Department of Surgery, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Center for Engineering in Medicine, Boston, MA 02114, USA.
Cell Transplant. 2010;19(6):783-9. doi: 10.3727/096368910X508898. Epub 2010 Jun 23.
Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.
先前的研究表明，消除猪器官中的半乳糖-α(1,3)半乳糖（Gal）表达可预防超急性异种移植物排斥反应。然而，天然存在的针对非 Gal 表位的抗体可激活内皮细胞，导致急性体液性异种移植物排斥反应。尽管如此，尚不清楚缺乏α(1,3)半乳糖基转移酶（GalT）的异种猪肝窦内皮细胞（LSEC）是否会受到抗体和补体介导的机制损伤。本研究检测了 GalT 缺陷型和野生型猪 LSEC 的异种抗体反应。将分离的野生型和 GalT 猪 LSEC 暴露于人血清和狒狒血清中；通过流式细胞术分析 IgM 和 IgG 结合。通过酶联免疫吸附试验（ELISA）从暴露于血清的 LSEC 培养物中定量测定补体激活（C3a 和 CH50）。还通过荧光死活检测和测定 LDH 释放来确定补体激活的细胞毒性（CAC）水平。与野生型猪 LSEC 相比，GalT 敲除（KO）LSEC 上的 IgM 结合显著降低（80%的人血清和 87%的狒狒血清）。所有组的 IgG 结合均较低。此外，暴露于人或狒狒血清后释放的补体激活（C3a 和 CH50）水平显着降低（42%的人血清和 52%的狒狒血清），与野生型 LSEC 相比，GalT KO LSEC 中的 CAC 在人血清中降低了 60%，在狒狒血清中降低了 72%，LDH 释放水平分别降低了 37%和 57%。与野生型猪 LSEC 相比，GalT KO 猪 LSEC 在暴露于人血清时对体液诱导的细胞损伤表现出显著的保护作用。尽管不足以完全抑制异种反应，但转基因 GalT KO 在猪肝脏上的表达可能有助于与其他保护策略相结合，成功应用临床异种移植。
