HLA－G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

In order to investigate whether the non-classical HLA-G class I molecule protects the prcine endothelial cells(PECs)from the lysis mediated by human immune cells in pig to human discordant xenotransplantation,we have cloned HLA-G cDNA from a human placents by RT-PCR.Mammalian expression vector,pEFG-neo,was constructed by insertion of HLA-G cDNA in pEF-neo.We obtained efficiently expressed PECs by stable transfection.Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis.The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells.It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes.The reduction of lysis ranged between 59.1% and 88.9A%.These findings suggest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overconing the immune rejections in xenotransplantion,including delayed xenograft rejection and cell-mediated rejection.     

Immunoinhibitory effect of soluble HLA-G1 on the cytotoxicity of NK92 cells

HLA-G (human leukocyte antigen-G) is a non-classical HLA class I molecule, playing an important immuno-modulatory role in maintaining maternal immune tolerance of the semiallogenic fetus and organ transplantation. In this study, the cDNA sequence of extracellular domain of HLA-G1 was subcloned into the pET28a vector and a soluble 35 kD fusion protein (His-sHLA-G1) with six histine residues was obtained. In the 4 h 51Cr-release assay the fusion protein obviously inhibited the cytotoxicity of NK92 cells in a dose-dependent manner. These results indicated that sHLA-G1, as an activated immunoinhibitor, may provide an effective approach to overcoming the immune rejection of transplantation.     

Identification of the candidate genes associated with cellular rejection in pig-to-human xenotransplantation

To identify the genes associated with cellular rejection in pig-to-human xenotransplantation, the suppression subtractive hybridization (SSH) was used in screening the up-regulated genes from a co-culture of human peripheral blood mononuclear cells (PBMCs) and porcine vascular endothelial cell line PIEC. The up-regulated cDNAs were cloned into pGEM-T Easy vector and then sequenced. Nucleic acid homology searches were performed using the BLAST program. A subtracted cDNA library including about 300 clones with the expected up-regulated genes was obtained. Twenty-four of these clones were analyzed by sequencing and homology comparison was made. These clones represent the genes of human perforin (PRF1), proteasome, lymphocyte specific interferon regulatory factor/interferon regulatory factor 4 (LSIRF/IRF 4), muscleblind-like (MBNL) protein and a porcine expressed sequence tag (EST) which has 81% homology with human oxidative-stress responsive 1 (OSR 1). These genes might be the candidate genes which are associated with cellular rejection in pig-to-human xenotransplantation.     

A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity

Natural killer (NK) cells are classically viewed as lymphocytes that provide innate surveillance against virally infected cells and tumour cells through the release of cytolytic mediators and interferon (IFN)-gamma. In humans, blood CD56(dim) NK cells specialize in the lysis of cell targets. In the lymph nodes, CD56(bright) NK cells secrete IFN-gamma cooperating with dendritic cells and T cells in the generation of adaptive responses. Here we report the characterization of a human NK cell subset located in mucosa-associated lymphoid tissues, such as tonsils and Peyer's patches, which is hard-wired to secrete interleukin (IL)-22, IL-26 and leukaemia inhibitory factor. These NK cells, which we refer to as NK-22 cells, are triggered by acute exposure to IL-23. In vitro, NK-22-secreted cytokines stimulate epithelial cells to secrete IL-10, proliferate and express a variety of mitogenic and anti-apoptotic molecules. NK-22 cells are also found in mouse mucosa-associated lymphoid tissues and appear in the small intestine lamina propria during bacterial infection, suggesting that NK-22 cells provide an innate source of IL-22 that may help constrain inflammation and protect mucosal sites.     

HLA-G蛋白在不同孕期胎盘绒毛中的表达及意义

人类白细胞抗原-G(HLA-G)主要表达于胎盘组织中,在维持母胎界面的免疫耐受过程中发挥着关键作用。为研究HLA-G在正常胎盘形成过程中的表达模式,实验检测了妊娠不同阶段的胎盘绒毛组织中HLA-G蛋白的表达水平。实验结果显示HLA-G蛋白在孕6周与7周的绒毛中表达量最高,从孕8周开始,表达量逐渐下降,至足月时,表达量达到最低水平。由此推测HLA-G在妊娠早、中期与胎盘形成有关,在妊娠晚期可能参与妊娠维持。     

角膜移植及其排斥反应的内皮改变——角膜内皮显微镜观察

本文报告用角膜内皮显微镜观察兔穿透性角膜移植术前、术后和角膜移植排斥反应时的角膜内皮改变结果。发现在角膜移植术前,正常角膜的中央区和周边区的内皮细胞密度、细胞面积基本相同,没有显著差异。在角膜移植术后,术眼的植片和受主角膜的内皮细胞均出现密度减低、细胞面积增大,与术前比较有显著差异。在排斥反应期间,植片内皮细胞出现细胞肿胀、边界不清、甚至模糊不能窥见等异常改变,而受主角膜内皮细胞则表现正常。本文认为兔角膜内皮细胞的形态、密度等与人角膜内皮细胞基本相同。穿透性角膜移植术对角膜内皮有较大损伤,植片内皮细胞在排斥反应中遭受严重损害,提出在角膜移植术时,要注意保护角膜内皮;发现排斥反应时,要及时治疗,防止角膜内皮的过多损伤。     

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 cell (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 for 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-α 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 human Mo/NK-PAEC interactive mechanisms.     

人穿孔素羧基端肽段的表达纯化与活性鉴定

穿孔素,即成孔蛋白（pore forming protein,PFP),其溶细胞作用与免疫调节和自身免疫病以及其它多种疾病过程中的免疫性病理损伤相关。为得到足够量的PFP建立与之相关的免疫学研究手段用于基础和临床研究,在已克隆人PFP cDNA的基础上,用基因工程方法表达了人PFP C端124个氨基酸肽段（hPFP-C),并通过谷胱甘肽琼脂糖新和层析获得纯化的GST/hPFP-C融合蛋白,经凝血酶酶切和再次北极和层析去除GST部分,得到了纯化的hPFP-C蛋白。纯化的hPFP-C蛋白与兔红细胞共育,呈现钙依赖的溶血活性。     

Variable expression of Ia antigens on the vascular endothelium of mouse skin allografts

Ia antigens are membrane-bound glycoproteins that play a part in antigen recognition and subsequent cell-cell interactions in the immune response. In the mouse they are coded for by the I region of the major histocompatibility complex H-2 and have been demonstrated on B lymphocytes, monocytes, activated T cells, macrophages and dendritic cells, including Langerhans cells. Ia-like antigens have also been detected on the vascular endothelium in man and on epidermal keratinocytes in rats but expression on the latter cells was induced by a graft-versus-host reaction or by contact hypersensitivity. In the mouse, previous studies have suggested that Ia antigens in skin are restricted to epidermal Langerhans cells and it was thought that these were the targets for Ia-dependent rejection of skin allografts. The results presented here show that Ia antigens in mouse allografts are also present on the vascular endothelium but their expression is variable and dependent on the immunological status of the recipient. These findings suggest that vascular endothelial cells can act as targets in Ia-incompatible skin allograft rejection.     

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-sclectin 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 endothelial 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 kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.     

重组人组织型纤溶酶原激活剂缺失变体基因的克隆及工程菌的构建与鉴定

利用RT-PCR技术,从人脐带静脉上皮细胞中克隆人组织型纤溶酶原激活剂基因,将其接入TA克隆载体PCR2．1中,经DNA序列测定后,以该重组质粒DNA为模板,用PCR方法获得了人组织型纤溶酶原激活剂缺失变体(K2tPA)基因,将其转入pET29a,构建了重组表达质粒pET29a／K2tPA,并转化至大肠杆菌BL21(DE3)中,构成工程菌．经IPTG诱导表达,在40kDa处有一明显表达条带,表达量为约占菌体总蛋白的20％．该菌种在贮存与复苏及传代过程中具有良好的质粒稳定性和表达稳定性．     

Cellular immune responses to HIV

The cellular immune response to the human immunodeficiency virus, mediated by T lymphocytes, seems strong but fails to control the infection completely. In most virus infections, T cells either eliminate the virus or suppress it indefinitely as a harmless, persisting infection. But the human immunodeficiency virus undermines this control by infecting key immune cells, thereby impairing the response of both the infected CD4+ T cells and the uninfected CD8+ T cells. The failure of the latter to function efficiently facilitates the escape of virus from immune control and the collapse of the whole immune system.     

Cloning of murine BRI3 gene and study on its function for inducing cell death

To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.     

A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells

Metastatic progression of cancer is a complex and clinically daunting process. We previously identified a set of human microRNAs (miRNAs) that robustly suppress breast cancer metastasis to lung and bone and which display expression levels that predict human metastasis. Although these findings revealed miRNAs as suppressors of cell-autonomous metastatic phenotypes, the roles of non-coding RNAs in non-cell-autonomous cancer progression processes remain unknown. Here we reveal that endogenous miR-126, an miRNA silenced in a variety of common human cancers, non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. It suppresses metastatic endothelial recruitment, metastatic angiogenesis and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1 and MERTK--novel pro-angiogenic genes and biomarkers of human metastasis. Insulin-like growth factor binding protein 2 (IGFBP2) secreted by metastatic cells recruits endothelia by modulating IGF1-mediated activation of the IGF type-I receptor on endothelial cells; whereas c-Mer tyrosine kinase (MERTK) receptor cleaved from metastatic cells promotes endothelial recruitment by competitively antagonizing the binding of its ligand GAS6 to endothelial MERTK receptors. Co-injection of endothelial cells with breast cancer cells non-cell-autonomously rescues their miR-126-induced metastatic defect, revealing a novel and important role for endothelial interactions in metastatic initiation. Through loss-of-function and epistasis experiments, we delineate an miRNA regulatory network's individual components as novel and cell-extrinsic regulators of endothelial recruitment, angiogenesis and metastatic colonization. We also identify the IGFBP2/IGF1/IGF1R and GAS6/MERTK signalling pathways as regulators of cancer-mediated endothelial recruitment. Our work further reveals endothelial recruitment and endothelial interactions in the tumour microenvironment to be critical features of metastatic breast cancer.     

Growth potential of human hepatocarcinoma cells in the liver of neonatal immunocompetent mice and its relation to immunological tolerance

To determine the pathological behavior of human hepatocarcinoma cells in the liver microenvironment of neonatal non-immunode-ficient mice, three human hepatocarcinoma cell lines (Bel7402, HepG2, and SK-Hep-1), traced by DiI, were transplanted into the intrahepatic or subcutaneous tissue of neonatal and adult Kunming mice. Histopathological observations showed that cells in the adult liver induced a severe immune response as early as the second day after the implantation, while the subcutaneous neoplasm underwent extensive necrosis by the end of the study. Only the cells injected into the neonatal liver underwent a delayed immunologic rejection in the organ microenvironment. These cells retained recognizable tumor features over the first seven days, and displayed an intrahepatic invasive pattern. The expression of tumor markers including alpha-fetoprotein and survivin was maintained. The quantitative ELISA for the expression patterns of IL-2 and IL-10 also confirmed that the intrahepatic immunity was non-susceptive during this period. The high serum alpha-fetoprotein level was inversely correlated with the change in immune response. Our study provided a bio-system for the research of immune responses to xenografts in the liver.     

人类超氧化物歧化酶在酿酒酵母系统中的高效表达

超氧化物歧化酶(SOD)是一种在生物界广泛存在的抗氧化酶,在抗衰老、抗肿瘤、抗免疫疾病和电磁辐射上都起着重要的作用．对一从人类胎肝cDNA文库中筛到的SOD基因片段进行重组、克隆,并得到带有启动子PADHZ—GAPDH和终止子TADHI的高效稳定的酿酒酵母表达载体pHC11-hSOD．转化酿酒酵母DCO4后获得工程菌DCO4／pHC11-hSOD．表达产物经破壁后SDS-PAGE电泳检测为20ku的条带;酶活性测定结果表明发酵液有40万U／L的hSOD活性．此外,还研究了工程菌的发酵条件和hSOD产物的纯化方法．纯化产物在SDS-PAGE上和Superdex分子筛检测显示,所得产物的纯度已达95％以上．     

胚胎停育绒毛中HLA-G mRNA和蛋白水平的表达及差异

研究HLA-G mRNA及蛋白在早孕绒毛和胚胎停育绒毛中的表达及意义,在50例正常早期妊娠妇女和50例胚胎停育妇女绒毛中,半定量RT-PCR法检测HLA-G mRNA的水平。S-P免疫组织化学染色法检测HLA-G蛋白的水平。两组标本中HLA-G mRNA水平差异不具有统计学意义(P>0.05);胚胎停育绒毛标本中HLA-G蛋白水平降低,与正常早孕绒毛标本相比较,差异具有统计学意义(P<0.05)。说明HLA-G分子可能参与了胚胎停育的发生发展。     

Therapeutic angiogenesis induced by human hepatocyte growth factor (HGF) gene in rat myocardial ischemia models

Coronary arteriosclerotic cardiopathy is also named myocardial ischemia, which severely threatens humanhealth. Following the economic development and change of life style in China, population blood pressure, weight index and serum cholesterol level all rise. This prophesies incidence rate of coronary arteriosclerotic cardiopathy and stroke will increase year by year. Angioplasty and surgical bypass, the primary interventional therapies for these in-dividuals, are temporally limited by the prob…