【作者】 刘琼；

【导师】 叶秀峰； 张徽；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的:通过观察熊果酸(Ursolic acid,UA)对人脐静脉血管内皮细胞(HUVEC) ERK1、c-Jun、c-Myc、Cyclin D1表达的影响,以及熊果酸对胶质瘤裸鼠移植瘤生长和对胶质瘤裸鼠移植瘤ERK1、c-Jun、c-Myc、Cyclin D1和CD34的影响,探讨熊果酸抗胶质瘤生长的机制。方法:1.体外培养HUVEC,用熊果酸和PD98059处理HUVEC 48h,以及熊果酸按浓度梯度(0、31.5、62.5、125、250、500ug/ml)处理HUVEC 24h和125ug/ml按时间梯度(0、12、24、48h)处理HUVEC,然后通过RT-PCR检测ERK1、c-Jun、c-Myc、Cyclin D1 mRNA水平,Western blot检测ERK1、c-Jun、c-Myc、Cyclin D1蛋白表达情况。2.皮下移植术复制胶质瘤裸鼠皮下移植瘤模型,然后将其分为三组:熊果酸组(熊果酸50mg.kg-1.d-1,腹腔注射,连续用药20天);PD98059组(PD98059 2mg.kg-1.d-1,腹腔注射,连续用药7天);空白对照组(生理盐水0.2ml.d-1,腹腔注射,共20天)。用免疫组化技术检测移植瘤组织ERK1、C-Jun、C-Myc、Cyclin D1和CD34蛋白表达变化;用原位杂交技术观察ERK1 mRNA表达变化。结果:RT-PCR结果显示,经熊果酸和PD98059处理后,HUVEC中ERK1、C-Jun、C-Myc、Cyclin D1 mRNA表达水平都有明显减弱,且熊果酸不同浓度、不同时间处理HUVEC后,HUVEC中ERK1、C-Myc、C-Jun和Cyclin D1 mRNA表达随熊果酸浓度增加和时间的增加逐渐减少(p<0.05);Western blot结果表明,熊果酸和PD98059处理后,HUVEC中ERK1、C-Jun、C-Myc、Cyclin D1蛋白表达也有不同程度下调,熊果酸处理后ERK1、C-Jun、C-Myc、Cyclin D1蛋白表达下调程度呈浓度-时间依赖性((p<0.05);免疫组化显示,经熊果酸和PD98059处理后的恶性胶质瘤细胞株C6裸鼠移植瘤中ERK1、C-Jun、C-Myc、Cyclin D1和CD34蛋白表达比空白对照组明显减少;原位杂交结果显示,熊果酸组和PD98059组ERK1 mRNA表达水平比对照组显著减少;结论:熊果酸可抑制HUVEC中ERK1、C-Jun、C-Myc、Cyclin D1 mRNA和蛋白表达,呈浓度-时间依赖性(p<0.05);熊果酸可抑制恶性胶质瘤细胞株C6裸鼠移植瘤ERK1、C-Jun、C-Myc、Cyclin D1和CD34蛋白表达及ERK1 mRNA表达。研究表明:熊果酸可通过抑制ERK信号转导通路抑制胶质瘤血管内皮细胞及胶质瘤细胞增殖,抑制胶质瘤生长。本课题为胶质瘤治疗的新途径提供了重要的实验依据。更多还原

【Abstract】 Objective: To investigate the effects of the expression of ERK1、C-Jun、C-Myc、Cyclin D1 of Human Umbilical Vein Endothelial Cells (HUVEC) in ursolic acid,and the effects of ursolic acid on the expression of ERK1、C-Jun、C-Myc、Cyclin D1 and CD34 in transplanlationumor of malignant glioblastoma cell line C6 in nude mice ,and to explore the mechanism of in effect of ursolic acid on glioma.Methods: 1. HUVEC was treated with ursolic acid and PD98059 or with ursolic acid at 0、31. 5、62.5、125、250、500ug/ml for 24h,or with ursolic acid at 125ug/ml for 0、12、24、48h for the course assay in vitro.RT-PCR was performed to measure the endogenous levels of ERK1、C-Jun、C-Myc、Cyclin D1 mRNA,Western blotting was use to detect the protein expression of ERK1、C-Jun、C-Myc、Cyclin D1. 2. C6 glioblastoma cells were injected into subcutaneously nude mice to establish the subcutaneous model of glioblastoma in nude mice,then the mouse models were assigned 3 groups: blank comtrol group,Ursolic acid group(50 mg·kg-1·d-1, intra-abdominal injection,20 days), PD98059 group (2 mg·kg-1·d-1, intra-abdominal injection,7 days). Living of nude mice,growth and histopathological changes of the tumors were observed. The expression of ERK1、C-Jun、C-Myc and Cyclin D1 and CD34 and the expression of ERK1 mRNA of the tumor tissues were detected by immunohistochemical technique(IHC) and in suit hybridization(ISH), respectively.Results: RT-PCR results showed that the mRNA level of ERK1、C-Jun、C-Myc、Cyclin D1 were decreased obviously after treated with ursolic acid and PD98059,and the degression is in a dose- and time-dependent manner after treated with ursolic acid(p<0.05).Western blotting results showed that the protein expression of ERK1、C-Jun、C-Myc、Cyclin D1 were all decreased obviously were decreased obviously after treated with ursolic acid and PD98059 ,and the descent is in a dose- and time-dependent manner after treated with ursolic acid(p<0.05).Immunohistochemical technique(IHC) results showed that the expression of the protein of ERK1、C-Jun、C-Myc、Cyclin D1 and CD34 of Ursolic acid group and PD98059 group were obviously lower than the control group(P<0.05) ,respectively.In suit hybridization(ISH) results showed the expression of ERK1 mRNA of Ursolic acid group and PD98059 group were also obviously lower than the control group(P<0.05).Conclusion: Ursolic acid could inhibited the expression of ERK1、C-Jun、C-Myc、Cyclin D1 mRNA and protein expression of HUVEC ,and those changes were dose- and time-dependent (p<0.05).Ursolic acid could also significantly inhibited the implanted glioma of C6 glioblastoma cells the protein expression of ERK1、C-Jun、C-Myc、Cyclin D1 and CD34 ,and the expression ERK1 mRNA.Our study demonstrated that Ursolic acid significantly inhibited the generation of Vascular endothelial cell of glioma and glioblastoma cells by down- regulating the expression of ERK1、C-Jun、C-Myc and Cyclin D1 of ERK signal transduction pathway.更多还原