Carboxylated ε-poly-L-lysine,a cryoprotective agent,is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.     

C1q Protein Binds to the Apoptotic Nucleolus and Causes C1 Protease Degradation of Nucleolar Proteins

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r₂C1s₂), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus.     

Protective effects of cynaroside on oxidative stress in retinal pigment epithelial cells

Cynaroside is a flavonoid compound proved to possess antioxidant activity, but its protective effect on age‐related macular degeneration still remains unclear. In this study, the protective effects of cynaroside on oxidative stress and apoptosis in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H₂O₂) were investigated. Results showed that cynaroside effectively attenuated the decrease of cell activity induced by H₂O₂. The total reactive oxygen species can be remitted by decreasing malondialdehyde level, as well as increasing glutathione level, and superoxide dismutase and catalase activities. In addition, Western blot analysis indicated that cynaroside protected ARPE‐19 cells from apoptosis through downregulation of caspase‐3 protein activation which was controlled by the upstream proteins Bcl‐2 and Bax. It was finally proved that cynaroside could enhance the antioxidant and antiapoptotic ability in ARPE‐19 cells by promoting the expression of p‐Akt.     

真盐生植物盐地碱蓬根系边缘细胞在耐盐中的作用初探

以黄河三角洲潮间带盐地碱蓬种子生成的幼苗为材料,研究了NaCl胁迫对盐地碱蓬生长与根系边缘细胞的影响。盐地碱蓬的第一个边缘细胞几乎与根尖同步产生,当根长达到13mm时,边缘细胞数目达到最大值。NaCl胁迫抑制边缘细胞的活性,但低浓度的NaCl处理增加边缘细胞的数目。低浓度NaCl处理时果胶甲基酯酶（PME）的活性比对照有明显增加,超氧化物歧化酶（SOD）活性随着NaCl浓度的增加呈现先上升后下降的趋势,低浓度NaCl可以增加盐地碱蓬根内过氧化氢酶（CAT）的活性,NaCl处理时间和处理浓度都对过氧化物酶（POD）活性的影响不明显。这些结果表明,盐地碱蓬至少部分通过增加调控活性氧（ROS）水平增加PME活性及根系边缘细胞数目来抵抗NaCl胁迫。     

Pluripotency of male germline stem cells

The ethical issues and public concerns regarding the use of embryonic stem (ES) cells in human therapy have motivated considerable research into the generation of pluripotent stem cell lines from non-embryonic sources. Numerous reports have shown that pluripotent cells can be generated and derived from germline stem cells (GSCs) in mouse and human testes during in vitro cultivation. The gene expression patterns of these cells are similar to those of ES cells and show the typical self-renewal and differentiation patterns of pluripotent cells in vivo and in vitro. However, the mechanisms underlying the spontaneous dedifferentiation of GSCs remain to be elucidated. Studies to identify master regulators in this reprogramming process are of critical importance for understanding the gene regulatory networks that sustain the cellular status of these cells. The results of such studies would provide a theoretical background for the practical use of these cells in regenerative medicine. Such studies would also help elucidate the molecular mechanisms underlying certain diseases, such as testicular germ cell tumors.     

达格列净对高渗诱导的血管内皮细胞衰老的干预研究

目的:探讨SGLT2i类药物达格列净(dapagliflozin)对高渗诱导的人脐静脉内皮细胞(HUVECs)衰老的影响。方法:将HUVECs分为空白组(Blank组)、高渗330组(M-330组)、高渗350组(M-350组)、达格列净+高渗组(DAPA+M-350组),高渗培养环境由甘露醇诱导。衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老情况;免疫荧光染色检测SGLT2表达变化;Western blot检测SGLT2、细胞衰老标志物p21的表达变化,JC-1染色试剂盒检测线粒体膜电位的变化。结果:免疫荧光染色和western blot结果显示,Blank组,M-330组及M-350组细胞上均存在SGLT2受体蛋白表达,且Blank组,M-330组及M-350组的SGLT2表达依次显著增加。与Blank组相比,M-350组SA-β-Gal胞质蓝染、染色阳性率、衰老蛋白p21及SGLT2表达显著增加,并伴有线粒体膜电位的显著下降(P0.05);DAPA+M-350组与M-350组相比,SA-β-Gal胞质蓝染、染色阳性率和p21表达显著下降,并伴有线粒体膜电位的显著上升(P0.05)。结论:HUVECs上存在SGLT2受体蛋白,且在300-350 m Osm/L范围内随着渗透压的升高而增加,达格列净可改善高渗所诱导的血管内皮细胞衰老,其机制可能与达格列净改善高渗导致的线粒体功能障碍有关。     

AP-1在AngⅡ正反馈调节其前体基因表达中的作用

血管紧张素Ⅱ(AngⅡ)可诱导其前体基因在血管平滑肌细胞(vascularsmoothmusclecells,VSMC)中进行表达,其作用机制与促进转录激活蛋白-1(activatingprotein-1,AP-1)的基因调控区中存在的AP-1位点结合有关.为进一步明确AngⅡ调节AP-1结合活性的分子机制,用放线菌酮(cycloheximide,CHX)作为c-Jun磷酸化抑制剂,经DNA-蛋白质相互作用和蛋白质印迹实验,探讨AngⅡ对AP-1结合活性的影响并探讨其分子机制.结果表明,受AngⅡ刺激的VSMC,其核蛋白中AP-1的组成亚基之一c-Jun水平明显升高.免疫细胞化学染色显示,在被AngⅡ处理的细胞中,c-Jun主要定位于细胞核,胞浆中几乎检测不出该转录激活蛋白的存在.用丝氨酸磷酸化抗体检测证实,AngⅡ可诱导c-Jun磷酸化.电泳迁移率改变分析(electrophoreticmobilityshiftassay,EMSA)显示,c-Jun的磷酸化水平与AP-1结合血管紧张素原基因顺式元件的活性,和对该基因的转录激活作用呈正相关关系,CHX通过阻断c-Jun磷酸化抑制AngⅡ诱导的AP-1结合活性,但是不影响c-Jun的表达水平.上述结果提示,AP-1的磷酸化活化是AngⅡ正反馈调节其前体基因表达的重要机制之一,首次发现CHX是c-Jun磷酸化的抑制剂.     

Morphological profiling using machine learning reveals emergent subpopulations of interferon-γ–stimulated mesenchymal stromal cells that predict immunosuppression

Background

Although a preponderance of pre-clinical data demonstrates the immunosuppressive potential of mesenchymal stromal cells (MSCs), significant heterogeneity and lack of critical quality attributes (CQAs) based on immunosuppressive capacity likely have contributed to inconsistent clinical outcomes. This heterogeneity exists not only between MSC lots derived from different donors, tissues and manufacturing conditions, but also within a given MSC lot in the form of functional subpopulations. We therefore explored the potential of functionally relevant morphological profiling (FRMP) to identify morphological subpopulations predictive of the immunosuppressive capacity of MSCs derived from multiple donors, manufacturers and passages.

小鼠胚胎干细胞建系技术研究进展

目前,对小鼠胚胎干细胞的研究较为深入,并已成为研究细胞分化及信号转导、新基因发现及功能鉴定、器官发生、人类疾病和药物开发等的有效手段。胚胎干细胞建系是一项基础性工作。虽然技术日趋成熟,有些品系小鼠的胚胎干细胞建系已是常规技术,但不同品系小鼠胚胎干细胞的建系效率仍有很大差异,建系途径和方法各有特点,一个品系胚胎干细胞的建系方法不一定都适用于其他品系。本文从小鼠胚胎干细胞建系的途径、分离操作技术、培养体系等方面进行综述,并就与之相关的有些问题提出思考和对策。