Inhibitory effect of a modified adenovirus type 5 E1A gene on the NF-kB activity in porcine aortic endothelial cells induced by TNF-α

The transcription factor nuclear factor kB (NF-kB) plays a key role in the delayed xenograft rejection (DXR). One of the important objects in the field is how to inhibit the NF-kB activity at optimal level. Thus, a modified E1A gene (E1AD) containing function domain (1—80 aa) and nuclear localization domain (139—243 aa) was used and cloned into an eucaryotic expression vector pcDNA3 to transfect the porcine aortic endothelial cells (PAEC). The stable transfectants were screened with G418. E1AD gene was able to be stably expressed in the PAEC and could not affect the growth of PAEC as analyzed by RT-PCR and cell growth rate. Reporter gene assay demonstrated that E1AD was capable of inhibiting NF-kB activity in the PAEC in-duced by TNF-a without sensitizing to apoptosis, and the rate of inhibition was 53%. Furthermore, E1AD inhibited the expression of a NF-kBdependent inflammatory gene E-selectin in the cells, and the rate of inhibition was 63%. In summary, the usage of E1AD gene may be a new strategy to overcome DXR in the xenotransplantation.

Adhesion of subsets of human blood mononuclear cells to porcine endothelial cells

Cellular immune response is a major barrier to xenotransplantation, and cell adhesion is the first step in intercellular recognition. Flow-cytometric adhesion assay has been used to investigate the differential adhesions of monocyte (Mo), natural killer celi (NK) and T lymphocyte (T) present within human peripheral blood mononuclear cells (PBMC) to porcine aortic endothelial cells (PAEC), and to demonstrate the effect of human interferon-γ (hIFN-γ) or/and tumor necrosis factor-α (hTNF-α) pretreatment of PAEC on their adhesiveness f or different PBMC subsets. The preferential sequence for PBMC subset binding to resting PAEC is Mo, NK and T cells, among which T cells show the slightest adherence; hTNF- a can act across the species, and augment Mo, NK and T cell adhesion ratios by 40%, 110% and 3 times, respectively. These results confirm at the cell level that host Mo and NK cells are major participants in the cellular xenograft rejection, thereby, providing a prerequisite for further studying the

Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endo-thelial cells (PAEC) in vitro. rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine

载脂蛋白CⅢ刺激猪主动脉血管内皮细胞差异表达基因的研究

本研究旨在探究载脂蛋白CⅢ(ApoCⅢ)刺激猪主动脉血管内皮细胞前后的差异基因表达谱,从而揭示ApoCⅢ的功能。利用酶解法成功分离猪主动脉血管内皮细胞并进行体外培养,采用高通量测序技术筛选出ApoCⅢ刺激前后的差异表达基因。结果表明,ApoCⅢ刺激猪主动脉血管内皮细胞前后共有647个差异表达基因,包括390个上调表达基因和257个下调表达基因。实时荧光定量PCR(qRT-PCR)检测表明,高通量测序数据结果正确可靠。GO及Pathway分析结果显示,差异表达基因的功能涉及免疫应答、细胞凋亡及死亡等。这表明ApoCⅢ可通过炎症反应、细胞黏附、细胞凋亡等分子通路影响猪主动脉血管内皮细胞的生理功能,为进一步解析ApoCⅢ引发动脉粥样硬化发生的分子机制提供了理论基础。

猪血管内皮细胞α-Gal对人外周血淋巴作用初探

超急性排斥反应克服后，异种移植物将面临血管性和细胞性排斥。血管内皮是免疫排斥的主要场所，异种移植免疫中血管内皮细胞的作用以及其携带的αＧａｌ在超急性排斥后免疫反应中的作用研究尚少。通过混合淋巴细胞反应（ＭＬＲ），以同种异体ＭＬＲ为对照，对异种（内江猪→人）外周血淋巴细胞（ＰＢＬＣ）混合反应和内江猪血管内皮细胞（ＰＡＥＣ）人ＰＢＬＣ混合反应进行研究，同时考察α半乳糖基酶消化后ＰＡＥＣ对人ＰＢＬＣ的刺激性。结果表明：异种ＭＬＲ增殖较相应的同种异体ＭＬＲ弱，可能与在异种周围淋巴细胞ＭＬＲ中以间接呈递为主有关；ＰＡＥＣ作为刺激细胞的ＭＬＲ增殖极强，可能与ＰＡＥＣ本身是抗原呈递细胞且抗原呈递以直接呈递为主有关；αＧａｌ引起增殖，可能与其在体内数量多、与生物大分子结合可成为全抗原有关，提示该抗原可能也是超急性排斥后排斥反应的靶抗原；去除ＰＡＥＣ表面αＧａｌ后，仍可诱发人Ｔ细胞反应，提示可能还存在其它超急性排斥后抗原。

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.

一种新的非Gal异种移植抗原——FAM234A的鉴定

目的探讨FAM234A是否为一种新的非Gal异种移植抗原。方法用α-1,3-半乳糖基转移酶基因敲除(GTKO)小型猪的原代猪主动脉内皮细胞(PAEC)免疫食蟹猴。将免疫的猴血清与PAEC孵育,其中经免疫产生的猴抗猪细胞抗体能够识别并结合PAEC表面的未知抗原。采用流式细胞术测定PAEC表面结合的猴抗猪细胞抗体的平均荧光强度(MFI),用以表示PAEC表面抗原的含量。利用慢病毒介导的短发夹核糖核酸(shRNA)敲减PAEC中的FAM234A基因,检测该细胞结合的猴抗猪细胞抗体含量是否减少,以确定该基因是否为潜在的移植抗原。结果 PAEC中的非Gal抗原能够在猴体内引发大量的猴抗猪细胞抗体。在GTKO小型猪的PAEC中用shRNA敲减FAM234A基因后,PAEC结合的猴IgG抗体减少。结论 FAM234A是一种新的非Gal异种移植抗原。

原代猪肝细胞及主动脉内皮细胞的提取和培养

目的建立有效高活力的原代猪肝细胞和主动脉内皮细胞提取和培养的方法。为异种移植排斥反应的靶细胞——血管内皮细胞和肝细胞的研究提供基础。方法从野生巴马猪分离肝脏和主动脉血管,通过蠕动泵灌注Ⅱ型胶原酶消化的方法提取猪肝细胞,低速离心和差速贴壁法纯化肝细胞,过碘酸雪夫(PAS)染色、白蛋白与肝细胞核因子4α免疫荧光染色对原代肝细胞进行鉴定;利用Ⅰ型胶原酶血管管腔灌注消化法提取猪主动脉内皮细胞,并通过检测因子Ⅷ相关抗原vWF及内皮细胞吞噬乙酰化低密度脂蛋白的方法进行鉴定。结果通过本文方法提取到了大量高活力的原代猪肝细胞和主动脉内皮细胞,猪肝细胞可以连续培养一周,内皮细胞可进行传代培养和冻存。两种细胞分别表达肝细胞和内皮细胞标志性蛋白。结论本研究提供的原代猪内皮细胞和肝细胞提取方法是制备大量高活力的原代内皮细胞及肝细胞的可靠方案。