Midkine拮抗核素碘131治疗耐药的通路机制研究

Radioactive isotope iodine 131 therapeutic resistance is a very serious problem for clinical nuclear medicine. In our preliminary work, which is published in Life Sciences, we prove that over-expression of midkine (MK) is positively related with the malignant degree of differentiated thyroid cancer (DTC). MK measurement can be used for differential diagnosis between benign and malignant thyroid nodules, and can also be used for the prediction of metastases in DTC. Iodine 131 therapeutic resistance happens when DTC malignant degree increases. In this research, we use small interference RNA and MK antibody to suppress MK in cancer cells, and we use MK-gene carrying plasmids and MK recombinant protein to over-express or enhance MK in cancer cells. Then cell survival assay is performed. By Western blot, the changes of MK, MK receptors, key proteins in MAPK and PI3K pathways, and key apoptotic proteins are assessed. The changes of iodine-processing proteins and thyroid transcription factors are analyzed by real-time RT-PCR at mRNA level and by Western blot at protein level. The changes of iodine 131 uptake ability influenced by MK suppression are determined by iodine 131 uptake experiment at cellular level and tumor-bearing nude mice models in vivo imaging experiment. In vivo therapeutic effects are to be monitored in nude mice experiment in order to determine the difference between combining iodine 131 with MK suppression and any mono-therapy. Clinical study will determine the values of serum MK for iodine 131 therapeutic effects, prognosis and surveillance of DTC.

核素碘131治疗耐药是临床核医学面临的难题。发表于Life Sciences的本研究前期工作证实midkine（MK）过表达与分化型甲状腺癌（DTC）恶性程度正相关：MK测定可鉴别甲状腺结节良恶性、也可判断DTC是否存在转移灶。随DTC恶性程度的增高会出现碘131治疗耐药。本研究拟用小干扰RNA和MK抗体抑制癌细胞MK，通过转染表达MK质粒和给予MK重组蛋白增强癌细胞MK；做存活分析实验；用Western法检测MK、MK受体、MAPK和PI3K通路关键蛋白及凋亡关键蛋白变化；从基因水平（PCR法）和蛋白水平（Western法）检测碘处理关键蛋白和甲状腺转录因子变化；用体外细胞水平摄碘131实验和荷瘤裸鼠模型活体显像研究抑制MK对癌细胞摄碘131能力的影响；观测体内实验疗效、明确联合碘131和抑制MK与单独用药的区别。临床研究明确血MK水平对DTC碘131治疗疗效、预后和病情监测的价值。

本研究证明中期因子（Midkine、MK）在甲状腺癌细胞内的高表达，可以被siRNA和MK抑制剂（iMDK）有效抑制。抑制MK后甲状腺癌细胞的增殖可以受到抑制，促进凋亡的蛋白增多、抑制凋亡的蛋白减少。这样的变化，是因为抑制MK会对PI3K通路发挥抑制的效果，但是，无论siRNA和iMDK，都没有对MAPK通路产生影响，也没有对碘代谢产生明显影响。对甲状腺癌细胞进行转染MK表达质粒、做过表达的实验，发现PI3K通路蛋白表达增高，促进凋亡的蛋白减少、抑制凋亡的蛋白增多；加上PI3K通路抑制剂可以逆转这样的变化。但是，对MAPK通路没产生影响，也没有对碘代谢产生明显影响。癌细胞的摄131I实验也表明，siRNA-3抑制、iMDK抑制和MK质粒转染过表达均对摄131I无明显影响。动物体内研究证明，MK抑制治疗虽然可以抑制甲状腺癌的移植瘤的增长，但是，这并不是通过对131I摄取能力的提高来达到的效果，而是因为拮抗MK可以抑制PI3K通路，进而抑制癌细胞的增殖，而获得的疗效。临床研究发现MK可被用作甲状腺乳头状癌的疾病监测的血清学指标，Cox回归分析显示如果首次131I治疗后10-12个月时候的MK水平比131I治疗前MK水平降低，但未达到正常水平，存在转移的风险是MK能降低到正常水平的3.006倍；如果首次131I治疗后10-12个月时候的MK水平比131I治疗前MK水平升高，存在转移的风险是5.030倍。临床研究还发现对于甲状腺球蛋白抗体阳性，甲状腺球蛋白不适合做血清学标记物的时候，MK可作为预测分化型甲状腺癌转移的有效血清学标志物。