EGF抑制人脑胶质母细胞瘤BT325细胞生长的相关基因分离、克隆及其意义探讨
摘要
脑胶质瘤是危害人类健康的常见恶性肿瘤之一，在各种原发性中枢神经系统肿瘤中发病率最高。目前，胶质瘤特别是恶性胶质瘤的治疗还是一个非常棘手的问题。与正常胶质细胞相比，胶质瘤细胞中许多基因发生了结构和表达的改变，其中主要有癌基因的激活和抑癌基因的失活以及大量相关基因的表达变化。这些基因改变是胶质瘤恶性生物学行为的基础，但对此的了解还不是很清楚。体外培养的肿瘤细胞系的刺激、诱导常能提供有关肿瘤发生发展及药物、生物治疗的有用信息。分离、克隆胶质瘤发生发展和分化相关的cDNA，能够丰富人们对胶质瘤的分子机理认识，可以提供诊断、治疗的新线索，在理论和应用上具有重要的价值。
脑胶质瘤，特别是胶质母细胞瘤最常见而突出的分子特征为表皮生长因子受体(EGFR)的突变和(或)过表达，并与胶质瘤的恶性程度和生物学行为密切相关。为此，我们用其相应的配基EGF刺激诱导人脑胶质母细胞瘤BT325细胞，分析这一过程中所发生的基因表达谱变化；探寻胶质瘤发生发展的相关基因，以期发现可用作胶质瘤治疗的靶基因和(或)分期分型的分子标志。
分别给予每毫升0.1ng，1ng，10ng，100ng EGF刺激BT325细胞，发现并证实随着剂量的增大，BT325细胞生长增殖明显的抑制。EGF可抑制胶质瘤细胞生长这一现象，尚未引起人们的关注，文献中主要是EGF刺激胶质瘤细胞增殖和浸润的报道，但也仍未对其分子机制进行深入的研究。
应用新近发展的mRNA差异显示分析(DDRT-PCR)技术，分析BT325细胞生长抑制后的差异表达基因。进行不同的锚定引物和随机引物配对的DDRT-PCR反应，经变性聚丙烯酰胺长凝胶电泳，PCR产物的回收、扩增，分离了61个差异片段，大小介于200-500bp之间。为进一步鉴定分离出的片段所代表的基因的差异表达情况，把这些片段点膜制成cDNAarray，采用生长抑制组和对照组RNA反转录标记的cDNA第一条链的混合探针，分别同时对所得的差异片段进行反向Northern斑点杂交。根据斑点杂交的结果挑选出6个表达明显上调的差异表达片段进行克隆和测序。
The glioblastoma is one of the common malignant tumors endangering human health and it has the highest incidence among the primary tumors in center nervous system. At present time, the therapy of glioblastoma, especially the malignant glioblastoma is very difficulty. Compared with those in normal glial cell, many genes in glioblastoma cell have been changed in structure and expression, including activated oncogenes, inactivated tumor suppressor genes and many associating genes. Such gene expression changes are the molecular basis of biological behaviors of malignant glioblastoma, but still not well known. The stimulation and induction of in vitro tumor cell culture can provide useful information about the cancer genesis, drug and biological therapy. The isolation and cloning of cDNA associating with glioblastoma developing and differentiation can enrich our understanding molecular mechanism of glioblastoma formation, provide the clue to diagnosis and treatment and have important theoretical and practical values.
The most common and outstanding molecular characteristic of Glioma, especially the glioblastoma, is the mutation and /or over-expression of Epidermal Growth Factor Receptor (EGFR), closely associating with malignancy and biological behavior. So we stimulate and induce human glioblastoma BT325 cells with Epidermal Growth Factor to analyses and detect the gene spectrum changes related to glioma growth and progression, anticipating to find target moleculars which can be use as glioma gene therapy and/or molecular markers of glioma staging and classification.
We use the EGF 0.1ng/ml, 1ng/ml, 10ng/ml; 100ng/ml to stimulate BT325 cells separatively and find that with increased dosage, the growth and proliferation of BT325 was apparently inhibited. EGF inhibiting glioma cellgrowth has not been caused to attention. There were some papers
脑胶质瘤，特别是胶质母细胞瘤最常见而突出的分子特征为表皮生长因子受体(EGFR)的突变和(或)过表达，并与胶质瘤的恶性程度和生物学行为密切相关。为此，我们用其相应的配基EGF刺激诱导人脑胶质母细胞瘤BT325细胞，分析这一过程中所发生的基因表达谱变化；探寻胶质瘤发生发展的相关基因，以期发现可用作胶质瘤治疗的靶基因和(或)分期分型的分子标志。
分别给予每毫升0.1ng，1ng，10ng，100ng EGF刺激BT325细胞，发现并证实随着剂量的增大，BT325细胞生长增殖明显的抑制。EGF可抑制胶质瘤细胞生长这一现象，尚未引起人们的关注，文献中主要是EGF刺激胶质瘤细胞增殖和浸润的报道，但也仍未对其分子机制进行深入的研究。
应用新近发展的mRNA差异显示分析(DDRT-PCR)技术，分析BT325细胞生长抑制后的差异表达基因。进行不同的锚定引物和随机引物配对的DDRT-PCR反应，经变性聚丙烯酰胺长凝胶电泳，PCR产物的回收、扩增，分离了61个差异片段，大小介于200-500bp之间。为进一步鉴定分离出的片段所代表的基因的差异表达情况，把这些片段点膜制成cDNAarray，采用生长抑制组和对照组RNA反转录标记的cDNA第一条链的混合探针，分别同时对所得的差异片段进行反向Northern斑点杂交。根据斑点杂交的结果挑选出6个表达明显上调的差异表达片段进行克隆和测序。
The glioblastoma is one of the common malignant tumors endangering human health and it has the highest incidence among the primary tumors in center nervous system. At present time, the therapy of glioblastoma, especially the malignant glioblastoma is very difficulty. Compared with those in normal glial cell, many genes in glioblastoma cell have been changed in structure and expression, including activated oncogenes, inactivated tumor suppressor genes and many associating genes. Such gene expression changes are the molecular basis of biological behaviors of malignant glioblastoma, but still not well known. The stimulation and induction of in vitro tumor cell culture can provide useful information about the cancer genesis, drug and biological therapy. The isolation and cloning of cDNA associating with glioblastoma developing and differentiation can enrich our understanding molecular mechanism of glioblastoma formation, provide the clue to diagnosis and treatment and have important theoretical and practical values.
The most common and outstanding molecular characteristic of Glioma, especially the glioblastoma, is the mutation and /or over-expression of Epidermal Growth Factor Receptor (EGFR), closely associating with malignancy and biological behavior. So we stimulate and induce human glioblastoma BT325 cells with Epidermal Growth Factor to analyses and detect the gene spectrum changes related to glioma growth and progression, anticipating to find target moleculars which can be use as glioma gene therapy and/or molecular markers of glioma staging and classification.
We use the EGF 0.1ng/ml, 1ng/ml, 10ng/ml; 100ng/ml to stimulate BT325 cells separatively and find that with increased dosage, the growth and proliferation of BT325 was apparently inhibited. EGF inhibiting glioma cellgrowth has not been caused to attention. There were some papers
引文
1. Funari FB, et al. Genetics and malignant progression of human brain tumors. Cancer Surv. 1995, 25: 233-275
2. Torp Sh, et al. Amplification of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci USA. 1987,84: 6899-6903
3. MulerisM, et al.Oncogene amplification in human gliomas: a molecular cytogenetic analysis. Oncogene 1994; 9: 2717-2422
4. Sehgal A.Molecular changes during the genesis of human gliomas. Seminar Surg Oncol. 1998, 14: 3-12
5. Hendrick SM, et al. Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature. 1984,308:149-153
6. Hubank M et al. Identifying differences in mRNA expression by representional difference analysis of cDNA. Nucleic Acids Res. 1994,22:5640-5648
7. Liang P, et al . Differential display of eucaryotic messenger RNA by means of the polymerase chain reaction . Science 1992,257:967-971
8. Liang P, et al. Differential display using one-base anchored Oligo(dT) primer. Nucleic Acids Res. 1994,22: 5763-5764
9. Sung YJ, et al. Use of two reverse transcriptases eliminates false-positive results in differential display. Biotechniques. 1997, 23: 462-464.
10. Petitot AS, et al. Cloning of two plant cDNAs encoding a beta-type proteasome subunit and a transformer-2-like SR-related protein: early induction of the corresponding genes in tobacco cells treated with cryptogein. Plant Mol Biol. 1997,35: 261-269.
11. Werner JA, et al. Expression of a novel mRNA in human head and neck squamous cell carcinoma cells. Mol Pathol. 1997,50: 82-86.
12. Schwechheimer K, et al. EGFR gene amplification-rearrangement in human glioblastomas. Int. J. Cancer, 1995:62:145-148
13. Canute GW, et al. The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme. Neurosurgery, 1998; 42:609-616
14. Negri DRM, et al. In vitro and in vivo stability and anti-tumor efficacy of an anti-EGFR/anti-CD3F(ab')_2 bispecific monoclonal antibody. British journal of cancer, 1995:72:928-933
15. Sugawa N, et al. An antisense EGFR oligodeoxynucleotide enveloped in lipofectin induces growth inhibition in human maligant gliomas in vitro. J Neurooncol, 1998:39:237-244
16. Pu P (蒲佩玉), et al. Inhibitory effect of antisense epidermal growth factor??RNA on the proliferation of rat C6 glioma cells in vitro and in vivo. J Neurosurg, 2000; 92:132-139
17．邵文钊等 人脑恶性胶质瘤细胞系BT325的细胞动力学研究。中华神经外科杂志 1988，第4卷：239-242
18．鄂征 组织培养技术，人民卫生出版社 1988
19．Sambrook J等著 分子克隆(第二版)科学出版社 1992
20. Zhang H, et al. Differential screening of gene expression difference by display. Nucleic Acid Res. 1996, 24:2454-2455
21. Liew CC, et al. A catalogue of genes in the cardiovascular system as identified by expressed sequence tags. Proc. Natl. Acad. Sci. USA. 1994,91:10645-10649
22. Tang P et al. The autocrine loop of TGF-alpha/EGFR and brain tumors. J Neurooncol, 1997; 35: 303-314
23. E. Van de Kelft. Moleculer pathogenesis of astrocytoma and glioblastoma multiforme. Acta Neurochir, 1997; 139:589-599
24. Alan Wells. EGF recertor. The international journal of biochemistry & cell biology, 1999; 31: 637-643
25. El-Obeid A, et al. Cell scattering and migration induced by autocrine transforming growth factor a in glioma cells in vitro. Cancer research, 1997; 57: 5598-5604
26. Michael R, et al. Modification of human glioma locomotion in vitro by cytokines EGF, bFGF, pDGFbb, NGF, and TNFα. Neurosurgery, 1995;36:1165-1171
27. Hamel W, et al. Growth factors in gliomas revisited. Acta N Neurochir, 2000; 142: 113-138
28. Jonsson JJ, et al. From mutation mapping to phenotype cloning. Proc. Natl. Acad. Sci. USA, 1995;92:83-90
29. Seghal A, et al。Isolation of differentially expressed cDNAs durilsolation and Characterization of a novel gene from human glioblastoma multiforme tumor tissue。 Int J Cancer 1997; 71:565-572
30. Sun ZS, et al. A putative mammalian ortholog of the Drosophila period gene. Cell, 1997, 90:1003-1011
31. http://www.ncbi.nlm.nih.gov/dbEST
32. Sutcliffe JG, et al. Control of neuronal gene expression. Science, 1984, 225:1308-1315
33. Milner RJ, et al. Brain-specific genes have identifier sequences in their introns. Proc. Natl. Acad. Sci. USA, 1984;81:713-717
34. Banki K, et al. Glutathione levels and sensitivity to apoptosis are regulated by changes in transadolase expression. The journal of biological chemistry, 1996; 271:32994-33001
35. Banki K, et al. Elevation of mitochondrial transmembrane potential and reactive oxygen intermediate levels are early events and occur indepently from activation??of caspases in fas signaling. The journal of immunology, 1999:162:1466-1479
36. Boguski MS, et al. Gene discovery in dbEST. Science 1994;265:1993-4
37.Hillier LD, et al. Generation and analysis of 280000 human expressed sequence tags. Genome Res 1996;6:807-28
38.Delseny M, et al. The arabidopsis thaliana cDNA sequencing projects. FEBS Lett 1997;405:129-32
39.Hartl DL, et al. ESTI,ESTII,ESTIII Bioessays 1996;18:1021-4
40.Wells TN, et al. The chemochine information source: identification and characterization of novel chemochine using the WWW and expressed sequence tags database. J Leukoc Biol 1997;61:545-60
41. Pan G, et al. The receptor for the cytoxic ligand TRAIL. Science 1997;276:111-3
42. Sheridan JP, et al. Contral of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:816-21
43. Pan G, et al. An antagonist decoy receptor and a death-domain containing receptor for TRAIL . Scince 1997;277:815-8
44.Neuwald AF, et al. Extracting protein alignment models from the sequence database. Nucleic Acids Res 1997;25:1665-77
45.Fannon MR, et al. Gene expression in normal and disease states-identification of therapeutic targets. Trends Biotechnol 1996; 14:294-8
46. Wan JS, et al. Cloning differentially expressed mRNAs. Nat Biotechnol 1996;14:1685-91
47. Heller RA, et al. Discovery and analysis of inflammatory disease-related genes using cDNA microarray. Proc Natl Acad Sci USA 1997;94:2150-55
48.Schena M, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microassay. Science 1995;270:467070
49. Schuler GD, et al. A gene map of the human genome. Science 1996,274:540-46
50.Fishel R, et al. The human mutator gene homolog MSH2 and its association with hereditary colon cancer. Cell 1993 ;75:1027-3 8
51. Scott WK, et al. Genetic analysis of complex disease. Science 1997;275:1327
52.Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science, 1995;267:1456-1462
53. Wyllie AH. Apoptosis: an overview. British Medical Bulletin, 1997, 53:451-465
54. Thomas T, et al. Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: alterations in polyamine biosynthesis and the expression of p21/CIPl/WAF1. Journal of cellular physiology, 1999; 179: 257-266
55. Armstrong DK, et al. Epidermal growth factor-mediated apoptosis of MDA-MB-468 human breast cancer cells. Cancer research, 1994;54:5280-5283
56.Gulli Lf, et al. Epidermal growth factor induced apoptosis in A431 cells can be reversed by reducing the tyrosine kinas activity. Cell Growth Differ,1996;7:173-178
57. Stone R, et al. Moleculer' surgery" for brain tumors. Sciences, 1992; 256: 1513
58.Fueyo J, et al. Molecular control of the cellular cycle and apoptosis: new treatments for gliomas. Neurologia,1998;13: 349-355
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2. Torp Sh, et al. Amplification of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci USA. 1987,84: 6899-6903
3. MulerisM, et al.Oncogene amplification in human gliomas: a molecular cytogenetic analysis. Oncogene 1994; 9: 2717-2422
4. Sehgal A.Molecular changes during the genesis of human gliomas. Seminar Surg Oncol. 1998, 14: 3-12
5. Hendrick SM, et al. Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature. 1984,308:149-153
6. Hubank M et al. Identifying differences in mRNA expression by representional difference analysis of cDNA. Nucleic Acids Res. 1994,22:5640-5648
7. Liang P, et al . Differential display of eucaryotic messenger RNA by means of the polymerase chain reaction . Science 1992,257:967-971
8. Liang P, et al. Differential display using one-base anchored Oligo(dT) primer. Nucleic Acids Res. 1994,22: 5763-5764
9. Sung YJ, et al. Use of two reverse transcriptases eliminates false-positive results in differential display. Biotechniques. 1997, 23: 462-464.
10. Petitot AS, et al. Cloning of two plant cDNAs encoding a beta-type proteasome subunit and a transformer-2-like SR-related protein: early induction of the corresponding genes in tobacco cells treated with cryptogein. Plant Mol Biol. 1997,35: 261-269.
11. Werner JA, et al. Expression of a novel mRNA in human head and neck squamous cell carcinoma cells. Mol Pathol. 1997,50: 82-86.
12. Schwechheimer K, et al. EGFR gene amplification-rearrangement in human glioblastomas. Int. J. Cancer, 1995:62:145-148
13. Canute GW, et al. The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme. Neurosurgery, 1998; 42:609-616
14. Negri DRM, et al. In vitro and in vivo stability and anti-tumor efficacy of an anti-EGFR/anti-CD3F(ab')_2 bispecific monoclonal antibody. British journal of cancer, 1995:72:928-933
15. Sugawa N, et al. An antisense EGFR oligodeoxynucleotide enveloped in lipofectin induces growth inhibition in human maligant gliomas in vitro. J Neurooncol, 1998:39:237-244
16. Pu P (蒲佩玉), et al. Inhibitory effect of antisense epidermal growth factor??RNA on the proliferation of rat C6 glioma cells in vitro and in vivo. J Neurosurg, 2000; 92:132-139
17．邵文钊等 人脑恶性胶质瘤细胞系BT325的细胞动力学研究。中华神经外科杂志 1988，第4卷：239-242
18．鄂征 组织培养技术，人民卫生出版社 1988
19．Sambrook J等著 分子克隆(第二版)科学出版社 1992
20. Zhang H, et al. Differential screening of gene expression difference by display. Nucleic Acid Res. 1996, 24:2454-2455
21. Liew CC, et al. A catalogue of genes in the cardiovascular system as identified by expressed sequence tags. Proc. Natl. Acad. Sci. USA. 1994,91:10645-10649
22. Tang P et al. The autocrine loop of TGF-alpha/EGFR and brain tumors. J Neurooncol, 1997; 35: 303-314
23. E. Van de Kelft. Moleculer pathogenesis of astrocytoma and glioblastoma multiforme. Acta Neurochir, 1997; 139:589-599
24. Alan Wells. EGF recertor. The international journal of biochemistry & cell biology, 1999; 31: 637-643
25. El-Obeid A, et al. Cell scattering and migration induced by autocrine transforming growth factor a in glioma cells in vitro. Cancer research, 1997; 57: 5598-5604
26. Michael R, et al. Modification of human glioma locomotion in vitro by cytokines EGF, bFGF, pDGFbb, NGF, and TNFα. Neurosurgery, 1995;36:1165-1171
27. Hamel W, et al. Growth factors in gliomas revisited. Acta N Neurochir, 2000; 142: 113-138
28. Jonsson JJ, et al. From mutation mapping to phenotype cloning. Proc. Natl. Acad. Sci. USA, 1995;92:83-90
29. Seghal A, et al。Isolation of differentially expressed cDNAs durilsolation and Characterization of a novel gene from human glioblastoma multiforme tumor tissue。 Int J Cancer 1997; 71:565-572
30. Sun ZS, et al. A putative mammalian ortholog of the Drosophila period gene. Cell, 1997, 90:1003-1011
31. http://www.ncbi.nlm.nih.gov/dbEST
32. Sutcliffe JG, et al. Control of neuronal gene expression. Science, 1984, 225:1308-1315
33. Milner RJ, et al. Brain-specific genes have identifier sequences in their introns. Proc. Natl. Acad. Sci. USA, 1984;81:713-717
34. Banki K, et al. Glutathione levels and sensitivity to apoptosis are regulated by changes in transadolase expression. The journal of biological chemistry, 1996; 271:32994-33001
35. Banki K, et al. Elevation of mitochondrial transmembrane potential and reactive oxygen intermediate levels are early events and occur indepently from activation??of caspases in fas signaling. The journal of immunology, 1999:162:1466-1479
36. Boguski MS, et al. Gene discovery in dbEST. Science 1994;265:1993-4
37.Hillier LD, et al. Generation and analysis of 280000 human expressed sequence tags. Genome Res 1996;6:807-28
38.Delseny M, et al. The arabidopsis thaliana cDNA sequencing projects. FEBS Lett 1997;405:129-32
39.Hartl DL, et al. ESTI,ESTII,ESTIII Bioessays 1996;18:1021-4
40.Wells TN, et al. The chemochine information source: identification and characterization of novel chemochine using the WWW and expressed sequence tags database. J Leukoc Biol 1997;61:545-60
41. Pan G, et al. The receptor for the cytoxic ligand TRAIL. Science 1997;276:111-3
42. Sheridan JP, et al. Contral of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997;277:816-21
43. Pan G, et al. An antagonist decoy receptor and a death-domain containing receptor for TRAIL . Scince 1997;277:815-8
44.Neuwald AF, et al. Extracting protein alignment models from the sequence database. Nucleic Acids Res 1997;25:1665-77
45.Fannon MR, et al. Gene expression in normal and disease states-identification of therapeutic targets. Trends Biotechnol 1996; 14:294-8
46. Wan JS, et al. Cloning differentially expressed mRNAs. Nat Biotechnol 1996;14:1685-91
47. Heller RA, et al. Discovery and analysis of inflammatory disease-related genes using cDNA microarray. Proc Natl Acad Sci USA 1997;94:2150-55
48.Schena M, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microassay. Science 1995;270:467070
49. Schuler GD, et al. A gene map of the human genome. Science 1996,274:540-46
50.Fishel R, et al. The human mutator gene homolog MSH2 and its association with hereditary colon cancer. Cell 1993 ;75:1027-3 8
51. Scott WK, et al. Genetic analysis of complex disease. Science 1997;275:1327
52.Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science, 1995;267:1456-1462
53. Wyllie AH. Apoptosis: an overview. British Medical Bulletin, 1997, 53:451-465
54. Thomas T, et al. Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: alterations in polyamine biosynthesis and the expression of p21/CIPl/WAF1. Journal of cellular physiology, 1999; 179: 257-266
55. Armstrong DK, et al. Epidermal growth factor-mediated apoptosis of MDA-MB-468 human breast cancer cells. Cancer research, 1994;54:5280-5283
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