Suyama K, Li H, Zhu A
Cell Biochemistry Laboratory, The Lindsley F. Kimball Research Institute of the New York Blood Center, New York, New York 10021, USA.
Transfusion. 2000 Feb;40(2):214-21. doi: 10.1046/j.1537-2995.2000.40020214.x.
To gain insight into the formation of the Rh complex during erythroid differentiation, the ways in which Rh30 and Rh-related glycoproteins, especially Rh50, were produced in a modified two-phase liquid culture system were studied.
A mononuclear cell fraction from fresh peripheral blood was first cultured in a medium supplemented with conditioned medium collected from the culture of a bladder carcinoma cell line (5637) for 7 days. Nonadherent cells were then collected for culture in a secondary medium containing 2 U per mL of erythropoietin to initiate erythroid differentiation. The expression of Rh30 and Rh50 during secondary culture (16 days) was monitored by flow cytometry.
D+ cells appeared after Day 4 and increased to 70 percent by Day 8. On Day 12, 90 percent of the total cells became D+ and remained so until the end of the culture. A similar expression profile was obtained for Rh50. As determined from mean fluorescence intensities recorded in flow cytometry, the number of both D and Rh50 antigenic sites per cell increased as the differentiation progressed. Rh-related glycoprotein, CD47, had expression patterns significantly different from those of Rh30 and Rh50. In addition, the cultured cells produced partially glycosylated protein (approx. 32 kDa) in Rh50.
Expressions of Rh30 and Rh50 occur simultaneously during erythroid differentiation, and both proteins are most actively synthesized at the last stage of the differentiation. In contrast, CD47 may be involved in expression of Rh30 in a different manner from Rh50. The two-phase liquid culture system will be an excellent model for studying the interaction among the components of the Rh complex during protein synthesis and complex assembly on the cell membrane.
为深入了解红系分化过程中Rh复合物的形成，研究了在改良的两相液体培养系统中Rh30和Rh相关糖蛋白（尤其是Rh50）的产生方式。
首先将新鲜外周血的单个核细胞部分在补充有从膀胱癌细胞系（5637）培养物中收集的条件培养基的培养基中培养7天。然后收集非贴壁细胞，在含有每毫升2单位促红细胞生成素的二次培养基中培养以启动红系分化。通过流式细胞术监测二次培养（16天）期间Rh30和Rh50的表达。
第4天后出现D +细胞，到第8天增加到70%。在第12天，90%的总细胞变为D +并一直保持到培养结束。Rh50获得了类似的表达谱。根据流式细胞术中记录的平均荧光强度确定，随着分化的进行，每个细胞的D和Rh50抗原位点数量均增加。Rh相关糖蛋白CD47的表达模式与Rh30和Rh50的表达模式明显不同。此外，培养的细胞在Rh50中产生了部分糖基化蛋白（约32 kDa）。
Rh30和Rh50的表达在红系分化过程中同时发生，并且两种蛋白在分化的最后阶段合成最为活跃。相比之下，CD47可能以与Rh50不同的方式参与Rh30的表达。两相液体培养系统将是研究细胞膜上蛋白质合成和复合物组装过程中Rh复合物各成分之间相互作用的优秀模型。
