山楂酸减少L02细胞脂质累积作用的研究(英文)

非酒精性脂肪性肝在发达国家和发展中国家中是一种常见的肝脏疾病。引起非酒精性脂肪性肝病的最常见原因有肥胖、糖尿病和高胆固醇。尽管非酒精性脂肪性肝病的发病率日益升高,事实上至今尚未发现可以用于治疗的药物。山楂酸是一种五环三萜化合物,已报道具有多种药理特性,包括抗炎、抗氧化以及免疫调节作用。我们的前期研究表明,山楂酸能够抑制高脂饲料诱导大鼠非酒精性脂肪性肝病的生成。本研究中,我们将探讨山楂酸体外对肝细胞(LO2)脂质累积的抑制作用。结果表明,山楂酸可抑制游离脂肪酸(FFA)诱导的LO2细胞脂质累积,进一步研究表明,山楂酸可抑制LO2细胞的SCAP mRNA水平和蛋白水平的表达。     

Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial‐to‐mesenchymal transition and immune modulation

Mesenchymal stem cells (MSCs) have been shown to improve the outcome of acute renal injury models; but whether MSCs can delay renal failure in chronic kidney disease (CKD) remains unclear. In the present study, the were cultured in media containing various concentrations of basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2‐phosphate to investigate whether hepatocyte growth factor (HGF) secretion could be increased by the stimulation of these growth factors. Then, TGF‐β1‐treated renal interstitial fibroblast (NRK‐49F), renal proximal tubular cells (NRK‐52E) and podocytes were co‐cultured with conditioned MSCs in the absence or presence of ascorbic acid 2‐phosphate to quantify the protective effects of conditioned MSCs on renal cells. Moreover, male Sprague‐Dawley rats were treated with 1 × 10⁶ conditioned MSCs immediately after 5/6 nephrectomy and every other week through the tail vein for 14 weeks. It was found that basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2‐phosphate promoted HGF secretion in MSCs. Besides, conditioned MSCs were found to be protective against TGF‐β1 induced epithelial‐to‐mesenchymal transition of NRK‐52E and activation of NRK‐49F cells. Furthermore, conditioned MSCs protected podocytes from TGF‐β1‐induced loss of synaptopodin, fibronectin induction, cell death and apoptosis. Rats transplanted with conditioned human MSCs had a significantly increase in creatinine clearance rate, decrease in glomerulosclerosis, interstitial fibrosis and increase in CD4⁺CD25⁺Foxp3⁺ regulatory T cells counts in splenocytes. Together, our studies indicated that conditioned MSCs preserve renal function by their anti‐fibrotic and anti‐inflammatory effects. Transplantation of conditioned MSCs may be useful in treating CKD.     

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.     

Imaging of primary human hepatocytes performed with micron-sized iron oxide particles and clinical magnetic resonance tomography

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepa-tocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron-sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose-finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5-day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO-labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO-labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation.     

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease

Abstract Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl₄)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl₄-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.     

Leukemia inhibitory factor contributes to hepatocyte-like differentiation of human bone marrow mesenchymal stem cells

The current majority of protocols for hepatocyte differentiation of mesenchymal stem cells (MSCs) are conducted using oncostatin M (OSM) as an inducer of hepatocyte-like maturation. As leukemia inhibitory factor (LIF) and OSM share similar signaling pathways, we examined whether LIF could play a role in the hepatocyte differentiation process. A differentiation protocol was designed using LIF as a maturation cytokine and this was compared with standard and control protocols applied to human MSCs of bone marrow origin. We observed that mesenchymal-derived hepatocyte-like cells (MDHLCs) acquired similar morphological changes when exposed to LIF or to OSM. Using protein and gene expression assays, we noticed a comparable hepatic marker expression in both differentiation conditions. Furthermore, LIF and OSM allowed the acquisition of equivalent levels of hepatocyte-like functionality as attested by evaluation of urea secretion and glycogen deposition. However, no increase in the expression of hepatocyte-like features could be observed in MDHLCs after a combined exposition to LIF and OSM. In conclusion, we demonstrated that LIF can play a similar role as OSM in the hepatocyte differentiation process of human MSCs.     

Mechanisms determining phenotypic heterogeneity of hepatocytes

This review summarizes results of biochemical and immunohistochemical studies indicating the existence of functional heterogeneity of hepatocytes depending on their localization in the hepatic acinus; this determines characteristic features of metabolism of carbohydrates, lipids, and xenobiotics. The physiological significance of hepatocyte heterogeneity is discussed. According to the proposed model of intercellular communication, the metabolic specialization of hepatocytes is determined by secretory activity of hepatic resident macrophages (Kupffer cells) localized mainly in the periportal zone of the liver acinus. Macrophages participate in secretion of a wide spectrum of intercellular mediators (cytokines, prostaglandins, growth factors) and also in metabolism of numerous blood metabolites and biologically active substances (hormones, lipoproteins, etc.). In the sinusoid and in the space of Disse (also known as perisinusoidal space) they form a concentration gradient of regulatory factors and metabolites inducing the phenotypic differences between hepatocytes.     

Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.     

人肝细胞生长因子(hdHGF)基因在毕赤酵母中的分泌表达

研究了hdHGF基因在毕赤酵母中的表达 .以人胎盘mRNA为模板 ,经逆转录、重叠PCR获得hdHGF全长和成熟基因片段 .将该基因片段克隆到pPIC9载体上 ,将重组表达质粒转化巴斯德毕赤酵母 (Pichiapastoris)GS115,筛选mut+ 表型 ,经甲醇诱导可实现rhdHGF的分泌表达 .经摇瓶培养筛选出 4株表达水平较高的酵母工程菌株 ,SDS PAGE分析和Western印迹试验表明 ,产物分子量约为80kD ,5L发酵罐高密度培养已使生物量达 13 5g L(干重 ) ,发酵液上清总蛋白量为 8 0g L ,电泳结果表明rhdHGF表达水平为总蛋白的 12 3 % .