RGD修饰的肿瘤抑素19肽的合成与鉴定及其对肝癌SK-Hep-1细胞增殖、迁移和侵袭的抑制作用  

Synthesis and identification of RGD-modified tumstatin peptide 19 and its inhibitory effect on proliferation,migration,and invasion of liver cancer SK-Hep-1 cells

作　　者：王顺 于佳琪 胡月 赵正林 牛淑冬[3] 贾迪 杨超[1] 衣同辉[4] 李淑艳[1] WANG Shun;YU Jiaqi;HU Yue;ZHAO Zhenglin;NIU Shudong;JIA Di;YANG Chao;YI Tonghui;LI Shuyan(Department of Biochemistry,Qiqihar Medical University,Qiqihar 161006,Heilongjiang,China;Department of Medical Oncology of the Third Affiliated Hospital,Qiqihar Medical University,Qiqihar 161006,Heilongjiang,China;Department of Physiology,Qiqihar 161006,Heilongjiang,China;Health Inspection Center,Qiqihar Medical University,Qiqihar 161006,Heilongjiang,China;Clinical Laboratory of Longquan People’s Hospital,Longquan 323700,Zhejiang,China)

机构地区：[1]齐齐哈尔医学院生物化学教研室,黑龙江齐齐哈尔161006 [2]齐齐哈尔医学院附属第三医院肿瘤内科,黑龙江齐齐哈尔161006 [3]齐齐哈尔医学院生理学教研室,黑龙江齐齐哈尔161006 [4]齐齐哈尔医学院卫生检验中心,黑龙江齐齐哈尔161006 [5]龙泉市人民医院检验科,浙江龙泉323700

出　　处：《中国肿瘤生物治疗杂志》2024年第9期849-856,共8页Chinese Journal of Cancer Biotherapy

基　　金：齐齐哈尔市科学技术局面上研究项目(No.LSFGG-2023030)。

摘　　要：目的:探讨精氨酸-甘氨酸-天冬氨酸(RGD)修饰对肿瘤抑素19肽(T-19)抗肝癌活性的影响,比较分析T-19及RGD修饰的T-19(RGD-T-19)对肝癌SK-Hep-1细胞增殖、侵袭和迁移能力的影响。方法:用Fmoc固相法合成T-19及RGD-T-19,用高效液相色谱仪和质谱进行分离、鉴定。常规培养SK-Hep-1细胞,用0、50、100、150、200、250μg/mL的T-19及RGD-T-19分别处理细胞,分为0μg/mL(对照)组、50μg/mL组、100μg/mL组、150μg/mL组、200μg/mL组、250μg/mL组。CCK-8法、克隆形成实验、划痕愈合实验和Tanswell小室实验、WB法和q PCR法分别检测SK-Hep-1细胞的增殖、迁移、侵袭能力,以及环氧合酶-2(COX-2)、基质金属蛋白酶-2(MMP-2)、MMP-9、组织基质金属蛋白酶抑制剂-1(TIMP-1)、TIMP-2蛋白和MMP-1、MMP-2 mRNA的表达。结果:经质谱鉴定,用Fmoc固相法合成的T-19及RGD-T-19纯度高。T-19和RGD-T-19均能显著抑制SK-Hep-1细胞的增殖、迁移、侵袭能力,抑制COX-2蛋白、MMP-2和MMP-9蛋白及mRNA的表达、促进TIMP-1、TIMP-2蛋白的表达(P <0.05, P <0.01, P <0.001),RGD-T-19的抑制或促进效应均明显强于T-19(均P <0.05)。结论:利用Fmoc固相法合成了纯度高、活性好的T-19及RGD-T-19,两种肽均能抑制SK-Hep-1细胞增殖、侵袭和迁移能力,RGD-T-19作用明显强于T-19。Objective:To analyze the effects of arginine-glycine-aspartic acid(RGD)modification on anti-hepatocarcinoma activity of tumstatin peptide 19(T-19)and to comparatively analyze the effects of tumor suppressor peptide 19(T-19)and RGD modified-T-19(RGD-T-19)on the proliferation,invasion,migration of on liver cancer SK-Hep-1 cells.Methods:T-19 and RGD-T-19 were synthesized by Fmoc solid-phase method and separated and identified using high-performance liquid chromatography and mass spectrometry.SK-Hep-1 cells were routinely cultured and treated with 0,50,100,150,200,and 250 μg/mL of T-19 and RGD-T-19,respectively.The cells were divided into control group(0 μg/mL),50 μg/mL group,100 μg/mL group,150 μg/mL group,200 μg/mL group,and 250 μg/mL group.CCK-8 assay and clone formation test were used to detect the effects of T-19 and RGD-T-19 on the viability and proliferation of SK-hep-1 cells.The invasion and migration of SK-Hep-1 cells were observed by scratch and Transwell test.The mRNA expression of cellular matrix metalloproteinases MMP-2 and MMP-9 was detected by qPCR.The protein expression of COX-2,MMP-2,MMP-9,TIMP-1,and TIMP-2 was detected by Western blot.Results:The synthesized T-19 and RGD-T-19 were identified to be of high purity by mass spectroscopy.Both T-19 and RGD-T-19 significantly inhibited the proliferation,migration,and invasion abilities of SK-Hep-1 cells,suppressed the protein expression of COX-2 and both the mRNA and protein expression of MMP-2,and MMP-9,but promoted the protein expression of TIMP-1 and TIMP-2(P<0.05,P<0.01,P<0.001).Notably,the inhibitory or promoting effects of RGD-T-19 were significantly stronger than those of T-19(P<0.05).Conclusion:T-19 and RGD-T-19 synthesized by Fmoc solid-phase method were highly pure and eligible.Both T-19 and RGD-T-19 can inhibit the proliferation,invasion,migration of SK-Hep-1 cells,with better effects of RGD-T-19 than T-19.

关 键 词：肿瘤抑素 RGD序列 肝癌 SK-Hep-1细胞 增殖 侵袭 迁移 肝癌 

分 类 号：R735.7[医药卫生—肿瘤] R730.5[医药卫生—临床医学] R979.1