胶质瘤相关新基因的研究
摘要
第一部分:胶质瘤相关基因的反向多点杂交研究
目标 利用反向多点杂交技术检测胶质瘤相关基因谱在胶质瘤组织中的表达情况,为这些基因在胶质瘤恶性进展中的作用提供依据,并找出具有进一步研究价值的基因。
方法 分别提取正常脑和胶质瘤标本中的总RNA,并反转录为cDNA,随机引物法标记探针,与点于特殊尼龙膜上的基因片断进行反向杂交。用复日SmartView图像分析软件进行处理,将各个基因的表达强度和β—actine进行比对,根据比值作相关基因的半定量分析。
结果 与正常脑组织比较,2级胶质瘤中表达上调的基因38条,下调24条;3级胶质瘤中有45条基因上调,17条基因下调;4级胶质瘤中有32条上调,29条下调,1条差异不明显。经方差分析,按病理级别分组,差异具有统计学意义(p<0.05)的有Caspasel、CDK4、c13、A20、EWS、Caspase3和EPS8。将恶性胶质瘤(WHOⅢ级和WHOⅣ级胶质瘤)基因表达水平与WHOⅡ级胶质瘤相比较,我们发现CDK4、MMP2、TOP1、c13和RAD17表达水平存在统计学差异(p<0.05)。
结论 反向多点杂交可以同时检测数十个基因的表达,并能对表达量高低进行半定量分析。我们通过这种方法发现,我们发现两条(A20、eps8)与胶质瘤相关的新基因。
第二部分:A20、EPS8、RAD17在胶质瘤中表达的研究
目的:第一部分中我们已经利用反向多点杂交技术筛选到A20、EPS8、RAD17等有研究价值的基因。我们利用RT-PCR技术对A20、EPS8、RAD17进行进一步的研究。一方面在更大的样本范围内明确以上基因的表达改变情况,另一方面通过对这些基因在不同级别星形胶质细胞瘤标本中表达水平的半定量分析为进一步的功能研究提供依据。
方法:提取6例正常脑组织和16例中枢神经系统胶质瘤标本的总RNA,并反转录为cDNA。加入相应的引物后PCR扩增。扩增产物电泳后,按预期产物条带
Objective: To analyze the glioma relative gene expressions states and find potential gene target used in the future by reverse dot-blot.Method: total RNA was extracted from gilomas and normal brain tissues, and reversely transcribed into cDNA. The probe was marked randomly and the dot blot was performed with the gene fragment which had been added onto nylon membrane. Using smart view image analysis system we contrasted the glioma development relative genes with β - actine, and made semiquantitation analysis by their ratios. Result: Contrast with the normal brain, there were 38 genes up-regulated and 24 genes down-regulated in grade two gliomas, 45 genes up-regulated and 17 genes down-regulated in grade three gliomas, 32 genes up-regulated -. 29 genes down-regulated and one unchanged in grade four glioblastomas. Grouped by pathology classification, Caspasel CDK4 c13 A20 EWS Caspase3 and EPS8 were significant in statistic. Compared maglinant gliomas with grade 2 gliomas, CDK4 MMP2 TOP1 c13 and RAD 17 were singificant in statistic. Conclusion: The expressions of genes in different tissues can be analysed semiquantitively by reverse dot blot. Using this way ,the overexpressions of two novel glioma relative genes (A20, EPS8) were found in glioma.Part II: Measure EPS8 A20 RAD17 expressions states in gliomasObjective: In the first part, we find three novel giloma relative genes (A20 EPS8 RAD 17) by reverse dot blot. Then, We will use RT-PCR to do more research about these genes expression states in different grade gliomas, which can confirm EPS8
目标 利用反向多点杂交技术检测胶质瘤相关基因谱在胶质瘤组织中的表达情况,为这些基因在胶质瘤恶性进展中的作用提供依据,并找出具有进一步研究价值的基因。
方法 分别提取正常脑和胶质瘤标本中的总RNA,并反转录为cDNA,随机引物法标记探针,与点于特殊尼龙膜上的基因片断进行反向杂交。用复日SmartView图像分析软件进行处理,将各个基因的表达强度和β—actine进行比对,根据比值作相关基因的半定量分析。
结果 与正常脑组织比较,2级胶质瘤中表达上调的基因38条,下调24条;3级胶质瘤中有45条基因上调,17条基因下调;4级胶质瘤中有32条上调,29条下调,1条差异不明显。经方差分析,按病理级别分组,差异具有统计学意义(p<0.05)的有Caspasel、CDK4、c13、A20、EWS、Caspase3和EPS8。将恶性胶质瘤(WHOⅢ级和WHOⅣ级胶质瘤)基因表达水平与WHOⅡ级胶质瘤相比较,我们发现CDK4、MMP2、TOP1、c13和RAD17表达水平存在统计学差异(p<0.05)。
结论 反向多点杂交可以同时检测数十个基因的表达,并能对表达量高低进行半定量分析。我们通过这种方法发现,我们发现两条(A20、eps8)与胶质瘤相关的新基因。
第二部分:A20、EPS8、RAD17在胶质瘤中表达的研究
目的:第一部分中我们已经利用反向多点杂交技术筛选到A20、EPS8、RAD17等有研究价值的基因。我们利用RT-PCR技术对A20、EPS8、RAD17进行进一步的研究。一方面在更大的样本范围内明确以上基因的表达改变情况,另一方面通过对这些基因在不同级别星形胶质细胞瘤标本中表达水平的半定量分析为进一步的功能研究提供依据。
方法:提取6例正常脑组织和16例中枢神经系统胶质瘤标本的总RNA,并反转录为cDNA。加入相应的引物后PCR扩增。扩增产物电泳后,按预期产物条带
Objective: To analyze the glioma relative gene expressions states and find potential gene target used in the future by reverse dot-blot.Method: total RNA was extracted from gilomas and normal brain tissues, and reversely transcribed into cDNA. The probe was marked randomly and the dot blot was performed with the gene fragment which had been added onto nylon membrane. Using smart view image analysis system we contrasted the glioma development relative genes with β - actine, and made semiquantitation analysis by their ratios. Result: Contrast with the normal brain, there were 38 genes up-regulated and 24 genes down-regulated in grade two gliomas, 45 genes up-regulated and 17 genes down-regulated in grade three gliomas, 32 genes up-regulated -. 29 genes down-regulated and one unchanged in grade four glioblastomas. Grouped by pathology classification, Caspasel CDK4 c13 A20 EWS Caspase3 and EPS8 were significant in statistic. Compared maglinant gliomas with grade 2 gliomas, CDK4 MMP2 TOP1 c13 and RAD 17 were singificant in statistic. Conclusion: The expressions of genes in different tissues can be analysed semiquantitively by reverse dot blot. Using this way ,the overexpressions of two novel glioma relative genes (A20, EPS8) were found in glioma.Part II: Measure EPS8 A20 RAD17 expressions states in gliomasObjective: In the first part, we find three novel giloma relative genes (A20 EPS8 RAD 17) by reverse dot blot. Then, We will use RT-PCR to do more research about these genes expression states in different grade gliomas, which can confirm EPS8
引文
1.黄强,董军,王爱东等.建立胶质瘤恶性进展相关基因表达谱.中华肿瘤杂志,2003,25(5):437-440.
2.孙立军,黄强,王爱东等.建立胶质瘤细胞诱导分化相关基因谱.中华肿瘤杂志,2002,24(3):222-225.
3. T.yoshioka, T.Matsutani, S.Iwagami,et al. Quantitative analysis of the usage of human T cell receptor α and β chain variable regions by reberse dot blot hybridization.J Immu Meth. 1997,201:145-155
4.丁峰,高建声,马旭等.人乳头瘤病毒反向点杂交检测方法的研究.中国优生与遗传杂志,2002,10(6):14-16.
5. Ehrmann J Jr, Rihakova P, Hlobilkova A, et al. The expression of apoptosis-related proteins and the apoptotic rate in glial tumors of the brain. Neoplasma. 2000;47(3):151-5.
6. Kondo S, Barna BP, Morimura T, et al. Interleukin-1 beta-converting enzyme mediates cisplatin-induced apoptosis in malignant glioma cells. Cancer Res. 1995 Dec 15;55(24):6166-71.
7. Shinoura N, Muramatsu Y, Yoshida Y, et al. Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells. Exp Cell Res. 2000, May 1 ;256(2):423-33.
8. Hueber A, Winter S, Weller M, et al. Chemotherapy primes malignant glioma cells for CD95 ligand-induced apoptosis up-stream of caspase 3 activation. Eur J Pharmacol. 1998, Jul 3 ;352(1): 111-5.
9. Holland EC, Hively WP, Depinho RA, et al. A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes Dev. 1998 Dec 1; 12(23):3675-85.
10. Koschny R, Koschny T, Froster UG, et al. Comparative genomic hybridization in glioma: a meta-analysis of 509 cases. Cancer Genet Cytogenet. 2002, 135(2):147-59.
11. Lam PY, Di Tomaso E, Ng HK, et al. Expression of pl9INK4d, CDK4, CDK6 in glioblastomas multiforme. Br J Neurosurg, 2000, 14(1): 28-32.
12. Schmidt U, Fiedler U, Pilarsky CP, et al. Identification of a novel gene on chromosome 13 between BRCA-2 and RB-1. Prostate. 2001 May 1;47(2):91-101.
13. Dorte W, Helle M, Marja J, et al. TNF-induced mitochondrial changes and activation of apoptotic porteases are inhibited by A20. Free Rad Bio, 1998, 25(1): 57-65.
14. Grey ST, Arvelo WM, Hasenkamp FH, et al. adenovirus-mediated gene transfer of the anti-apoptotic protein A20 in rodent islets inhibits IL-1 induced NO release. Trans Pro, 1999, 31: 789-790.
15. Torchia EC, Jaishankar S, Baker SJ. Ewing tumor fusion proteins block the differentiation of pluripotent marrow stromal cells. Cancer Res. 2003 Jul 1;63(13):3464-8.
16. Tanaka K , Iwakuma T , Harimaya K , et al. EWS-Flil antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J. Clin. Invest. 1999, 99: 239-247.
17. Hahm KB, Cho K, Lee C, et al. Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLIl oncoprotein. Nat Genet. 1999, Oct;23(2):222-7.
18. Rousseau-Merck M, Versteege I, Zattara-Cannoni H, et al. Fluorescence in situ hybridization determination of 22q12-q13 deletion in two intracerebral ependymomas. Cancer Genet Cytogenet. 2000 ,Sep;121(2):223-7.
19. Ino Y, Silver JS, Blazejewski L, et al. Common regions of deletion on chromosome 22q12.3-q13.1 and 22q13.2 in human astrocytomas appear related to malignancy grade. J Neuropathol Exp Neurol. 1999, 58(8):881-5.
20. G. Scita, J. Nordstrom, R. Carbone, P. Tenca, G,et al. Eps8 and E3B1 transduce signals from Ras to Rac: Nature .1999 ,401 (6750): 290-293.
21.L.anzetti, V. Rybin, M.G. Malabarba, S. Christoforidis, G, et al. The Eps8
protein coordinates EGF receptor signaling through Rac and trafficking through Rab5. Nature, 2000 ,408 (6810):374-377.
22. B. Matoskova, W.T. Wong, A.E. Salcini, P.G. Pelicci, et al.,Constitutive phosphorylation of Eps8 in tumor cell lines: elevance to malignant transformation. Mol. Cell. Biol.1995 15 (7) :3805-3812.
23. Raithatha SA, Muzik H, Muzik H, et al. Localization of gelatinase-A and gelatinase-B mRNA and protein in human gliomas. Neuro-oncol. 2000 , 2(3):145-50.
24. Nakagawa T, Kubota T, Kabuto M, et al. Secretion of matrix metalloproteinase-2 (72 kD gelatinase/type IV collagenase = gelatinase A) by malignant human glioma cell lines: implications for the growth and cellular invasion of the extracellular matrix. J Neurooncol. 1996 , 28(1): 13-24.
25. Zhao C, Yasui K, Lee CJ, et al. Elevated expression levels of NCOA3, TOP1, and TFAP2C in breast tumors as predictors of poor prognosis. Cancer. 2003, 98(1):18-23.
26. Weller M, Winter S, Schmidt C, et al. Topoisomerase-I inhibitors for human malignant glioma: differential modulation of p53, p21, bax and bcl-2 expression and of CD95-mediated apoptosis by camptothecin and beta-lapachone. Int J Cancer. 1997 ,73(5):707-14.
27. Lamond JP, Mehta MP, Boothman DA, et al. The potential of topoisomerase I inhibitors in the treatment of CNS malignancies: report of a synergistic effect between topotecan and radiation. J Neurooncol. 1996 ,30(1):1-6.
28. Kataoka A, Sadanaga N, Minmori K, et al. Overexpression of Hradl7 mRNA in human breast cancer: correlation with lymphnode metastasis. Clin Cancer Res, 2001, 7(9): 2815-2820.
29. Yu, D, Jing, T., Liu, B, et al. Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cipl, which inhibits p34Cdc2 kinase. Molec. Cell, 1998, 2: 581-591
30. Josef matousek, et al. Ribonucleases and their antitumor activity. Comparative Biochemistry and physiology. 2001, 129:175-191.
2.孙立军,黄强,王爱东等.建立胶质瘤细胞诱导分化相关基因谱.中华肿瘤杂志,2002,24(3):222-225.
3. T.yoshioka, T.Matsutani, S.Iwagami,et al. Quantitative analysis of the usage of human T cell receptor α and β chain variable regions by reberse dot blot hybridization.J Immu Meth. 1997,201:145-155
4.丁峰,高建声,马旭等.人乳头瘤病毒反向点杂交检测方法的研究.中国优生与遗传杂志,2002,10(6):14-16.
5. Ehrmann J Jr, Rihakova P, Hlobilkova A, et al. The expression of apoptosis-related proteins and the apoptotic rate in glial tumors of the brain. Neoplasma. 2000;47(3):151-5.
6. Kondo S, Barna BP, Morimura T, et al. Interleukin-1 beta-converting enzyme mediates cisplatin-induced apoptosis in malignant glioma cells. Cancer Res. 1995 Dec 15;55(24):6166-71.
7. Shinoura N, Muramatsu Y, Yoshida Y, et al. Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells. Exp Cell Res. 2000, May 1 ;256(2):423-33.
8. Hueber A, Winter S, Weller M, et al. Chemotherapy primes malignant glioma cells for CD95 ligand-induced apoptosis up-stream of caspase 3 activation. Eur J Pharmacol. 1998, Jul 3 ;352(1): 111-5.
9. Holland EC, Hively WP, Depinho RA, et al. A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes Dev. 1998 Dec 1; 12(23):3675-85.
10. Koschny R, Koschny T, Froster UG, et al. Comparative genomic hybridization in glioma: a meta-analysis of 509 cases. Cancer Genet Cytogenet. 2002, 135(2):147-59.
11. Lam PY, Di Tomaso E, Ng HK, et al. Expression of pl9INK4d, CDK4, CDK6 in glioblastomas multiforme. Br J Neurosurg, 2000, 14(1): 28-32.
12. Schmidt U, Fiedler U, Pilarsky CP, et al. Identification of a novel gene on chromosome 13 between BRCA-2 and RB-1. Prostate. 2001 May 1;47(2):91-101.
13. Dorte W, Helle M, Marja J, et al. TNF-induced mitochondrial changes and activation of apoptotic porteases are inhibited by A20. Free Rad Bio, 1998, 25(1): 57-65.
14. Grey ST, Arvelo WM, Hasenkamp FH, et al. adenovirus-mediated gene transfer of the anti-apoptotic protein A20 in rodent islets inhibits IL-1 induced NO release. Trans Pro, 1999, 31: 789-790.
15. Torchia EC, Jaishankar S, Baker SJ. Ewing tumor fusion proteins block the differentiation of pluripotent marrow stromal cells. Cancer Res. 2003 Jul 1;63(13):3464-8.
16. Tanaka K , Iwakuma T , Harimaya K , et al. EWS-Flil antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J. Clin. Invest. 1999, 99: 239-247.
17. Hahm KB, Cho K, Lee C, et al. Repression of the gene encoding the TGF-beta type II receptor is a major target of the EWS-FLIl oncoprotein. Nat Genet. 1999, Oct;23(2):222-7.
18. Rousseau-Merck M, Versteege I, Zattara-Cannoni H, et al. Fluorescence in situ hybridization determination of 22q12-q13 deletion in two intracerebral ependymomas. Cancer Genet Cytogenet. 2000 ,Sep;121(2):223-7.
19. Ino Y, Silver JS, Blazejewski L, et al. Common regions of deletion on chromosome 22q12.3-q13.1 and 22q13.2 in human astrocytomas appear related to malignancy grade. J Neuropathol Exp Neurol. 1999, 58(8):881-5.
20. G. Scita, J. Nordstrom, R. Carbone, P. Tenca, G,et al. Eps8 and E3B1 transduce signals from Ras to Rac: Nature .1999 ,401 (6750): 290-293.
21.L.anzetti, V. Rybin, M.G. Malabarba, S. Christoforidis, G, et al. The Eps8
protein coordinates EGF receptor signaling through Rac and trafficking through Rab5. Nature, 2000 ,408 (6810):374-377.
22. B. Matoskova, W.T. Wong, A.E. Salcini, P.G. Pelicci, et al.,Constitutive phosphorylation of Eps8 in tumor cell lines: elevance to malignant transformation. Mol. Cell. Biol.1995 15 (7) :3805-3812.
23. Raithatha SA, Muzik H, Muzik H, et al. Localization of gelatinase-A and gelatinase-B mRNA and protein in human gliomas. Neuro-oncol. 2000 , 2(3):145-50.
24. Nakagawa T, Kubota T, Kabuto M, et al. Secretion of matrix metalloproteinase-2 (72 kD gelatinase/type IV collagenase = gelatinase A) by malignant human glioma cell lines: implications for the growth and cellular invasion of the extracellular matrix. J Neurooncol. 1996 , 28(1): 13-24.
25. Zhao C, Yasui K, Lee CJ, et al. Elevated expression levels of NCOA3, TOP1, and TFAP2C in breast tumors as predictors of poor prognosis. Cancer. 2003, 98(1):18-23.
26. Weller M, Winter S, Schmidt C, et al. Topoisomerase-I inhibitors for human malignant glioma: differential modulation of p53, p21, bax and bcl-2 expression and of CD95-mediated apoptosis by camptothecin and beta-lapachone. Int J Cancer. 1997 ,73(5):707-14.
27. Lamond JP, Mehta MP, Boothman DA, et al. The potential of topoisomerase I inhibitors in the treatment of CNS malignancies: report of a synergistic effect between topotecan and radiation. J Neurooncol. 1996 ,30(1):1-6.
28. Kataoka A, Sadanaga N, Minmori K, et al. Overexpression of Hradl7 mRNA in human breast cancer: correlation with lymphnode metastasis. Clin Cancer Res, 2001, 7(9): 2815-2820.
29. Yu, D, Jing, T., Liu, B, et al. Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cipl, which inhibits p34Cdc2 kinase. Molec. Cell, 1998, 2: 581-591
30. Josef matousek, et al. Ribonucleases and their antitumor activity. Comparative Biochemistry and physiology. 2001, 129:175-191.