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、250 mg/mL的T-19及RGD-T-19分别处理细胞， 分为0 mg/mL（对照）组、50 mg/mL组、100 mg/mL组、150 mg/mL组、200 mg/mL组、250 mg/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qPCR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 mg/mL of T-19 and RGD-T-19, respectively. The cells were divided into control group (0 mg/mL), 50 mg/mL group, 100 mg/mL group, 150 mg/mL group, 200 mg/mL group, and 250 mg/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, and migration of SK-Hep-1 cells, with better effects of RGD-T-19 than T-19.

齐齐哈尔市科学技术局面上研究项目（No.LSFGG-2023030）