重组人核心蛋白聚糖对白血病K562细胞体外影响的实验研究

【作者】 景刚；

【导师】 王桂琴；

【作者基本信息】 山西医科大学 ， 免疫学， 2010， 硕士

【摘要】 目的：观察重组人核心蛋白聚糖(rhDCN)对白血病K562细胞生长抑制、凋亡的影响,并探讨可能的作用机制。方法：1.将已构建好的pcDNA3.1(+)-DCN真核表达载体经EcoRI、NotI双酶切、测序鉴定。2.鉴定正确的重组质粒大量纯化扩增后,通过LipofectamineTM2000脂质体介导转染对数生长期的白血病K562细胞,提取转染后48h细胞的总RNA,并进行RT-PCR鉴定。3.试验分为0.9% NaCl溶液组、pcDNA3.1(+)/K562组、pcDNA3.1(+)-DCN/K562组和脂质体组。4.经瑞特染色观察转染后细胞形态的改变,MTT法检测细胞增殖活性,碘化丙啶(PI)染色,流式细胞术分析细胞周期及凋亡。5.Western blot检测细胞凋亡相关蛋白BCL-XL、Mcl-l及Bax的表达。结果：1.重组质粒双酶切后,所得基因片断与预期基因片断大小相符；测序结果与Genebank中的DCN基因序列完全相符,无突变。2.通过脂质体转染法将pcDNA3.1(+)-DCN重组质粒转入K562细胞后,RT-PCR结果证实转染成功。3.pcDNA3.1 (+)-DCN/K562组瑞特染色呈现典型的凋亡形态学改变,其他各组无明显凋亡形态学改变。4.MTT结果显示,pcDNA3.1 (+)-DCN/K562组细胞增殖抑制率(转染24h为16±1.08%,转染48h为14±1.01%,转染72h为20±1.19%)明显高于对应时间点其他组增殖抑制率,差别有统计学意义(P<0.05)；FCM检测结果显示,pcDNA3.1 (+)-DCN/K562组凋亡率(20.15±1.31%)、细胞的G0/G1期细胞百分率(51.15±0.57%)与其他各组结果比较增加明显,差别有统计学意义(P<0.05)。5.Western blot结果显示pcDNA3.1 (+)-DCN/K562组与其他各组比较BCL-XL、Mcl-1蛋白表达减低,Bax蛋白表达升高。结论：rhDCN重组质粒可有效抑制K562细胞增殖,并诱导其凋亡。rhDCN对细胞周期及凋亡相关蛋白BCL-XL、Mcl-1、Bax的影响可能是其发挥作用的机制,为白血病生物治疗提供了新的实验依据。目的：研究重组人核心蛋白聚糖(rhDCN)联合多柔比星(ADM)对白血病K562细胞生长的影响,并分析其可能机制。方法：用含10%胎牛血清的RPMI1640培养液体外培养K562细胞,经脂质体介导转染对数生长期的K562细胞,试验分为0.9% NaCl溶液组、pcDNA3.1(+)-DCN/K562组、ADM/K562组、pcDNA3.1(+)-DCN加ADM/K562组。瑞特染色观察细胞形态学改变；采用MTT法检测细胞增殖活性；流式细胞术(FCM) Annexinv/PI双染色,分析细胞凋亡；采用RT-PCR法分析各处理组K562细胞TGF-β1mRNA水平表达的变化。结果：pcDNA3.1(+)-DCN加ADM/K562组细胞比单独DCN和ADM组细胞,染色呈现更显著的凋亡形态学改变；MTT结果显示联合组细胞增殖抑制率(61±1.32%)明显高于单独干预组(DCN组20±1.9%；ADM组47±1.04%)(P<0.05),FCM检测结果显示联合组细胞凋亡率(61.30±0.9%)与单独干预组(DCN组28.25±1.3%；ADM组31.85±1.5%)比较增加明显(P<0.05),RT-PCR结果显示联合组细胞TGF-β1 mRNA的转录减少。结论：rhDCN可以明显增强ADM对K562细胞的杀伤作用,提高肿瘤细胞的凋亡率。对TGF-β1 mRNA转录的影响可能是发挥协同效应的原因,其具体机制有待进一步研究。更多还原

【Abstract】 Objective:To investigate the anti-tumorigenesis function of rhDCN on the leukemia K562 cells in vitro and analyze the possible mechanism.Methods:1. pcDNA3.1(+)-DCN eukaryotic expression vector was verified by EcoRI, NotI double digestion and sequencing.2.Verified recombinant plasmids were amplified. After 48h, total RNA was extracted from liposome-mediated transfection K562 cells and carried out RT-PCR identification.3.They were divided into physiological saline group,pcDNA3.1(+) vector group,pcDNA3.1(+)-DCN group and liposome group. Morphology change of K562 cells was detected by Wright stain.4.Cell proliferation activity was detected by MTT. Cell cycle and apoptosis of K.562 cells was assessed by propidium iodide (PI) staining FCM.5. The expression of apoptosis-related protein, BCL-XL, Mcl-1 and Bax was detected by Western blot.Results:1. The size of gene fragment obtained by double digestion was the same to expected gene fragment size. Sequencing results completely consist with the DCN in Genebank sequences.2. pcDNA3.1 (+)-DCN recombinant plasmids were transformed into K562 cells by liposome, RT-PCR results confirmed the successful transfection.3.Wright stain showed that typical apoptotic morphological change of K562 cells in transfected group. There were no morphological changes of apoptosis in other groups.4.MTT method results showed that the transfected cell proliferation inhibition rate(24h,16±1.08%; 48h,14±1.01%; 72h,20±1.19%)was higher than that of the other corresponding groups (P<0.05). FCM results showed that the apoptosis index (20.15±1.31%)of the transfected group was higher than that of the other groups (P<0.05), cells were arrested in the G0/G1 phase (51.15±0.57%) (P<0.05).5. Western blot showed the expression levels of Bax were increased and that of BCL-XL and Mcl-1 was decreased in pcDNA3.1(+)-DCN/K562 group.Conclusion:rhDCN can inhibit the growth of K562 cells and induce the apoptosis, the effect of cell cycle and apoptosis-related protein BCL-XL, Mcl-1, Bax may play a role in its mechanism. rhDCN has provided a new way for the biological treatment of leukemia.Objective:To investigate the effect of rhDCN and ADM on suppression, apoptosis and TGF-β1 mRNA expression of leukemic K562 cell line. Methods:K562 cells were cultured by containing 10% fetal calf serum RPMI1640 culture fluid.K562 cells in Logarithmic growth phase were divided into Saline group, pcDNA3.1(+)-DCN group, ADM group, pcDNA3.1(+)-DCN-ADM group. Morphology change of cell was detected by Wright stain, cell proliferation activity was assessed by MTT. The apoptosis index of K562 cells was assessed by FCM. TGF-β1 mRNA of cell was assessed by RT-PCR.Results:Wright stain showed that typical apoptotic morphological change of K562 cells in combined group. MTT method results showed that the proliferation inhibition rate of the combined group was (61±1.32%) higher than that of individual intervention group (DCN group,20±1.9%;ADM group,47±1.04%)(P<0.05). FCM results showed that the apoptosis index of the combined group was (61.30±0.9%) higher than that of individual intervention group(DCN group,28.25±1.3%;ADM group,31.85±1.5%) (P＜0.05). TGF-β1 mRNA synthesis of combined group cell was significantly decreased.Conclusions:rhDCN can markedly enhance cytotoxicity of ADM on K562 cells, and the mechanisms of apoptosis may be down-regulation of TGF-β1 mRNA. Specific mechanisms will be further studied.更多还原