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目的 克隆肿瘤转移相关基因。方法 应用倍比稀释法对人肺巨细胞癌PG进行单细胞克隆 ,并应用裸鼠异种接种及自发性转移体内实验对各亚系的转移潜能进行鉴定。以具有不同转移潜能的癌细胞亚系为研究对象 ,应用mRNA差异显示技术克隆差异片段 ,sanger双脱氧末端终止法测序及自动双向测序获得其碱基序列 ,与GenBank数据库进行同源性比较。在两组具有不同转移潜能的癌细胞亚系 (人肺癌 ,前列腺癌 )中Northern杂交验证表达差异。结果 两个具有不同转移潜能的亚系BE1和LH7,在完全相同的遗传背景下 ,亚系BE1在裸鼠体内 10 0 %成瘤 ,10 0 %转移 ;亚系LH7在裸鼠体内 10 0 %成瘤 ,无一例转移。二者转移表型的显著差异必然反映了转移相关基因表达与调控的差异。因此该系统是mRNA差异显示的良好模型。应用mRNA差异显示技术克隆差异片段 ,一个84 9bp片段在高转移亚系BE1中低表达 ,而在不转移亚系LH7高表达 ,二者差异显著 ,在重复实验中得到了验证。成功的回收该片段 ,连接入pGEM T载体 ,经sanger双脱氧末端终止法测序及自动双向测序获得其碱基序列 ,经GenBankBlastn检索 ,与G3BP(U32 519)同源性高达 99%。Northern杂交显示G3BP在LH7高表达 (约 3 3kb) ,而在BE1中低表达 ,验证了差异显示的结果 ,排除了假阳性的可能性
Objective To clone tumor metastasis-related genes. Methods Single cell cloning of human pulmonary giant cell carcinoma (PGC) was performed by the double dilution method. The metastatic potential of each subline was identified by xenogeneic inoculation and spontaneous metastasis in nude mice. The subpopulations of cancer cells with different metastatic potentials were used as research subjects. mRNA differential display technology was used to clone differential fragments, sanger dideoxy end termination sequencing and automatic two-way sequencing to obtain their base sequences, and compared with GenBank database for homology. Northern blots were used to verify differential expression in two subsets of cancer cells with different metastatic potentials (human lung cancer, prostate cancer). Results The two sublineages BE1 and LH7 with different metastatic potential have 100% tumorigenicity and 100% metastasis of subtype BE1 in nude mice in the same genetic background. The subline LH7 is 10% in nude mice. Into the tumor, no case of metastasis. The significant difference between the two metastatic phenotypes necessarily reflects the differences in the expression and regulation of metastasis-associated genes. Therefore, this system is a good model for differential display of mRNA. Using mRNA differential display technology to clone differential fragments, a 84 9 bp fragment was lowly expressed in the highly metastatic subtype BE1, but was not highly expressed in the non-metastatic subtype LH7. The difference was significant and was verified in repeated experiments. The fragment was successfully recovered, ligated into the pGEM T vector, sequenced by sanger dideoxy end termination method, and sequenced automatically by two-way sequencing. The GenBank Blastn search revealed a homology of 99% with G3BP (U32 519). Northern hybridization showed that G3BP was highly expressed in LH7 (about 3 3 kb), but was lowly expressed in BE1, which verified the difference in the displayed results, excluding the possibility of false positives.