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近几年来我所已建立了人肝癌裸鼠原位移植转移模型,并获得了稳定的高转移肿瘤系 LCI-D20及低转移肿瘤系 LCI-D35。本工作比较研究了 LCI-D20和 LCI-D35二个肝癌系中 c-fos、c-jun,N-ras、H-ras,CD44,nm23-H1的基因表达以及 p53基因第七外显子249位点突变,以期深入探求 LCI-D20株高转移的原因。材料和方法一、LCI-D20,LCI-D35肝癌取材和 mRNA 提取分别取生长35天的 LCI-D20及 LCI-D35模型中新鲜肝癌组织各100mg,采用异硫氰酸胍-酚-氯仿一步法提取 mRNA,提取物分别溶于50μl DEPC 水中。二、RT-PCR 半定量分析取 mRNA 提取物10μl 70℃变性10分钟,以 oligo-dT 为引物,42℃逆转录1小时,反应后95℃变性10分钟。应用 PCR 扩增六种基因的 cDNA 产物,引物见附
In recent years, we have established a human orthotopic liver transplantation model of human hepatocellular carcinoma in nude mice, and obtained a stable high-metastatic tumor line LCI-D20 and a low-metastatic tumor line LCI-D35. This work compared the expression of c-fos, c-jun, N-ras, H-ras, CD44, and nm23-H1 in the LCI-D20 and LCI-D35 liver cancer lines and the exon seventh exon 249 of the p53 gene. Site mutations, with a view to explore the reasons for high metastasis of LCI-D20 strains. Materials and Methods 1. LCI-D20, LCI-D35 liver cancer extracts and mRNA extraction Take LCI-D20 for 35 days and 100 mg for fresh HCC tissues in LCI-D35 model respectively, using one-step method of guanidinium isothiocyanate-phenol-chloroform mRNA was extracted and the extracts were separately dissolved in 50 μl of DEPC water. Second, semi-quantitative RT-PCR analysis Take mRNA extract 10μl denaturation at 70 °C for 10 minutes, using oligo-dT as a primer, 42 °C reverse transcription for 1 hour, 95 minutes after the reaction for 10 minutes. PCR amplification of cDNA products of six genes, primer attachment