Transgenic Pig Expressing the Red Fluorescent Protein Kusabira-Orange as a Novel Tool for Preclinical Studies on Hepatocyte Transplantation
Transgenic Pig Expressing the Red Fluorescent Protein Kusabira-Orange as a Novel Tool for Preclinical Studies on Hepatocyte Transplantation
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DOI：
10.1016/j.transproceed.2013.01.017
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发表时间：
2013-06-01
影响因子：
0.9
通讯作者：
Nagashima, H.
中科院分区：
文献类型：
--
作者：
Shigeta, T.;Hsu, H. -C.;Nagashima, H.
导论.肝细胞移植作为肝移植的替代或补充治疗的研究正在取得进展。然而，为了推进临床试验，必须建立对该技术的信心，并且必须通过使用与人类大小相当的动物（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。供体肝细胞从转基因Kusabira-Orange（KO）表达猪（年龄，41天;体重，10 kg）中分离，所述转基因Kusabira-Orange（KO）表达猪通过使用Matsunari等人的转基因克隆KO猪的精子和家猪的卵子进行体外受精产生。肝细胞移植受体为非转基因、KO阴性同窝仔。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。采用胶原酶灌注法分离转基因雄性猪的KO阳性供体肝细胞。通过套管移植肝细胞（1 × 10（9）个细胞）。为了评估同种异体免疫原性，将来自同一供体的全层皮肤（3 x 3 cm）原位移植到受体的颈部区域。未进行免疫抑制治疗。分别于移植后第7天和第39天处死受体猪，摘取肺、心、肝、胰和肾等器官。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检测到强烈的红色荧光。灌注后第7天，在受体猪的肝脏和肺中检测到移植细胞。第39天肝细胞仍残留在肝和肺中，但数量较第7天减少。表达荧光蛋白KO的转基因猪在临床前研究中用作细胞移植的有用模型。
Introduction. 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.Materials and Methods. 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 x 10(9) cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 x 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.Results. 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.Conclusion. Transgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.