摘要
目的:研究新型溶瘤腺病毒GP73-SphK1sR-Ad5对肝癌细胞生长的抑制作用。创新点:GP73-SphK 1sR-Ad5是一种新型的溶瘤腺病毒,可以特异及有效地抑制肝癌细胞生长,为肝癌的临床治疗提供新思路。方法:通过整合高尔基体蛋白73(GP73)及鞘氨基醇激酶1(SphK 1)构建了GP73-SphK1sR-Ad5腺病毒,进而转染肝癌Huh7细胞及正常HL7702肝细胞。通过实时定量PCR和蛋白印记实验检测Sph K1和E1A基因的表达;通过四氮唑盐比色分析法(MTT法)检测细胞活力;通过流式细胞术检测细胞凋亡率。构建Huh7异种移植小鼠模型,并注射GP73-SphK1sR-Ad5腺病毒。20天后,记录小鼠肿瘤体积和重量,以及存活时间。用苏木精-伊红(HE)染色法和透射电镜(TEM)观察肿瘤组织的病理变化。结果:GP73-SphK 1sR-Ad5转染显著上调了Huh7细胞中E1A的表达,下调了SphK 1的表达,降低了细胞活性,并提高了凋亡率,然而对HL7702细胞无明显影响。Huh7异种移植小鼠模型内注射GP73-SphK 1sR-Ad5显著降低了肿瘤的体积和重量,延长了小鼠的存活时间,并降低了肿瘤组织中的肿瘤浸润面积、血管密度及核变形和染色质浓缩的细胞数。结论:新型溶瘤腺病毒GP73-Sph K1s R-Ad5可以特异和有效地抑制肝癌进展。
Objective: To evaluate the inhibitory role of a novel oncolytic adenovirus(OA), GP73-SphK1 sR-Ad5, on the growth of hepatocellular carcinoma(HCC). Methods: GP73-SphK1 sR-Ad5 was constructed by integrating Golgi protein 73(GP73) promoter and sphingosine kinase 1(SphK1)-short hairpin RNA(shRNA) into adenovirus serotype 5(Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1(E1 A) was detected by quantitative real-time PCR(qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK 1 sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM). Results: Transfection of GP73-SphK1 sR-Ad5 significantly upregulated E1 A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1 sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1 sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues. Conclusions: GP73-SphK1 sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.
基金
Project supported by the Shandong Provincial Science and Technology Development Plan Project(No.2013GSF11853),China
