双光子ROCK蛋白质荧光探针的研制及在癌症病理机理研究中的应用

Rho-associated coiled-coil forming protein kinase (ROCK) belongs to serine/threonine kinase family, involved in the regulation of cell cytoskeletal structure and plenty of cell biologic behavior regulations, and therefore it is an important key protein for understanding of various biological and pathological mechanisms. This project gained to study two-photon ROCK protein fluorescent probes, the reason is unable to study ROCK vivid behavior directly in live cells and live tissues by current ROCK assay methods. Two-photon microscopy (TPM) has become a tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. However, the application always is limited because of scarcely enough necessary befit two-photon probes. In recent years, the design and synthesis of two-photon probes has been a hot research topic. This project team summarized the current two-photon probes research and designed several fluophores, which are 1,4-distyrylbenzene derivatives containg dicyano groups or its bioisostere derivatives. The ROCK antibody will has fluorescence by conjugation with the fluophore. The series of fluorescent antibody compounds will carried out reaction ability test with ROCK protein and related indicators detection steps to select two or three candidates, and thereafter will be applied to study some biological and phathological mechanisms, thus new and ideal two-photon ROCK protein fluorescent probes will have been demonstrated.

Rho相关卷曲螺旋形成蛋白激酶(ROCK)属于丝氨酸／苏氨酸蛋白激酶家族成员，它可通过多种途径调解细胞骨架，参与各种细胞生物学行为的调控，是一种弄清各种生理和病理机制的关键性蛋白质。目前测定ROCK的方法都不能直接在活细胞或活组织中观察ROCK的行为，所以本课题拟研究开发双光子ROCK蛋白质荧光探针。双光子荧光显微镜具有长时间、无损伤活体成像的能力，但其应用往往是无适合双光子荧光探针而受限制。设计和合成双光子荧光探针是近几年才兴起的一个崭新的热门课题。本项目组总结了目前双光子荧光探针的研究状况，设计出若干个荧光团，每个荧光团是含两个氰基的双苯乙烯基苯衍生物或其生物电子等排体衍生物，这些荧光团与抗体上的氨基反应使抗体具有双光子荧光特性，然后通过与ROCK蛋白质的反应能力与各种指标的测定，确定2-3个候选化合物，再应用于生理和病理机制的研究中，以期获得新的理想的双光子ROCK蛋白质荧光探针。

双光子荧光显微镜具有长时间、无损伤活体成像的能力，但其应用往往受无适合双光子荧光探针而受限制，而此双光子荧光探针的成功问世离不开性能好的荧光团的支持，所以本项目研究的关键是寻找一些性能良好的双光子荧光团。双光子效应的强弱取决于分子的极化程度，极化程度越高，双光子吸收截面越大，双光子荧光效果越佳。已报道的各类双光子化合物中高吸收截面的化合物大多数是含氰基的双苯乙烯类衍生物。本项目组总结了目前双光子荧光探针的研究状况，设计出若干个荧光团，每个荧光团是含两个氰基的双苯乙烯类衍生物或其生物电子等排体。在研究双光子荧光团的过程中，作为反应的中间体或其生物电子等排体，合成了诸多二苯乙烯类衍生物，其中包括双键上含氰基的或直接将双键替换成杂环结构的双苯乙烯类，并测定了其抗肿瘤活性。结果，2,5-二氰基-1-(4’-二甲氨苯乙烯基)-4-(4’’-甲酸琥珀酰亚胺酯苯乙烯基)苯等荧光团具有良好的双光子荧光性能，而且通过细胞和动物实验筛选出50余种对癌细胞和正常细胞有选择性抑制活性的潜在抗癌药物，是一些新观念抗癌药物的候选物质，有待进一步研究和开发。