【作者】 阮晶晶；

【导师】 陈飞虎；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 硕士

【摘要】 目的:本研究观察维甲酸衍生物对白血病细胞株K562和NB4细胞的增殖的影响和诱导分化活性,并筛选出有具有药理学活性的衍生物进行药效学和诱导分化机制的研究。方法:1.新型维甲酸类衍生物作用于K562和NB4细胞后,通过MTT法检测细胞的增殖;瑞氏染色法在倒置相差显微镜下观察加药处理前后细胞形态学变化。NBT还原实验法分析细胞的分化指标。FCM检测分析细胞周期和细胞表面分化抗原CD_11b、CD₁₃变化。2.筛选出具有活性的化合物后,采用细胞计数方法观察白血病细胞在不同浓度的药物作用下生长曲线的改变, NBT还原实验法分析细胞在不同浓度作用下的分化指标,FCM检测分析细胞周期及K562细胞表面分化抗原CD₄₅、CD₇₁、CD₁₁₇和NB4细胞表面分化抗原CD_11b、CD₁₃、CD₁₄的变化。RT-PCR检测细胞周期相关因子Cyclin D1、Cyclin E、PCNA、CDK2、CDK4、CDK6、P21^cip1、P27^kip1、P57^kip2 mRNA表达改变。Western Blot法检测cyclin D1、CDK4蛋白、PML/RARα融合蛋白和RARα蛋白表达的改变。结果:1.新型维甲酸衍生物作用3d后,4a-02,5a-02抑制K562和NB4细胞增殖作用呈剂量依赖性增加,10^-5 mol/L抑制作用最强。维甲酸衍生物(10^-5 mol/L)的诱导分化活性则表现在油镜下观察白血病细胞向粒系分化成熟的改变,2a-03,4a-02,5a-02,6a-02作用后NBT阳性细胞率增加,K562细胞表面分化抗原CD11b在2a-03,3a-02,4a-01,5a-02作用后表达量增加,NB4细胞在2a-01,2a-02,2a-03,4a-01,5a-01,5a-02作用后,CD11b增加同时CD13表达则减少。通过对细胞周期的分析,发现2a-03,4a-01,4a-02,5a-02对K562和NB4细胞周期均有影响,表现为G0/G1期细胞表达量增加,S期细胞表达量减少,呈G1期阻滞。2.经过活性筛选发现,维甲酸衍生物4-氨基-2-三氟甲基苯基维甲酸酯（4-amino-2-trifluoromethyl-phenyl retinate, ATPR）是抑制增殖和诱导分化活性较强的衍生物,进行药效学研究发现,ATPR呈浓度依赖和时间性依赖抑制K562和NB4细胞增殖的作用。ATPR诱导分化活性表现为NBT阳性细胞率增加,NB4细胞表面分化抗原CD11b表达增加,CD13表达减少,CD14表达增加,作用与阳性对照ATRA相类似。并随作用时间延长而变化加大。ATPR作用3d后G0/G1期细胞表达量增加,S期细胞表达量减少,呈G1期阻滞。周期相关因子Cyclin D1、Cyclin E、CDK2、CDK4、CDK6、mRNA在ATPR作用3d后表达有减少改变,PCNA mRNA在ATPR作用7d后有减少改变。P57^kip2mRNA有明显增加的变化,但P21^cip1、P27^kip1mRNA不但没有增加,反而有略减少的趋势。cyclin D1和CDK4蛋白表达减少,PML/RARα融合蛋白和RARα蛋白表达也呈减少的改变。结论:1.维甲酸衍生物（2a-03、4a-02、5a-02）是抑制增殖和诱导分化活性较强的衍生物,提示以氨基三氟甲基苯基维甲酸酯为出发点开发具有更好的诱导肿瘤细胞分化和抑制增殖活性的维甲酸类药物具有重大意义。2. ATPR（4a-02）有较强的抑制K562和NB4细胞增殖并诱导其分化的活性,并通过上调P57kip2mRNA,抑制Cyclin-CDK激酶复合物,发挥细胞周期阻滞的作用。更多还原

【Abstract】 Objective:To investigate the proliferation inhibition and differentiation induction activities of 10 retionoic acid derivatives in K562 and NB4 cells ,screening of the derivatives with pharmacological activity ,and investigate their pharmacodynamics and mechanism of inducing differentiation .Methods: 1、Cell proliferation was assessed by MTT assay before and after the treatment with retionoic acid derivatives in vitro.Morphologic changes were observed after Wright’s staining using inverted phase contrast microscope.Cell differentiation indexs were analyzed by NBT reduction test.Cell cycle and the expression of the maturation specific cell surface markers CD_11b, CD₁₃ were determined by FCM analysis.2、After screening of the derivatives with pharmacological activity,the changes of growth curve induced by different concentrations of derivatives were analysed by cell counting. Cell differentiation indexs were analyzed by NBT reduction test.Cell cycle,the expression of the maturation specific cell surface markers CD₄₅、CD₇₁、CD₁₁₇ of K562 cells and CD11b、CD₁₃、CD₁₄ of NB4 cells were detected by FCM.The expression of cell cycle-related factors such as Cyclin D1、Cyclin E、PCNA、CDK2、CDK4、CDK6、P21^cip1、P27^kip1、P57^kip2 mRNA was measured by RT-PCR.Western blotting was performed to detect the expression of protein cyclin D1, CDK4 protein, PML / RARαand RARα.Results:1.The growth of K562 and NB4 cells was inhibited in a dose-dependent manner after the treatment with retinoic acid derivatives for 3 days.The changes of differentiation and maturation of leukemic cells induced by 10 retinoic acid derivatives at the concentration of 10-5 mol/L were observed through immersion objective.NBT reduction test indicated that the retinoic acid derivatives could induce differentiation of leukemic cells and increase the positive cell ratio.The expression level of the maturation specific cell surface marker CD11b was increased while the expression level of CD13 was decreased.The proportion of cells in G0/G1 phase was increased.Cell cycle progression was blocked in the G1 phase.2. Through the above analysis and comparison of experimental results, found that 4 -amino-2-trifluoromethyl-phenyl ester of retinoic acid （4-amino-2-trifluoromethyl-phenyl retinate, ATPR） has powerful activity in inbibiting proliferation and inducing differentiation . After the treatment with ATPR,the growth of leukemic cells was inhibited in a dose-dependent and time-dependent manner.The NBT positive cell ratio,the expression level of the maturation specific cell surface marker CD11b、CD14 of NB4 cells were increased while the expression level of CD13 was decreased,which was similar to ATRA.The longer, the greater the change.The proportion of cells in G0/G1 phase increased while S phase cells decreased. Cell cycle progression was blocked in the G1 phase. The expression level of Cyclin D1、Cyclin E、CDK2、CDK4、CDK6、mRNA decreased after 3 days, while PCNA mRNA decreased after 7 days treatment with ATPR and the expression level of P57 kip2 mRNA was significantly increased.Conclusion:1. Above of the all,the retinoic acid derivatives retinoid 2a-03, 4a-02 and 5a-02 have powerful activity in inhibiting proliferation and inducing cell differentiation., suggesting that it is significant to develop a better retinoic acid-type drug with amino trifluoromethyl phenyl retinate as the starting point.2. ATPR （4a-02） has powerful activity in inbibiting proliferation and inducing differentiation through increasing the expression level of P57 kip2 mRNA to inhibit Cyclin-CDK kinase which plays a role in cell cycle arrest.更多还原