Notch信号在S-沙利度胺作用于人U266细胞中的作用
|
摘要
目的:多发性骨髓瘤(multiple myeloma,MM)是血液系统比较常见的恶性肿瘤。但是到目前为止,无论是常规化疗还是大剂量化疗,MM仍然不可治愈。为克服对化疗的抵抗并改善疗效,已经或正在研制新的治疗策略。其中包括针对经典靶位点如DNA或细胞骨架、靶向调节细胞增殖与生存的分子途径等方面。近年来沙利度胺被应用于MM的治疗中,并取得了巨大成功。但其治疗机制仍不清楚。沙利度胺用量大,不良反应重,多数病人无法耐受,阻碍了它的广泛应用。新的研究认为Notch信号是血液恶性肿瘤及实体瘤潜在的治疗靶点。我们将从探讨沙利度胺是否通过Notch信号传导途径对骨髓瘤细胞发生作用为着手点,为其治疗MM的机制及基因治疗和新药的研究作一实验基础。
材料方法:1、人骨髓瘤U266细胞、人成纤维细胞系(human bone marrow fibroblastoid stromal cell line,HFCL)细胞的培养及生长特性:试验分为四组:A:U266细胞组;B:U266细胞+S-Thd组;C:U266细胞+HFCL组;D:U266细胞+HFCL+S-Thd组。HFCL细胞70-80%融合时,给予~(60)C。照射25Gy。B、D两组各孔分别加入2.5ulS-Thd原液,即S-Thd的终浓度为100ug/ml。A、C两组各孔分别加入2.5ulDMSO作为对照。培养24、48、72小时后,各组U266细胞分别经台盼兰染色后绘制生长曲线。2、S-Thd诱导U266细胞凋亡:A、B、C、D组U266细胞经72小时培养后通过吉姆萨染色、TUNEL法及检测DNA Ladder的方法检测是否发生凋亡。3、S-Thd改变U266细胞Notch基因的表达水平:Notch基因的激活间接表现为Hes-1基因的过度表达。通过竞争RT-PCR半定量的方法,检测A、B、C、D组U266细胞经72小时培养后Hes-1基因的表达变化。
结果:1.各组U266细胞的生长具有时间依赖性(P<0.001);但在试验24小时内,各组U266细胞的生长情况并没有显著的差异(P>0.1);试验24小时后,U266细胞的生长受到S-Thd、HFCL的抑制(与A组相比,B、C、D组的U266细胞数降低,P<0.001);S-Thd和HFCL对U266细胞的抑制作用无差异(B组与C组相比,P>0.05)。2.吉姆萨染色法可见在加S-Thd的两个试验组中部分U266细胞皱缩、胞质和染色质浓缩、核染色质呈新月状的典型的凋亡细胞形态;TUNEL
Objection: Multiple myeloma(MM) is a common tumor in the hematological system. But until now,MM has not been cured by either routine chemotherapy or large doses chemotherapy.To overcome the resistance to chemotherapy and amend curative effect,people have been developing new therapeutic strategies,including aiming at classical targets as DNA or framework of cells and modulating directionally molecule approach of proliferation and survival of cells. Recent years thalidomide (Thd) has been applied for MM and succeeds in treating MM. Its mechanism is still unknown. Most scientists can't tolerate thalidomide, because its large doses and toxic and side reactions. New studies have implied Notch signal as a treated target in hematological and solid tumors. We will discuss whether thalidomide has an impact on myeloma cells by Notch signal, and provide a laboratory foundation for mechanism of thalidomide and gene therapy and new drugs.Methods: 1) human myeloma U266 cells and HFCL cells were cultured together,and protracted their growth curve.there were four groups: A: U266 cells group. B: U266 cells+S-Thd group. C: U266 cells+ HFCL. D: U266 cells+ HFCL+S-Thd group. When HFCL cells inosulated to 70-80%, irradiation at dose of 25 Gy was given. Each pore in B and D groups was given 2.5ul S-Thd original liquid respectively, namely final concentration of S-Thd was 100ug/ml. Each pore in A and C groups was given 2.5ul DMSO as control. After 24、 48、 72hours of culturing,U266 cells of each group were stained by typan blue, then protracted their growth curve. 2) S-Thd induced U266 cells apoptositic: After 72hours of culturing, U266 cells of each group were detected whether apoptosis occurred by Giemsa staining. TUNEL and DNA ladder methods. 3) S-Thd can change the expression of Notch2. Overexpression of Hes-1 indicates activation of Notch indirectly. By competitive RT-PCR method, we detected the changes of expression of Hes-1 of U266 cells in four groups after 72 hours.
材料方法:1、人骨髓瘤U266细胞、人成纤维细胞系(human bone marrow fibroblastoid stromal cell line,HFCL)细胞的培养及生长特性:试验分为四组:A:U266细胞组;B:U266细胞+S-Thd组;C:U266细胞+HFCL组;D:U266细胞+HFCL+S-Thd组。HFCL细胞70-80%融合时,给予~(60)C。照射25Gy。B、D两组各孔分别加入2.5ulS-Thd原液,即S-Thd的终浓度为100ug/ml。A、C两组各孔分别加入2.5ulDMSO作为对照。培养24、48、72小时后,各组U266细胞分别经台盼兰染色后绘制生长曲线。2、S-Thd诱导U266细胞凋亡:A、B、C、D组U266细胞经72小时培养后通过吉姆萨染色、TUNEL法及检测DNA Ladder的方法检测是否发生凋亡。3、S-Thd改变U266细胞Notch基因的表达水平:Notch基因的激活间接表现为Hes-1基因的过度表达。通过竞争RT-PCR半定量的方法,检测A、B、C、D组U266细胞经72小时培养后Hes-1基因的表达变化。
结果:1.各组U266细胞的生长具有时间依赖性(P<0.001);但在试验24小时内,各组U266细胞的生长情况并没有显著的差异(P>0.1);试验24小时后,U266细胞的生长受到S-Thd、HFCL的抑制(与A组相比,B、C、D组的U266细胞数降低,P<0.001);S-Thd和HFCL对U266细胞的抑制作用无差异(B组与C组相比,P>0.05)。2.吉姆萨染色法可见在加S-Thd的两个试验组中部分U266细胞皱缩、胞质和染色质浓缩、核染色质呈新月状的典型的凋亡细胞形态;TUNEL
Objection: Multiple myeloma(MM) is a common tumor in the hematological system. But until now,MM has not been cured by either routine chemotherapy or large doses chemotherapy.To overcome the resistance to chemotherapy and amend curative effect,people have been developing new therapeutic strategies,including aiming at classical targets as DNA or framework of cells and modulating directionally molecule approach of proliferation and survival of cells. Recent years thalidomide (Thd) has been applied for MM and succeeds in treating MM. Its mechanism is still unknown. Most scientists can't tolerate thalidomide, because its large doses and toxic and side reactions. New studies have implied Notch signal as a treated target in hematological and solid tumors. We will discuss whether thalidomide has an impact on myeloma cells by Notch signal, and provide a laboratory foundation for mechanism of thalidomide and gene therapy and new drugs.Methods: 1) human myeloma U266 cells and HFCL cells were cultured together,and protracted their growth curve.there were four groups: A: U266 cells group. B: U266 cells+S-Thd group. C: U266 cells+ HFCL. D: U266 cells+ HFCL+S-Thd group. When HFCL cells inosulated to 70-80%, irradiation at dose of 25 Gy was given. Each pore in B and D groups was given 2.5ul S-Thd original liquid respectively, namely final concentration of S-Thd was 100ug/ml. Each pore in A and C groups was given 2.5ul DMSO as control. After 24、 48、 72hours of culturing,U266 cells of each group were stained by typan blue, then protracted their growth curve. 2) S-Thd induced U266 cells apoptositic: After 72hours of culturing, U266 cells of each group were detected whether apoptosis occurred by Giemsa staining. TUNEL and DNA ladder methods. 3) S-Thd can change the expression of Notch2. Overexpression of Hes-1 indicates activation of Notch indirectly. By competitive RT-PCR method, we detected the changes of expression of Hes-1 of U266 cells in four groups after 72 hours.
引文
1.陈世伦 武永吉主编,《多发性骨髓瘤》,人民卫生出版社出版,2004年4月第1版,第235-260页.
2. Kojika, S. Griffin, J. D. . Notch Receptors and Hematopoiesis. Experimental Hematology. Experimental Hematology 2001; 29: 1041-1052.
3. Radtke F, Wilson A, Mancini SJ, et al. Notch regulation of lymphocyte development and function. Nature immunology 2004; 5 (3) : 247-253.
4. Laurie A. Milner. Notch signaling: a key to the pathogenesis of multiple myeloma? Blood 2004; 103 (9) : 3253-3254.
5. Zagouras P, Stifani S, Blaumueller CM, et al. Alterations in Notch sig nalingin neoplastic lesions of the human cervix. Proc Natl Acad Sci USA 1995; 92 (14) : 6414-6418.
6. Leethanakul C, Patel V, Gillespie J, et al. Distinct pattern of expression of differentiation and growth related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays. Oncogene 2000; 19 (28) : 3220-3224.
7. Enlund F, Behboudi A, Andren Y, et al. Altered Notch signaling resulting from expression of a WAMTP12MAML2 gene fusion in mucoepidermoid carcinomas and benign Warthin' s tumors. Exp Cell Res 2004; 292: (1) : 212-218.
8. Jundt F, Schulze Proebsting KS, Anagnostopoulos I, et al. Jaggedl-induced Notch signaling drives proliferation of multiple myeloma cells. Blood 2004; 103 (9) : 3511—3515.
9. Chauhan D, Uchiyama H, Akbarali Y, et al. Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B. Blood 1996; 87: 1104-1112.
10. Faid L, Van Riet I, De Waele M, et al. Adhesive interaction between tumour cells and bone marrow stromal elements in human multiple myeloma. Eur J Haematol 1996; 57: 349-358.
11. Eriksson T, Bjorkman S, et al. clinical pharmacology of thalidomide. Eur J Clin Pharmacol 2001; 57: 365-376.
12. Thomas D, Kantarjian H. The revitalization of thalidomide. Ann Oncol 2001; 12: 885-886.
13. William S. Dalton. The tumor microenvironment: focus on myeloma. Cancer Treatment Reviews 2003; 24 (Suppl 1) : 11-19.
14. Boris tin, Laurence Catley, et el. Patupilone(epothilone B) inhibits growth and survival of multiple myeloma cells in vitro and in vivo. Blood 2005; 105 (1) : 350-357.
15. Toshiaki Hayashi, Teru Hideshima, Aaron N. Nguyen. Transforming Growth Factor β Receptor I Kinase Inhibitor Down-Regulates Cytokine Secretion and Multiple Myeloma Cell Growth in the Bone Marrow Microenvironment. Clin Cancer Res 2004; 10 (22) : 7540-7546.
16. Y Nefedova, TH handowski, et al. Bone marrow stromal-derived soluble factors and direct cell contact contribute to denovo drug resistance of myeloma cells by distinct mechanism. Leukemia 2003; 17: 1175-1182.
17.靳雪琴 白庆咸等,正常人骨髓成纤维基质细胞对骨髓瘤细胞RPMI8226增殖的影响,中国实验血液学杂志 2004;12(4):475-479.
18. Yulia Nefedova, Pingyan Cheng, Melissa Alsina. Involvement of Notch-1 signaling in bone marrow stroma-mediated de novo drug resistance of myeloma and other malignant lymphoid cell lines. Blood 2004; 103 (9) : 3503-3510.
19.来茂德主编,《医学分子生物学》,人民卫生出版社,1999年9月第1版,第10-94页.
20.薛绍白 柳惠图等主编,《细胞生物学》,北师大出版社,1998年第2版,第1-733页.
21. Nicholas M, constantine S, et al. Apoptotic signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma
2. Kojika, S. Griffin, J. D. . Notch Receptors and Hematopoiesis. Experimental Hematology. Experimental Hematology 2001; 29: 1041-1052.
3. Radtke F, Wilson A, Mancini SJ, et al. Notch regulation of lymphocyte development and function. Nature immunology 2004; 5 (3) : 247-253.
4. Laurie A. Milner. Notch signaling: a key to the pathogenesis of multiple myeloma? Blood 2004; 103 (9) : 3253-3254.
5. Zagouras P, Stifani S, Blaumueller CM, et al. Alterations in Notch sig nalingin neoplastic lesions of the human cervix. Proc Natl Acad Sci USA 1995; 92 (14) : 6414-6418.
6. Leethanakul C, Patel V, Gillespie J, et al. Distinct pattern of expression of differentiation and growth related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays. Oncogene 2000; 19 (28) : 3220-3224.
7. Enlund F, Behboudi A, Andren Y, et al. Altered Notch signaling resulting from expression of a WAMTP12MAML2 gene fusion in mucoepidermoid carcinomas and benign Warthin' s tumors. Exp Cell Res 2004; 292: (1) : 212-218.
8. Jundt F, Schulze Proebsting KS, Anagnostopoulos I, et al. Jaggedl-induced Notch signaling drives proliferation of multiple myeloma cells. Blood 2004; 103 (9) : 3511—3515.
9. Chauhan D, Uchiyama H, Akbarali Y, et al. Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B. Blood 1996; 87: 1104-1112.
10. Faid L, Van Riet I, De Waele M, et al. Adhesive interaction between tumour cells and bone marrow stromal elements in human multiple myeloma. Eur J Haematol 1996; 57: 349-358.
11. Eriksson T, Bjorkman S, et al. clinical pharmacology of thalidomide. Eur J Clin Pharmacol 2001; 57: 365-376.
12. Thomas D, Kantarjian H. The revitalization of thalidomide. Ann Oncol 2001; 12: 885-886.
13. William S. Dalton. The tumor microenvironment: focus on myeloma. Cancer Treatment Reviews 2003; 24 (Suppl 1) : 11-19.
14. Boris tin, Laurence Catley, et el. Patupilone(epothilone B) inhibits growth and survival of multiple myeloma cells in vitro and in vivo. Blood 2005; 105 (1) : 350-357.
15. Toshiaki Hayashi, Teru Hideshima, Aaron N. Nguyen. Transforming Growth Factor β Receptor I Kinase Inhibitor Down-Regulates Cytokine Secretion and Multiple Myeloma Cell Growth in the Bone Marrow Microenvironment. Clin Cancer Res 2004; 10 (22) : 7540-7546.
16. Y Nefedova, TH handowski, et al. Bone marrow stromal-derived soluble factors and direct cell contact contribute to denovo drug resistance of myeloma cells by distinct mechanism. Leukemia 2003; 17: 1175-1182.
17.靳雪琴 白庆咸等,正常人骨髓成纤维基质细胞对骨髓瘤细胞RPMI8226增殖的影响,中国实验血液学杂志 2004;12(4):475-479.
18. Yulia Nefedova, Pingyan Cheng, Melissa Alsina. Involvement of Notch-1 signaling in bone marrow stroma-mediated de novo drug resistance of myeloma and other malignant lymphoid cell lines. Blood 2004; 103 (9) : 3503-3510.
19.来茂德主编,《医学分子生物学》,人民卫生出版社,1999年9月第1版,第10-94页.
20.薛绍白 柳惠图等主编,《细胞生物学》,北师大出版社,1998年第2版,第1-733页.
21. Nicholas M, constantine S, et al. Apoptotic signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma