含精氨酸-甘氨酸-天冬氨酸肽的白细胞介素-24突变体真核表达载体的构建及其体外抗肝癌作用

目的 构建含精氨酸-甘氨酸-天冬氨酸(RGD)肽的白细胞介素(IL)-24突变体(RGD-IL-24)真核表达质粒,观察其对肝癌细胞的抑制作用.方法 利用重叠PCR技术在IL-24cDNA中插入GGT三个碱基,获得RGD-IL-24 cDNA.将其克隆入真核表达载体pcDNA3.1(+),构建pcDNA3.1/RGD-IL-24.将pcDNA3-1/RGD-IL-24及作为对照的pcDNA3.1/IL-24转染肝癌细胞HepG2,逆转录-聚合酶链反应(RT-PCR)检测IL-24 mRNA表达;Western blot检测IL-24蛋白表达;噻唑蓝(MTT)检测细胞增殖;FITC-Annexin V染色检测细胞凋亡;Western blot检测bax、bcl-2表达.结果 测序证明pcDNA3.1/RGD-IL-24构建成功.RT-PCR、Western blot证实转染pcDNA3.1/RGD-IL-24的肝癌HepG2细胞表达IL-24.pcDNA3.1/RGD-IL-24、pcDNA3.1/IL-24处理组HepG2细胞存活率分别为(0.382±0.064、0.563±0.038),凋亡细胞阳性率分别为(0.346±0.049、0.163±0.087),两组差异均有统计学意义.Western blot证实pcDNA3.1/RGD-IL-24促进bax表达、抑制bcl-2表达作用强于pcDNA3.1/IL-24.结论 RGD-IL-24真核表达质粒不仅不影响IL-24表达,且能显著增强IL-24诱导肝癌细胞凋亡及抑制肿瘤生长作用.

Construction of IL-24 mutant expression vector and research its therapic effect on HepG2 cells

Objective To construct an expression vector of IL-24 mutant RGD-IL-24, which a Glycine residue was inserted into the mda-7/IL-24 to form a Arg-Gly-Asp (RGD)motif and research its therapic effect on HepG2 cells.Methods Constructed RGD-IL-24 by means of overlapping PCR, which resulted in the extra codon GTT encoding the Glycine between Arg164 and Asp165 to form a RGD motif.The RGD-IL-24 was inserted inframe into pCDNA3.1 (+) expression vector to construct the pCDNA3.1/RGD-IL-24.HepG2 cells were transfected with pCDNA3.1/RGD-IL-24, as compare with pCDNA3.1/IL-24,which were collected at the expected time.IL-24 mRNA expression and protein expressions were observed by RT-PCR analysis and Western blot respectively.The proliferation and apoptosis were detected by MTT assays and FITC-Annexin V assays respectively.The pro-apoptotic protein bax and anti-apoptotic protein bcl-2 were detected by Western blot.Results The fragement encoded RGD-IL-24 was confirmed by nucleotide sequencing.IL-24 mRNA was detected by RT-PCR analysis 24 hours after transfected.IL-24 protein was detected by western blot analysis 48 hours after transfected.Transfected with pCDNA3.1/IL-24 and pCDNA3.1/RGD-IL-24 48 hours later, FITC-annexin V staining assays showed high levels of FITC-Annexin V positive cells in pCDNA3.1/RGD-IL-24 group.The significantly up-regulated pro-apoptotic protein bax and down-regulated anti-apoptotic protein bcl-2 were detected in pCDNA3.1/RGD-IL-24 treatment group than pCDNA3.1/IL-24 treatment group.Conclusion RGD-IL-24 significantly enhanced IL-24 therapeutic efficacy in HepG2 cells in vitro.