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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 platelet membrane receptor glycoprotein (GP) Ib-IX-V complex plays a key role in platelet activation. Therefore, its structure and function have been the hot spots and difficult points. Due to the lack of nuclei in platelets, researchers usually need to transfect wild-type or mutant GPIb-IX-V genes into other mammalian cell lines (eg, CHO, HEK 293T, etc.) to study their structure and function. Therefore, GPIb-IX -V complex in these cell lines can faithfully reflect its platelet situation is to decide whether these cell lines suitable for GPIb-IX-V complex research. In this study, by detecting the expression of GPIb-IX-V complex in different cell lines, it was clarified whether cell lineage might interfere with the expression of the complex, and then to find out the optimal cell line for GPIb-IX-V complex. The subunits of GPIb-IX-V complex were singly or co-transfected in common cell lines (CHO, HEK 293T, HeLa, etc.) of different species and different tissue sources, and GPIb-IX- V The amount of complex expressed on the membrane surface. The results showed that the expression of GPV on the cell membrane surface after transient transfection and stable transfection depends on its interaction with GPIb-IX complex, which is consistent with that in platelets. In contrast, expression of GPV on the cell membrane surface was no longer dependent on the GPIb-IX complex in the HEK 293T cell line, although the expression of the GPIb-IX complex was consistent with that in CHO cells and platelets; further, in HeLa, MES13 and HUVEC In cell lines, GPV was expressed independently on HeLa and MES13 cell membranes but not on HUVEC cells, suggesting a dependency between GPIb-IX-V complex subunits (and hence membrane surface expression Amount) varies in different cell lines. Conclusion: This study is of guiding significance for the future study of GPIb-IX-V complex, especially in cell line selection. The HEK 293T cell line most likely biases the results and is therefore not the best choice for studying GPIb-IX-V complexes, especially GPV subunits.