细胞骨架和GFAP基因在NDGA诱导人恶性胶质瘤细胞分化过程中的作用
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摘要
恶性胶质瘤具有生长迅速和高度侵袭等生物学特性，其机制尚未阐明，也给治疗带来很大难度。近些年发现，细胞骨架的构成和不同成分之间的相互作用与肿瘤的生物学特性可能有密切联系。我们既往研究也发现，NDGA对恶性胶质瘤具有诱导分化治疗作用，而在这一过程中，作为细胞骨架重要成分之一的中间丝增多，其结构蛋白GFAP表达明显上调，GFAP基因甲基化状态也发生改变。然而，NDGA对胶质瘤细胞整个骨架系统的影响和GFAP基因在诱导分化中的作用及其意义仍然不清。
本研究分四个部分，分别从恶性胶质瘤细胞骨架成分和NDGA的影响、正义GFAP cDNA真核表达载体和反义GFAP逆转录病毒表达载体的构建与鉴定及其对胶质瘤细胞生物学特性的影响，以及NDGA对反义GFAP cDNA封闭后的胶质瘤细胞GFAP基因表达的诱导作用等方面，探讨了细胞骨架蛋白和GFAP基因功能状态在恶性胶质瘤增殖、分化和NDGA诱导分化中的可能作用及其意义。主要研究内容和结果如下：
1．应用免疫荧光细胞化学、激光共聚焦显微镜、原位包埋、选择性抽提和高压透射电镜技术，首次对两种不同分化程度的胶质瘤细胞系CHG-5和SHG-44细胞骨架系统(胞质骨架和核骨架)的定位及分布特征进行了比较研究，并观察了NDGA诱导分化过程中的细胞骨架蛋白的变化及其与恶性表型逆转的关系。结果显示，两种细胞微管蛋白的定位和分布具有共性，而微丝、中间丝及核骨架的结构和排布方式有所不同，并与瘤细胞的分化程度相关，提示细胞骨架蛋白的分布与构型上的差异构成了CHG-5和SHG-44细胞生物学特性差异的结构基础，并在判定胶
质瘤细胞分化程度和恶性行为上有重要价值。NDGA可改变胶质瘤细
胞骨架蛋白表达与分布，且这种作用与NDGA诱导分化和抑制增殖效
应相一致，提示改善胞质骨架及核骨架构型是NDGA促进瘤细胞分化
作用的重要途径之一。
2．应用基因工程技术分别构建了反义GFAP逆转录病毒载体和携带绿色
荧光蛋白（GFP）的正义 GFAP CDNA真核表达载体，并分别将其成功
导入两种胶质瘤细胞。RT－PCR、ISH和 ICC结果显示，反义 GFAP CDNA
封闭CHG－5细胞内源性GFAP表达后，GFAP mRNA及其蛋白表达减
弱甚至缺失，瘤细胞异型性增加，突起缩短，细胞增殖速度加快。与之
相反，正义 GFAP cDNA转染 SHG－44细胞后，GFAP mRNA及其蛋白
、表达增强，瘤细胞形态趋向成熟，突起增多变细，细胞增殖速度显著降
低，群体倍增时间延长，细胞骨架蛋白表达增加。它们对细胞周期也有
截然不同的干预作用。提示GFAP基因功能状态可能是决定胶质瘤细胞
分化程度及恶性行为的重要环节。这一结果在恶性胶质瘤治疗策略上可
能有重要意义。
3．本研究首次探讨了NDGA对反义GFAP CDNA存在下的GFAP基因表
达的诱导作用。结果显示，反义 GFAP CDNA封闭的 CHG－5细胞经
NDGA （10 u M）作用后，GFAP mRNA及其蛋白重新高表达，且在
时间和强度上与NDGA诱导细胞分化、抑制细胞增殖及增加细胞骨架
蛋白含量等作用呈正相关。这些结果提示，上调GFAP基因及其蛋白表
达是NDGA诱导胶质瘤细胞恶性逆转作用的主要靶点。
4．利用活细胞分子探针一GFP基因标记了中国人胶质瘤细胞系SHG－44并
获表达及传代，为体外和在体进一步动态研究胶质瘤细胞生长、分化及
侵袭过程提供了新方法和新材料。
上述研究结果对于深入认识细胞骨架和 GFAP基因在胶质瘤生物
－学特性和 NDGA诱导分化机制中的作用具有重要意义。
Malignant gliomas are characterized by rapid growth and high invasiveness, which result in the difficulties in treatment. Recently, it has been found that cytoskeleton components and their relationship might be closely related to biologic characteristics of tumors. Our previous studies have shown that nordihydroguaiaretic acid (NDGA) possessed a marked differentiation inducement effect on human glioma cells, which outstandingly presented as an increase in intermediate filaments, up-regulation of GFAP expression, as well as changes of GFAP gene methylation pattern. However, the effects of NDGA on cytoskeleton system and the exact roles of GFAP gene in NDGA-induced differentiation remain obscure.
In the present study, cytoskeletal components in malignant glioma cells and the effects of NDGA on them were observed. Meanwhile, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed. The effects of eliminating or reinforcing endogenous GFAP expression on the biological features were studied. Moreover, reversion of malignant phenotype in GFAP-deficient CHG-5 cells, which induced by NDGA simultaneously, was investigated in this experimental cell system. Our aim was to explore the possible roles of cytoskeleton proteins and GFAP gene in glioma cell proliferation, differentiation and NDGA-induced differentiation. The main results and conclusions are as follows:
1. The localization and distribution of cytoskeletal components, including cytoplasmic cytoskeleton and nuclear cytoskeleton, in two glioma cells and its
relationship with malignant grade were detected using immunofluorescence cytochemistry combined with confocal laser scanning microscopy and whole-mount embedded, selective extraction combined together high voltage transmission electron microscopy. The result showed that the localization and distribution of -tubulin, a subunit of microtubules, was identical in two glioma cell lines CHG-5 and SHG-44. In contrast, the structure and arrangement pattern of microfilaments, intermediate filaments as well as nuclear matrix in cell line CHG-5 were distinct from those of SHG-44, and these were also related to their degrees of differentiation and certain malignant phenotype. These results suggested that the difference of organization of cytoplasmic and nuclear cytoskeleton might provide inherent constructive foundation, and, might also provide an accessory evaluation in degrees of differentiation and malignant grade of glioma cells. It was also found that the skeleton protein expression and distribution could be altered by NDGA, along with its effects of growth inhibition and differentiation. This result suggested that enhancement of cytoskeletal protein synthesis and improvement of cytoplasmic and nuclear cytoskeleton organization might be one of the mechanisms of NDGA-induced differentiation in human glioma cells.
2. Using gene recombination technique, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed respectively. The results of RT-PCR, in situ hybridization and immunocytochemistry showed that the expression of GFAP mRNA and its protein was reduced or even eliminated amongst CHG-5 cells infected with antisense GFAP retrovirus. GFAP-negative CHG-5 cells had marked cellular atypia and enhanced proliferative potential. In contrast with above, ithe expression of GFAP mRNA and its protein was markedly increased in SHG-44 cells after stably transfected with an expression vector containing GFAP cDNA
in the sense orientation. The profound morphological changes in these cells, including exhibited stellate, abundant and thin cytoplasmic process formation, reduction of atypia occurred. And cell proliferation rate was markedly reduced. The contents of cytoskeletal proteins were also significantly increased. Cell cycle analysis results also revealed the differ
本研究分四个部分，分别从恶性胶质瘤细胞骨架成分和NDGA的影响、正义GFAP cDNA真核表达载体和反义GFAP逆转录病毒表达载体的构建与鉴定及其对胶质瘤细胞生物学特性的影响，以及NDGA对反义GFAP cDNA封闭后的胶质瘤细胞GFAP基因表达的诱导作用等方面，探讨了细胞骨架蛋白和GFAP基因功能状态在恶性胶质瘤增殖、分化和NDGA诱导分化中的可能作用及其意义。主要研究内容和结果如下：
1．应用免疫荧光细胞化学、激光共聚焦显微镜、原位包埋、选择性抽提和高压透射电镜技术，首次对两种不同分化程度的胶质瘤细胞系CHG-5和SHG-44细胞骨架系统(胞质骨架和核骨架)的定位及分布特征进行了比较研究，并观察了NDGA诱导分化过程中的细胞骨架蛋白的变化及其与恶性表型逆转的关系。结果显示，两种细胞微管蛋白的定位和分布具有共性，而微丝、中间丝及核骨架的结构和排布方式有所不同，并与瘤细胞的分化程度相关，提示细胞骨架蛋白的分布与构型上的差异构成了CHG-5和SHG-44细胞生物学特性差异的结构基础，并在判定胶
质瘤细胞分化程度和恶性行为上有重要价值。NDGA可改变胶质瘤细
胞骨架蛋白表达与分布，且这种作用与NDGA诱导分化和抑制增殖效
应相一致，提示改善胞质骨架及核骨架构型是NDGA促进瘤细胞分化
作用的重要途径之一。
2．应用基因工程技术分别构建了反义GFAP逆转录病毒载体和携带绿色
荧光蛋白（GFP）的正义 GFAP CDNA真核表达载体，并分别将其成功
导入两种胶质瘤细胞。RT－PCR、ISH和 ICC结果显示，反义 GFAP CDNA
封闭CHG－5细胞内源性GFAP表达后，GFAP mRNA及其蛋白表达减
弱甚至缺失，瘤细胞异型性增加，突起缩短，细胞增殖速度加快。与之
相反，正义 GFAP cDNA转染 SHG－44细胞后，GFAP mRNA及其蛋白
、表达增强，瘤细胞形态趋向成熟，突起增多变细，细胞增殖速度显著降
低，群体倍增时间延长，细胞骨架蛋白表达增加。它们对细胞周期也有
截然不同的干预作用。提示GFAP基因功能状态可能是决定胶质瘤细胞
分化程度及恶性行为的重要环节。这一结果在恶性胶质瘤治疗策略上可
能有重要意义。
3．本研究首次探讨了NDGA对反义GFAP CDNA存在下的GFAP基因表
达的诱导作用。结果显示，反义 GFAP CDNA封闭的 CHG－5细胞经
NDGA （10 u M）作用后，GFAP mRNA及其蛋白重新高表达，且在
时间和强度上与NDGA诱导细胞分化、抑制细胞增殖及增加细胞骨架
蛋白含量等作用呈正相关。这些结果提示，上调GFAP基因及其蛋白表
达是NDGA诱导胶质瘤细胞恶性逆转作用的主要靶点。
4．利用活细胞分子探针一GFP基因标记了中国人胶质瘤细胞系SHG－44并
获表达及传代，为体外和在体进一步动态研究胶质瘤细胞生长、分化及
侵袭过程提供了新方法和新材料。
上述研究结果对于深入认识细胞骨架和 GFAP基因在胶质瘤生物
－学特性和 NDGA诱导分化机制中的作用具有重要意义。
Malignant gliomas are characterized by rapid growth and high invasiveness, which result in the difficulties in treatment. Recently, it has been found that cytoskeleton components and their relationship might be closely related to biologic characteristics of tumors. Our previous studies have shown that nordihydroguaiaretic acid (NDGA) possessed a marked differentiation inducement effect on human glioma cells, which outstandingly presented as an increase in intermediate filaments, up-regulation of GFAP expression, as well as changes of GFAP gene methylation pattern. However, the effects of NDGA on cytoskeleton system and the exact roles of GFAP gene in NDGA-induced differentiation remain obscure.
In the present study, cytoskeletal components in malignant glioma cells and the effects of NDGA on them were observed. Meanwhile, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed. The effects of eliminating or reinforcing endogenous GFAP expression on the biological features were studied. Moreover, reversion of malignant phenotype in GFAP-deficient CHG-5 cells, which induced by NDGA simultaneously, was investigated in this experimental cell system. Our aim was to explore the possible roles of cytoskeleton proteins and GFAP gene in glioma cell proliferation, differentiation and NDGA-induced differentiation. The main results and conclusions are as follows:
1. The localization and distribution of cytoskeletal components, including cytoplasmic cytoskeleton and nuclear cytoskeleton, in two glioma cells and its
relationship with malignant grade were detected using immunofluorescence cytochemistry combined with confocal laser scanning microscopy and whole-mount embedded, selective extraction combined together high voltage transmission electron microscopy. The result showed that the localization and distribution of -tubulin, a subunit of microtubules, was identical in two glioma cell lines CHG-5 and SHG-44. In contrast, the structure and arrangement pattern of microfilaments, intermediate filaments as well as nuclear matrix in cell line CHG-5 were distinct from those of SHG-44, and these were also related to their degrees of differentiation and certain malignant phenotype. These results suggested that the difference of organization of cytoplasmic and nuclear cytoskeleton might provide inherent constructive foundation, and, might also provide an accessory evaluation in degrees of differentiation and malignant grade of glioma cells. It was also found that the skeleton protein expression and distribution could be altered by NDGA, along with its effects of growth inhibition and differentiation. This result suggested that enhancement of cytoskeletal protein synthesis and improvement of cytoplasmic and nuclear cytoskeleton organization might be one of the mechanisms of NDGA-induced differentiation in human glioma cells.
2. Using gene recombination technique, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed respectively. The results of RT-PCR, in situ hybridization and immunocytochemistry showed that the expression of GFAP mRNA and its protein was reduced or even eliminated amongst CHG-5 cells infected with antisense GFAP retrovirus. GFAP-negative CHG-5 cells had marked cellular atypia and enhanced proliferative potential. In contrast with above, ithe expression of GFAP mRNA and its protein was markedly increased in SHG-44 cells after stably transfected with an expression vector containing GFAP cDNA
in the sense orientation. The profound morphological changes in these cells, including exhibited stellate, abundant and thin cytoplasmic process formation, reduction of atypia occurred. And cell proliferation rate was markedly reduced. The contents of cytoskeletal proteins were also significantly increased. Cell cycle analysis results also revealed the differ
引文
1. Randolph TR. Acute promyelocytic leukemia (AML-M3), Part 1: Pathophysiology, clinical diagnosis and differentiation therapy. Clin Lab Sci. 2000; 2:98-105
2. Pawlak G, Helfman DM. Cytoskeletal changes in cell transformation and tumorigenesis. Curr Opin Genet Dev. 2001; 11(1): 41-47
3. Intermediate filament proteins during carcinogenesis and apoptosis. Int J Oncol. 1999; 14(3): 563-570
4．卞修武，史景泉，辛榕，等．去甲二氢愈创木酸对人恶性胶质瘤细胞系SHG-44生长和分化的影响，中华病理学杂志，1997；26(5)：285-288
5．郭德玉，陈意生，史景泉，等．去甲二氢愈创木酸对恶性胶质瘤细胞增殖和分化的影响．第三军医大学学报，1999；21(1)：9-12
6．陈自强，卞修武，辛榕，等．人脑胶质瘤细胞系CHG-5的建立及其生物学特性分析．第三军医大学学报，1999；21(12)：880-883
7．卢佳友，卞修武．NDGA对人恶性胶质瘤细胞系CHG-5和SHG-44甲基转移酶的影响及意义．全国第8届神经病理学术会议论文集，2001；27
8．杜子威，徐庚达，王尧，等．人脑恶性胶质瘤细胞系SHG-44的建立及其特征．中华肿瘤杂志，1984；6(4)：241-243
9．谢锦玉．现代细胞化学技术及其在中西医药中的应用．北京：中医古籍出版社，1998；102-151
10. Gajkowska B, Cholewinski W, Gniadecki R. Structure of cytomatrix and nuclear matrix revealed by embedment-free electron microscopy. Acta Neurobiol Exp Warsz. 2000; 60(2): 147-158
11．马文丽，薛杜普．一种实用有效的整装电镜制样法—K562细胞核基质-中间纤维的研究．中国医学科学院学报，1994；2
12. Sheetz MP. Cell control by membrane-cytoskeleton adhesion. Nat Rev Cell
Biol. 2001; 2(5): 392-396
13. Wang TH, Popp DM, Wang HS, et al. Microtubule dysfunction induced by paclitaxel intiates apoptosis through both c-jun N-terminal kinase (JNK)-dependent and independent pathways in ovarian cancer cells. J Biol Chem. 1999; 274(12): 8208-8216
14．李祖国，丁彦青，邓永华，等．CD44V6表达对大肠癌细胞骨架的影响．中华病理学杂志，1998；27(4)：273，275
15. Sabine H, Patricia C, Laila S, et al. IL-2-dependent expression of genes involved in cytoskeleton organization, oncogene regulation, and transcriptional control. J Immunol. 1999; 162(6): 3280-3288
16．韩亚玲，张志谦，葛险峰，等．生长特性差别的胃癌MGC803细胞亚系间细胞骨架、细胞通讯与癌基因表达的比较研究．实验生物学报，1996；29(1)：13-17
17. Hill PB, Dora KA, Hughes AD. The involvement of intracellular Ca~(++) in 5-HT(1B/1D) receptor-mediated contraction of the rabbit isolated renal artery. Br J Pharmacol. 2000; 130(4): 835-842
18．王立安，赵俊霞．钙对细胞骨架的调控及其在生命活动中的重要作用．生物学杂志，1999；16(4)：6-7
19. Steime PA, Hoffert JD, Adey NB, et al. Polyphosphoinositides inhibit the interaction of vinculin with actin filaments. J Biol Chem. 1999; 274(26): 18414-18420
20. Ben-zeer A. Cytoskeletal and adhesion proteins as tumor suppressors. Curr Opin Cell Biol. 1997; 9(1): 99-108
21. Puck TT, Krystosek A. Role of the cytoskeleton in gene regulation and cancer. Int Rev Cytol. 1992; 132:75-81
22. Fuchs E, and Weber K. Intermediate filaments: structure, dynamics, function, and disease. Ann Rev Biochem. 1994; 63:345-382
23. Fuchs E, and Cleveland DW. A structural scaffolding of intermediate
filaments in health and disease. Science. 1998; 279:514-519
24. Baba H, Nakahira K, Morita N, et al. GFAP gene expression during development of astrocyte. Dev Neurosci. 1997; 19(1): 49-57
25. Absza MS, Shaban F, Narayan RK, et al. Human glioma associated intermediate filament proteins: over-expression, co-localization and cross-reactivity. Anticancer Res. 1998; 18(2B): 1333-1340
26．卞修武，史景泉，柳凤轩．243例胶质瘤免疫组织化学和超微结构的研究与预后探讨．中华病理学杂志，1992；21(3)：171-173
27. Wilson DE, Anderson KM, Seed TM. Ultra-structural evidence for differentiation in a human glioblastoma cell line treated with inhibitors of sicosanoid metabolism. Neurosurgery. 1990; 27(4): 523-531
28. Moody TW, Leyton J, Martinez A, et al. Lipoxygenase inhibitors prevent lung carcinogenesis and inhibit non-small cell lung cancer growth. Exp Lung Res, 1998; 24(4): 617-628
29. Cunningham DC, Harrison LY, Shultz TD. Proliferative responses of normal human mammary and MCF-7 breast cancer cells to linoleic acid, conjugated linoleic acid and eicosanoid synthesis inhibitors in culture. Anticancer Res. 1997; 17(1A): 197-203
30. Earashi M, Noguchi M, Kinoshita K, et al. Effects of eicosanoid synthesis inhibitors on the in vitro growth and prostaglandin E and leukotriene B secretion of a human breast cancer cell line. Oncology. 1995; 52(2): 150-155
31. Su W, Tseng LL, Lin MC, et al. Effect of nordihydriguaiaretic acid on intracellular Ca++ concentrations in C6 glioma cells. Neurochem Interna. 2002; 40:249-254
32. Biswal SS, Datta K, Shaw SD, et al. Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells. Toxicol Sci.
2000; 53:77-83
33．鞠文君，王端顺．细胞骨架与早期基因mRNA定位的相关性研究．科学通报，1997；42(13)：1440-1444
34. Chintala SK, Sawaya R, Aggarwal BB, et al. Induction of matrix metalloproteinase-9 requires a polymerized actin cytoskeleton in human malignant glioma cells. J Biol Chem. 1998; 273(22): 13545-13551
35. Anesini C, Ferraro G, Lopez P. Different intracellular signals coupled to the antiproliferative action of aqueous crude extract from Larrea divaricata Cav. and nordihydroguaiaretic acid on a lymphoma cell line. Phytomedicine. 2001; 8(1): 1-7
36. Inagaki M, Nakamura Y, Takeda M, et al. Glial fibrillary acidic protein: dynamic property and regulation by phosphorylation. Brain Pathol. 1994; 4: 239-243
37. Liu F, Verin AD, Borbiev T, et al. Role of cAMP-dependent protein kinase activity in endothelial cell cytoskeletaon rearrangement. Am J Physiol Lung Mol Physiol. 2001; 280(6): L1309-1317
38. Gniadecki R, Olszewska H, Gajkowska B. Changes in the ultra-structure of cytoskeleton and nuclear matrix during HaCaT keratinocyte differentiation. Exp Dermatol. 2001; 10(2): 71-79
39. Stein GS, Montecino M, van Wijnen AJ, et al. Nuclear structure-gene expression interrelationships: implications for aberrant gene expression in cancer. Cancer Res. 2000; 60(8):2067-2076
40. Wang ZH, Yam HF, Or PC, et al. Effects of all-trans-retinoic acid on human esophageal carcinoma cells and their nuclear matrixes. Anticancer Res. 1999; 19(1A):563-568
41．李娟，罗绍凯，洪文德，慢性粒细胞性白血病急性变时核基质的改变及其意义．肿瘤，1999；19(1)：37-40
42. Palu G, Parolin C, Takeuchi Y. Progress with retroviral gene vectors. Rev
Med Virol. 2000; 10(3): 185-202
43. Naylor LH. Reporter gene technology: the future looks bright. Biochem Pharmacol. 1999; 58:749-757
44．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯．分子克隆实验指南．第二版．北京，科学出版社，1992
45．F．奥斯伯，R．布伦特，R．E．金斯顿，等．精编分子生物学实验指南．第一版．北京，科学出版社，1998
46. Meng YG, Liang J, Wong WL, et al. Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene. 2000; 242:201-207
47．于丽莉，陈诗书．绿色荧光蛋白标记基因在肿瘤研究中的应用．中国肿瘤生物治疗杂志，1999；6(2)：81-82
48. Salgia R, Li JH, Ewanink DS. Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility. Oncogene. 1999; 18(1): 67-77
49. Galipeau J, Li H, Paquin A, et al. Vesicular stomatitis virus G pseudo-typed retro-vector mediates effective in vivo suicide gene delivery in experimental brain cancer. Cancer Res. 1999; 59(10): 2384-2394
50. Schamhart DH, and Westerhof AC. Strategies for gene cloning. Urol Res. 1999; 27(2): 87-96
51. Vile RG, Russell SJ. Retriviruses as vectors. British Med Bull. 1995; 51(1): 12-18
52. Jaffer EM, Dranoff G, Cohen LK, et al. High efficiency gene transfer into primary human tumor exptant without cell selection. Cancer Res. 1993; 53(10suppl): 2221-2226
53. Bodine DM, McDonagh KT, Brandt KT, et al. Development of a high titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells. Proc Natl Acada Sci USA. 1990; 87:3738-3744
54. Morling FJ, Ressell SJ. Enhanced transduction efficiency of retroviral vectors co-precipitated with calium phosphate. Gene Therapy. 1995; 2: 504-508
55. Wiltecnson GWG, Lowenstein PR. Introduction to gene transfer: viral vectors. Gene therapy. 1995; 2(suppl): S1
56．苏慧慈，主编．原位杂交．第一版．北京，中国科学技术出版社，1994
57．蔡文琴，王伯云，主编．实用免疫细胞化学与核酸分子杂交技术．第一版．成都，四川科学技术出版社，1994
58. Jen KY, Gewirtz AM. Suppression of gene expression by targeted disruption of mRNA: available options and current strategies. Stem Cells. 2000; 18(5): 307-319
59．王升启，朱元晓．反义核酸技术，现代生物化学理论与技术．孙志贤，主编．北京，军事医学出版社，1995
60. Weiss B, Daividkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci. 1999; 55(3): 321-348
61. Alemany R, Gomez MC, Balague C. Gene therapy for gliomas: molecular targets, adenoviral vectors, and oncolytic adenoviruses. Exp Cell Res. 1999; 252(1): 1-12
62. Rutka JT, and Smith SL. Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity. Cancer Res. 1993; 53:3624-3631
63. Sadatomo T, Yoshida J, Wakabayashi T, et al. New approach for the treatment of medulloblastoma by transfection with glial fibrillary acidic protein gene. Surg Oncol. 1996; 5(2): 69-75
64. Pekny M, Eliasson C, Chien CL, et al. GFAP-deficient astrocytes are capable of stellation in vitro when co-cultured with neurons and exhibit a reduced amount of intermediate filaments and an increased cell saturation
density. Exp Cell Res. 1998; 239:332-343
65. Rurka JT, Hubbard SL, Fukuyama K, et al. Effects of antisense glial fibrillary acidic protein complementary DNA on the growth, invasion, and adhesion of human astrocytoma cells. Cancer Res. 1994; 54:326-3272
66. Garbuglia M, Verzini M, Sorci G, et al. The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type Ⅲ intermediate filaments. Braz J Med Biot Res. 1999; 32:1177-1185
67．卞修武．胶质纤维酸性蛋白基因调控及其在胶质瘤诱导分化中的作用．中华病理学杂志，1999；28(3)：1-2
68. Goldman RD, Khuon S, Chou YH, The function of intermediate filaments in cell shape and cytoskeletal integrity. J Cell Biol. 1996; 134:971-983
69. Steinbock FA, and Wiche G. Plectin: a cytolinker by design. Biol Chem. 1999; 380:151-158
70．鄂征．对细胞周期学说中矛盾的探讨．中华肿瘤杂志，1994；16(2)：156-158
71．姚莉，卞修武，张树辉，等。去甲二氢愈创木酸对恶性胶质瘤细胞基因组甲基化水平的影响及其意义．第三军医大学学报，2001；22(3)：251-253
72. Ansar S, Iqbal M, Athar M. Nordihydroguairetic acid is a potent inhibitor of ferric-nitrilotriacetate-mediated hepatic and renal toxicity, and renal tumor promotion in mice. Carcinogenesis. 1999; 20:599-606
73. Ramasamy S, Drummond GR, Ahn J, et al. Modulation of expression of endothelial nitric oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant in cultured endothelial cells. Mol Pharmacol. 1999; 56: 116-123
74. Ramoner R, Rieser C, Bartsch G, et al. Nordihydroguaiaretic acid blocks secretory and endocytic pathways in human dendritic cells. J Leuko Biol. 1998; 64:747-752
75. Fujiwara T, Takami N, Misumi Y, et al. Nordihydroguaiaretic acid blocks protein transport in the srcretory pathway causing redistribution of Golgi proteins into the endoplasmic reticulum. J Biol Chem. 1998; 273: 3068-3075
76. Barnaby JW, Styles AR, Cockerell CJ. Actinic keratoses: differential diagnosis and treatment. Drugs Aging. 1997; 11(3): 186-205
77. Damtew B, Spagnuolo PJ. Tumor cell-endothelial cell interactions: evidence for roles for lipoxygenase products of arachidonic acid in metastasis. Prost Leukot Essent Fatty Acids. 1997; 56(4): 295-300
78. Ingalill AM, Jett M, Boyle T, et al. Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J Clin Invest. 1996; 97(3): 806-813
79. Wilson DE, DiGianfilippo A, Ondrey FG, et al. Effects of nordihydroguaiaretic acid on cultured rat and human glioma cell proliferation. J Neurosurg. 1989; 71:551-557
80．郭德玉，陈意生，卞修武，等．bcl-2、c-myc基因表达在去甲二氢愈创木酸诱导人恶性胶质瘤细胞凋亡中的意义．第三军医大学学报．2001；22(3)：260-263
81．孙慧勤，卞修武，陈意生，等．去甲二氢愈创木酸对胶质瘤细胞SHG-44迁移的影响．中华病理学杂志，2000；29(1)：30-33
82．孙慧勤，陈意生，史景泉，等．去甲二氢愈创木酸对活体血管生成作用的实验研究．第三军医大学学报．2001；22(3)：276-279
83. Condorcelli DF, Dell'Albani P, Conticello SG, et al. Aneural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene. Dev Neurosci. 1997; 19:446-456
84. Barresi V, Condorelli DF, Giuffrida-Stella AM. GFAP gene methylation in different neural cell types from rat brain. Int J Dev Neurosci. 1999; 17(8): 821-828
85.Park S, Lee DK, Yang CH. Inhibition of fos-jun-DNA complex formation by dihydroguaiaretic acid and in vitro cytotoxic effects on cancer cells. Cancer Lett. 1998; 127(1-2): 23-28
2. Pawlak G, Helfman DM. Cytoskeletal changes in cell transformation and tumorigenesis. Curr Opin Genet Dev. 2001; 11(1): 41-47
3. Intermediate filament proteins during carcinogenesis and apoptosis. Int J Oncol. 1999; 14(3): 563-570
4．卞修武，史景泉，辛榕，等．去甲二氢愈创木酸对人恶性胶质瘤细胞系SHG-44生长和分化的影响，中华病理学杂志，1997；26(5)：285-288
5．郭德玉，陈意生，史景泉，等．去甲二氢愈创木酸对恶性胶质瘤细胞增殖和分化的影响．第三军医大学学报，1999；21(1)：9-12
6．陈自强，卞修武，辛榕，等．人脑胶质瘤细胞系CHG-5的建立及其生物学特性分析．第三军医大学学报，1999；21(12)：880-883
7．卢佳友，卞修武．NDGA对人恶性胶质瘤细胞系CHG-5和SHG-44甲基转移酶的影响及意义．全国第8届神经病理学术会议论文集，2001；27
8．杜子威，徐庚达，王尧，等．人脑恶性胶质瘤细胞系SHG-44的建立及其特征．中华肿瘤杂志，1984；6(4)：241-243
9．谢锦玉．现代细胞化学技术及其在中西医药中的应用．北京：中医古籍出版社，1998；102-151
10. Gajkowska B, Cholewinski W, Gniadecki R. Structure of cytomatrix and nuclear matrix revealed by embedment-free electron microscopy. Acta Neurobiol Exp Warsz. 2000; 60(2): 147-158
11．马文丽，薛杜普．一种实用有效的整装电镜制样法—K562细胞核基质-中间纤维的研究．中国医学科学院学报，1994；2
12. Sheetz MP. Cell control by membrane-cytoskeleton adhesion. Nat Rev Cell
Biol. 2001; 2(5): 392-396
13. Wang TH, Popp DM, Wang HS, et al. Microtubule dysfunction induced by paclitaxel intiates apoptosis through both c-jun N-terminal kinase (JNK)-dependent and independent pathways in ovarian cancer cells. J Biol Chem. 1999; 274(12): 8208-8216
14．李祖国，丁彦青，邓永华，等．CD44V6表达对大肠癌细胞骨架的影响．中华病理学杂志，1998；27(4)：273，275
15. Sabine H, Patricia C, Laila S, et al. IL-2-dependent expression of genes involved in cytoskeleton organization, oncogene regulation, and transcriptional control. J Immunol. 1999; 162(6): 3280-3288
16．韩亚玲，张志谦，葛险峰，等．生长特性差别的胃癌MGC803细胞亚系间细胞骨架、细胞通讯与癌基因表达的比较研究．实验生物学报，1996；29(1)：13-17
17. Hill PB, Dora KA, Hughes AD. The involvement of intracellular Ca~(++) in 5-HT(1B/1D) receptor-mediated contraction of the rabbit isolated renal artery. Br J Pharmacol. 2000; 130(4): 835-842
18．王立安，赵俊霞．钙对细胞骨架的调控及其在生命活动中的重要作用．生物学杂志，1999；16(4)：6-7
19. Steime PA, Hoffert JD, Adey NB, et al. Polyphosphoinositides inhibit the interaction of vinculin with actin filaments. J Biol Chem. 1999; 274(26): 18414-18420
20. Ben-zeer A. Cytoskeletal and adhesion proteins as tumor suppressors. Curr Opin Cell Biol. 1997; 9(1): 99-108
21. Puck TT, Krystosek A. Role of the cytoskeleton in gene regulation and cancer. Int Rev Cytol. 1992; 132:75-81
22. Fuchs E, and Weber K. Intermediate filaments: structure, dynamics, function, and disease. Ann Rev Biochem. 1994; 63:345-382
23. Fuchs E, and Cleveland DW. A structural scaffolding of intermediate
filaments in health and disease. Science. 1998; 279:514-519
24. Baba H, Nakahira K, Morita N, et al. GFAP gene expression during development of astrocyte. Dev Neurosci. 1997; 19(1): 49-57
25. Absza MS, Shaban F, Narayan RK, et al. Human glioma associated intermediate filament proteins: over-expression, co-localization and cross-reactivity. Anticancer Res. 1998; 18(2B): 1333-1340
26．卞修武，史景泉，柳凤轩．243例胶质瘤免疫组织化学和超微结构的研究与预后探讨．中华病理学杂志，1992；21(3)：171-173
27. Wilson DE, Anderson KM, Seed TM. Ultra-structural evidence for differentiation in a human glioblastoma cell line treated with inhibitors of sicosanoid metabolism. Neurosurgery. 1990; 27(4): 523-531
28. Moody TW, Leyton J, Martinez A, et al. Lipoxygenase inhibitors prevent lung carcinogenesis and inhibit non-small cell lung cancer growth. Exp Lung Res, 1998; 24(4): 617-628
29. Cunningham DC, Harrison LY, Shultz TD. Proliferative responses of normal human mammary and MCF-7 breast cancer cells to linoleic acid, conjugated linoleic acid and eicosanoid synthesis inhibitors in culture. Anticancer Res. 1997; 17(1A): 197-203
30. Earashi M, Noguchi M, Kinoshita K, et al. Effects of eicosanoid synthesis inhibitors on the in vitro growth and prostaglandin E and leukotriene B secretion of a human breast cancer cell line. Oncology. 1995; 52(2): 150-155
31. Su W, Tseng LL, Lin MC, et al. Effect of nordihydriguaiaretic acid on intracellular Ca++ concentrations in C6 glioma cells. Neurochem Interna. 2002; 40:249-254
32. Biswal SS, Datta K, Shaw SD, et al. Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells. Toxicol Sci.
2000; 53:77-83
33．鞠文君，王端顺．细胞骨架与早期基因mRNA定位的相关性研究．科学通报，1997；42(13)：1440-1444
34. Chintala SK, Sawaya R, Aggarwal BB, et al. Induction of matrix metalloproteinase-9 requires a polymerized actin cytoskeleton in human malignant glioma cells. J Biol Chem. 1998; 273(22): 13545-13551
35. Anesini C, Ferraro G, Lopez P. Different intracellular signals coupled to the antiproliferative action of aqueous crude extract from Larrea divaricata Cav. and nordihydroguaiaretic acid on a lymphoma cell line. Phytomedicine. 2001; 8(1): 1-7
36. Inagaki M, Nakamura Y, Takeda M, et al. Glial fibrillary acidic protein: dynamic property and regulation by phosphorylation. Brain Pathol. 1994; 4: 239-243
37. Liu F, Verin AD, Borbiev T, et al. Role of cAMP-dependent protein kinase activity in endothelial cell cytoskeletaon rearrangement. Am J Physiol Lung Mol Physiol. 2001; 280(6): L1309-1317
38. Gniadecki R, Olszewska H, Gajkowska B. Changes in the ultra-structure of cytoskeleton and nuclear matrix during HaCaT keratinocyte differentiation. Exp Dermatol. 2001; 10(2): 71-79
39. Stein GS, Montecino M, van Wijnen AJ, et al. Nuclear structure-gene expression interrelationships: implications for aberrant gene expression in cancer. Cancer Res. 2000; 60(8):2067-2076
40. Wang ZH, Yam HF, Or PC, et al. Effects of all-trans-retinoic acid on human esophageal carcinoma cells and their nuclear matrixes. Anticancer Res. 1999; 19(1A):563-568
41．李娟，罗绍凯，洪文德，慢性粒细胞性白血病急性变时核基质的改变及其意义．肿瘤，1999；19(1)：37-40
42. Palu G, Parolin C, Takeuchi Y. Progress with retroviral gene vectors. Rev
Med Virol. 2000; 10(3): 185-202
43. Naylor LH. Reporter gene technology: the future looks bright. Biochem Pharmacol. 1999; 58:749-757
44．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯．分子克隆实验指南．第二版．北京，科学出版社，1992
45．F．奥斯伯，R．布伦特，R．E．金斯顿，等．精编分子生物学实验指南．第一版．北京，科学出版社，1998
46. Meng YG, Liang J, Wong WL, et al. Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene. 2000; 242:201-207
47．于丽莉，陈诗书．绿色荧光蛋白标记基因在肿瘤研究中的应用．中国肿瘤生物治疗杂志，1999；6(2)：81-82
48. Salgia R, Li JH, Ewanink DS. Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility. Oncogene. 1999; 18(1): 67-77
49. Galipeau J, Li H, Paquin A, et al. Vesicular stomatitis virus G pseudo-typed retro-vector mediates effective in vivo suicide gene delivery in experimental brain cancer. Cancer Res. 1999; 59(10): 2384-2394
50. Schamhart DH, and Westerhof AC. Strategies for gene cloning. Urol Res. 1999; 27(2): 87-96
51. Vile RG, Russell SJ. Retriviruses as vectors. British Med Bull. 1995; 51(1): 12-18
52. Jaffer EM, Dranoff G, Cohen LK, et al. High efficiency gene transfer into primary human tumor exptant without cell selection. Cancer Res. 1993; 53(10suppl): 2221-2226
53. Bodine DM, McDonagh KT, Brandt KT, et al. Development of a high titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells. Proc Natl Acada Sci USA. 1990; 87:3738-3744
54. Morling FJ, Ressell SJ. Enhanced transduction efficiency of retroviral vectors co-precipitated with calium phosphate. Gene Therapy. 1995; 2: 504-508
55. Wiltecnson GWG, Lowenstein PR. Introduction to gene transfer: viral vectors. Gene therapy. 1995; 2(suppl): S1
56．苏慧慈，主编．原位杂交．第一版．北京，中国科学技术出版社，1994
57．蔡文琴，王伯云，主编．实用免疫细胞化学与核酸分子杂交技术．第一版．成都，四川科学技术出版社，1994
58. Jen KY, Gewirtz AM. Suppression of gene expression by targeted disruption of mRNA: available options and current strategies. Stem Cells. 2000; 18(5): 307-319
59．王升启，朱元晓．反义核酸技术，现代生物化学理论与技术．孙志贤，主编．北京，军事医学出版社，1995
60. Weiss B, Daividkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci. 1999; 55(3): 321-348
61. Alemany R, Gomez MC, Balague C. Gene therapy for gliomas: molecular targets, adenoviral vectors, and oncolytic adenoviruses. Exp Cell Res. 1999; 252(1): 1-12
62. Rutka JT, and Smith SL. Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity. Cancer Res. 1993; 53:3624-3631
63. Sadatomo T, Yoshida J, Wakabayashi T, et al. New approach for the treatment of medulloblastoma by transfection with glial fibrillary acidic protein gene. Surg Oncol. 1996; 5(2): 69-75
64. Pekny M, Eliasson C, Chien CL, et al. GFAP-deficient astrocytes are capable of stellation in vitro when co-cultured with neurons and exhibit a reduced amount of intermediate filaments and an increased cell saturation
density. Exp Cell Res. 1998; 239:332-343
65. Rurka JT, Hubbard SL, Fukuyama K, et al. Effects of antisense glial fibrillary acidic protein complementary DNA on the growth, invasion, and adhesion of human astrocytoma cells. Cancer Res. 1994; 54:326-3272
66. Garbuglia M, Verzini M, Sorci G, et al. The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type Ⅲ intermediate filaments. Braz J Med Biot Res. 1999; 32:1177-1185
67．卞修武．胶质纤维酸性蛋白基因调控及其在胶质瘤诱导分化中的作用．中华病理学杂志，1999；28(3)：1-2
68. Goldman RD, Khuon S, Chou YH, The function of intermediate filaments in cell shape and cytoskeletal integrity. J Cell Biol. 1996; 134:971-983
69. Steinbock FA, and Wiche G. Plectin: a cytolinker by design. Biol Chem. 1999; 380:151-158
70．鄂征．对细胞周期学说中矛盾的探讨．中华肿瘤杂志，1994；16(2)：156-158
71．姚莉，卞修武，张树辉，等。去甲二氢愈创木酸对恶性胶质瘤细胞基因组甲基化水平的影响及其意义．第三军医大学学报，2001；22(3)：251-253
72. Ansar S, Iqbal M, Athar M. Nordihydroguairetic acid is a potent inhibitor of ferric-nitrilotriacetate-mediated hepatic and renal toxicity, and renal tumor promotion in mice. Carcinogenesis. 1999; 20:599-606
73. Ramasamy S, Drummond GR, Ahn J, et al. Modulation of expression of endothelial nitric oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant in cultured endothelial cells. Mol Pharmacol. 1999; 56: 116-123
74. Ramoner R, Rieser C, Bartsch G, et al. Nordihydroguaiaretic acid blocks secretory and endocytic pathways in human dendritic cells. J Leuko Biol. 1998; 64:747-752
75. Fujiwara T, Takami N, Misumi Y, et al. Nordihydroguaiaretic acid blocks protein transport in the srcretory pathway causing redistribution of Golgi proteins into the endoplasmic reticulum. J Biol Chem. 1998; 273: 3068-3075
76. Barnaby JW, Styles AR, Cockerell CJ. Actinic keratoses: differential diagnosis and treatment. Drugs Aging. 1997; 11(3): 186-205
77. Damtew B, Spagnuolo PJ. Tumor cell-endothelial cell interactions: evidence for roles for lipoxygenase products of arachidonic acid in metastasis. Prost Leukot Essent Fatty Acids. 1997; 56(4): 295-300
78. Ingalill AM, Jett M, Boyle T, et al. Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J Clin Invest. 1996; 97(3): 806-813
79. Wilson DE, DiGianfilippo A, Ondrey FG, et al. Effects of nordihydroguaiaretic acid on cultured rat and human glioma cell proliferation. J Neurosurg. 1989; 71:551-557
80．郭德玉，陈意生，卞修武，等．bcl-2、c-myc基因表达在去甲二氢愈创木酸诱导人恶性胶质瘤细胞凋亡中的意义．第三军医大学学报．2001；22(3)：260-263
81．孙慧勤，卞修武，陈意生，等．去甲二氢愈创木酸对胶质瘤细胞SHG-44迁移的影响．中华病理学杂志，2000；29(1)：30-33
82．孙慧勤，陈意生，史景泉，等．去甲二氢愈创木酸对活体血管生成作用的实验研究．第三军医大学学报．2001；22(3)：276-279
83. Condorcelli DF, Dell'Albani P, Conticello SG, et al. Aneural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene. Dev Neurosci. 1997; 19:446-456
84. Barresi V, Condorelli DF, Giuffrida-Stella AM. GFAP gene methylation in different neural cell types from rat brain. Int J Dev Neurosci. 1999; 17(8): 821-828
85.Park S, Lee DK, Yang CH. Inhibition of fos-jun-DNA complex formation by dihydroguaiaretic acid and in vitro cytotoxic effects on cancer cells. Cancer Lett. 1998; 127(1-2): 23-28