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该研究在前期重组日本七鳃鳗RGD3毒素蛋白(recombinant Lampetra japonica RGD3,rLj-RGD3)工作基础上,设计并人工合成分子克隆,获得去纯化标签的rLj-RGD4。该论文采用小鼠黑色素瘤(B16)为肿瘤细胞模型,对rLj-RGD4是否抑制B16细胞增殖、迁移、浸润与凋亡进行了研究。研究结果表明,采用不同浓度的rLj-RGD4经MTT方法检测发现,rLj-RGD4对B16细胞的增殖有抑制作用,IC50为9.6μmol/L;使用Transwell法和细胞划痕实验结果证实,rLj-RGD4对B16细胞的迁移和浸润都具有抑制作用,且呈剂量依赖方式;B16细胞经鬼笔环肽-FITC染色结果显示,rLj-RGD4肽对B16细胞骨架发生破坏;Hoechst 33258法和TUNEL-FITC法结果显示,rLj-RGD4可诱导B16细胞发生凋亡;运用Western blot对其诱导B16细胞发生凋亡的作用机制研究结果显示,随着rLj-RGD4浓度的增加cleaved-caspase-8和cleaved-caspase-3的水平显著增加,B淋巴细胞瘤-2(B cell lymphoma/leukaemia-2,Bcl-2)基因水平量显著减少。由此可见,rLj-RGD4具有显著促B16细胞凋亡的作用,具有抑制增殖与侵袭的功能,有可能成为一个具有抗肿瘤作用的候选药物。
Based on the previous work on recombinant Lampetra japonica RGD3 (rLj-RGD3), we designed and synthesized molecular cloning to obtain purified rLj-RGD4. In the present study, murine melanoma (B16) was used as a tumor cell model to investigate whether rLj-RGD4 inhibits the proliferation, migration, invasion and apoptosis of B16 cells. The results showed that rLj-RGD4 could inhibit the proliferation of B16 cells with different concentration of rLj-RGD4 by MTT assay and the IC50 was 9.6μmol / L. Transwell assay and cell scratch assay showed that rLj-RGD4 B16 cell migration and invasion were inhibited in a dose-dependent manner; B16 cells by phalloidin-FITC staining showed that rLj-RGD4 peptide destruction of B16 cytoskeleton; Hoechst 33258 and TUNEL-FITC method The results showed that rLj-RGD4 could induce apoptosis in B16 cells. The mechanism of apoptosis induced by rLj-RGD4 in B16 cells was studied. The results showed that cleaved-caspase-8 and cleaved-caspase- 3 significantly increased the level of B lymphoblastoma -2 (B cell lymphoma / leukaemia-2, Bcl-2) gene levels were significantly reduced. Thus, rLj-RGD4 has a significant role in promoting apoptosis of B16 cells, has the function of inhibiting proliferation and invasion, and may become a candidate drug with anti-tumor effect.