血小板膜受体糖蛋白Ib-IX-V复合物在瞬时转染的HEK293T细胞中的异常表达

Abnormal Expression of the Platelet Membrane Receptor Glycoprotein Ib-IX-V Complex in Transiently Transfected HEK 293T Cells

血小板膜受体糖蛋白(glycoprotein,GP)Ib-IX-V复合物在血小板活化过程中起到关键作用,因此其结构及功能一直是研究的热点和难点。由于血小板缺乏细胞核,研究者通常需将野生型或突变型的GPIb-IX-V基因转染至其他哺乳动物细胞系中(如CHO、HEK 293T等)以研究其结构与功能,因此GPIb-IX-V复合物在这些细胞系中是否能如实的反映其在血小板中的情况,是决定这些细胞系是否适合GPIb-IX-V复合物研究的关键。本研究通过检测GPIb-IX-V复合物在不同细胞系中的表达情况,明确细胞系差异是否可能干扰复合物的表达,进而找出研究GPIb-IX-V复合物的最适细胞系。在不同种属、不同组织来源的常用细胞系(如CHO、HEK 293T、HeLa等)中单独或共转染GPIb-IX-V复合物的亚基,并用流式细胞术检测GPIb-IX-V复合物在膜表面的表达量。结果发现:CHO细胞在瞬时转染与稳定转染后GPV在细胞膜表面表达依赖于其与GPIb-IX复合物的相互作用,与血小板中的情况一致。相反,在HEK 293T细胞系中,尽管GPIb-IX复合物的表达情况与CHO细胞及血小板中一致,但是GPV在细胞膜表面的表达不再依赖于GPIb-IX复合物;进一步在HeLa、MES13及HUVEC细胞系中的研究发现,GPV可以在HeLa及MES13细胞膜上独立表达,但是在HUVEC细胞表面不表达,提示GPIb-IX-V复合物各亚基之间的依赖性(进而表现为膜表面的表达量)在不同的细胞系中有所不同。结论:本研究为今后对于GPIb-IX-V复合物的研究具有一定的指导意义,尤其是在细胞系的选择上。HEK 293T细胞系极可能对研究结果造成偏倚,因而并非研究GPIb-IX-V复合物尤其是GPV亚基的最佳选择。

The structure and function of the glycoprotein （GP） Ib-IX-V complex has been extensively investigated over the decades due to its vital role in platelet activation. For the lack of nucleus in platelets, researchers usually need to study the GPIb-IX-V complex by transfecting wild type or mutant GPIb-IX-V plasmids into other mammalian cell lines, such as CHO or HEK 293T. Therefore, whether the characteristics of the GPIb-IX-V complex in these cell lines can tru- ly represent that in platelets is pivotal to determine whether these cell lines are appropriate for GPIb-IX-V complex stud- ies. In order to determine the most appropriate cell line to study the GPIb-IX-V complex, the surface expression level of the complex in different cell lines was detected and whether difference among cell lines will affect expression of the com- plex was explored in the present study. The different combinations of the GPIb-IX-V subunits were transfected into cell lines from different species or different tissues, such as CHO, HEK293T and HeLa, and the surface expression levels of the complex were detected by flow cytometry. The results indicated that in both transiently and stably transfected CHO cells, surface expression of GPV depended on the presence of the GPIb-IX complex, which is consistent with that in hu- -man platelets. In contrast, GPV could be efficiently expressed on surface in HEK 293T cells even in the absence of GPIb-IX, although the inter-subunit dependence within the GPIb-IX complex is still similar to that in CHO cells or hu- man platelets. Further studies in HeLa, MES13 and HUVEC cell lines revealed that GPV could be efficiently expressed on the surface by itself in HeLa and MES13 cells, but not in HUVEC, suggesting different behaviors of the GPIb-IX-V complex in difference cell lines. It is concluded that this study provides some guidance and advice to future GPIb-IX-V complex studies, especially to the choice of suitable cell line. HEK 293T cell line, for example, is likely to provide mis- leading results since it could not represent the fact in human platelets, thus is not the optimal choice for the GPlb-IX-V complex, particularly the GPV subunit.