Shigeta T, Hsu H-C, Enosawa S, Matsuno N, Kasahara M, Matsunari H, Umeyama K, Watanabe M, Nagashima H
Transplantation Center, National Center for Child Health and Development, Tokyo, Japan.
Transplant Proc. 2013 Jun;45(5):1808-10. doi: 10.1016/j.transproceed.2013.01.017.
Research on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as pigs. We used transgenic pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells.
Donor hepatocytes were isolated from transgenic Kusabira-Orange (KO)-expressing pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a transgenic-cloned KO pig by Matsunari et al. and ova from a domestic pig. The hepatocyte transplant recipients were the nontransgenic, KO-negative littermates. In these recipient pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the transgenic male pig were isolated using collagenase perfusion. Hepatocytes (1 × 10(9) cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys.
Strong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the transgenic male donor pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7.
Transgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.
肝细胞移植作为肝移植的替代或补充治疗方法的研究正在取得进展。然而，要推进到临床试验阶段，必须建立对该技术的信心，并通过使用与人类体型相当的动物（如猪）进行实验来验证其安全性。我们使用表达红色荧光蛋白的转基因猪来研究移植细胞的分布和存活情况。
供体肝细胞取自通过体外受精培育的表达红色荧光蛋白（KO）的转基因猪（年龄41天；体重10千克），精子来自松成等人培育的转基因克隆KO猪，卵子来自家猪。肝细胞移植受体为非转基因、KO阴性的同窝仔猪。在这些受体猪中，将双腔插管插入肠系膜上静脉以进入肝门区域。使用胶原酶灌注法从转基因雄性猪中分离出KO阳性供体肝细胞。将肝细胞（1×10⁹个细胞）通过插管进行移植。为评估同种异体免疫原性，将来自同一供体的全层皮肤（3×3厘米）原位移植到受体猪的颈部区域。未实施免疫抑制治疗。在移植后7天和39天对受体猪实施安乐死，并采集包括肺、心脏、肝脏、胰腺和肾脏在内的器官。
通过荧光显微镜在转基因雄性供体猪的实质和非实质肝细胞中均检测到强烈的红色荧光。灌注后7天在受体猪的肝脏和肺中检测到移植细胞。在第39天，肝细胞仍存在于受体猪的肝脏和肺中，但数量比第7天少。
表达荧光蛋白KO的转基因猪可作为临床前研究中细胞移植的有用模型。
