水栀子及京尼平苷抑制RBL-2H3细胞脱颗粒作用的代谢组学研究

目的 基于细胞代谢组学的方法研究水栀子Gardenia jasminoside及其主要有效成分京尼平苷对RBL-2H3细胞脱颗粒模型的干预作用。方法 利用UPLC-QTOF-MS高分辨质谱检测RBL-2H3细胞的代谢轮廓改变，并应用主成分分析（PCA）和正交偏最小二乘判别分析（OPLS-DA）筛选出引起这种差异的代谢标志物；同时应用MEV软件对差异标志物水平的变化进行聚类分析和热图绘制。结果 分别得到与水栀子和京尼平苷作用相关的代谢标志物54和46个，其中共有标志物31个，且在2个给药组中变化趋势相同。由共有代谢标志物富集得到了受干扰的5条代谢通路：甘氨酸、天冬氨酸、谷氨酸代谢，谷胱甘肽代谢，组胺代谢，能量代谢，烟酰胺代谢。结论 水栀子和京尼平苷通过调节组胺代谢、氧化应激和能量代谢而抑制RBL-2H3细胞脱颗粒过程，且京尼平苷是水栀子发挥作用的主要药效物质基础之一。     

Control the fate of human umbilical cord mesenchymal stem cells with dual‐enzymatically cross‐linked gelatin hydrogels for potential applications in nerve regeneration

Stem‐cell‐based therapy is a promising strategy to treat challenging neurological diseases, while its application is hindered primarily by the low viability and uncontrolled differentiation of stem cell. Hydrogel can be properly engineered to share similar characteristics with the target tissue, thus promoting cell viability and directing cell differentiation. In this study, we proposed a new dual‐enzymatically cross‐linked and injectable gelatin hydrogel for regulating survival, proliferation, and differentiation of human umbilical cord mesenchymal stem cells (hUC‐MSCs) in a three‐dimensional matrix. This injectable gelatin hydrogel was formed by oxidative coupling of gelatin–hydroxyphenyl acid conjugates catalyzed by hydrogen horseradish peroxidase (HRP) and choline oxidase (ChOx). Modulus and H₂O₂ release can be well controlled by ChOx activity. Results from calcein‐AM/PI staining and Ki67 immunofluorescence tests demonstrated that the survival and proliferation behavior of hUC‐MSCs were highly enhanced in HRP_1UChOx_0.25U hydrogel with lower modulus and less H₂O₂ release compared with other groups. Attractively, the expression of neuron‐specific markers β‐III tubulin, neurofilament light chain (NFL), and synapsin‐1 was significantly increased in HRP_1UChOx_0.25U hydrogel as well. Additionally, in vitro hemolysis test and in vivo HE staining data highlighted the good biocompatibility. Undoubtedly, this injectable gelatin hydrogel's ability to control hUC‐MSCs' fate holds enormous potentials in nervous disorders' therapy and nerve regeneration.     

Polyethylene glycol 35 ameliorates pancreatic inflammatory response in cerulein-induced acute pancreatitis in rats

BACKGROUND Acute pancreatitis(AP) is a sudden inflammatory process of the pancreas that may also involve surrounding tissues and/or remote organs. Inflammation and parenchymal cell death are common pathological features of this condition and determinants of disease severity. Polyethylene glycols(PEGs) are nonimmunogenic, non-toxic water-soluble polymers widely used in biological, chemical, clinical and pharmaceutical settings.AIM To evaluate the protective effect of a 35-k Da molecular weight PEG(PEG35) on the pancreatic damage associated to cerulein-induced acute pancreatitis in vivo and in vitro.METHODS Wistar rats were assigned at random to a control group, a cerulein–induced AP group and a PEG35 treatment group. AP was induced by five hourly intraperitoneal injections of cerulein(50 μg/kg/bw), while the control animals received saline solution. PEG35 was administered intraperitoneally 10 minutes before each cerulein injection in a dose of 10 mg/kg. After AP induction, samples of pancreatic tissue and blood were collected for analysis. AR42 J pancreatic acinar cells were treated with increasing concentrations of PEG35 prior to exposure with tumor necrosis factor α(TNFα), staurosporine or cerulein. The severity of AP wasdetermined on the basis of plasma levels of lipase, lactate dehydrogenase activity, pancreatic edema and histological changes. To evaluate the extent of the inflammatory response, the gene expression of inflammation-associated markers was determined in the pancreas and in AR42 J-treated cells. Inflammation-induced cell death was also measured in models of in vivo and in vitro pancreatic damage.RESULTS Administration of PEG35 significantly improved pancreatic damage through reduction on lipase levels and tissue edema in cerulein-induced AP rats. The increased associated inflammatory response caused by cerulein administration was attenuated by a decrease in the gene expression of inflammation-related cytokines and inducible nitric oxide synthase enzyme in the pancreas. In contrast, pancreatic tissue m RNA expression of interleukin 10 was markedly increased. PEG35 treatment also protected against inflammation-induced cell death by attenuating lactate dehydrogenase activity and modulating the pancreatic levels of apoptosis regulator protein BCL-2 in cerulein hyperstimulated rats. Furthermore, the activation of pro-inflammatory markers and inflammationinduced cell death in pancreatic acinar cells treated with TNFα, cerulein or staurosporine was significantly reduced by PEG35 treatment, in a dose-dependent manner.CONCLUSION PEG35 ameliorates pancreatic damage in cerulein-induced AP and AR42 J-treated cells through the attenuation of the inflammatory response and associated cell death. PEG35 may be a valuable option in the management of AP.     

Cyclical strain improves artificial equine tendon constructs in vitro

Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo environment, in this study, we have utilised a novel and affordable, custom‐made bioreactor to apply a cyclical strain to tendon‐like constructs generated in three‐dimensional (3D) culture by equine tenocytes. Dynamic shear analysis (DSA), dynamic scanning calorimetry (DSC) and Fourier‐transform infrared (FTIR) spectroscopy were used to determine the mechanical and chemical properties of the resulting tendon‐like constructs. Our results demonstrate that equine tenocytes exposed to a 10% cyclical strain have an increased amount of collagen gel contraction after 7 and 8 days of culture compared with cells cultured in 3D in the absence of external strain. While all the tendon‐like constructs have a very similar chemical composition to native tendon, the application of strain improves their mechanical properties. We envisage that these results will contribute towards the development of improved biomimetic artificial tendon models for the development of novel strategies for equine regenerative therapies.     

Effects and long‐term follow‐up of using umbilical cord blood–derived mesenchymal stromal cells in pediatric patients with severe BK virus‐associated late‐onset hemorrhagic cystitis after unrelated cord blood transplantation

This is a retrospective study to evaluate the efficacy and safety of umbilical cord blood–derived mesenchymal stromal cells (MSCs) for the treatment of pediatric patients with severe BK virus–associated late‐onset hemorrhagic cystitis (BKV‐HC) after unrelated cord blood transplantation (UCBT). Thirteen pediatric patients with severe BKV‐HC from December 2013 to December 2015 were treated with MSCs. The number of MSCs transfused in each session was 1 × 10⁶/kg once a week until the symptoms improved. The median follow‐up time was 1432 (89‐2080) days. The median frequency of MSC infusion was 2 (1‐3), with eight cured cases and five effective cases; the total efficacy rate was 100%. The copy number of urine BKV DNA was 4.43 (0.36‐56.9) ×10⁸/mL before MSC infusion and 2.67 (0‐56.3) ×10⁸/mL after MSC infusion; the difference was not significant (P = .219). There were no significant differences in the overall survival, disease‐free survival, and the incidence of relapse and acute and chronic graft‐versus‐host disease between the MSC infusion group and non‐MSC infusion group. There was also no significant difference in the cytomegalovirus, Epstein‐Barr virus (EBV), and fungal and bacterial infection rates between the two groups. Although umbilical cord blood–derived MSCs do not reduce the number of BKV DNA copies in the urine, the cells have a high efficacy rate and minimal side effects in treating severe BKV‐HC after UCBT among pediatric patients. MSCs do not affect the rates of relapse, long‐term infection, or survival of patients with leukemia.     

Beta-adrenergic agonist protects retinal pigment epithelium against hydroxycholoroquine toxicity via c AMP-PKA signal pathway

AIM: To test our hypothesis that activation of protein kinase A(PKA) signal pathway by β-adrenergic agonist plays an important role in the protecting of cultured retinal pigment epithelial(RPE) cells against the hydroxychloroquine(HCQ) toxicity. METHODS: Cultured human RPE cells were treated with 1) HCQ, 2) HCQ with salbutamol(a β2-adrenergic receptor agonist), and 3) HCQ with salbutamol and a PKA inhibitor, and compared these to 4) untreated cells(controls). After treated for 24 h, cell vacuolation, cells viability, PKA and PKA kinase activity levels were determined by the measurement of the size of vacuoles using Image J software, the cell counting with a dye-exclusion testing, Western blot and PKA kinase detection, respectively. RESULTS: Cell vacuolation and cell death of cultured RPE cells were significantly increased by the treatment of HCQ. Salbutamol significantly elevated PKA and PKA activity levels and this was associated with the inhibition of the vacuolation and cell death. The PKA inhibitor significantly decreased the PKA levels and eliminated the protective effects of salbutamol on HCQ-treated RPE cells. CONCLUSION: The PKA pathway plays an important role in the protective effects of β2-adrenergic agonist on the RPE cells against HCQ toxicity. These findings reveal a novel potential strategy against HCQ retinopathy by treatment with PKA activating medications.     

The secrets of human stem cell-derived transfusable RBC for targeted large-scale production and clinical applications: A fresh look into what we need most and lessons to be learned

Blood transfusion, using the safest conventional blood bioproducts, is an irreplaceable part of substitution therapy. It is considered the most essential supportive clinical intervention aimed to restore the health of patients in need. Nevertheless, numerous unresolved problems are still associated with current blood substitution therapy. To alleviate our dependency on blood donors, many investigators have been focusing on the quest for stem cell-derived blood cells in line with major developments in the field of regenerative medicine. The main objective is to provide a safe and highly standardized universal cultured red cell concentrate [CRBC] for all clinical applications, regardless of blood groups. Currently, we are close to overcoming some of the main obstacles in culturing cells. This concise report is a prelude to the immortalized cell lines that are ready for in vivo clinical trials. It is only through the sharing of experimental ideas and knowledge-based strategies that we will be able to achieve such an enormous task and better understand ‘’the one for all concept’’ of CRBCs and their universal usage in all clinical settings.     

PNPLA3对肝细胞和肝癌细胞脂质代谢的影响*

PNPLA3基因在人类和哺乳动物体内都有表达,并且在肝癌细胞中也有表达。PNPLA3在人体和小鼠体内突变后可导致肝细胞内甘油三酯的蓄积,肝细胞发生脂肪变性。在人类和大鼠肝癌细胞使突变型PNPLA3过表达,与过表达野生型PNPLA3细胞比,也会导致肝癌细胞脂质的聚集。本文将归纳总结以往关于PNPLA3基因对肝细胞和肝癌细胞脂质代谢的影响,并分别讨论了PNPLA3对人类肝细胞和肝癌细胞、大鼠和小鼠肝细胞及其肝癌细胞的影响。     

Cyclin D1启动子在养精胶囊促进小鼠精原干细胞增殖中的作用※

目的 研究养精胶囊促进小鼠精原干细胞（SSCs）增殖的分子机制。方法 将不同浓度的养精胶囊提取物加入SSCs中培养48 h，CCK-8检测细胞的增殖活性，流式细胞仪检测细胞周期，荧光素酶报告基因检测Cyclin D1启动子的活性，qRT-PCR以及免疫荧光检测Cyclin D1的表达。之后进行阻断实验，在加入养精胶囊前预先加入siRNA-Cyclin D1，同样的方法检测细胞的增殖活性、细胞周期、Cyclin D1启动子的活性以及Cyclin D1的表达情况。结果 低、中、高浓度的养精胶囊可以促进SSCs的增殖，提高S期细胞的比例，增强Cyclin D1启动子的活性，促进Cyclin D1的表达。阻断Cyclin D1后，SSCs的增殖活性降低，S期细胞比例减少，Cyclin D1启动子的活性降低，Cyclin D1的表达减少。结论 养精胶囊通过增强Cyclin D1启动子的活性提高Cyclin D1的转录和翻译水平，进而促进SSCs增殖。