雌二醇诱导人成骨样细胞差异表达基因筛查

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

目的 快速筛查 17β雌二醇 (E2 )诱导人成骨肉瘤MG 6 3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 (cDNARDA)分离E2 干预MG 6 3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 6 3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 6 3细胞 ;共得到 6 0 0余个含有阳性插入片段的cDNA克隆 ,点杂交筛查得到 12 0个差异表达克隆。选 2 0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 cDNARDA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 6 3细胞某些基因表达上调。

Objective To obtain a serial differentially expressed cDNA fragments from human osteoblast like osteosarcoma MG 63 cells induced with 17β 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 17β 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 α32 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 17β 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 17β 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 17β estradiol