【作者】 杨啊晶；

【导师】 陈虎；

【作者基本信息】 中国人民解放军军事医学科学院 ， 内科学， 2005， 硕士

【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⁶⁰C。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更多还原

【Abstract】 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.更多还原