摘要：淋巴瘤是一种淋巴细胞来源的实体肿瘤，分为霍奇金淋巴瘤和非霍奇金淋巴瘤两大类。侵袭性B细胞非霍奇金淋巴瘤中最常见的是弥漫性大B细胞淋巴瘤。研究发现淋巴瘤病人高表达ID1蛋白。ID1蛋白属于螺旋-环-螺旋(helix-loop-helix，HLH)转录因子家族，但不含DNA结合结构域。本课题使用能够下调ID1蛋白的抑制剂哌咪清（Pimozide）在淋巴瘤细胞中去研究ID1蛋白低表达在B细胞淋巴瘤中的作用与机制。实验结果表明：哌咪清对Raji和Raji-4RH的增殖都有抑制作用，并且可以诱导Raji的凋亡，还可以影响Raji和Raji-4RH的细胞周期。Pimozide与阿霉素和依托泊苷具有协同效应，表明ID1低表达可以提高细胞对这两种化疗药物的敏感性。而Pimozide与顺铂和盐酸吉西他滨具有拮抗效应，表明ID1低表达不能提高细胞对这两种化疗药物的敏感性。

关键词：ID1蛋白；复发难治性B细胞淋巴瘤；化疗药物；机制

The study on the effect and mechanism of ID1 protein inhibitor Pimozide in the recurrent refractory B-cell lymphoma

Abstract: Lymphoma is a kind of solid tumor from lymphoid cell, which can be pided into two categories, hodgkin's lymphoma and non-hodgkin's lymphoma. Diffuse large B-cell lymphoma is the most common form of invasive B-cell non-hodgkin's lymphoma. High expression of ID1 protein was found in patients with lymphoma. ID1 protein belongs to the family of helix-loop-helix(HLH) transcription factor, which does not contain basic DNA binding domain. To study the effect and mechanism of ID1 protein inhibitor in the recurrent refractory B-cell lymphoma, we used Pimozide to down-regulate the expression level of ID1 protein in lymphoma cells. The results showed that Pimozide inhibited the proliferation of Raji and Raji-4RH, induced apoptosis of Raji, and changed the cell cycle of Raji and Raji-4RH. The combination of Pimozide and chemotherapy drugs Dox/etoposide has the synergistic effect indicating that inhibition of ID1 can improve the sensitivity of cells to both etoposide and Dox. However, Pimozide combined with cisplatin/HCl-gemcitabine shows the antagonistic effect which suggest that low expression of ID1 could not improve the sensitivity of the cells to cisplatin and HCl-gemcitabine.

Key words: ID1 protein;Recurrent refractory B-cell lymphoma;chemotherapeutics;mechanism

目  录

摘要2

关键词2

Abstract2

Key words2

引言3

1材料与方法4

1.1实验材料与仪器 4

1.1.1实验材料4

1.1.2仪器设备4

1.2实验方法 4

1.2.1慢病毒质粒构建4

1.2.2 慢病毒质粒转化、培养及抽提5

1.2.3 293T、Raji和Raji-4RH的复苏、培养和传代6

1.2.4 转染293T——磷酸钙法6

1.2.5 感染Raji-4RH建立稳定细胞系7

1.2.6 蛋白质免疫印迹实验（Western Blot）7

1.2.7 细胞增殖实验9

1.2.8 检测哌咪清对Raji和Raji-4RH的生长抑制作用9

1.2.9 流式细胞术检测细胞凋亡9

1.2.10流式细胞术检测细胞周期9

1.2.11检测哌咪清与四种化疗药物的相互作用9

2结果与分析9

2.1哌咪清对Raji和Raji-4RH的生长抑制实验结果9

2.2细胞凋亡及细胞周期实验结果10

2.3哌咪清与四种化疗药物相互作用实验结果11

2.4293T转染结果——荧光观察13

3讨论14

致谢15

参考文献16

ID1蛋白抑制剂哌咪清（Pimozide）在复发难治B细胞淋巴瘤治疗中的作用与机制研究

引言

淋巴瘤是原发于淋巴细胞、淋巴结等淋巴造血系统的恶性肿瘤。由于其分型众多，且在形态、病理特征等方面具有很高的异质性，淋巴瘤的分类经历了一系列的变化，并且在不断更新。2016年，WHO发布了最新版淋巴瘤分类[1]。其中，淋巴瘤主要分为两大类：霍奇金淋巴瘤（hodgkin’s lymphoma,HL）和非霍奇金淋巴瘤（non-hodgkin’s lymphoma, NHL）。HL分型相对较少，而NHL分型非常多。在NHL中按照恶性程度可分为低度、中度和高度恶性淋巴瘤；按照细胞来源可分为成熟T细胞淋巴瘤、成熟NK细胞瘤和成熟B细胞淋巴瘤。其中B细胞淋巴瘤占比最大。