以Mac-1（CD11b/CD18）受体为靶点毛细管电泳快速筛选药物新方法研究

Mac-1（CD11b/CD18） receptor is an extremely hydrophobic receptor. However, as to extremely hydrophobic receptors, there exists a serious problem, i.e., isolating and purifying these receptors from the cell membrane will typically results in loss of native conformation. As such, most of the binding studies for hydrophobic receptors are limited to functional assay and radioligand-binding assay (RLBA). But, the functional assay has big measurement errors, low accuracy, and low sensitivity in quantitative analysis. RLBA is hazardous, costly, and time-consuming. In the present study, the transient transfection was performed by using electroporation in HEK-293 cells with pEGFP-CD11b plus pDsRed-CD18, under the physiological condition or approximately physiological conditions, pEGFP-CD11b, and pDsRed-CD18. The CD11b, CD18 and Mac-1 receptors expressing cells will be coated on the inner wall of a capillary column as the stationary phase in HPCE for the first time, respectively, which will be used to study the interactions of lactose derivatives Gu-4 with CD11b, CD18 and Mac-1 receptors, respectively. A series of new technologies will be explored, the kinetic parameters of interaction between Gu-4 and Mac-I, CD11b, CD18 are calculated by NLC model. And the new HPCE screening platform will be established, which will be used to screen rapidly a series of the new synthetic carbohydrate derivatives and the natural products. Furthermore, the ICAM-1 ligand expressing cells will be coated firstly on the inner wall of a capillary as the stationary phase in HPCE, which will be used to investigated the adhesion activities of HEK-293-ICAM-1 cells and HEK-293-Mac-1 cells under the buffer solution contained the different levels of Gu-4. And the new HPCE method will be established on the determination of the adhesion activities between the cells. This will supply scientific evidence for the further studying the antitumor metastasis and anti-atherosclerosis efficacy of the compounds with strong cellular adhesion activity by animal model experiments. This will lay the foundation for discovering the source of innovative drugs and establish a new method for screening anti-tumor metastasis drugs.

Mac-1受体是疏水性受体，从细胞膜中纯化疏水性受体会引起其活性结构改变，对于以此类受体为靶点药物筛选研究只限于功能实验和放射配基结合实验，但重现性差、毒性大、成本高、耗时长。本项目旨在采用前人未曾报道的方法，将CD11b、CD18真核表达质粒共转染及单独转染HEK293细胞，并在生理或接近生理条件下，将高表达上述受体细胞固定在毛细管内壁作为固定相，研究乳糖衍生物Gu-4与三种受体的相互作用，利用NLC模型计算Gu-4与受体相互作用动力学参数，建立CE筛选平台，筛选合成糖类衍生物和天然产物。将高表达ICAM-1配体的HEK-293细胞固定在毛细管内壁作为固定相，研究乳糖衍生物Gu-4对高表达Mac-1受体细胞与高表达配体ICAM-1细胞的粘附活性的影响，建立全新CE研究细胞粘附活性的方法。为进入体内抗肿瘤转移和动脉粥样硬化药效研究提供科学依据，为发现源头创新药物奠定基础并建立新筛选方法。

Mac-1受体在白细胞捕捉内皮细胞过程中起重要作用，阻断其与配体结合可调节宿主炎症反应强度、抑制白细胞与内皮细胞粘附、减轻粒细胞介导组织损伤，已成为一项新治疗策略。Mac-1受体和ICAM-1配体是疏水性的蛋白质，从细胞膜中纯化疏水性受体会引起其活性结构改变，对于以此类受体/配体为靶点药物筛选研究只限于功能实验和放射配基结合实验，但重现性差、毒性大、成本高、耗时长。本项目采用前人未曾报道的方法，分别构建并在HEK-293细胞表达ICAM-1-EGFP、Mac-1-EGFP、CD11b-EGFP、CD18-DsRed蛋白。在接近体内生理条件下，优化了毛细管电泳各项条件，将高表达上述受体细胞固定在毛细管内壁作为固定相，定性定量研究Mac-1、CD11b和CD18受体与乳糖衍生物Gu-4和DMSO的相互作用，计算动力学参数（容量因子、结合常数、结合速率常数、解离速率常数），建立以高表达受体的全细胞为固定相，以Mac-1、CD11b、CD18为靶点的全新ICCE筛选平台。筛选乳糖衍生物及天然药物，为进一步设计及合成的一系列具有新颖结构的化合物、分离纯化的天然产物，提供相互作用及构效关系的数据。同时将高表达ICAM-1配体的HEK-293细胞在毛细管中进行贴壁培养并固定，在流动相缓冲溶液中加入乳糖衍生物Gu-4、乳糖和DMSO，通过对高表达Mac-1受体的HEK-293细胞、HEK-293细胞与高表达ICAM-1配体的HEK-293细胞的相互作用的观察，在线定性定量研究Gu-4拮抗细胞粘附能力，建立全新毛细管电泳研究拮抗细胞粘附活性化合物的平台。最后针对有强结合活性化合物Gu-4进行了细胞粘附实验、细胞划痕实验及体内抗肿瘤转移实验研究，证明了毛细管电泳筛选药物新方法的有效性和可行性。在项目执行期间，申请了2项国家发明专利，获得了1项中国发明专利的授权，培养了博士研究生3名，硕士研究生10名,发表了科研论文9篇，其中SCI收录论文6篇。