美洲大蠊多肽对血管生成的影响
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摘要
目的:探讨美洲大蠊多肽对血管生成的作用,探讨其机制。方法:以人脐静脉内皮细胞HUVECs和人肝癌Hep G2细胞为研究对象,采用噻唑蓝(MTT)比色法和细胞划痕实验,观察不同质量浓度(6. 25,12. 5,25,50,100 mg·L-1)的美洲大蠊多肽(PAP-1～3),脱脂膏和美洲大蠊多肽前体物CⅡ-3对HUVECs增殖、迁移的影响,另设正常组和沙利度胺组;采用小管形成实验检测各组HUVECs小管形成能力;通过细胞间黏附实验,观察不同质量浓度(25,50,100 mg·L-1)的美洲大蠊提取物处理后人肝癌Hep G2细胞同HUVECs之间黏附能力;利用免疫细胞化学染色和酶联免疫吸附测定(ELISA)检测各组HUVECs中血管内皮生长因子(VEGF)蛋白表达。结果:MTT比色法结果显示,多肽均能抑制HUVECs的增殖(P <0. 05),呈剂量依赖关系。不同质量浓度多肽分别作用24,48,72 h,PAP-2的抑制作用优于PAP-1,PAP-3(P <0. 05)。不同质量浓度的CⅡ-3和脱脂膏作用,HUVECs的存活率明显升高。细胞划痕实验结果显示多肽均能抑制HUVECs迁移(P <0. 05),且随药物浓度的增加,抑制迁移的作用也越强。其中PAP-2的作用优于PAP-1和PAP-3(P <0. 05); CⅡ-3在一定程度上能抑制HUVECs迁移,但是剂量越大,抑制作用越弱;脱脂膏作用后,HUVECs迁移能力增强。小管生成实验结果显示,多肽均能抑制HUVECs小管形成(P <0. 05),且随着给药浓度的增大,抑制作用越强。其中,PAP-2对小管形成的抑制作用优于PAP-1和PAP-3(P <0. 05)。CⅡ-3和脱脂膏一定程度上促进HUVECs小管的形成。细胞间黏附实验结果显示,多肽均能阻断Hep G2同HUVECs之间的黏附(P <0. 05),其中PAP-2对Hep G2同HUVECs间黏附的阻断作用强于PAP-1和PAP-3(P <0. 05)。相反,CⅡ-3和脱脂膏在一定程度上却能促进Hep G2同HUVECs间黏附。免疫细胞化学染色和ELISA实验结果显示,多肽均能够下调HUVECs细胞中VEGF含量(P <0. 05),且呈浓度依赖性。其中,PAP-2对VEGF的下调作用优于PAP-1和PAP-3(P <0. 05)。而CⅡ-3和脱脂膏能上调HUVECs中VEGF的表达。结论:美洲大蠊多肽对HUVECs的侵袭和转移,分化形成小管的能力有一定抑制作用,并能下调细胞中VEGF蛋白的表达。其中,美洲大蠊多肽的作用优于CⅡ-3和脱脂膏,多肽中PAP-2的作用较优。
Objective: To investigate the effect of the Periplaneta Americana polypeptide on the angiogenesis. Method: Methylthiazolyldiphenyl-tetrazolium bromide( MTT) assay and cell scratch assay were used to observe effect of different concentration( 6. 25,12. 5,25,50,100 mg·L-1) of the Periplaneta Americana polypeptide,CⅡ-3 and skimmed cream on the proliferation and migration of human umbilical vein endothelial cells( HUVECs),and a normal group and a thalidomide group were also established in this study. The tubule formation assay was used to detect the effect of different concentration( 25,50,100 mg·L-1) of the Periplaneta Americana extracts on the formation of tubules in HUVECs cells. The adhesion between HepG2 cells and HUVECs cells was observed by cell adhesion assay. The expression of vascular endothelial growth factor( VEGF) proteins in HUVECs was detected by immunocytochemical staining and enzyme linked immunosorbent assay( ELISA). Result: MTT results showed that the Periplaneta Americana polypeptide could inhibit the proliferation of HUVECs in a dosedependent manner( P < 0. 05). The effect of different concentrations of PAP-2 was better than that of PAP-1 and PAP-3 at 24,48 72 h( P < 0. 05). However,the survival rate of HUVECs was significantly increased after treatment with different concentrations of CⅡ-3 and skimmed cream in a time-dose dependent manner. Cell wound scratch assay indicated that migration of HUVECs could be inhibited by Periplaneta Americana polypeptide in a dose-dependent manner( P < 0. 05),and the effect of PAP-2 was better than that of PAP-1 and PAP-3( P <0. 05). C Ⅱ-3 could inhibit migration HUVECs to some extent,but the higher the dose was,the weaker the inhibition effect was. Skimmed cream promoted migration of HUVECs in a dose-dependent manner. Tube formation assay revealed that Periplaneta Americana polypeptide could inhibit tube formation of HUVECs,and the inhibitory effect of PAP-2 was better than that of PAP-1 and PAP-3( P < 0. 05). CⅡ-3 and skimmed cream promoted the tube formation of HUVECs to a certain extent. Intercellular adhesion assay stated clearly that Periplaneta Americana polypeptide could block the adhesion between HepG2 and HUVECs( P < 0. 05),and the effect of PAP-2 was better than that of PAP-1 and PAP-3( P < 0. 05). However,C Ⅱ-3 and skimmed cream could promote the adhesion between HepG2 and HUVECs. Results of ELISA assay and immunocytochemical staining indicated that Periplaneta Americana polypeptide could down-regulate the expression of VEGF of HUVECs in a dose-dependent manner( P < 0. 05),in which effect of PAP-2 was better than that of PAP-1 and PAP-3( P < 0. 05). However,CⅡ-3 and skimmed cream could up-regulate the expression of VEGF in HUVECs. Conclusion: The Periplaneta Americana polypeptide can inhibit the invasion,metastasis and tube formation of HUVECs,and down-regulate the expression of VEGF in HUVECs. The effect of Periplaneta Americana polypeptide is better than C Ⅱ-3 and skimmed cream,and the among the polypeptide,the effect of PAP-2 is superior to the other two.
Objective: To investigate the effect of the Periplaneta Americana polypeptide on the angiogenesis. Method: Methylthiazolyldiphenyl-tetrazolium bromide( MTT) assay and cell scratch assay were used to observe effect of different concentration( 6. 25,12. 5,25,50,100 mg·L-1) of the Periplaneta Americana polypeptide,CⅡ-3 and skimmed cream on the proliferation and migration of human umbilical vein endothelial cells( HUVECs),and a normal group and a thalidomide group were also established in this study. The tubule formation assay was used to detect the effect of different concentration( 25,50,100 mg·L-1) of the Periplaneta Americana extracts on the formation of tubules in HUVECs cells. The adhesion between HepG2 cells and HUVECs cells was observed by cell adhesion assay. The expression of vascular endothelial growth factor( VEGF) proteins in HUVECs was detected by immunocytochemical staining and enzyme linked immunosorbent assay( ELISA). Result: MTT results showed that the Periplaneta Americana polypeptide could inhibit the proliferation of HUVECs in a dosedependent manner( P < 0. 05). The effect of different concentrations of PAP-2 was better than that of PAP-1 and PAP-3 at 24,48 72 h( P < 0. 05). However,the survival rate of HUVECs was significantly increased after treatment with different concentrations of CⅡ-3 and skimmed cream in a time-dose dependent manner. Cell wound scratch assay indicated that migration of HUVECs could be inhibited by Periplaneta Americana polypeptide in a dose-dependent manner( P < 0. 05),and the effect of PAP-2 was better than that of PAP-1 and PAP-3( P <0. 05). C Ⅱ-3 could inhibit migration HUVECs to some extent,but the higher the dose was,the weaker the inhibition effect was. Skimmed cream promoted migration of HUVECs in a dose-dependent manner. Tube formation assay revealed that Periplaneta Americana polypeptide could inhibit tube formation of HUVECs,and the inhibitory effect of PAP-2 was better than that of PAP-1 and PAP-3( P < 0. 05). CⅡ-3 and skimmed cream promoted the tube formation of HUVECs to a certain extent. Intercellular adhesion assay stated clearly that Periplaneta Americana polypeptide could block the adhesion between HepG2 and HUVECs( P < 0. 05),and the effect of PAP-2 was better than that of PAP-1 and PAP-3( P < 0. 05). However,C Ⅱ-3 and skimmed cream could promote the adhesion between HepG2 and HUVECs. Results of ELISA assay and immunocytochemical staining indicated that Periplaneta Americana polypeptide could down-regulate the expression of VEGF of HUVECs in a dose-dependent manner( P < 0. 05),in which effect of PAP-2 was better than that of PAP-1 and PAP-3( P < 0. 05). However,CⅡ-3 and skimmed cream could up-regulate the expression of VEGF in HUVECs. Conclusion: The Periplaneta Americana polypeptide can inhibit the invasion,metastasis and tube formation of HUVECs,and down-regulate the expression of VEGF in HUVECs. The effect of Periplaneta Americana polypeptide is better than C Ⅱ-3 and skimmed cream,and the among the polypeptide,the effect of PAP-2 is superior to the other two.
引文
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[2] WU C H,LAN C H,WU K L,et al. Hepatocellular carcinoma-targeted nanoparticles for cancer therapy[J].Int J Oncol,2018,52(2):389-401.
[3] XIONG Y X,REN L,WANG Z Q,et al. The role of angiogenesis inhibitors re-challenge in colorectal cancer previously treated with bevacizumab:a Meta-analysis of randomized controlled trials[J]. Eur Rev Med Pharmacol Sci,2017,21(7):1489-1494.
[4]杨传玉,曾荣耀,刘荣亮,等.姜黄素对化学诱导大鼠肝癌缺氧模型血管生成的影响[J].中国肿瘤生物治疗杂志,2018,25(2):137-141.
[5]赵川,李俊萱,刘雪梅,等.肿瘤血管生成机制的研究进展[J].中华中医药学刊,2017,35(1):130-132.
[6]章凡,张献全,钱程. Foxg1抑制肝癌细胞血管形成及其机制[J].第三军医大学学报,2018,40(6):494-499.
[7]张艳,刘威,李军红,等.鱼藤素对胆管癌TFK-1细胞诱导肿瘤新生血管生成的抑制作用[J].中国实用医刊,2016,43(11):31-34.
[8]唐国建,沈璇,李长福.肿瘤血管生成与肝癌的研究进展[J].中国社区医师,2017,33(10):9-10.
[9]杜光明,赵元贵,金荣敏,等.康复新对虚证老人的超氧化物岐化酶细胞免疫临床及实验研究[J].云南中医杂志,1989,10(3):32-33.
[10]胡艳芬,吕小满,王玉梅,等.美洲大蠊药用价值研究进展[J].医学综述,2008,14(18):2822-2824.
[11]焦春香,刘光明,周萍.天然药物康复新的研究进展[J].时珍国医国药,2008,19(11):2623-2624.
[12]甘平,张旭强,何旭,等.美洲大蠊醇提物对小鼠急性肝损伤的保护作用[J].现代药物与临床,2011,26(2):123-128.
[13]杜一民,陈鸿珊,李树楠,等.治疗乙型肝炎新药肝龙胶囊的药效学初步研究[J].时珍国医国药,2006,17(8):1369-1371.
[14]李武,段丽芳,何贵清,等.美洲大蠊提取物对实验性肝纤维化的影响[J].时珍国医国药,2010,21(5):1137-1138.
[15]谢荣慧,刘云华,赵玉蓉.肝龙胶囊治疗慢性乙型肝炎疗效分析[J].中西医结合肝病杂志,2003,13(16):366-366.
[16]杨永荣,缪新权,梅光涛,等.肝龙胶囊治疗HBe Ag阳性慢性乙型肝炎的疗效观察[J].云南医药,2008,29(2):182-183.
[17]马家骅,蒋巧梅,徐华,等.美洲大蠊抗CCl4急性肝损伤作用的研究[J].中药药理与临床,2008,24(3):79-80.
[18]高永翔,扈晓宇,钟森,等.蟑螂提取物抗肝损伤及抗病毒作用探讨[J].国际中医中药杂志,2006,28(3):155-158.
[19]何正春,彭芳,宋丽艳,等.美洲大蠊化学成分及药理作用研究进展[J].中国中药杂志,2007,32(21):2326-2331.
[20]吴少辉,彭芳,高鹏飞,等.膜超滤分离美洲大蠊抗肿瘤活性部位小分子肽的研究[J].安徽农业科学,2012,40(4):2041-2042,2089.
[21]胡艳芬,吕小满,刘光明,等.美洲大蠊提取物对3株人肺癌细胞的体外抑制作用[J].大理学院学报,2009,8(12):1-3.
[22]何旭,普小菲,李娇,等.美洲大蠊提取物对S180荷瘤小鼠肿瘤抑制作用及免疫功能的影响[J].中国实验方剂学学志,2012,18(15):179-182.
[23]张蕊,袁发璐,李婷,等.美洲大蠊提取物对人肝癌Hep G2细胞的作用机制研究[J].中国现代医学杂志,2017,27(12):1-8.
[24]陈俊雅,耿玲,张旭强,等.美洲大蠊提取物CⅡ-3对H22肝癌小鼠血管生成作用的研究[J].肿瘤学杂志,2012,18(4):274-276.
[25]孙珺,白倩,周红梅.沙利度胺抗肿瘤血管生成的作用[J].临床口腔医学杂志,2009,25(6):379-380.
[26]普小菲,罗亦佳,彭丽,等.美洲大蠊提取物CⅡ-3体内外抗HSV-2实验研究[J].大理学院学报,2014,13(10):5-9.
[27]李洪文,耿玲,刘光明,等.美洲大蠊脱脂膏及其活性炭脱色物体外抗菌活性研究[J].中国实验方剂学杂志,2012,18(11):159-161.
[28]王绍钧,王锐,朱冬波.实体瘤靶向药物的研究进展[J].中国药房,2017,28(8):1149-1152.
[29]杨传玉,刘荣亮,柯恩明,等.小管形成实验在肿瘤微血管形成中的应用研究[J].现代肿瘤医学,2018,26(4):634-637.
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