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目的:研究二氯化钴(CoCl2)对缺氧反应元件调控E1AE1B表达的条件复制型腺病毒载体(Ad-5HRE-E1AE1B-RFP)和缺乏缺氧反应元件的复制缺陷型腺病毒载体(Ad-EGFP)在肿瘤细胞内表达和复制的影响。方法:肿瘤细胞经不同浓度的CoCl2处理后,Western印迹法检测缺氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的表达;倒置荧光显微镜、FCM法及空斑形成实验等观察经CoCl2处理后,感染条件复制型腺病毒和(或)复制缺陷型腺病毒的肿瘤细胞中外源基因的表达水平和病毒复制情况;小动物成像仪观察缺氧调控的条件复制型腺病毒在肿瘤内提高复制缺陷型腺病毒表达的效率。结果:适当浓度的CoCl2(0.4和0.08μg/mL)能使胃癌细胞株(SGC7901)中HIF-1α蛋白稳定表达并积聚,可较好地模拟缺氧状态;在0.4μg/mLCoCl2作用下,Ad-5HRE-E1AE1B-RFP对肿瘤细胞的感染效率明显提高,表现为外源基因RFP阳性表达的百分率和荧光强度均明显提高,但空斑形成实验显示Ad-5HRE-E1AE1B-RFP并没有复制。0.4μg/mLCoCl2也能提高其他非缺氧调控的复制缺陷型腺病毒如Ad-EGFP、Ad-Luc的感染效率和表达水平;Ad-5HRE-E1AE1B-RFP与复制缺陷型腺病毒Ad-Luc联合注射裸鼠肿瘤能显著提高Ad-Luc的表达。结论:CoCl2能明显提高腺病毒的基因表达水平,其作用机制不仅与CoCl2诱导缺氧有关,也可能与影响基因转录有关。
OBJECTIVE: To investigate the effect of cobalt chloride (CoCl2) on the replication-deficient adenovirus vector Ad-5HRE-E1AE1B-RFP which regulates the expression of E1AE1B by hypoxia-responsive elements and the replication-deficient adenovirus vector Ad- EGFP) expression and replication in tumor cells. Methods: The tumor cells were treated with different concentrations of CoCl2. The expression of hypoxia inducible factor-1α (HIF-1α) was detected by Western blotting. The expression of HIF-1α was detected by inverted fluorescence microscope, FCM and plaque formation assay. After infection with replication-competent adenovirus and / or replication-deficient adenovirus in tumor cells, the expression level of foreign genes and viral replication; small animal imaging device to observe hypoxia regulated replication-competent adenovirus in the tumor to improve replication The efficiency of defective adenovirus expression. Results: CoCl2 (0.4 and 0.08 μg / mL) could stably express and accumulate HIF-1α protein in gastric cancer cell line SGC7901, which could simulate hypoxia well. Under the action of 0.4μg / The infection rate of tumor cells by -5HRE-E1AE1B-RFP obviously increased, which showed that the percentage of RFP positive expression and fluorescence intensity of exogenous gene were significantly increased, but plaque formation experiments showed that Ad-5HRE-E1AE1B-RFP did not replicate. 0.4μg / mL of LCOOCl2 also increased the infection efficiency and expression level of other non-hypoxia-regulated replication-deficient adenovirus such as Ad-EGFP and Ad-Luc; Ad-5HRE-E1AE1B-RFP combined with replication-deficient adenovirus Ad-Luc Injecting tumor of nude mice can significantly improve the expression of Ad-Luc. CONCLUSION: CoCl2 can significantly increase the gene expression of adenovirus. The mechanism of action is not only related to the induction of hypoxia by CoCl2, but also may be related to the influence of gene transcription.