雌二醇诱导人成骨样MG-63细胞差异表达基因的分离和鉴定
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摘要
目的 筛查17β雌二醇(E_2)诱导人成骨肉瘤MG-63细胞株差异表达cDNA片段，寻找MG-63细胞中雌激素相关基因，探讨雌激素在绝经后骨质疏松症的发病中可能的保护作用和雌激素替代治疗绝经后骨质疏松症的作用机制。
方法 用改良的cDNA代表性差异分析法同时对雌激素干预组和对照组MG-63细胞来源的cDNA进行正向和反向消减杂交、动力性富集、选择性PCR扩增，分别得到E_2干预MG-63细胞株表达上调和下调的cDNA片段。差异表达的cDNA片段经纯化回收后制备混合探针行Southern杂交分析和快速Northern杂交分析证实其来源后，克隆到pGEM-T easy载体，转化JM109感受态细菌并铺Amp~+／X-gal／IPTG皿得到差异表达的细菌文库，经蓝白筛选后挑取单菌落培养。培养的白色阳性克隆经PCR扩增鉴定后点成4张完全一样的尼龙阵列膜，分别与未消减的“检测”、“驱赶”扩增子及双向消减后的第四轮差异产物行斑点杂交，显影后挑取阳性差异表达cDNA克隆抽提质粒DNA，测序并行同源比较分析。最后选取点杂交筛查得到的部分阳性差异表达克隆经培养扩增后抽提重组体质粒DNA，EcoR I酶切后回收插入片段经α-~(32)P-dATP标记探针行Northern印迹杂交分析。
结果 (1)经过4轮消减杂交和动力性富集后分别得到3个E_2诱导人成骨肉瘤MG-63细胞表达上调的cDNA条带和2个E_2诱导MG-63细胞表达下调的cDNA条带，Southern杂交证明这些差异表达的cDNA片段分别来自作为正向和反向消减杂交时的“检测”扩增子。(2)快速Northern杂交证明这些差异表达片段确实在雌激素干预后表达上调或下调。(3)分别将这些差异表达片段克隆到pGEM-T easy载体后转化JM109感受态细菌并铺Amp~+／X-gal／IPTG皿即得到消
中南大学 博士学位论文
减CDNA细菌文库，蓝白筛选后随机从中挑取近600个白色菌落，经PCR扩增
鉴定均含有阳性插入片段。O）点杂交筛查得到 120个差异表达阳性 CDNA克
隆，分别选20个表达上调和 20个表达下调克隆测序，共得到 36个序列，其中
表达上调者有17个序列，表达下调者19个序列。17个表达上调序列分别代表
13个不同的基因，其中8个与已知基因同源，5个序列未比到同源序列，可能
为新基因：19个表达下调序列分别代表14个不同基因，均与已知基因高度同
源。共选 5个序列（4个上调，l个下调）经 Northem印迹杂交分析，证实 3
个经EZ干预后表达上调，l个表达上调不明显，l个表达下调。
结论CDNA代表性差异分析法能有效筛查差异表达CDNA片段，联合CDNA
阵列点杂交方法可快速高通量筛查差异表达基因。EZ诱导人成骨肉瘤MG－63
细胞中与骨基质组成、能量供应、类固醇类激素代谢、基因转录、炎症反应和
肿瘤发生等相关基因差别表达，可能从分子水平提供一些关于雌激素在绝经后
骨质疏松症发病中所起保护作用的新依据。
Objective To obtain a series of differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced by 17beta-estradiol, to identify some estrogen-responsive genes and to provide a new molecular basis for the protective effects of estrogens against the development of postmenopausal osteoporosis (PMOP) and the mechanisms of estrogen replacement therapy. Methods Modified cDNA representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol. The sources of differentially expressed cDNA fragments were proved by Southern blot and "shotgun" Northern blot analysis. The fragments were then cloned into the pGEM-T easy vector and subtractive cDNA libraries were prepared in E.coli JM109 cells. The cDNA libraries were plated on LB/Amp+/X-gal/IPTG plates and white colonies were picked up randomly and individually grown in LB/amp+ medium in 96-we
ll plates. After PCR, colonies were individually blotted onto a Hybond N membrane. Membranes were hybridized with -32P-labeled subtracted or unsubtracted cDNA. Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones and vice-versa were selected for DNA sequencing and homologue analysis. Northern blot analysis was performed after release of the pGEM-T easy insert with EcoR I.
Results Through cDNA RDA, three upregulated and two downregulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced by 17beta-estradiol and cDNA from untreated MG-63 cells as
"tester" amplicon, respectively. These fragments were proved to really come from the "tester" amplicons by Southern blotting and proved to be really upregulated or downregulated expressed after E2-treated through "shotgun" Northern blotting analysis. We obtained nearly 600 cDNA clones with positive insert from MG-63 cells induced by E2 and 120 differentially expressed clones through dot blotting. Forty clones including 20 upregulated and 20 downregulated were sequenced and 36 sequences (17 upregulated and 19 downregulated ones) obtained, some of them highly homologous to known genes, with three of them proved to be upregulated expressed, one downregulated and one's expression without obvious change through Northern blotting.
Conclusion cDNA RDA is one of the most effective methods to isolate differentially expressed genes and we have screened differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays. Some of the genes of human osteoblast-like osteosarcoma MG-63 cells were differentially expressed induced by 17beta-estradiol. The results show that 17 -E2 regulates the expression of genes associated with bone matrix, supplying energy, steroid hormone metabolism, gene transcription, inflammatory reaction and tumorigenesis. These data might provide a new molecular basis for the protective effects of estrogens against the development of PMOP.
方法 用改良的cDNA代表性差异分析法同时对雌激素干预组和对照组MG-63细胞来源的cDNA进行正向和反向消减杂交、动力性富集、选择性PCR扩增，分别得到E_2干预MG-63细胞株表达上调和下调的cDNA片段。差异表达的cDNA片段经纯化回收后制备混合探针行Southern杂交分析和快速Northern杂交分析证实其来源后，克隆到pGEM-T easy载体，转化JM109感受态细菌并铺Amp~+／X-gal／IPTG皿得到差异表达的细菌文库，经蓝白筛选后挑取单菌落培养。培养的白色阳性克隆经PCR扩增鉴定后点成4张完全一样的尼龙阵列膜，分别与未消减的“检测”、“驱赶”扩增子及双向消减后的第四轮差异产物行斑点杂交，显影后挑取阳性差异表达cDNA克隆抽提质粒DNA，测序并行同源比较分析。最后选取点杂交筛查得到的部分阳性差异表达克隆经培养扩增后抽提重组体质粒DNA，EcoR I酶切后回收插入片段经α-~(32)P-dATP标记探针行Northern印迹杂交分析。
结果 (1)经过4轮消减杂交和动力性富集后分别得到3个E_2诱导人成骨肉瘤MG-63细胞表达上调的cDNA条带和2个E_2诱导MG-63细胞表达下调的cDNA条带，Southern杂交证明这些差异表达的cDNA片段分别来自作为正向和反向消减杂交时的“检测”扩增子。(2)快速Northern杂交证明这些差异表达片段确实在雌激素干预后表达上调或下调。(3)分别将这些差异表达片段克隆到pGEM-T easy载体后转化JM109感受态细菌并铺Amp~+／X-gal／IPTG皿即得到消
中南大学 博士学位论文
减CDNA细菌文库，蓝白筛选后随机从中挑取近600个白色菌落，经PCR扩增
鉴定均含有阳性插入片段。O）点杂交筛查得到 120个差异表达阳性 CDNA克
隆，分别选20个表达上调和 20个表达下调克隆测序，共得到 36个序列，其中
表达上调者有17个序列，表达下调者19个序列。17个表达上调序列分别代表
13个不同的基因，其中8个与已知基因同源，5个序列未比到同源序列，可能
为新基因：19个表达下调序列分别代表14个不同基因，均与已知基因高度同
源。共选 5个序列（4个上调，l个下调）经 Northem印迹杂交分析，证实 3
个经EZ干预后表达上调，l个表达上调不明显，l个表达下调。
结论CDNA代表性差异分析法能有效筛查差异表达CDNA片段，联合CDNA
阵列点杂交方法可快速高通量筛查差异表达基因。EZ诱导人成骨肉瘤MG－63
细胞中与骨基质组成、能量供应、类固醇类激素代谢、基因转录、炎症反应和
肿瘤发生等相关基因差别表达，可能从分子水平提供一些关于雌激素在绝经后
骨质疏松症发病中所起保护作用的新依据。
Objective To obtain a series of differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced by 17beta-estradiol, to identify some estrogen-responsive genes and to provide a new molecular basis for the protective effects of estrogens against the development of postmenopausal osteoporosis (PMOP) and the mechanisms of estrogen replacement therapy. Methods Modified cDNA representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol. The sources of differentially expressed cDNA fragments were proved by Southern blot and "shotgun" Northern blot analysis. The fragments were then cloned into the pGEM-T easy vector and subtractive cDNA libraries were prepared in E.coli JM109 cells. The cDNA libraries were plated on LB/Amp+/X-gal/IPTG plates and white colonies were picked up randomly and individually grown in LB/amp+ medium in 96-we
ll plates. After PCR, colonies were individually blotted onto a Hybond N membrane. Membranes were hybridized with -32P-labeled subtracted or unsubtracted cDNA. Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones and vice-versa were selected for DNA sequencing and homologue analysis. Northern blot analysis was performed after release of the pGEM-T easy insert with EcoR I.
Results Through cDNA RDA, three upregulated and two downregulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced by 17beta-estradiol and cDNA from untreated MG-63 cells as
"tester" amplicon, respectively. These fragments were proved to really come from the "tester" amplicons by Southern blotting and proved to be really upregulated or downregulated expressed after E2-treated through "shotgun" Northern blotting analysis. We obtained nearly 600 cDNA clones with positive insert from MG-63 cells induced by E2 and 120 differentially expressed clones through dot blotting. Forty clones including 20 upregulated and 20 downregulated were sequenced and 36 sequences (17 upregulated and 19 downregulated ones) obtained, some of them highly homologous to known genes, with three of them proved to be upregulated expressed, one downregulated and one's expression without obvious change through Northern blotting.
Conclusion cDNA RDA is one of the most effective methods to isolate differentially expressed genes and we have screened differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays. Some of the genes of human osteoblast-like osteosarcoma MG-63 cells were differentially expressed induced by 17beta-estradiol. The results show that 17 -E2 regulates the expression of genes associated with bone matrix, supplying energy, steroid hormone metabolism, gene transcription, inflammatory reaction and tumorigenesis. These data might provide a new molecular basis for the protective effects of estrogens against the development of PMOP.
引文
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2. Rickard DJ, Subramaniam M, Spelsberg TC. Molecular and cellular mechanisms of estrogen action on the skeleton. J Cell Biochem 1999; Suppl 32-33: 123-132
3. Oursler MJ. Estrogen regulation of gene expression in osteoblasts and osteoclasts. Clin Rev Eukaryot Gene Expr 1998; 8: 125-140
4. Notelovitz M. Overview of bone mineral density in postmenopausal women. J Reprod Med 2002; 47(1 Suppl): 71-81
5. Hofbauer LC, Khosla S, Dunstan CR, et al. Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinol 1999; 140: 4367-4370
6. Ramalho AC, Jullienne A, Couttet P, et al. Effect of oestradiol on cytokine production in immortalized human marrow stromal cell lines. Cytokine 2001; 21; 16: 126-130
7. Kassem M, Okazaki R, Harris SA, et al. Estrogen effects on insulin-like growth factor gene expression in a human osteoblastic cell line with high levels of estrogen receptor. Calcif Tissue Int 1998; 62: 60-66
8. Wang X, Schwartz Z, Yaffe P, et al. The expression of transforming growth factor-β and intetleukin-1 β mRNA and the response to 1, 25(OH)2D3, 17 β -estradiol, and testosterone is age-dependent in primary cultures of mouse-derived osteoblasts in vitro. Endocrine 1999; 11: 13-22
9. Kassem M, Harris SA, Spelsberg TG, et al. Estrogen inhibits interleukin-6 production and gene expression in a human osteoblastic cell line with high levels of estrogen receptor. J Bone Miner Res 1996; 11: 193-199
10. Rao LG, Kung Sutherland MS, Muzaffar SA, et al. Positive interaction between 17 beta-Estradiol and parathyroid hormone in normal human osteoblasts cultured
long term in the presence of dexamethasone. Osteoporos Int 1996; 6: 111-119
11. Kirkwood KL, Homick K, Dragon MB, et al. Cloning and characterization of the type Ⅰ inositol 1, 4, 5-trisphosphate receptor gene promoter. Regulation by 17beta-estradiol in osteoblasts. J Biol Chem 1997; 272: 22425-22431
12. Bodine PV, Henderson RA, Green J, et al. Estrogen receptor-alpha is developmentally regulated during osteoblast differentiation and contributes to selective responsiveness of gene expression. Endocrinology 1998; 139: 2048-2057
13. Rickard DJ, Waters KM, Ruesink TJ, et al. Estrogen receptor isoform-specific induction of progesterone receptors in human osteoblasts. J Bone Miner Res 2002; 17: 580-592
14. Liao EY, Luo XH, Deng XG, et al. Effects of 17beta-estradiol on the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in human osteoblastic MG-63 cell cultures. J Endocrinol Invest 2001; 24: 876-881
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