cRGD肽二聚体对B16黑色素瘤细胞的体外抑制作用及在荷瘤鼠体内分布及显像研究

The Inhibition Effects of c(RGD)_2 Peptides on Proliferation in B16 Melanoma Cells and the Its Imaging and Biodistribution Study in Tumor-bearing Mice

目的探讨cRGD肽二聚体对B16黑色素瘤细胞的体外抑制作用及其在荷B16黑色素瘤细胞小鼠动物模型体内的分布及显像研究。方法用MTT法检测不同浓度及作用时间cRGD肽二聚体对B16黑色素瘤细胞体外增殖能力的影响。采用直接标记法标记^(99)Tc^m-c(RGD)_2。建立荷B16黑色素瘤株动物模型,待肿瘤体积为1.0~1.5cm^3左右时,分别于30min、1h、2h、3h、4h、5h及6h时,进行荷瘤鼠体内生物分布及动态显像研究。结果 cRGD肽二聚体浓度为500mg/L、作用48h时,对B16黑色素瘤细胞的增殖具有明显的抑制作用。室温下、ρ(SnCl_2·2H_2O)=1 g/L、反应时间为30 min时,^(99)Tc^m-c(RGD)_2的标记率可达(87.42±3.21)%,标记产物经Sephadex G10分离纯化后放射化学纯度大于95%;静脉注射后30min行小鼠全身SPECT显像,肿瘤部位可见明显显像剂聚集,但肿瘤与周围组织的对比度较低;延迟至6小时肿瘤仍清晰可见,且随时间延迟肿瘤与周围组织的对比度增高,此时肿瘤/血液为2.15±0.24,肿瘤/肌肉为5.07±0.03。结论该结构cRGD肽二聚体对B16黑色素瘤细胞株具有一定的体外抑制作用,且动物体内肿瘤组织摄取率高,显像清晰,证明其可进一步应用于聚合物多肽靶向药物的研发。

Objective To investigate the inhibitory effects of c （RGD）2 peptide on proliferation of B16 melanoma cells in vitro and the imaging and biodistribution study with ^99Tc^m- c（RGD） 2 tracer in a xenograft B16 melanoma bearing mice model. Methods The cell proliferation was assessed by MTT assay. We labeled ^99Tc^m-c（RGD）2 peptide with the direct labeling method. The strain B16 melanoma cells were cultured to build the animal model. To generated solid tumors, 5 × 10^6 melanoma cells were injected s. c. into the armpit of KM mice. When the tumors reached 1.0 - 1.5cm^3, the imaging and biodistribution experiment were performed at 30min, 1,2,3,4,5, and 6 hour respectively after intravenous injection in tumor- bearing mice. Results With 500mg/L c（RGD）2 after 48h, the proliferation of B16 melanoma cells was significantly decreased. With 1 g/L SnC12 · 2H2O and the 30 min of reaction time, Labeling efficiency of ^99TC^m- c（RGD）2 reached （87.42 ±3.21 ）%. After Sephadex G10 purification, the radiochemical purity was no less than 95 %. SPECT imaging clearly revealed at 30min post-injection, even though the contrast was poor. At 6 hours time point, tumor was still clearly visible, and the contrast was getting sharper with time lasted, with tumor/blood was 2.15 ± 0.24, tumor/muscle was 5.07 ± 0.03. Conclusion The results in vitro showed the inhibition effect of c（RGD）2 on B16 melanomao And in vivo ^99Tc^m-c（RGD）2 could be well located in tumor o The data demonstrated that c （RGD）2 was suitable for the further development of polymer-conjugated RGD peptide targeted-drugs.