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目的 观察TGFβ1正、反义基因转染6 0 Coγ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 2 76bp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pMAMneo TGFβ1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6 0 Coγ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
Objective To observe the effect of TGFβ1 positive and antisense gene transfection with HELF irradiated by 60Co γ ray on the expression of TGFβ1 mRNA and type Ⅰ procollagen mRNA. Methods Lipofectamine was used to transfect the gene stably. The transfected cells were identified by PCR, DNA dot blot identification and RNA dot blot analysis. Results 5Gy irradiated cells were selected. TGFβ1 positive and antisense gene expression vectors pMAMneo TGFβ1 and pMAMneo AntiTGFβ1 were transfected into HELF by LipofectAMINE. The transfected cells were screened by G418 resistance and cultured under the induction of glucocorticoid dexamethasone. The RNA and chromosomal DNA of cultured cells were extracted. The transfected cells were positive for digoxigenin-labeled neo-specific probe, and the positive fragment of 76bp was amplified by neo gene-specific PCR primers. The digoxigenin-labeled TGFβ1 probe and α1 (Ⅰ) oligonucleotide probe of the extracted RNAs showed that the level of TGFβ1 mRNA decreased in transfected pMAMneo AntiTGFβ1 cells, while the level of TGFβ1 mRNA in transfected pMAMneo TGFβ1 cells Rise. For type I procollagen mRNA, the former was lower than the untransfected cells, while the latter was slightly higher. Conclusion Transfection of TGFβ1 antisense gene into human embryonic lung fibroblasts irradiated with 60 Co γ-rays can decrease the level of TGFβ1 mRNA and decrease the expression of type Ⅰ procollagen mRNA