In vivo detection and imaging of phosphatidylserine expression during programmed cell death

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.

程序性细胞死亡（programmed cell death）最早出现的标志性事件之一，是磷脂酰丝氨酸（phosphatidylserine）的外翻——这种膜磷脂通常仅局限于脂质双层（lipid bilayer）的内层小叶。Annexin V是一种内源性人类蛋白质，对膜结合的磷脂酰丝氨酸具有高亲和力，可在体外（in vitro）用于检测早于其他已被充分描述的程序性细胞死亡相关形态学或细胞核变化的细胞凋亡（apoptosis）。本研究测试了外源性给予的放射性标记Annexin V在体内（in vivo）靶向聚集于细胞凋亡部位的能力。使用肼烟酰胺（hydrazinonicotinamide）进行衍生化修饰后，Annexin V可被锝99m（technetium 99m）标记。我们在三种动物模型中测试了锝99m-肼烟酰胺-Annexin V的体内定位效果：一是向BALB/c小鼠注射抗Fas抗体（anti-Fas antibody）诱导的暴发性肝凋亡；二是移植异位PVG心脏同种异体移植物的ACI大鼠发生的急性排斥反应；三是采用环磷酰胺（cyclophosphamide）处理移植的38C13小鼠B细胞淋巴瘤模型。放射性核素成像显示，三种模型的凋亡部位中，放射性标记Annexin V的摄取量均提升2至6倍。对心脏同种异体移植物进行外源性Annexin V免疫组化染色（immunohistochemical staining）结果显示，单核细胞浸润区域周边的大量心肌细胞呈现强阳性染色，而通过末端脱氧核苷酸转移酶介导的UTP缺口末端标记法（terminal deoxynucleotidyltransferase-mediated UTP end labeling，TUNEL法）仅能检测到少量阳性凋亡细胞核。上述结果表明，放射性标记的Annexin V可作为一种无创手段，在体内对发生程序性细胞死亡的组织与器官进行检测及连续成像。