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目的研究携带组织特异性E2F-1启动子的重组腺病毒载体的肿瘤细胞靶向性作用。方法利用AdEasy-1系统,构建含有E2F-1启动子的重组腺病毒并对重组病毒中E2F-1启动子基因进行PCR及测序鉴定。将分别携带E2F-1启动子和CMV启动子并且可以表达绿色荧光蛋白报告基因GFP基因的重组腺病毒分别转染LS174T和H292细胞,观察GFP基因表达情况;以携带肿瘤杀伤基因vpr基因的靶向性重组腺病毒rvAdE2F-1/vpr分别转染人二倍体细胞L-02、H292和肿瘤细胞SMMC-7721、LS174T,MTT法检测rvAdE2F-1/vpr对不同细胞的抑制增殖的作用;用蛋白免疫印迹的方法检测肿瘤细胞和二倍体细胞中靶蛋白E2F蛋白的表达。结果在相同的培养条件下,E2F-1启动子可使下游的GFP基因在肿瘤细胞LS174T中大量表达而在二倍体细胞H292中微量表达,CMV启动子下游的GFP基因表达量则无明显差异;且在LS174T细胞中两种启动子转录活性相似(P>0·05);E2F-1启动子调控下vpr基因的表达可以选择性的抑制肿瘤细胞增殖而对二倍体细胞无抑制作用;E2F-1蛋白在肿瘤组织中高表达而在正常组织中的表达不能用WesternBlot检测到。结论以E2F-1为启动子的重组腺病毒载体具有肿瘤细胞靶向性,并且有足够的启动效率调控杀伤基因作用于肿瘤细胞,因此是一个具有应用价值的靶向治疗体系。
Objective To study the tumor cell targeting effect of recombinant adenovirus vector carrying tissue-specific E2F-1 promoter. Methods AdEasy-1 system was used to construct the recombinant adenovirus containing E2F-1 promoter. The recombinant adenovirus E2F-1 promoter was identified by PCR and sequencing. The recombinant adenovirus carrying the E2F-1 promoter and CMV promoter and expressing the green fluorescent protein reporter gene GFP gene was transfected into LS174T and H292 cells respectively to observe the expression of GFP gene. The targeting of the vpr gene carrying the tumor-killing gene The recombinant adenovirus rvAdE2F-1 / vpr were transfected into human diploid cells L-02, H292 and tumor cells SMMC-7721, LS174T, MTT assay rvAdE2F-1 / vpr on proliferation inhibition of different cells; Immunoblotting was used to detect the expression of target protein E2F protein in tumor cells and diploid cells. Results Under the same culture condition, the E2F-1 promoter could make the downstream GFP gene express abundantly in the LS174T tumor cells and slightly in the diploid cells H292, while there was no significant difference in the GFP gene expression downstream of the CMV promoter (P> 0.05). The expression of vpr gene under the regulation of E2F-1 promoter could selectively inhibit the proliferation of tumor cells and had no inhibitory effect on diploid cells in LS174T cells. E2F-1 protein is overexpressed in tumor tissues but not in normal tissues. CONCLUSION: The recombinant adenovirus vector with E2F-1 as promoter has tumor cell targeting and has sufficient start-up efficiency to regulate killer genes on tumor cells. Therefore, it is a targeted therapeutic system with application value.