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目的:观察转化生长因子β1(TGFβ1)转入对人肝癌细胞系癌基因表达的影响。方法:pL(TGFβ1)SN和相应重组病毒上清用脂质体转染包装细胞及肝癌细胞,经G418筛选后,进行免疫组化染色,观察癌基因蛋白表达的改变。结果:提取的TGFβ1cDNA连接物经酶切鉴定,证明有正向插入及反向插入两类。病毒上清TGFβ1含量在16~41ng/ml,套式PCR后均出现290bp条带。转基因后肝癌细胞癌基因蛋白免疫组化染色结果:c-myc,c-met及ras癌基因表达,反向TGFβ1转入者为(+)~(),而正向转入均为(-)。免疫组化检测其TGFβ1表达蛋白,空白对照染色(±),正向则为()。结论:正向插入的TGFβ1cDNA可使癌基因c-myc,c-met及ras表达减少,提示它在体外对肝癌细胞具有负调控作用。
Objective: To observe the effect of transforming growth factor β1 (TGFβ1) on the expression of oncogene in human hepatocellular carcinoma cell line. Methods: The pL (TGFβ1) SN and corresponding recombinant virus supernatants were transfected into packaging cells and hepatoma cells with liposome. After screening by G418, immunohistochemical staining was performed to observe the changes of oncogene protein expression. RESULTS: The extracted TGFβ1 cDNA conjugates were identified by enzymatic digestion and demonstrated positive insertion and reverse insertion. The viral supernatant TGFβ1 content was between 16 and 41 ng/ml, and a 290 bp band appeared after nested PCR. The results of immunohistochemical staining of hepatocellular carcinoma oncogene proteins after transgene: c-myc, c-met and ras oncogene expression, reverse TGFβ1 transduction were (+)~(), and positive transduction was (- ). The expression of TGFβ1 protein was detected by immunohistochemistry, blank control staining (±), positive (则). Conclusion: Positively inserted TGFβ1 cDNA can reduce the expression of oncogene c-myc, c-met and ras, suggesting that it has a negative regulatory effect on hepatoma cells in vitro.