| 刘纯杰,王德文,张兆山,龙建银,王会信,高亚兵,熊呈琦,彭瑞云,崔雪梅,宋良文.TGFβ1正、反义基因转染60Co照射HELF对TGFβ1及I型前胶原mRNA表达调控影响[J].中华放射医学与防护杂志,2001,21(2):103-106 |
| TGFβ1正、反义基因转染60Co照射HELF对TGFβ1及I型前胶原mRNA表达调控影响 |
| Gene transfection using lipid mediated TGFβ1 sense and antisense gene expression vectors and its effects on TGFβ1 and procollagen I mRNA expression in 60Co-irradiated human embryo lung fibroblasts |
| 投稿时间：2000-06-18 |
| DOI： |
| 中文关键词: TGFβ1 基因转染 |
| 英文关键词:Transforming growth factor beta 1 Gene transfection |
| 基金项目:全军九五指令基金资助项目(96L011) |
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| 中文摘要: |
| 目的 观察TGFβ1正、反义基因转染60Coγ射线照射的HELF后对其TGFβ1mRNA及Ⅰ型前胶原mRNA表达调控的影响。方法 采用脂质体介导法进行基因稳定转染,转染细胞经PCR,DNAdotblot鉴定和RNAdotblot分析。结果 选择5Gy照射细胞,采用LipofectAMINE将TGFβ1正、反义基因表达载体pMAMneo-TGFβ1和pMAMneo-AntiTGFβ1转入HELF,转染细胞经G418抗性筛选出来,并且在糖皮质激素地塞米松诱导下培养。提取培养细胞的RNA和染色体DNA,转染细胞DNA与地高辛标记的neo特异探针杂交阳性,且可用neo基因特异的PCR引物扩增出276bp的阳性片段。对提取的RNA用地高辛标记的TGFβ1探针和α1(Ⅰ)寡核苷酸探针经RNAdotblot分析表明,转染pMAMneo-AntiTGFβ1的细胞其TGFβ1mRNA水平下降,而转染pMAMneoTGFβ1的细胞其TGFβ1mRNA水平则升高。对于Ⅰ型前胶原mRNA,前者表达水平比未转染细胞低,后者则略高。结论 TGFβ1反义基因转染60Coγ射线照射的人胚肺成纤维细胞后可以使细胞中TGFβ1mRNA含量水平减少,使Ⅰ型前胶原mRNA表达水平降低。 |
| 英文摘要: |
| Objective To investigate the effects on gene expression of 60Co-irradiated human embryo lung fibroblasts after gene transfection using lipid mediated TGFβ1 sense and antisense gene expression vectors. Methods TGFβ1 sense and antisense gene expression vectors were transfected using a lipid mediated method.Gene expression was analysed by RNA dot blot. Results HELFs irradiated with 5 Gy were transfected with an expression vector encoding the human TGFβ1 sense or antisense gene under control of the mouse mammary tumor virus long terminal repeat(MMTV-LTR)promotor/enhance sequence(pMAMneo-TGFβ1,or pMAMneo-anti TGFβ1).The transfected cells elected by G418 resistance were cultured in DMEM containing dexamethasone.The chromosomal DNA and RNA were extracted.Positive reaction was showed from chromosomal DNA by a PCR method of neo-specific primers and DNA dot blot with Dig-labelling neo-specific probe.RNA dot blot analysis showed that TGFβ1 mRNA level of the cells transfected with pMAM neo-anti TGFβ1 decreased,but that of transfected with pMAM neo-TGFβ1 increased.For procollagen Ⅰ mRNA,the transfected pMAM neo-anti TGFβ1 was lower than untransfected cells and the transfected pMAM neo-TGFβ1 was higher. Conclusion After TGFβ1 sense and antisense gene transfection,TGFβ1 mRNA level of the cells transfected with TGFβ1 antisense gene decreased,but that with TGFβ1 sense gene increased.For procollagen Ⅰ mRNA,the cells transfected with TGFβ1 antisense gene was lower than untransfected cells and the cells transfected with TGFβ1 sense gene was higher than untransfected cells. |
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