淋巴瘤细胞系Raji细胞血管内皮细胞生长因子表达及基质胶中ECV304细胞血管形成与雷公藤内酯醇的干预*☆

崔国惠，陈卫华，张纯，陈燕

课题背景：课题为国家自然科学基金资助项目（30472267）的部分实验内容。课题包括雷公藤内酯醇对核孔蛋白的调节作用、对血管内皮细胞生长因子及肿瘤血管生成的影响、对SDF-1/CXCR4生物学轴效应的作用等。

应用要点：实验利用细胞培养以及基质胶中血管形成实验证实雷公藤内酯醇具有抑制淋巴瘤Raji细胞表达及分泌血管内皮细胞生长因子，并抑制其介导的血管形成。

同行评价：雷公藤内酯醇作为雷公藤一个有效成分单体，其作用明确。本文对其影响淋巴瘤系Raji细胞血管内皮细胞生长因子表达合成分泌及相关血管生成机制进行观察，文章条理清楚，思路清晰，结果具有较强的实际意义。

摘要
目的:观察雷公藤内酯醇对淋巴瘤细胞系Raji细胞血管内皮细胞生长因子（vascular endothelial growth factor, VEGF）的表达、合成分泌以及对基质胶中ECV304细胞血管形成的影响。
方法：实验于2005-08/2006-02在华中科技大学同济医学院公共卫生学院分子生物学实验室完成。①MTT法检测3.125，6.25，12.5，25，50 nmol/L的雷公藤内酯醇在作用12，24，36，48，60，72 h对Raji细胞增殖的影响。②RT-PCR法检测12.5，25，50 nmol/L雷公藤内酯醇处理Raji细胞24 h后，细胞VEGFmRNA表达的变化。③ELISA法检测25 nmol/L雷公藤内酯醇处理Raji细胞24 h后，细胞培养上清VEGF的分泌量。④采用血管形成实验检测雷公藤内酯醇对ECV304细胞在基质胶中血管形成的影响。
结果：①雷公藤内酯醇以时间和剂量依赖方式抑制Raji细胞增殖，24 h半数抑制浓度为25 nmol/L。②Raji细胞内VEGFmRNA主要为VEGF165和VEGF121，雷公藤内酯醇明显抑制其表达，呈剂量依赖方式。③雷公藤内酯醇明显抑制Raji细胞VEGF的分泌量(P < 0.01)。④Raji细胞培养上清、10 μg/L VEGF处理的Raji细胞培养上清可促进ECV304细胞在基质胶中形成血管，而25 nmol/L雷公藤内酯醇处理的Raji细胞培养上清加入基质胶则未见血管形成。
结论：雷公藤内酯醇以时间和剂量依赖性方式抑制淋巴瘤细胞增殖、VEGF的表达分泌及血管形成。
关键词：雷公藤内酯醇；血管内皮细胞生长因子；血管形成��

崔国惠，陈卫华，张纯，陈燕. 淋巴瘤细胞系Raji细胞血管内皮细胞生长因子表达及基质胶中ECV304细胞血管形成与雷公藤内酯醇的干预 [J].中国组织工程研究与临床康复，2008，12(11):2039-2042
[www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2039(ps).pdf]

华中科技大学同济医学院附属协和医院血液病研究所，湖北省武汉市 430022

崔国惠☆，女，1963年生，湖北省宜昌市人，汉族，1986年白求恩医科大学毕业，博士，副教授，主要从事恶性血液病分子生物学方面研究。
ghcui@medmail.
com.cn

通讯作者：陈燕，博士，教授，华中科技大学同济医学院附属协和医院血液病研究所，湖北省武汉市 430022

国家自然科学基金资助项目
(30472267)*

中图分类号:R329.114
文献标识码:A
文章编号:1673-8225
(2008)11-02039-04

收稿日期：2007-07-24
修回日期：2008-01-22 (07-50-7-3991/N·Q)

Effects of triptolide on the expression of vascular endothelial growth factor in Raji cells and angiogenesis of ECV304 cells in matrigel

Abstract

AIM：
AIM：To investigate the effect of triptolide on the expression of vascular endothelial growth factor (VEGF) in Raji cell line and on angiogenesis in cell line (ECV304).
METHODS: The experiment was performed at the Molecular Biology Laboratory of Public Health Constitute, Tongji Medical College, Huazhong University of Science and Technology from August 2005 to February 2006. ①The antiproliferative effects of triptolide (3.125, 6.25, 12.5, 25 and 50 nmol/L) against Raji cells were determined by the MTT method at 12, 24, 36, 48, 60 and 72 hours. ②After 24 hours incubations treated by different concentrations of triptolide (12.5, 25, 50 nmol/L), the mRNA level of VEGF by Raji cells was measured by reverse transcription-polymerase chain reaction (RT-PCR). ③After 24 hours treatment with 25 nmol/L triptolide, VEGF concentrations in cell culture supernatants were determined by enzyme-labeled immunosorbent assay (ELISA). ④Effect of triptolide on angiopoiesis of ECV304 cells in matrigel was detected by angiopoiesis test.
RESULTS: ①The triptolide significantly inhibited the proliferation of Raji cells in time- and dose-dependent manner. IC50 value was 25 mmol/L. ②The mRNA expression of VEGF165 and VEGF121 (containing 165 and 121 amino acid residues, respectively) was detected in all groups. Triptolide reduced the mRNA expression of VEGF165 and VEGF121 in a dose-dependent manner. ③Triptolide significantly suppressed VEGF expression in Raji cells (P < 0.01). ④There was blood vessel formation on matrigel in the supernatants from Raji culture group and VEGF group. No network and cord formed were seen in control and triptolide groups.
CONCLUSION: Triptolide could inhibit the proliferation of Raji cells in time- and dose-dependent manners as well as the expressions of VEGF in Raji cells and angiogenesis in ECV304 cells.

Cui GH, Chen WH, Zhang C, Chen Y. Effects of triptolide on the expression of vascular endothelial growth factor in Raji cells and angiogenesis of ECV304 cells in matrigel.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(11):2039-2042(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2039(ps).pdf]

Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China

Cui Guo-hui☆ Doctor, Associate professor, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
ghcui@medmail.
com.cn

Correspondence to: Chen Yan, Doctor, Professor, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China

Supported by: the National Natural Science Foundation of China, No. 30472267*

Received: 2007-07-24
Accepted: 2008-01-22