【作者】 王颖；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 药理学， 2004， 博士

【摘要】 第一部分目的 探讨全反式维甲酸（all-trans-retinoicacid,ATRA）对小细胞肺癌细胞的诱导分化作用。方法 细胞培养至对数生长期，给予不同浓度不同时间的全反式维甲酸诱导分化，MTT法检测细胞体外增殖能力，电镜形态学的观察，流式细胞仪进行细胞周期分析，角蛋白表达作为检测小细胞肺癌分化的特异性指标，RT-PCR法维甲酸受体亚型基因分析。结果 全反式维甲酸浓度为 20μmol/L, 10μmol/L, 5μmol/L，2.5μmol/L, 1.25μmol/L时诱导分化小细胞肺癌NCI-H446细胞株，24小时 OD值分别为0.498,0.502,0.503,0.507,0.510，各浓度间无显著性差别（P>0.05）；48小时 OD值分别为0.292, 0.342,0.467,0.498,0.521, ATRA20μmol/L与5μmol/L有显著性差别（P<0.01）； 72小时 OD值分别为0.206,0.312, 0.415, 0.482，0.509，ATRA20μmol/L与10μmol/L有显著性差别（P<0.05）；96小时 OD值分别为0.189,0.251,0.380,0.487,0.506。由此可见随浓度的增加，作用时间的延长，OD值降低，活细胞数目减少，细胞存活力明显下降； 在一定的药物浓度范围内，经过一定的作用时间，ATRA对细胞的抑制增殖作用呈量效关系，ATRA10μmol/L为最佳药物浓度，<WP=10>ATRA干预最佳时间为72h。 ATRA10μmol/L诱导分化细胞24h,48h,72h,凋亡形成率为4.9%, 8.8%, 11.45%; 而对照组为4.08%, 4.10%, 4.18%。72h二组有显著性差别（P<0.05）,更多的细胞被阻止于G1/G0期,提示静止期细胞增多，DNA合成受到抑制。药物组光镜可见细胞增殖减慢，细胞折光性减弱，胞质中可见空泡，细胞形态不规则，细胞间空隙增大。透射电镜观察细胞内超微结构可见细胞核明显变小，核/浆比值减少，细胞核成分叶状，部分细胞核固缩，染色质浓集，呈凋亡细胞特征，并可见凋亡小体。免疫组化检测角蛋白表达明显下降，提示细胞向良性分化；RT-PCR电泳结果进行计算机图象分析，测定各条带的灰度值，药物组RARβ,RXRα为143.58,78.89; 对照组为105.33,45.37。显示维甲酸诱导分化后受体亚型RARβ,RXRα基因表达上调。结论 全反式维甲酸分化诱导能使小细胞肺癌NCI-H446细胞株维甲酸受体基因表达上调，并有效的抑制细胞的增殖。更多还原

【Abstract】 PART ONEOBJECTIVE TO study the differentiation and proliferation of the Small Cell Lung Cancer that were treated by all-trans-retinoic acid(ATRA). METHODS the cells were treated by ATRA with different concentration. After that,the proliferation and differentiation of NCI-H446 cells were examined by the methods of MTT , the electron microscope,and cytometry analysis.The expression of cornifin protein was tested by IHC, The primers of were designed based on the sequence of gene bank and the express of RARS and RXRS were detected with RT-PCR .RESULT ATRA could obviously inhibit the growth and the differentiation of NCI-H446 cells. When the ATRA concentration were 20μmol/L,10μmol/L,5μmol/L, 2.5μmol/L after 24h, the results of OD were 0.498,0.502,0.503,0.507,0.510. the difference is not significant(P>0.05).the results of OD were0.292, 0.342, 0.467,0.498,0.521 after 48h. the difference <WP=15>is significant(P<0.01) between ATRA20μmol/L group and 5μmol/L group; the results of OD were 0.206，0.312，0.415,0.482,0.509 after 72h, the difference is significant(P<0.05) between ATRA20μmol/L group and 10μmol/L group; the results of OD were 0.189,0.251,0.380,0.487,0.506 after 96h. Therefor the higher ATRA concentration was, the plus the rate of growth inhibition was. ATRA10μmol/L is the best concentration of treatment. The apoptosis of the cells treated by ATRA after 24h,48h,72h were 4.9%, 8.8%, 11.45%; the balance were 4.08%, 4.10%, 4.18%。the difference is significant(P<0.05) after 72h. There were more ATRA-NCI cell arrested in G1/G0 phase. The electron microscope observes that the ATRA cell nuclears diminish, the rates of nuclear/plasma reduce, and the apoptosis cells increase. The expression of cornifin protein decreased in ATRA group, the difference is significant(P<0.05); RT-PCR Electrophoresis graydegree of RARβ and RXRαin ATRA group after 72h treatment were 143.58,78.89; the balance were 105.33,45.37。The positive rate of RARS and RXRS was higher in ATRA cell than non-ATRA cell,the difference is significant (P<0.05).CONCLUSIONS ATRA could inhibit the proliferation and induce differention of the Small Cell Lung Cancer.更多还原