【作者】 周峰；

【导师】 许小平；

【作者基本信息】 复旦大学 ， 内科学， 2011， 硕士

【摘要】 目的：建立耐高三尖杉酯碱SKM-1细胞系,初步探讨该细胞系的生物学特性及多药耐药机制。方法：通过较大剂量间断冲击诱导法(pulsatile treatment)逐渐增加药物浓度的方法建立耐高三尖杉酯碱SKM-1细胞系。光学显微镜观察耐药细胞系和亲本细胞系的一般形态学变化,MTT法测定倍增时间和耐药指数,绘制生长曲线；利用流式细胞仪检测细胞周期分布及细胞内柔红霉素含量；常规遗传学方法R显带后进行核型分析；观察两种细胞在裸鼠成瘤的差异；多药耐药相关基因(mdr-1及MRP)及topo-IIa的表达采用RT-PCR方法检测。结果：历经7个月的培养,建立了耐药细胞系SKIM-1/HHT,并对耐药细胞进行生物学特性观察。形态上,耐药细胞与亲本细胞相似,悬浮生长,细胞大小不均一,体积偏大,核浆比例高,胞浆及胞核内见空泡；耐药及亲本细胞免疫表型近似：SKM-1细胞为CD2-、CD3-、CD4-、CD5-、CD7-、CD8-、CD11b+(42.28%)、CD13+(65.35%)、CD14-、CD33+(99.45%),SKM-1/HHT为CD2-、CD3-、CD4-、CD5-、CD7-、CD8-、CD11b+(46.81%)、CD13+(83.37%)、CD14-、CD33+(99.61%)：R显带核型分析示耐药细胞与亲本细胞大体一致但有部分差异；细胞周期分布检测发现耐药细胞G1期细胞增多,S期和G2期细胞减少；两种细胞裸鼠成瘤率为0%；耐药细胞系与亲本细胞耐药谱分析比较,KM-1/HHT对HHT耐药为17.94倍,对长春新碱(Vincristine, VCR)、柔红霉素(Danuorubicin, DNR)、依托泊苷(Etoposide, VP-16)耐药指数分别为8.78倍、5.99倍、13.76倍；流式细胞仪检测耐药细胞内DNR荧光量明显低于亲本细胞；半定量real-time PCR示：耐药细胞系mdr-1表达显著增高(20.1倍),MRP轻度升高(3.56倍),topo-Ⅱa表达下降(1：0.619)。结论：建立了对HHT耐药的MDS转白血病细胞系SKM-1/HHT,其耐药机制主要涉及mdr-1的过度表达而导致的细胞外排增强,MRP及topo-Ⅱa的改变也可能部分参与了这一过程。更多还原

【Abstract】 Objective:To estabilish a homoharringtonine(HHT)-resistant SKM-1 cell line and explore the biologic characteristics and mechanisms for drug resistance.Methods:The HHT-resistant SKM-1 cell line was established by repeatedly exposing the cells to comparatively large doses of HHT with a short-time duration, and gradually elevating the drug concentration to an endurable level.The morphology of the resistant and parental cell lines were observed through optical microscope.The MTT assay was used to determine the doubling time,the resistance index,and to draw growth curve.The immunophenotype, cell cycle distribution and DNR accumulation between SKM-1 and SKM-1/HHT were analyzed by flow cytometry and the karyotypes by R-banding.Both cell lines were compared in the nude mice nodulation.To evaluate the expression level of mdr-1, MRP and topo-IIa, semi-quantitative real-time PCR was performed.Results:Through 7-month drug induction, the HHT-resistant cell line,SKM-1/HHT,was eventually established. In morphology, the resistant cel line was the same to the parental cell line, both growing in suspension with big volume, size heterogeneity and large nuclear-plasma ratio. Vacuoles were seen in both the cytoplasma and nucleus cavity. The immunophenotpyes of two cell types were similar, SKM-1:CD2-、CD3-、CD4-、CD5-、CD7-、CD8-、CD11b+(42.28%)、CD13+(65.35%)、CD14-、CD33+(99.45%), SKM-1/HHT:CD2-、CD3-、CD4-、CD5-、CD7-、CD8-、CD11b+(46.81%), CD13+(83.37%). CD14-、CD33+(99.61%).The karyotypes were roughly the same, but with some differences.The resistant cell line had more G1 phase cells, less S phase and G2 phase cells compared to the parental cell line.Both cell lines cannot get nodulation in nude mice within 30 days.The drug resistance of SKM-1/HHT cell to HHT,VCR, DNR, and etoposide all increased. The resistance indices of HHT, VCR, DNR, and etoposide were 17.94,8.78,5.99, and 13.76,respectively.DNR accumulation was impaired in SKM-1/HHT. Both mdr-1 and MRP increased at gene level, though mdr-1 was prominent (20.1-fold). And there was a slightly decrease of topo-IIa (1:0.619) Conclusion:A HHT-resistant MDS-AML cell line,SKM-1/HHT,was established. The prominent overexpression of mdr-1 may be the main cause for multidrug resistance.更多还原