Immortalization of porcine hepatocytes with a alpha-1,3-galactosyltransferase knockout background
Immortalization of porcine hepatocytes with a alpha-1,3-galactosyltransferase knockout background
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α-1,3-半乳糖基转移酶敲除背景下猪肝细胞的永生化
DOI：
10.1111/xen.12550
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发表时间：
2020
影响因子：
3.9
通讯作者：
Dou Kefeng
中科院分区：
文献类型：
--
作者：
Wang Quancheng;Zhang Xuan;Wang Bo;Bai Ge;Pan Dengke;Yang Peijun;Tao Kaishan;Li Xiao;Dou Kefeng
背景与心脏或肾脏异种移植相比，体内猪肝异种移植的临床前试验似乎效率较差，因为异种排斥反应，包括凝血病，特别是血小板减少症。相反，离体猪肝（野生型）灌注系统已被证明是有效的"桥接"肝衰竭患者，直到随后的肝同种异体移植，和转基因（人CD55/CD59）的修改，甚至延长了猪肝灌注的持续时间。尽管事实上肝细胞系也已被提议用于急性肝衰竭条件下的体外血液循环，但猪肝细胞系，特别是GalT-KO背景，尚未在该领域开发和应用。在此，我们建立了永生化野生型和GalT-KO猪肝细胞系，可用于人工肝支持系统、细胞移植，甚至异种移植的体外研究。方法用SV40 LT慢病毒转染GalT-KO和野生型猪的原代肝细胞，建立永生化GalT-KO猪肝细胞（GalT-KO-hep）和野生型猪肝细胞（WT）。通过免疫荧光、高碘酸-希夫染色、吲哚菁绿色（ICG）摄取、生化分析、ELISA和RT-PCR评估肝细胞生物标志物和功能相关基因。并检测永生化细胞的致瘤性。此外，使用GalT-KO-hep和WT细胞进行补体依赖性细胞毒性（CDC）试验。结果GalT-KO和野生型猪肝细胞成功永生化，并保持了原代猪肝细胞的特征，包括白蛋白分泌、ICG摄取、尿素和糖原产生、肝细胞标志蛋白和特异性代谢酶的表达。确认GalT-KO-hep和WT细胞无致瘤性。此外，GalT-KO-hep细胞在补体和异种血清中的存活率高于WT细胞，凋亡率也低于WT细胞。结论成功建立了GalT-KO和野生型背景的两种猪肝细胞永生化细胞系。GalT-KO-hep细胞表现出更高的活力和对异种免疫应答的损伤抗性。
BackgroundIn vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in “bridging” liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT‐KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild‐type and GalT‐KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation.MethodsPrimary hepatocytes extracted from GalT‐KO and wild‐type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT‐KO porcine hepatocytes (GalT‐KO‐hep) and wild‐type porcine hepatocytes (WT). Hepatocyte biomarkers and function‐related genes were assessed by immunofluorescence, periodic acid‐Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT‐PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement‐dependent cytotoxicity (CDC) assay was performed with GalT‐KO‐hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK‐8 assay.ResultsGalT‐KO and wild‐type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT‐KO‐hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT‐KO‐hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum.ConclusionsTwo types of immortalized cell lines of porcine hepatocytes with GalT‐KO and wild‐type backgrounds were successfully established. GalT‐KO‐hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.