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目的建立三氧化二砷(As2O3)的耐药细胞株,进一步从分子水平研究其耐药机制。方法采用高度短时间接触培养方法建立耐As2O3的白血病细胞系(SHI-1/AS2)。细胞倍增时间和耐药倍数测定采用MTT法;细胞周期和细胞免疫表型测定采用流式细胞分析术;常见的10个耐药基因检测和比较采用实时定量PCR(RQ-PCR)。结果耐As2O3的白血病细胞系SHI-1/AS2的细胞倍增时间与SHI-1相似,差异无统计学意义(P>0.05)。SHI-1/AS2细胞G0/G1期细胞少于SHI-1细胞,而G2/M、S期细胞则多于SHI-1细胞,差异均有高度统计学意义(均P<0.01)。SHI-1/AS2细胞对As2O3的耐药倍数是SHI-1的2.7倍,差异有统计学意义(P<0.01)。SHI-1/AS2细胞的集落形成率高于SHI-1细胞。SHI-1/AS2细胞的耐药相关基因Bcl-2有明显上调,Bax、Fas及MGMT下调,而其他耐药相关基因无明显变化。结论建立一株对As2O3产生耐药的白血病细胞系SHI-1/AS2,耐药倍数是SHI-1的2.7倍,其耐药机制与Bcl-2、Bax和Fas基因介导的细胞凋亡调控有关。
Objective To establish an arsenic trioxide (As2O3) resistant cell line and to further study its drug resistance mechanism at the molecular level. Methods Arsenic trioxide-resistant leukemia cell line (SHI-1 / AS2) was established by highly short-term contact culture. The cell doubling time and the multiple of drug resistance were determined by MTT method. The cell cycle and cell phenotype were determined by flow cytometry. The common 10 resistance genes were detected and compared by real-time quantitative PCR (RQ-PCR). Results The cell doubling time of SHI-1 / AS2 cells was similar to that of SHI-1 cells in As2O3-resistant leukemia cell line. The difference was not statistically significant (P> 0.05). The number of G0 / G1 phase cells in SHI-1 / AS2 cells was less than that in SHI-1 cells, while the number in G2 / M and S phase cells was higher than that in SHI-1 cells. The difference was statistically significant (all P <0.01). The multiple of resistance to As2O3 in SHI-1 / AS2 cells was 2.7 times that of SHI-1, with statistical significance (P <0.01). The SHI-1 / AS2 cells had higher colony formation rate than SHI-1 cells. The resistance-related genes Bcl-2, Bax, Fas and MGMT in SHI-1 / AS2 cells were significantly down-regulated while the other resistance-related genes did not change significantly. Conclusions The establishment of SHI-1 / AS2 cell line resistant to As2O3 is 2.7-fold more resistant than SHI-1, and its mechanism of drug resistance is related to the regulation of apoptosis mediated by Bcl-2, Bax and Fas genes related.