白血病患儿骨髓间充质干细胞对K562/AO_2细胞增殖和凋亡的影响

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其少,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自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/AO_2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例自血病患儿,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/AO_2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/AO_2细胞组单独悬浮培养处于对数生长期的K562/AO_2细胞:K562/AO_2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1×10~8 L~(-1)处于对数生长期的K562/AO_2细胞,24 h后去除未黏附的K562/AO_2细胞.主要观察指标:白血病患儿骨髓间充质干细胞对K562/AO_2细胞生长的影响,AnnexinV-FITC法检测阿霉素对K562/AO_2细胞凋亡的影响,流式细胞仪测定不同条件培养下的K562/AO_2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/AO_2细胞比较,K562/AO_2细胞+骨髓间充质干细胞共培养组的K562/AO_2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/AO_2细胞明显增多,S期细胞减少;K562/AO_2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/AO_2细胞耐药与mdr1基因无关,而是通过黏附作用改变K562/AO_2细胞周期,进而逃避药物的促凋亡作用.

Effects of bone marrow mesenchymal stem cells from leukemia children on proliferation and apoptosis of K562/AO_2 cells

BACKGROUND:Little data have been available concerning the mechanism of drag resistance and anti-apoptosis in leukemic cells of leukemia children.The majority of studies focus on normal bone marrow mesenchymal stem cells (MSCs) and established stroma cells,but not interaction of MSCs and leukemic cells in leukemia children.OBJECTIVE:To explore the effect of MSCs in leukemia children on the proliferation and apoptosis of leukemic cell strain K562/AO_2.DESIGN,TIME AND SETTING:In vitro cytology experiment was performed at the laboratory of Department of Pediatrics,First Affiliated Hospital of Guangzhou Medical College from December 2007 to August 2008.MATERIALS:MSCs were provided by 30 leukemia children admitted to First Affiliated Hospital of Guangzhou Medical College,including 22 acute lymphoblastic leukemia and 8 acute myeloblastic leukemia.Written informed content was obtained from all families.K562/AO_2 was provided by Tianjin Institute of Hematopathy.METHODS:MSCs were isolated and cultured by Ficoll density gradient method.They were cultured in two conditions:the co-culture of MSCs and K562/AO_2 and K562/AO_2 suspension alone.In co-culture group,1×10~8 /L K562/AO_2 cells at log phase were added to confluent MSCs,and free floating K562/AO_2 cells were discarded after 24 hours.MAIN OUTCOME MEASURES:Effect of MSCs on the growth of K562/AO_2 cells was observed;effect of addamycln on K562/AO_2 cell apoptosis was detected by AnnexinV-FITC method.Cell cycle was determined by flow cytomtry,mdrl gene of K562/AO_2 cell was detected by Taqman-MGB probe real-time PCR.RESULTS:Compared with K562/AO_2 alone,the K562/AO_2 cell co-cultured with MSCs grew slower and the log phase of growth was not significant;the rate of apoptosis in earlier period was significantly decreased (P ＜ 0.05);co-cultured K562/AO_2 G_0-G_1 phase increased,but S phase decreased.No changes in mdr1 gene in cells were found between two culture conditions (P ＞ 0.05).CONCLUSION:In vitro cytology has demonstrated that leukemia children MSCs induce drug resistance of K562/AO_2 cells by changing K562/AO2 cell cycle through adhesion to avoid pro-apoptotic effect of drugs but not related with mdr1 gene.