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1、目 录1筛选VEGF和VEGFR高表达的肝癌细胞株及苦参碱的初步干预作用11.1中文摘要11.2英文摘要31.3前言61.41.51.6材料与方法8结果22讨论341.7结论391.8参考文献401.9英汉缩略词对照表432致谢443诱导肿瘤细胞凋亡的天然药物研究进展(综述)45筛选VEGF和VEGFR高表达的肝癌细胞株及苦参碱的初步干预作用摘 要 目的：原发性肝癌(hepatocellular carcinoma，HCC)是血管富集性的肿瘤，其生长及转移和血管生成密切相关。血管内皮生长因子(vascular endothelial growth factor，VEGF)是体内一种强有力的促血
2、管生成因子，通过与VEGF受体（VEGFR）结合，引起一系列信号传导，释放各种生长因子和细胞因子，促进内皮细胞的增殖与迁移，直接或间接参与血管生成，在肝癌的发生、发展及愈后中具有极其重要的地位。本研究通过筛选VEGF和VEGFR高表达的肝癌细胞株，观察苦参碱等中药有效成分的初步干预作用，旨在构建靶向VEGF/VEGFR信号通路抑制肝癌血管生成的细胞筛选体系，筛选靶向VEGF/VEGFR信号通路抑制肝癌血管生成的药物，为中药现代化及肝癌的药物治疗提供一个新的思路。方法：（1）筛选VEGF和VEGFR高表达的肝癌细胞株：体外培养人肝癌细胞株Bel-7402、HepG2和 SMMC-7721，然后采
3、用免疫细胞化学法检测该三种细胞的VEGF及其受体Flt-1、KDR 的表达情况，酶联免疫吸附试验法（Enzyme-Linked Immunosorbent Assay，ELISA）检测细胞培养上清液中VEGF的浓度，逆转录聚合酶链式反应（RT-PCR：Reverse Transcription Polymerase Chain Reaction）技术检测细胞中VEGF mRNA的表达；（2）苦参碱等中药有效成分的初步干预作用：用小檗碱、槲皮素和苦参碱分别干预经过上述实验筛选出VEGF和VEGFR高表达的肝癌细胞株Bel-7402 48h，采用CCK-8试剂盒 (cell counting ki
4、t-8)方法检测细胞的增殖，计算半抑制浓度（IC50）。苦参碱干预48h后，采用RT-PCR方法检测Bel-7402 VEGF mRNA的表达。结果：（1）筛选VEGF和VEGFR高表达的肝癌细胞株：免疫组化可检测HepG2和Bel-7402细胞均具有VEGF、Flt-1和KDR的表达，而SMMC-7721细胞则只具有VEGF和Flt-1受体的表达，而无KDR受体的表达。ELISA 检测Bel-7402、HepG2及SMMC-7721细胞VEGF的表达量分别为811.4780.97、744.9056.36和484.5470.69 ng/3105个细胞，VEGF表达Bel-7402强于HepG2
5、和SMMC-7721细胞(F=24.317，P=0.000)。RT-PCR检测Bel-7402、HepG2及SMMC-7721细胞均有VEGF mRNA表达，相对表达量分别为0.9700.006、0.9150.010、0.7630.006，Bel-7402的VEGF mRNA表达高于HepG2和SMMC-7721 (F=636.167，P=0.000)。据此，本研究选定VEGF和VEGFR均高表达的肝癌细胞株Bel-7402作为实验用细胞。（2）苦参碱等中药有效成分的初步干预作用：槲皮素、苦参碱和小檗碱对Bel-7402细胞均能起到抑制作用，且有随着药物浓度的增高抑制作用增强，通过CurveE
6、xpert 1.3软件计算，小檗碱、苦参碱和槲皮素的IC50分别为：221.12 g/mL、1186.58 g/mL和78.38 g/mL。RT-PCR方法检测结果表明，苦参碱作用于Bel-7402细胞48h后，VEGF mRNA的表达量明显减少，且呈现较好的浓度依赖关系，与阴性对照比较，F=1229.64，P=0.000。结论：通过免疫组化、ELISA及RT-PCR对人肝癌细胞株HepG2、SMMC-7721和Bel-7402的筛选，Bel-7402中VEGF和VEGFR均高度表达，可作为靶向VEGF/VEGFR信号通路抑制肝癌血管生成的细胞筛选体系。苦参碱可明显抑制Bel-7402细胞的增
7、殖，减少VEGF mRNA的表达，初步提示苦参碱可能具有靶向VEGF/VEGFR信号通路抑制肝癌细胞增殖和血管生成的作用，但苦参碱是否还作用于VEGF的受体Flt-1和KDR，尚需进一步研究证实。关键词：VEGF；VEGFR；肝癌；RT-PCR；苦参碱Screening Hepatocarcinoma Cell Line with High Expression of VEGF and VEGFR and the Preliminary Intervention of MatrineAbstract Objective: Hepatocellular carcinoma (HCC) is th
8、e enrichment of blood vessels of cancer, its growth and transfer are closely related with angiogenesis. Vascular endothelial growth factor (VEGF) is a powerful factor to promote angiogenic, through combination with VEGF receptor (VEGFR), cause a series of signal transmission, release growth factors
9、and cytokines, and promote endothelial cell proliferation and migration, directly or indirectly effect on angiogenesis, it plays an important role on occurrence and development of hepatocarcinoma. This study through screening hepatocarcinoma cell line with high expression of VEGF and VEGFR, and obse
10、rving preliminary intervention effect of matrine and some other effective ingredients of traditional Chinese medicine on the cell line, which is aimed at establishing the screening system targeted VEGF/VEGFR signaling pathways to inhibit angiogenesis of hepatocarcinoma, which is used to screen the d
11、rugs targeted VEGF /VEGFR signaling pathways to inhibit angiogenesis of hepatocarcinoma, and providing a new train of thought for the modernization of traditional Chinese medicine and drug treatments for hepatocarcinoma. Methods: (1) Screening hepatocarcinoma cell line with high expression of VEGF a
12、nd VEGFR: Culture human hepatocarcinoma cell lines Bel-7402, HepG2 and SMMC-7721 in vitro, then the expression of VEGF and its receptor Flt-1, KDR were detected by Immunohistochemistry detection. Using the ELISA(Enzyme-Linked Immunosorbent Assay, ELISA) method to detect the expression of VEGF in the
13、 cell supernatant, and taking the method of Reverse Transcription Polymerase Chain Reaction(RT-PCR) technology to detect the expression of VEGF mRNA; (2) The preliminary intervention effect of matrine and other traditional Chinese medicine: Berberine, quercetin and matrine, the traditional Chinese m
14、edicine, intervened Bel-7402 cell line 48 hours, which existed high expression of VEGF and VEGFR screened form above experiment. The proliferation of Bel-7402 cell was detected by CCK-8 kit (cell counting kit-8) method, then calculating the half inhibition concentration (IC50). After 48 hours interv
15、ened with matrine, the expression of Bel-7402 VEGF mRNA was tested by RT-PCR. Results:(1) Screening hepatocarcinoma cell line with high expression of VEGF and VEGFR: Immunohistochemical detection showed that the HepG2 and Bel-7402 cells had VEGF, Flt-1 and KDR expression, but SMMC-7721 cells only had VEGF and Flt-1 expression, with no KDR expression. ELISA test suggested that the expression of VEGF in the cell supernatant from Bel-7402, HepG2
