三种反义c-myc RNA 对肿瘤细胞顺铂敏感性的影响

C-myc is one of the most important members in oncogene families. It encodes nuclear phosphoproteins that appear to regulate cellular proliferation, differentiation, cell cycle, apoptosis and malignant transformation. Recently, c-myc have been identified as crucial to proliferative vascular diseases. C-myc is characteristic in gene structure, function and regulation. In order to investigate effects of different regions in c-myc molecule, determine the mechanism of c-myc transcription control, and to find out the best c-myc target region for the gene therapy of the proliferative diseases with c-myc overexpression, we used gene cloning technology to construct three antisense retrovirus expression vectors respectively for human c-myc exon 1,2 and 3, named pLNC-aM1, pLNC-aM2 and pLNC-aM3. Then, these three vectors were tranfered into three kinds of cell lines: vascular smooth muscle cell (VSMC), fibroblast cell NIH3T3 and myelocytic leukemia cell HL-60. The main results are as follows:1.All hree antisense c-myc RNA inhibited target gene expressions in three kinds of cell lines both in vitro and in vivo, but their inhibition intensity was different either between in vitro and in vivo, or among different cells. The effects of the transfected antisense RNA were variable, and seemed to mean the target cells could reduce the disturbance caused by the introduced gene. pLNC-aM2 displayed more powerful and stable inhibition effect , which suggested to block transcription activity regions (include AUG initiation code) usually can achieve more powerful and stable inhibition effect than to block transcription control regions or coding regions. It was also proved that target gene transcription involved in the mechanism of antisense RNA.2.pLNC-aM2 could prolong cell cycle of the target cell. However, in brief, antisense c-myc didn't induce extensive and remarkable changes in cell cycle progress or DNA synthesis. These appearances mentioned that cell cycle progress and DNA synthesis may not completely depend on c-Myc .3..PCNA expression level in target cell didn't change with the variety of Myc completely. These results suggested that PCNA doesn't wholly depend on the regulation of Myc . pLNC-aM2 downregulated PCNA expression in all experiments; pLNC-aM1 did so in NIH3T3 and SMC, but increased PCNA expression in HL-60; pLNC-aM3 inhibited PCNA expression in NIH3T3 and SMC in vivo, and increase it in other occasions, especially in HL-60..4.pLNC-aM2 could inhibit growth and proliferation of target cells in all tests, while the functions of pLNC-aM1 and pLNC-aM3 were unstable. The results also showed that not only growth of the malignant tumor cells and the benign tumor cells both with c-myc overexpression were inhibited by antisense c-myc RNA, but also growth of normal cells could be inhibited , which suggested local therapy or target therapy may be necessary for antisense c-myc gene therapy.5.Antisense c-myc transfected SMC could completely or partly transform into contractile phenotype from synthetic phenotype in vitro and in vivo. In HL-60, although reduced Myc expression might not be sufficient to commit the cells to differentiation, the presence of antisense c-myc appeared to change the developmental potential of HL-60 cells in the monocyte-macrophage pathway. Promotion of cell differentiation was accompanied with inhibition of cell proliferation and pLNC-aM2, having the strongest inhibition on cell proliferation, had the most significant effects on cell differentiation, both of which suggested that some of mechanisms may be similar or crossed between inhibiting proliferation and promoting differentiation, and that Myc may be at the crossed checkpoint.6.Antisense c-myc RNA, especially pLNC-aM3, could induce apoptosis of the cells. These results suggested that the specific DNA binding region is essential to regulate cell apoptosis. Furthermore, apoptosis induced by antisense c-myc RNA was often accompanied by PCNA high expression, suggesting a possibility that apoptosis-inducing agents may not induce cell mitosis sep

采用亚克隆技术构建三个反义c-myc重组逆转录病毒载体，分别载有c-myc的三个外显子，经病毒包装和滴度测定等，导入c-myc表达增高的大肠癌细胞、小细胞肺癌细胞和c-myc正常表达的正常肠腺细胞和肺上皮细胞中，观察三种反义基因导入后细胞在体内外对化疗药物顺铂的敏感性的变化，探讨反义c-myc调节细胞耐药性的机制并为其基因治疗的靶区选择提供实橐谰荨