Chouhan Manil, Puppi Juliana, Solanas Estela, Mitry Ragai R, Dhawan Anil, Hughes Robin D
Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London - UK.
Int J Artif Organs. 2012 Jun;35(6):450-7. doi: 10.5301/ijao.5000096.
Hepatocyte transplantation is a promising alternative to orthotopic liver transplantation, however, the fate of transplanted hepatocytes is not well defined. ⁹⁹mTc-galactosyl-serum albumin (⁹⁹mTc-GSA) is a clinical scintigraphic agent which is specifically taken up by the hepatocyte asialoglycoprotein receptor (ASGPR).
To investigate labeling of fresh and cryopreserved human hepatocytes and fresh rat hepatocytes in vitro using ⁹⁹mTc-GSA.
Human and rat hepatocytes were isolated from liver tissue by collagenase perfusion. The ASGPR were characterized using immunohistochemistry and RT-PCR. Hepatocytes were incubated with ⁹⁹mTc-GSA in suspension at 4°C and 37°C. Cell viability and function was determined using cell mitochondrial dehydrogenase (MTS) and sulphorhodamine B (SRB) assays.
Fresh and cryopreserved human hepatocytes expressed the ASGPR. Incubation of hepatocytes in suspension with ⁹⁹mTc-GSA reduced the viability of hepatocytes, but this was similar to unlabeled control cells. Greater loss of viability was seen on incubation at 37°C compared to 4°C, but there was a significantly greater uptake of ⁹⁹mTc-GSA at the physiological temperature (6.6 ± SE 0.6-fold increase, p<0.05) consistent with ASGPR-mediated endocytosis. MTS and SRB assays were not significantly affected by labeling with ⁹⁹mTc-GSA in all three cell types. A mean of 18.5% of the radioactivity was released over 120 min when ⁹⁹mTc-GSA -labeled hepatocytes were shaken in vitro at 37°C.
Human and rat hepatocytes can be labeled with ⁹⁹mTc-GSA, which may have potential application for in vivo imaging after hepatocyte transplantation.
肝细胞移植是原位肝移植一个很有前景的替代方案，然而，移植肝细胞的命运尚未明确界定。⁹⁹mTc-半乳糖基血清白蛋白（⁹⁹mTc-GSA）是一种临床闪烁显像剂，可被肝细胞去唾液酸糖蛋白受体（ASGPR）特异性摄取。
研究使用⁹⁹mTc-GSA在体外标记新鲜和冻存的人肝细胞以及新鲜大鼠肝细胞。
通过胶原酶灌注从肝组织中分离出人肝细胞和大鼠肝细胞。使用免疫组织化学和逆转录聚合酶链反应对ASGPR进行表征。肝细胞在4°C和37°C下于悬浮液中与⁹⁹mTc-GSA孵育。使用细胞线粒体脱氢酶（MTS）和磺酰罗丹明B（SRB）测定法测定细胞活力和功能。
新鲜和冻存的人肝细胞均表达ASGPR。肝细胞在悬浮液中与⁹⁹mTc-GSA孵育会降低肝细胞的活力，但这与未标记的对照细胞相似。与4°C孵育相比，37°C孵育时活力损失更大，但在生理温度下⁹⁹mTc-GSA的摄取明显更多（增加6.6±标准误0.6倍，p<0.05），这与ASGPR介导的内吞作用一致。在所有三种细胞类型中，MTS和SRB测定均未受到⁹⁹mTc-GSA标记的显著影响。当⁹⁹mTc-GSA标记的肝细胞在37°C下于体外振荡时，在120分钟内平均有18.5%的放射性释放。
人肝细胞和大鼠肝细胞可用⁹⁹mTc-GSA标记，这可能在肝细胞移植后的体内成像中具有潜在应用价值。
