Central nervous system and peripheral cell labeling by vascular endothelial cadherin-driven lineage tracing in adult mice

Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vascular cell types,we performed cell lineage tracing analysis using transgenic mice engineered to express a fluorescent marker following activation by tamoxifen in vascular endothelial cadherin promoter-expressing cells(VEcad-CreERT2;B6 Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze).Activation of target-cell labeling following 1.5 months of ad libitum feeding with tamoxifen-laden chow in 4–5 month-old mice resulted in the tracing of central nervous system and peripheral cells that include:cerebellar granule neurons,ependymal cells,skeletal myocytes,pancreatic beta cells,pancreatic acinar cells,tubular cells in the renal cortex,duodenal crypt cells,ileal crypt cells,and hair follicle stem cells.As Nestin expression has been reported in a subset of endothelial cells,Nes-CreERT2 mice were also utilized in these conditions.The tracing of cells in adult Nes-CreERT2 mice revealed the labeling of canonical progeny cell types such as hippocampal and olfactory granule neurons as well as ependymal cells.Interestingly,Nestin tracing also labeled skeletal myocytes,ileal crypt cells,and sparsely marked cerebellar granule neurons.Our findings provide support for endothelial cells as active contributors to adult tissue progenitor pools.This information could be of particular significance for the intravenous delivery of therapeutics to downstream endothelial-derived cellular targets.The animal experiments were approved by the Boise State University Institute Animal Care and Use Committee(approval No.006-AC15-018)on October 31,2018.     

精原干细胞体外转化和转分化的研究进展

精原干细胞(SSC)在睾丸曲细精管内微环境的精确调控下,只能单向分化为精子。然而,SSC可在特定条件下转化为多能干细胞,甚至直接重编程转分化为功能性终末分化细胞。该特殊潜能使其在再生和精准医学领域具有很好的运用前景。     

他克莫司对转化生长因子-β诱导的肾小管上皮细胞转分化的抑制作用及对核因子-κB活性的影响

目的:探讨他克莫司(FK506)对转化生长因子-β(TGF-β)诱导的肾小管上皮细胞转分化的作用,及其与核因子-κB(NF-κB)活性变化的关系.方法:以人类肾脏近曲小管上皮细胞株(Human kidney cell,HKC)为研究对象,分为以下4组:①阴性对照组;②TGF-β(8 ng/ml)组:③FK506(0.1、1、10、50 ng/ml)组:④TGF-β(8 ng/ml) FK506(0.1、1、10、50 ng/ml)组.应用形态学、间接免疫荧光法,免疫组化技术和酶联免疫吸附法观察FK506对TGF-β诱导的HKC细胞转分化的作用、细胞培养上清液纤连蛋白、Ⅰ型胶原的变化及FK506对HKC细胞NF-κB活性的影响.结果:FK506(1,10,50 ng/ml) TGF-β组比TGF-β组诱导的HKC细胞E-钙粘蛋白和细胞角蛋白表达有所增强,波形蛋白和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达比TGF-β组减弱(P＜0.05),同时HKC细胞NF-κB的活性比TGF-β组减弱(P＜0.05),培养上清液纤连蛋白及Ⅰ型胶原的浓度比TGF-β组降低(P＜0.05);FK506(1、10、50 ng/ml)使基础状态下HKC细胞NF-κB的活性明显下降(P＜0.05)和上清液中纤连蛋白及Ⅰ型胶原的水平明显降低(P＜0.05).结论:①FK506剂量依赖性地抑制TGF-β诱导的肾小管上皮细胞转分化和纤连蛋白及Ⅰ型胶原的形成及基础状态下和TGF-β诱导的HKC细胞NF-κB的表达.②FK506抑制TGF-β诱导的HKC细胞转分化很可能与NF-κB的表达下调有关,需要进一步研究.     

Endoplasmic reticulum stress participates in uric acid - induced phenotypic transformation of renal tubular epithelial cells

Objective To explore the effect of endoplasmic reticulum stress (ER stress) in uric acid-induced phenotypic change in renal tubular epithelial cells (HK-2). Methods (1) HK-2 cells were cultured with 0, 75, 150, 225, 300 mg/L uric acid for 24 h in vitro. (2) The cells were divided into normal control group, ER stress inhibitor 4-PBA (5 μmol/L) group, uric acid (150 mg/L) group and 4-PBA+uric acid group for 24 h. Morphological changes of HK-2 cells were observed under inverted microscope. MTT assay was used to detect the proliferation of HK-2 cells treated with 150 mg/L uric acid for 24, 48 and 72 h. The protein expressions of α-smooth muscle actin (α-SMA), vimentin, snail, glucose regulated protein 78 (GRP78) and the phosphorylation of eukaryotic initiation factor 2α (p-eIF2α) in HK-2 cells were measured by Western blotting. Results Compared with the control group, HK-2 cells in uric acid groups (150, 225, 300 mg/L) showed fibroblast-like appearance. The protein expressions of α-SMA, vimentin, snail, GRP78 and p-eIF2α in 150 mg/L and 225 mg/L uric acid groups were higher than those in the control group (all P＜0.05). The proliferation of HK-2 cells in 150 mg/L uric acid group was lower than that in control group at 48 and 72 h (all P＜0.01). Compared with the uric acid group, the cell morphology in 4-PBA+uric acid group was improved, and the protein expressions of α-SMA, vimentin, snail, GRP78 and p-eIF2α were decreased (all P＜0.05). Conclusions Uric acid may induce the phenotype transformation of renal tubular epithelial cell, and ER stress is involved. 4-PBA may inhibit the uric acid-induced ER stress response and phenotypic transformation, and may be beneficial in attenuating uric acid-induced renal tubular damage.     

The role of mRNA splicing in prostate cancer

Alternative splicing （AS） is a crucial step in gene expression. It is subject to intricate regulation, and its deregulation in cancer can lead to a wide array of neoplastic phenotypes. A large body of evidence implicates splice isoforms in most if not all hallmarks of cancer, including growth, apoptosis, invasion and metastasis, angiogenesis, and metabolism. AS has important clinical implications since it can be manipulated therapeutically to treat cancer and represents a mechanism of resistance to therapy. In prostate cancer （PCa） AS also plays a prominent role and this review will summarize the current knowledge of alternatively spliced genes with important functional consequences. We will highlight accumulating evidence on AS of the components of the two critical pathways in PCa： androgen receptor （AR） and phosphoinositide 3-kinase （PI3K）. These observations together with data on dysregulation of splice factors in PCa suggest that AR and PI3K pathways may be interconnected with previously unappreciated splicing regulatory networks. In addition, we will discuss several lines of evidence implicating splicing regulation in the development of the castration resistance.     

Immunohistochemical study for the origin of ductular reaction in chronic liver disease

The appearance of proliferating bile ductular structures, which is called the “atypical ductular reaction” is frequently observed in various chronic liver diseases associated. However, the origin of these increased bile ductules has been a matter of controversy. In this study, we investigated the origin of ductular cells as an aspect of relation between epithelial to mesenchymal transition (EMT) and epithelial members of liver parenchyme, such as hepatocyte and cholangiocyte by immunohistochemical staining of human liver. Thirteen specimens of surgically resected liver with biliary cirrhosis were selected. Three sets of double immunohistochemical stains were done; Hep-Par 1 - cytokeratin 19 (CK19), Hep-Par 1 - α-sm ooth mus cle actin (α-SMA) and CK19 - α-SMA. As a result, we investigated the dual expression of the markers of hepatocyte and cholangiocyte in the same cell; in ductular cell and surrounding hepatocyte. However, there seems to be no dual expression of markers for EMT with epithelial markers. This study suggests a possibility of phenotypic change of mature hepatocyte into cholangiocyte. Future studies will be necessary to determine the role that proliferating cholangiocytes play in the pathogenesis of biliary fibrosis and how cholangiocytes interact with other cell types of the liver such as hepatic stellate cells or Kupffer cells.     

PI3-K的激活在TGF-β1诱导肾小管上皮细胞间质转分化过程中的作用

目的探讨磷脂酰肌醇3-激酶（P1I3-K）的激活在转化生长因子岛（TGF-β1）体外诱导人肾小管上皮细胞（HKCs）间质转分化（EMT）过程中的作用及意义。方法将HKCs细胞株分为正常对照组（C）、不同浓度TGF-β1组、TGF-β1＋P13-K抑制剂Wortmannin组。选取不同时相,采用免疫印迹法（Western blot）测定总Akt（t-Akt）、磷酸化Akt（p-Akt）、α-平滑肌肌动蛋白（α-SMA）和E-钙黏素（E-cadherin）蛋白水平的表达变化;Akt磷酸化水平用磷酸化Akt与总Akt水平的比值表示。结果 正常体外培养的HKCs经10ng/ml TGF-β1刺激1h后p-Akt水平显著升高;mSMA蛋白表达水平在10ng／ml TGF-β1诱导48h后表达显著升高,而E-cadherin蛋白表达水平在TGF-β1诱导48h后表达显著下降。同时加用Wortmannin 10nmol／L组,p-Akt、α-SMA表达明显抑制,E-cadherin表达显著升高。结论PI3-K在TGF-β1诱导的EMT过程中被激活,并且参与TGF-β1诱导的EMT过程,特异性阻断PI3-K的激活可有效抑制TGF-β1介导的HK氏的EMT过程。