雌二醇诱导人成骨样细胞差异表达基因筛查

目的 快速筛查17β雌二醇(E2)诱导人成骨肉瘤MG—63细胞株差异表达cDNA片段,寻找雌激素相关基因。方法 改良cDNA代表性差异分析法(cDNA RDA)分离E2干预MG—63细胞株表达上调cDNA片段,Southern杂交证实后,制备阵列膜行点杂交,然后挑取阳性差异克隆测序,经同源比较分析,最后选取部分克隆标记探针行Northern印迹杂交。结果经过4轮消减和动力性富集后得到5个E2诱导人成骨肉瘤MG—63细胞表达上调的差异cDNA条带,Southern杂交证明这些表达上调的cDNA片段来自E2干预的MG—63细胞;共得到600余个含有阳性插入片段的cDNA克隆,点杂交筛查得到120个差异表达克隆。选20个克隆测序得到15个序列,其中1个经Northern杂交证实E2干预后表达上调。结论 cDNA RDA能有效筛查差异表达cDNA片段,联合cDNA阵列点杂交可快速筛查差异表达基因,E2诱导人成骨肉瘤MG—63细胞某些基因表达上调。

Isolation of differentially expressed genes in human osteoblast-like osteosarcoma cell line induced with 17beta-estradiol

OBJECTIVE: To obtain a serial differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced with 17beta-estradiol and to find some estrogen-responsive genes. METHODS: Optimized cDNA representational difference analysis (RDA) was performed to isolate up-regulated expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol. The sources of up-regulated expressed cDNA fragments were proved by Southern blot. The fragments were cloned into the pGEM-T easy vector and a cDNA library was prepared in E. coli JM109 cells. The cDNA library was plated on LB/Amp(+)/X-gal/IPTG plates and white colonies were picked up and individually grown in LB/Amp(+) medium in 96-well plates. After PCR, colonies were individually blotted onto a Hybond N membrane. Membranes were hybridized with alpha-(32)P-labeled subtracted or unsubtracted tester cDNA. Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones were selected for DNA sequencing, BLAST and Northern blot analysis after release of the pGEM-T easy insert with EcoR I. RESULTS: Five up-regulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced with 17beta-estradiol as tester amplicon and cDNA from untreated MG-63 cells as driver amplicon by cDNA RDA. These fragments were proved to be really coming from tester amplicon and down-regulated expressed in the untreated cell by Southern blotting. We obtained more than 600 cDNA clones with positive insert from MG-63 cells line induced with 17beta-estradiol and about 120 differentially expressed clones through dot blotting. Twenty clones were sequenced and we got 15 gene sequences. One of them was proved to be differentially expressed through Northern blotting. CONCLUSIONS: cDNA RDA is one of the most effective methods which can isolate differentially expressed genes and we can screen differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays. Some genes of human osteoblast-like osteosarcoma MG-63 cell line showed up-regulated expression after induction by 17beta-estradiol.