摘要
目的建立环磷酰胺诱导的结直肠癌耐药细胞株HCT 116、HT-29,并探讨磷脂酰丝氨酸外翻与结直肠癌细胞环磷酰胺耐药的相关性。方法用流式细胞仪检测环磷酰胺耐药患者和非耐药患者磷脂酰丝氨酸外翻的表达,实时荧光定量PCR检测耐药相关基因的表达。采用CCK-8测定环磷酰胺的半数抑制浓度值,并以此为筛选浓度,在体外间歇诱导下建立环磷酰胺诱导的耐药细胞株模型。实时PCR检测4种多药耐药基因MRP、MDR1、Topo II、LRP在耐药细胞和非耐药细胞中的表达;蛋白质免疫印迹法检测MDR1蛋白的表达。流式细胞仪检测结直肠癌环磷酰胺耐药细胞和非耐药细胞磷脂酰丝氨酸外翻表达阳性率。结果环磷酰胺耐药患者相对非耐药患者的组织的磷脂酰丝氨酸外翻表达率相对较高,且耐药相关基因的表达增高。筛选出的HCT116、HT-29耐药组细胞与非耐药细胞比较,其细胞活性显著提高(P<0.05)。实时PCR和蛋白质免疫印迹法检测耐药相关MDR1蛋白在HCT116、HT-29耐药细胞株表达增高,差异有统计学意义(P<0.05)。流式细胞检测结直肠癌HCT116、HT-29耐药细胞相对非耐药细胞磷脂酰丝氨酸外翻的表达增高。结论结直肠癌耐药组织或耐药细胞中耐药相关基因表达水平高于非耐药组织或非耐药细胞,且磷脂酰丝氨酸外翻的表达与耐药相关基因的表达成正比,对评估CRC环磷酰胺耐药具有一定参考意义。
Objective To establish cyclophosphamide induced colorectal cancer cell lines HCT 116 and HT-29,and to explore the relationship between phosphatidylserine valgus and cyclophosphamide resistance in colorectal cancer cells.Methods Flow cytometry was used to detect the expression of phosphatidylserine valgus in cyclophosphamide resistant and non-resistant patients,and the expression of resistance related genes was detected by real-time PCR.CCK-8 was used to determine the median inhibitory concentration of cyclophosphamide,which was used to the screen concentration to establish a cyclophosphamide induced drug-resistant cell line model in vitro.Real-time PCR detected the expression of 4 multidrug resistance genes MRP,MDR1,Topo II,and LRP in drug-resistant and non-resistant cells;Western blotting detected the expression of MDR1 protein.Flow cytometry was used to detect the positive rate of phosphatidylserine valgus in cyclophosphamide resistant and non-resistant colorectal cancer cells.Results The expression rate of phosphatidylserine in the tissues of cyclophosphamide-resistant patients was relatively higher than that of non-resistant patients,and the expression of resistance-related genes was increased.Compared with non-drug resistant cells,the cell viability of the selected HCT116 and HT-29 drug-resistant cells was significantly increased(P<0.05).Real-time PCR and Western blotting methods detected that the expression of drug resistance-related MDR1 protein increased in HCT116 and HT-29 resistant cell lines,and the difference was statistically significant(P<0.05).Flow cytometric detection of colorectal cancer HCT 116,HT-29 drug-resistant cells with increased expression of phosphatidylserine ectropion relative to non-resistant cells.Conclusion The expression level of drug resistance-related genes in colorectal cancer drug-resistant tissues or drug-resistant cells is higher than that in non-resistant tissues or non-resistant cells,and the expression of phosphatidylserine valgus is proportional to the expression of drug-resistant genes,which is useful for evaluating CRC Cyclophosphamide resistance and has certain reference significance.
作者
王维
韩光宇
宋国权
李海林
WANG Wei;HAN Guang-Yu;SONG Guo-Quan;LI Hai-Lin(Hongqi Hospital Affiliated to Mudanjiang Medical College,Mudanjiang 157011,China)
出处
《中国药物经济学》
2021年第1期107-111,124,共6页
China Journal of Pharmaceutical Economics
基金
牡丹江市科学技术计划项目(Z2015s0039)。
关键词
结直肠癌
磷脂酰丝氨酸外翻
环磷酰胺
耐药
Colorectal cancer
Phosphatidylserine exposure
Cyclophosphamide
Drug resistance
