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背景与目的:骨髓造血细胞中导入耐辐射基因是克服放疗对造血系统抑制作用的有效手段,但这也增加了肿瘤细胞的辐射耐受性。本实验旨在探索不明显增加肿瘤细胞对辐射抗拒性的同时,能特异性保护骨髓细胞免受辐射导致的损伤的方法。方法:构建以氨肽酶N(aminopeptidaseN,APN)基因骨髓特异性启动子调控的锰超氧化物歧化酶(manganesesuperoxidedismutase,MnSOD)基因逆转录病毒载体,并导入成骨髓细胞KG1a和肝癌细胞BEL7402中。用RT-PCR分析MnSODmRNA水平,测定细胞中MnSOD活性,以细胞存活实验检测骨髓细胞和肝癌细胞对X射线的敏感性,并用流式细胞术和断裂DNA电泳方法对辐射诱导的细胞凋亡进行分析。结果:导入基因的KG1a细胞中MnSODmRNA水平和酶活性明显升高。APN骨髓特异性启动子调控的MnSOD基因表达能有效抑制辐射引起的KG1a细胞凋亡。导入基因的KG1a细胞对辐射的耐受性提高,在用10Gy剂量照射时,KG1a细胞存活率比原来增加3.7倍。而导入MnSOD基因并未使BEL7402细胞中MnSODmRNA水平和酶活性提高,其放射敏感性未发生明显变化。结论:APN骨髓特异性启动子能控制MnSOD基因在骨髓细胞中高表达,而在癌细胞中低表达。在用X射线杀伤癌细胞的过程中,APN骨髓特异性启动子调控的MnSOD基因能特异性保护骨髓细胞。
BACKGROUND & OBJECTIVE: Introduction of anti-radiation gene into bone marrow hematopoietic cells is an effective way to overcome the inhibitory effect of radiotherapy on hematopoietic system, but it also increases the radiation tolerance of tumor cells. The aim of this experiment was to explore ways to specifically protect bone marrow cells from radiation-induced damage without significantly increasing the radioresistance of tumor cells. Methods: The recombinant retroviral vector carrying MnSOD gene regulated by bone marrow specific promoter of aminopeptidase N (APN) gene was constructed and introduced into myeloid cell KG1a and hepatocellular carcinoma BEL7402. MnSOD mRNA levels were assayed by RT-PCR, MnSOD activity in cells was measured, and cell viability assay was used to detect X-ray sensitivity of bone marrow cells and hepatoma cells. Radiation-induced apoptosis was analyzed by flow cytometry and DNA fragmentation assay . Results: MnSOD mRNA and enzyme activity of KG1a cells were significantly increased. The expression of MnSOD gene regulated by APN bone marrow-specific promoter can effectively inhibit the apoptosis of KG1a cells induced by radiation. KG1a cells transfected with the gene increased the radiation tolerance, and the survival rate of KG1a cells increased by 3.7 times when irradiated with the 10 Gy dose. The introduction of MnSOD gene did not increase MnSOD mRNA level and enzyme activity in BEL7402 cells, and its radiosensitivity did not change significantly. CONCLUSION: The bone marrow-specific promoter of APN can control the expression of MnSOD gene in bone marrow cells, but low in cancer cells. During x-ray killing of cancer cells, the MnSOD gene regulated by the APN bone marrow-specific promoter specifically protects bone marrow cells.