Mouneimne Y, Brown W C, Nicolau C, Tosi P F
Center for Blood Research Laboratories, Boston, Massachusetts.
Cytometry. 1993 Oct;14(7):764-71. doi: 10.1002/cyto.990140709.
Application of an electrical pulse field at a strength slightly below the value required for electroporation to a suspension of red blood cells in the presence of membrane xenoproteins leads to the insertion of those proteins in the erythrocyte plasma membrane. This observation is extended to nucleated cells. In the presence of glycophorin A, application of such pulses leads to the insertion of 10(4)-10(5) molecules of glycophorin A per cell in CEM-CM3, Hela S3, and bovine CD8+ T cells. Electroinserted glycophorin A is detected by flow cytometry using anti-glycophorin monoclonal antibodies. The survival of the cells subjected to electroinsertion was 55% for CEM-CM3 cells, 69% for Hela S3 cells, and 65% for CD8+ T cells. Cells cultured after electroinsertion lost the electroinserted glycophorin A, with two different rates, by a temperature and cell type-dependent mechanism. During the first 2 h after electroinsertion, the CD8+ T cells lost 12.5% of the inserted glycophorin A per h, the CEM-CM3 cells lost 7.7% per h, whereas the Hela S3 cells lost only 0.8% of the inserted protein per h. After 2 h, the rate increased substantially, to 41.7% per h for the CD8+ T cells, 13.5% for the CEM-CM3 cells, and 8.9% for the Hela S3 cells. Cytochalasin D efficiently inhibited the disappearance of electroinserted glycophorin A during the first 2 h after electroinsertion only.
在存在膜异种蛋白的情况下，向红细胞悬液施加强度略低于电穿孔所需值的电脉冲场，会导致这些蛋白质插入红细胞质膜。这一观察结果扩展到了有核细胞。在存在血型糖蛋白A的情况下，施加这样的脉冲会导致在CEM-CM3、Hela S3和牛CD8+ T细胞中每个细胞插入10⁴-10⁵个血型糖蛋白A分子。使用抗血型糖蛋白单克隆抗体通过流式细胞术检测电插入的血型糖蛋白A。接受电插入的细胞的存活率，CEM-CM3细胞为55%，Hela S3细胞为69%，CD8+ T细胞为65%。电插入后培养的细胞通过温度和细胞类型依赖性机制以两种不同速率丢失电插入的血型糖蛋白A。在电插入后的前2小时内，CD8+ T细胞每小时丢失12.5%的插入血型糖蛋白A，CEM-CM3细胞每小时丢失7.7%，而Hela S3细胞每小时仅丢失0.8%的插入蛋白。2小时后，速率大幅增加，CD8+ T细胞为每小时41.7%，CEM-CM3细胞为13.5%，Hela S3细胞为8.9%。细胞松弛素D仅在电插入后的前2小时有效抑制电插入的血型糖蛋白A的消失。
