Bautista Diana, Rodríguez Luz-Stella, Franco Manuel A, Angel Juana, Barreto Alfonso
Grupo de Inmunobiología y Biología Celular, Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia.
Cell Stress Chaperones. 2015 Jul;20(4):697-708. doi: 10.1007/s12192-015-0597-9. Epub 2015 May 15.
Previously, we showed that infecting human intestinal epithelial cells (Caco-2) with rotavirus (RV) increases the release of extracellular vesicles (EVs) with an immunomodulatory function that, upon concentration at 100,000×g, present buoyant densities on a sucrose gradient of between 1.10 to 1.18 g/ml (characteristic of exosomes) and higher than 1.24 g/ml (proposed for apoptotic bodies). The effect of cellular death induced by RV on the composition of these EV is unknown. Here, we evaluated exosome (CD63, Hsc70, and AChE) and apoptotic body (histone H3) markers in EVs isolated by differential centrifugation (4000×g, 10,000×g, and 100,000×g) or filtration/ultracentrifugation (100,000×g) protocols. When we infected cells in the presence of caspase inhibitors, Hsc70 and AChE diminished in EVs obtained at 100,000×g, but not in EVs obtained at 4000×g or 10,000×g. In addition, caspase inhibitors decreased CD63 and AChE in vesicles with low and high buoyant densities. Without caspase inhibitors, RV infection increased exosome markers in all of the EVs obtained by differential centrifugation. However, CD63 preferentially localized in the 100,000×g fraction and H3 only increased in EVs concentrated at 100,000×g and with high buoyant densities on a sucrose gradient. Thus, RV infection increases the release of EVs that, upon concentration at 100,000×g, are composed by exosomes and apoptotic bodies, which can partially be separated using sucrose gradients.
此前，我们发现用轮状病毒（RV）感染人肠道上皮细胞（Caco-2）会增加具有免疫调节功能的细胞外囊泡（EVs）的释放，这些囊泡在100,000×g离心浓缩后，在蔗糖梯度上的浮力密度介于1.10至1.18 g/ml（外泌体的特征）和高于1.24 g/ml（凋亡小体的特征）之间。RV诱导的细胞死亡对这些EVs组成的影响尚不清楚。在此，我们评估了通过差速离心（4000×g、10,000×g和100,000×g）或过滤/超速离心（100,000×g）方法分离的EVs中的外泌体标志物（CD63、Hsc70和AChE）和凋亡小体标志物（组蛋白H3）。当我们在存在半胱天冬酶抑制剂的情况下感染细胞时，在100,000×g获得的EVs中Hsc70和AChE减少，但在4000×g或10,000×g获得的EVs中没有减少。此外，半胱天冬酶抑制剂降低了低浮力密度和高浮力密度囊泡中的CD63和AChE。在没有半胱天冬酶抑制剂的情况下，RV感染增加了通过差速离心获得的所有EVs中的外泌体标志物。然而，CD63优先定位于100,000×g的组分中，而H3仅在100,000×g浓缩且在蔗糖梯度上具有高浮力密度的EVs中增加。因此，RV感染增加了EVs的释放，这些EVs在100,000×g浓缩后由外泌体和凋亡小体组成，它们可以使用蔗糖梯度进行部分分离。
