论文部分内容阅读
培养人高转移肺腺癌细胞系Anip973,构建其cDNA表达文库并转染小鼠成纤维细胞NIH3T3,将经药物筛选后的转染细胞克隆消化为单细胞,接种到软琼脂中培养2周,根据细胞明显的形态学变化挑选出有意义的细胞克隆,扩增再培养,提取DNA,PCR扩增插入片段并进行测序分析。结果表明:软琼脂中挑选出克隆100多个,PCR测序后,得到3个已知基因包括人类核糖体蛋白L23、人类假定蛋白FLJ22104和人类丝氨酸蛋白酶抑制因子6亚型以及一些氨基末端截短的核酸序列。进一步的研究表明转染人类核糖体蛋白L23的细胞与转染空载体细胞相比具有较高的侵袭能力(P<0.02)。利用cDNA文库在NIH3T3细胞中的表达,随后筛查鉴定在软琼脂中发生形态学变化的细胞,是一种寻找恶性转化和癌转移相关基因的有效方法。人类核糖体蛋白L23基因在细胞的运动和转移中发挥重要作用。
The human lung adenocarcinoma cell line Anip973 was cultured and its cDNA expression library was constructed and transfected into mouse fibroblasts NIH3T3. The transfected cells cloned by drug screening were digested into single cells, inoculated into soft agar for 2 weeks, According to the obvious morphological changes of the cells, the cell clones with significant significance were selected, amplified and cultured, and the DNA was extracted. The inserted fragment was amplified by PCR and sequenced. The results showed that more than 100 clones were selected in soft agar and three known genes including human ribosomal protein L23, human hypothetical protein FLJ22104 and human serine protease inhibitor 6 subtype and some amino-terminal truncated Nucleic acid sequence. Further studies showed that cells transfected with the human ribosomal protein L23 had a higher invasive capacity (P <0.02) than transfected empty vector cells. The use of cDNA library expression in NIH3T3 cells, followed by screening to identify cells that undergo morphological changes in soft agar, is an efficient way of finding genes involved in malignant transformation and cancer metastasis. The human ribosomal protein L23 gene plays an important role in cell motility and metastasis.