健脾解毒方对大肠癌肿瘤血管形成相关基因表达的影响

发布时间：2019-05-22 02:01

【摘要】：目的:探讨健脾解毒方对大肠癌肿瘤血管形成相关基因表达的影响。方法:采用水提法制作健脾解毒方粗提取物,MTT法检测健脾解毒方粗提取物对大肠癌HT29细胞增殖的影响,Graph Pad Prism 5软件计算IC50(50%inhibitory concentration)值,Affymetrix基因表达谱芯片检测健脾解毒方干预后有显著差异表达的基因并进行路径富集分析,基于哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路利用IPA(Ingenuity Pathway Analysis)软件对肿瘤血管形成的相关差异表达基因进行生物信息学分析并构建网路关系图,Western印迹法验证肿瘤血管形成相关差异表达的关键基因的蛋白表达水平。结果:健脾解毒方粗提取物能够抑制大肠癌HT29细胞增殖,处理24,48及72 h后的IC50值分别为13.060,9.646及8.448 mg/m L。Affymetrix基因表达谱芯片检测健脾解毒方干预后显著上调的基因218个,显著下调的基因252个,主要为生物合成、新陈代谢、细胞凋亡、抗原提取、血管生成等相关基因,其中肿瘤血管形成相关差异表达基因12个。IPA软件分析结果显示健脾解毒方干预后可使大肠癌中的鞘磷脂磷酸二酯酶3(sphingomyelin phosphodiesterase 3,SMPD3),VEGF,血管内皮生长因子A(vascular endothelial growth factor A,VEGFA),整合数α亚基1(integrin subunit alpha 1,ITGA1),组织蛋白酶B(cathepsin B,CTSB),组织蛋白酶S(cathepsin S,CTSS)等基因下调,GRB2相关结合蛋白2(GRB2 associated binding protein 2,GAB2),PLAUR(plasminogen activator,urokinase receptor)等基因上调。Western印迹检测显示健脾解毒方能明显下调磷酸化mTOR(phospho-mTOR,P-mTOR),信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3),缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和VEGF蛋白表达,明显上调磷酸化P53(phospho-P53,P-P53)蛋白表达。结论:健脾解毒方可能通过影响mTOR-HIF-1α-VEGF信号通路抑制大肠癌肿瘤血管形成,这可能是健脾解毒方治疗大肠癌的作用机制之一。
[Abstract]:Objective: to investigate the effect of Jianpi jiedu recipe on the expression of angiogenic genes in colorectal cancer. Methods: the crude extract of Jianpi jiedu recipe was prepared by water extraction. The effect of Jianpi jiedu recipe crude extract on the proliferation of colorectal cancer HT29 cells was detected by MTT method. The IC50 (50%inhibitory concentration) value was calculated by, Graph Pad Prism 5 software. Affymetrix gene expression profile microarray was used to detect the differentially expressed genes after the intervention of Jianpi jiedu recipe and the path enrichment analysis was carried out. Based on the mammal rapamicin target protein (mammalian target of rapamycin, MTOR) the signal pathway uses IPA (Ingenuity Pathway Analysis) software to analyze the differentially expressed genes related to tumor angiogenesis and construct the network diagram. Western staining was used to verify the protein expression of key genes related to tumor angiogenesis. Results: the crude extract of Jianpi jiedu recipe could inhibit the proliferation of colorectal cancer HT29 cells. The IC50 values of 24 h, 48 h and 72 h after treatment were 13.060. 9.646 and 8.448 mg/m L.Affymetrix gene expression profiles were used to detect 218 genes significantly upregulated and 252 genes down-regulated after Jianpi Jiedu recipe intervention, mainly biosynthesis. Metabolism, apoptosis, antigen extraction, angiogenesis and other related genes, Among them, 12 differentially expressed genes related to tumor angiogenesis were analyzed by IPA software. The results of IPA software analysis showed that the intervention of Jianpi jiedu recipe could make sphingomyosphatide phosphodiesterase 3 (sphingomyelin phosphodiesterase 3, SMPD 3), VEGF, in colorectal cancer. Vascular endothelial growth factor A (vascular endothelial growth factor A, VEGFA), integration number 伪 subunit 1 (integrin subunit alpha 1, cathepsin B (cathepsin B, CTSB), cathepsin S (cathepsin S, CTSS) were down-regulated. GRB2 related binding protein 2 (GRB2 associated binding protein 2, GABA 2), PLAUR (plasminogen activator,urokinase receptor) and other genes were up-regulated. Western blot analysis showed that Jianpi jiedu recipe could significantly down-regulate the phosphorylation of mTOR (phospho-mTOR,P-mTOR). The expression of signal transduction and transcription activating factor 3 (signal transducer and activator of transcription 3), hypoxia inducible factor 1 伪 (hypoxia inducible factor-1 伪 (HIF-1 伪) and VEGF protein were significantly up-regulated by phosphorylation of p53 (phospho-P53,P-P53) protein. Conclusion: Jianpi jiedu recipe may inhibit tumor angiogenesis of colorectal cancer by affecting mTOR-HIF-1 伪-VEGF signal pathway, which may be one of the mechanisms of Jianpi jiedu recipe in the treatment of colorectal cancer.
【作者单位】：中南大学湘雅二医院中西结合科;中南大学湘雅二医院普外科;中南大学湘雅二医院肿瘤科;湖南中医药大学药学院;中南大学湘雅二医院药剂科;湖南中医药大学研究生院;
【基金】：国家自然科学基金(81273722) 湖南省中医药管理局科研课题(201135)~~
【分类号】：R285

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