【作者】 邓守恒；

【导师】 陈崇宏；

【作者基本信息】 福建医科大学 ， 药理学， 2007， 博士

【摘要】 急性早幼粒细胞性白血病（APL）是急性白血病中的一种特殊类型，约占髓细胞性白血病的10％。95％以上的APL具有特征性的t（15；17）染色体异位，即位于15号染色体上的早幼粒细胞白血病（PML）基因与位于17号染色体上的维甲酸受体α（RARα）基因相互异位，所形成的PML-RARα融合基因表达的融合蛋白分子量约为110KD，具有一定的酪氨酸激酶活性，增高的酪氨酸激酶活性可使融合蛋白自身及细胞内多种底物分子磷酸化，进而募集和结合许多转接蛋白分子，从而激活许多相关的细胞信号通路，使细胞异常增殖，在APL的发病和治疗中起着关键作用。故而降低APL细胞内融合蛋白含量，抑制其酪氨酸激酶活性，阻断其可能的细胞信号传导途径已成为当前针对分子靶点进行治疗的策略。硒化壳聚糖是将壳聚糖与亚硒酸在酸性条件下，以混合金属离子为催化剂制备而成的一种低分子量有机硒，可有效克服无机硒不易被细胞吸收，活性和毒性范围较窄，致死量相对较小等缺点。硒和砷属同族，而砷在临床上早已做为一种抗APL药物使用。故硒化壳聚糖也应具有抗APL的作用，且毒副作用应该比无机硒和砷小得多。本课题以表达PML-RARα融合蛋白的NB4细胞为试验对象，全面观察硒化壳聚糖对NB4细胞增殖、凋亡和分化的影响，以及硒化壳聚糖与常用的化疗药合用对NB4细胞作用的特点，探索硒化壳聚糖对NB4细胞作用的最适浓度及对融合基因、融合蛋白、细胞周期蛋白以及相关信号传导通路的影响等，为硒化壳聚糖的临床应用奠定基础。研究结果表明，硒化壳聚糖可明显升高细胞内GSH-PX活力，降低MDA含量和SOD活性。在本实验条件下，50mg/L的硒化壳聚糖可诱导NB4细胞向正常成熟细胞方向分化，若增加剂量至100mg/L及以上，药物对细胞则主要产生诱导凋亡方面的作用；硒化壳聚糖和亚硒酸钠对NB4细胞都可产生明显的细胞毒作用，且呈量效和时效关系，它们的IC₅₀分别是103.7mg/L和16.7umol/L，以同等含硒量来比较其效应，则硒化壳聚糖的作用效果约为亚硒酸钠的三倍；以激光共聚焦显微镜、AO/EB荧光染色法、流式细胞仪检测、DNA片段化凝胶电泳测定以及电镜超微结构观察等方法分析表明，硒化壳聚糖可诱导NB4细胞凋亡，阻断细胞于G0/G1期，提示硒化壳聚糖对APL的治疗可能具有一定的价值。硒化壳聚糖与ADM同时合用对NB4细胞可产生单纯相加至增强的协同杀伤效果，尤其是硒化壳聚糖与低浓度ADM的协同作用更为显著。硒化壳聚糖与ADM序贯给药时二药可产生单向协同，但协同效果不如同时合用显著。本实验结果还显示，1、硒化壳聚糖可明显减少NB4细胞内PML-RARα融合蛋白含量，呈量效、时效关系，但硒化壳聚糖对PML-RARα融合基因的转录没有影响。2、硒化壳聚糖可降低NB4细胞内cyclinD1的表达。3、硒化壳聚糖可降低NB4细胞内c-jun和MEK-1信号分子的含量，从而抑制了Ras信号传导通路。4、硒化壳聚糖可减少NB4细胞核内NF-KB的含量，从而抑制了NF-KB信号传导通路。5、硒化壳聚糖可提高NB4细胞内cAMP含量，降低cGMP的含量，增加cAMP与cGMP的比值。6、硒化壳聚糖可使细胞内钙离子浓度呈剂量与时间依赖性提高。7、50mg/L的硒化壳聚糖可增加NB4细胞膜上CD11b抗原分子的表达。以上这些作用可能都与硒化壳聚糖能抑制细胞增殖，诱导细胞凋亡，促进细胞分化作用密切相关。综上所述，硒化壳聚糖可能通过减少NB4细胞内PML-RARα融合蛋白和cyclinD1蛋白的含量，抑制细胞内Ras信号途径和NF-KB信号途径，提高细胞内cAMP含量，降低cGMP的含量，增加cAMP与cGMP的比值以及升高细胞内钙离子浓度等途径来抑制急性早幼粒细胞性白血病细胞株NB4细胞增殖，诱导其凋亡；小剂量硒化壳聚糖尚可通过增加NB4细胞膜上CD11b表达来诱导其分化，提示硒化壳聚糖可能是一种能够替代无机硒的高效、低毒的APL治疗新药，具有良好的开发前景，硒化壳聚糖对APL的治疗作用值得进一步深入研究。更多还原

【Abstract】 Over 95% of cases of Acute Promyelocytic Leukemia（APL） isassociated with a reciprocal translocation between chromsomes 15 and17 that produces a PML-RARαfusion gene which encodes PML-RARαfusionprotein. This fusion protein exhibits high activity of tyrosine kinase,which is considered essential for malignant transformation. So.Decreasing the fusion protein contents, dowing regulation its tyrosinekinase activity and blocking its possible signal pathways appear to bean attractive therapeutic strategy.selenium chitosan, a potent antioxidant and chemopreventive agent,which is synthesized with selenous acid and chitosan, is a kind oforganic selenium, here we demonstrated that selenium chitosan and sodiumselenite all could inhibite proliferation in a dose and time dependentmanner in a PML-RARαfusion protein positive APL cell line NB4. theirIC₅₀ is 103.7mg/L and 16.7umol/L respectively. The effect of seleniumchitosan is three times high than that of sodim selenite with the sameamount of selenium. The effects of selenium chitosan on the cell cyclephases and on inducing apotosis were studied by using Laser scan confoulmicroscope, flow cytometry. AO/EB fluorescent staining, transmissionelectron microscope and DNA fragmentation assay with gelelectrophoresis analysis. Its promoting differentiation effect wasmeasured by NBT reduced method and CD11b expression by using flowcytometry.An exposure of NB4 cells to 50—100mg/L of selenium chitosan for 24h produced a dose and time dependent increase in G0-G1 phase cells,several hallmarks of apoptosis including DNA laddering, chromatincondensation and fragmentation were also observed after the cells weretreated with selenium chitosan. Besides this, we also observed that50mg/h selenium chitosan could promote NB4 cells differentiation andselenium chitosan could increase GSH-PX activity、decrease SODactivity and MDA content.The synergistic antitumor effects of selenium chitosan with ADM onNB4 cells in vitro were studied with MTT method and Jin’s formula wasused to analyse the effect of drug combination. It was found thatadminstration of selenium chitosan and ADM at the same time brought asynergistic effect but sequential administration of the drugs onlyproduced a undirectional synergistic effect.The PML-RARαfusion gene—encoded PML-NARαfusion protein whichpossesses high activity of tyrosine kinase, it is known to be a majorfactor for the cause and treatment of APL. In order to reveal themechanism by which selenium chitosan induces apoptosis and inhibitesproliferation. The effects of selenium chitosan on the expression ofPML-RARαfusion gene was studied by using RT-PCR method. The synthesisof PML-RARαfusion protein、cyclin D1、c-jun、MEK-1 and NF-KBwere studied by using western blot analysis using monoclonal antibodyagainst RARαprotein、cyclin D1 protein、c-junprotein, MEK-1 proteinand P65 protein respectively. The change of intracellular calcium、CD11b、cAMP、cGMP content were studied by using flow cytometry andradioimmunity method. It has been fond that selenium chitosanremarkably inhibited the expression of PML-RARαfusion protein in adose and time dependent manner in the same conditions above. But didnot affect the expression of PML-RARαfusion gene. selenium chitosan could inhibite the expression of cyclin D1、c-jun、MEK-1 and NF-KBprotein and down regualtion the Ras and NF-KB signal pathway. Moreover.selenium chitosan was able to stimulate the concentration ofintracellular calcium and CD11b, increase the cAMP content and decreasethe cGMP content in NB4 cells. These were possible mechanisms whichselenium chitosan could inhibite proliferation, induce apotosis andpromot differentiation.The results suggest that the inhibition of PML-RARαfusion proteinand intracellular cGMP. The stimulation of intracellular calcium andcAMP. the Ras and NF-KB signal pathways. Which are all involved inselenium chitosan mediated apoptotic cell death. The therapeuticpotential of selenium chitosan in human APL is worthy of furtherinvestigation.更多还原