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目的探讨GRP78对人肝癌细胞株SMMC7721细胞耐药性和Topo-Ⅱ表达的影响。方法用低浓度顺铂(cDDP)短期诱导SMMC-7721细胞产生耐药,并用脂质体转染不同浓度GRP78反义寡核苷酸(GRP78ASODN)以降低SMMC-7721细胞GRP78的表达,应用RT-PCR检测并筛选一个最有效的降低GRP78mRNA表达的GRP78ASODN浓度,然后用MTT法检测亲本SMMC-7721细胞、耐药细胞及筛选出的转染组细胞对cDDP的药物敏感性,得出各组的半数抑制浓度(IC50)及耐药指数,并用RT-PCR检测三组细胞Topo-ⅡmRNA的表达。结果RT-PCR显示转染GRP78ASODN浓度为100nmol/ml时,GRP78mRNA降低最为显著(P<0.01);耐药组Topo-ⅡmRNA的表达较亲本细胞相显著降低(P<0.01),转染组与耐药组相比显著升高(P<0.01)。经诱导得到的耐药细胞,IC50是亲本细胞IC50的1.91倍,转染后,其IC50与转染前相比其耐药性显著降低(P<0.01)。结论用低浓度cDDP短期诱导SMMC-7721细胞产生耐药;GRP78能提高SMMC-7721细胞的耐药性,并与Topo-Ⅱ有关;抑制GRP78可以降低SMMC-7721细胞的耐药性。
Objective To investigate the effect of GRP78 on drug resistance and Topo-II expression in human hepatocellular carcinoma cell line SMMC7721. Methods The drug resistance of SMMC-7721 cells was induced by low concentration of cisplatin (cDDP) for a short time, and GRP78 antisense oligonucleotide (GRP78ASODN) was transfected with liposome to reduce the expression of GRP78 in SMMC-7721 cells. RT- PCR detected and screened one of the most effective GRP78ASODN concentrations that reduced GRP78 mRNA expression. MTT assay was used to detect the sensitivity of parental SMMC-7721 cells, drug resistant cells, and selected transfection group cells to cDDP, and half of each group was obtained. Inhibition of concentration (IC50) and resistance index, and the expression of Topo-II mRNA in three groups of cells were detected by RT-PCR. Results RT-PCR showed that GRP78 mRNA was significantly decreased when GRP78ASODN concentration was 100 nmol/ml (P<0.01). The expression of Topo-II mRNA in the drug resistant group was significantly lower than that of the parental cells (P<0.01). The drug group was significantly higher (P<0.01). IC50 was 1.91 times higher than IC50 of the parental cells after induction. After transfection, IC50 was significantly lower than that before transfection (P<0.01). Conclusion The low dose of cDDP induces drug resistance in SMMC-7721 cells for a short time. GRP78 can increase the resistance of SMMC-7721 cells and is related to Topo-II. Inhibition of GRP78 can decrease the drug resistance of SMMC-7721 cells.