(P>0.05)。结论:体外细胞学实验结局证实,白血病患儿骨髓间充质干细胞诱导的K562/AO2细胞耐药与mdr1基因无关,而是通过黏附作用改变K562/AO2细胞周期,进而逃避药物的促凋亡作用。
BACKGROUND: There are few studies on the mechanism of drug-resistant and anti-apoptotic properties of leukemia in leukemia patients. Most studies focused on normal bone marrow-derived mesenchymal stem cells and established stromal cells, but not on children Bone marrow mesenchymal stem cells and leukemia cells interaction. OBJECTIVE: To set forth the theoretical hypothesis that the bone marrow mesenchymal stem cells from leukemia patients may have effects on the proliferation, proliferation and apoptosis of leukemia cell line K562 / AO2. DESIGN, TIME AND SETTING: The in vitro cytology experiment was performed at the Pediatric Laboratory, First Affiliated Hospital of Guangzhou Medical College from December 2007 to August 2008. MATERIALS: Bone marrow-derived mesenchymal stem cells (BMSCs) originated from 30 leukemia patients hospitalized in the First Affiliated Hospital of Guangzhou Medical College, including 22 children with acute lymphoblastic leukemia and 8 children with acute myeloid leukemia. Family members of the children signed informed consent book. K562 / AO2 cell line provided by the Tianjin Institute of Hematology. Methods: Ficoll density gradient method was used to isolate and culture bone marrow mesenchymal stem cells from children with leukemia. Two groups were established: K562 / AO2 cells alone were cultured in K562 / AO2 cells in logarithmic growth phase; K562 / AO2 cells + MSCs co-cultured with MSCs in the state of fusion, K562 / AO2 cells in the logarithmic growth phase were treated with 1 × 108 L-1, and non-adherent K562 / AO2 cells were removed after 24 h. MAIN OUTCOME MEASURES: The effect of bone marrow mesenchymal stem cells of leukemia on the growth of K562 / AO2 cells. Annexin V-FITC method was used to detect the effect of doxorubicin on the apoptosis of K562 / AO2 cells. Flow cytometry was used to determine the effect of K562 / / AO2 cell cycle, Taqman-MGB probe real-time fluorescent quantitative PCR detection of different conditions of the drug resistance gene mdr1 expression. Results: Compared with K562 / AO2 cells cultured in K562 / AO2 alone, K562 / AO2 cells and BMSCs co-cultured K562 / AO2 cells grew slowly and had no obvious logarithmic growth phase. The number of early apoptotic cells was significantly decreased (P <0.05). The expression of mdr1 gene in K562 / AO2 cells was not significantly different (P> 0.05). Conclusion: In vitro cytology results confirm that K562 / AO2 cells induced by bone marrow mesenchymal stem cells in children with leukemia have no relationship with mdr1 gene resistance, but change the cell cycle of K562 / AO2 by adhesion and thus avoid the pro-apoptotic effect .