IL-24重组载体的构建及其联合化疗药物对SKOV3细胞中TUBB3与ERCC-1基因表达的影响

doi:10.7503/cjcu20140975

摘要/Abstract

摘要：

通过基因重组技术将白介素24(IL-24)基因片段分别克隆成pcDNA3.0-OSP-1-IL-24和pcDNA3.0-IL-24载体, 利用逆转录PCR(RT-PCR)测定2种载体的表达. 将2种化疗药物紫杉醇(PTX)与顺铂(DDP)分别作用于正常SKOV3细胞及pcDNA3.0, pcDNA3.0-IL-24, pcDNA3.0-OSP-1和pcDNA3.0-OSP-1-IL-24稳定转染SKOV3细胞(卵巢癌细胞株), 通过噻唑蓝(MTT)法检测化疗药物联合IL-24基因对细胞的杀伤效果及细胞的增殖能力, 应用实时荧光定量PCR技术检测人β-微管蛋白3(TUBB3)与核苷酸切除修复交叉互补基因位1(ERCC1)基因在各组细胞中的表达. RT-PCR检测结果显示, 通过基因重组技术克隆了pcDNA3.0-IL-24与pcDNA3.0-OSP-1-IL-24载体, IL-24基因能提高SKOV3细胞对化疗药物顺铂和紫杉醇的敏感性. 其联合化疗药物对肿瘤药物的IC₅₀值分别下降了10倍和6倍, TUBB3和ERCC1基因在IL-24基因转染的SKOV3细胞内的表达水平与在正常SKOV3细胞内相比, 分别下降4倍和10倍多. 上述结果表明, IL-24联合化疗药物可明显下调TUBB3与ERCC1基因的表达, 该实验为临床治疗卵巢癌提供了重要的理论依据.

关键词: IL-24基因, 顺铂, 紫杉醇, SKOV3细胞

Abstract:

Gene fragments of IL-24 were cloned into pcDNA3.0-OSP-1-IL-24 and pcDNA3.0-IL-24 vector using gene recombination technology, the expression of two vectors was measured by RT-PCR methods. The two chemotherapy drugs of paclitaxel and cisplatin were applied into normal SKOV3 cells, stable transfected SKOV3 cells of pcDNA3.0, PcDNA3.0-IL-24, pcDNA3.0-OSP-1 and pcDNA3.0-OSP-1-IL-24, respectively. The cell killing effect and proliferation ability of IL-24 in combination with chemotherapy drugs were detected by 3-(4,5)-dimethylthiahiahiazo-(z-yl)-2,5-di-phenytetrazolium bromide(MTT) in SKOV3 cells. Gene expression of TUBB3 and ERCC-1 were detected by real-time fluorescent quantitative polymerase chain reaction(PCR) technology in cell of each group. RT-PCR results showed that the pcDNA3.0-IL-24 and pcDNA3.0-OSP-1-IL-24 vector were cloned successfully by gene recombination technology. IL-24 gene can increase the sensitivity of SKOV3 cells to chemotherapeutic drug of cisplatin and paclitaxel. Its combination with chemotherapy drugs for IC₅₀ of tumor drugs decreased 10 times and 6 times, respectively. Gene expression of TUBB3 gene and ERCC-1 decreased 4 times and 10 times, respectively. These results suggested that IL-24 in combination with chemotherapy drugs could reduce gene expression of TUBB3 and ERCC-1. This experiment provided an important theory basis for the clinical treatment of ovarian cancer.

Key words: Interleukin-24, Cisplatin, Paclitaxel, SKOV3 cell

中图分类号:

O629
R737.31

TrendMD:

祝贺, 杜珍武, 范丽梅, 高海成, 崔满华. IL-24重组载体的构建及其联合化疗药物对SKOV3细胞中TUBB3与ERCC-1基因表达的影响. 高等学校化学学报, 2015, 36(5): 927.

ZHU He, DU Zhenwu, FAN Limei, GAO Haicheng, CUI Manhua. Construction of Recombinant Vector on IL-24 and Its Effect in Combination with Chemotherapy Drugs for TUBB3 and ERCC-1 Expression in SKOV3 Cells^†. Chem. J. Chinese Universities, 2015, 36(5): 927.

图/表 5

参考文献 20

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Gene	Primer sequence	Base pair
ERCC-1	F: 5'-ACGCCGAATATGCCATCTCAC-3'	292 bp
	R: 5'-AGCCGCCCATGGATGTAGTCT-3'
TUBB3	F: 5'-TTCCAGCTGACCCACTCTCT-3'	226 bp
	R: 5'-ACAGGGCCTCGTTATCAATG-3'
GAPDH	F: 5'-CCACATCGCTCAGACACCAT-3'	196 bp
	R: 5'-ACGGTGCCATGGAATTTGCC-3'

Gene	Primer sequence	Base pair
ERCC-1	F: 5'-ACGCCGAATATGCCATCTCAC-3'	292 bp
	R: 5'-AGCCGCCCATGGATGTAGTCT-3'
TUBB3	F: 5'-TTCCAGCTGACCCACTCTCT-3'	226 bp
	R: 5'-ACAGGGCCTCGTTATCAATG-3'
GAPDH	F: 5'-CCACATCGCTCAGACACCAT-3'	196 bp
	R: 5'-ACGGTGCCATGGAATTTGCC-3'

Drug	IC₅₀/(μg·mL^-1)
Drug	SKOV3 cell	pcDNA3.0- SKOV3 cell	pcDNA3.0-OSP-1- SKOV3 cell	pcDNA3.0-IL-24- SKOV3 cell	pcDNA3.0-OSP-1- IL-24- SKOV3 cell
DDP	5.760±0.560	5.012±0.263	5.165±0.564	0.505±0.032^*	0.567±0.041^*
PTX	12.760±1.870	11.452±2.852	10.989±1.568	2.120±0.690^*	3.142±0.041^*

Drug	IC₅₀/(μg·mL^-1)
Drug	SKOV3 cell	pcDNA3.0- SKOV3 cell	pcDNA3.0-OSP-1- SKOV3 cell	pcDNA3.0-IL-24- SKOV3 cell	pcDNA3.0-OSP-1- IL-24- SKOV3 cell
DDP	5.760±0.560	5.012±0.263	5.165±0.564	0.505±0.032^*	0.567±0.041^*
PTX	12.760±1.870	11.452±2.852	10.989±1.568	2.120±0.690^*	3.142±0.041^*

Gene	IC₅₀/(μg·mL^-1)
Gene	SKOV3 cell	pcDNA3.0- SKOV3 cell	pcDNA3.0-OSP-1- SKOV3 cell	pcDNA3.0-IL-24- SKOV3 cell	pcDNA3.0-OSP-1- IL-24-SKOV3 cell
ERCC1	1.240±0.230	2.012±0.293	1.165±0.574	0.112±0.023^*	0.141±0.027^*
TUBB3	1.540±0.130	1.452±0.872	1.989±0.569	0.426±0.023^*	0.445±0.074^*