A Comparison of Methods for Isolation of Limbal Niche Cells: Maintenance of Limbal Epithelial Stem/Progenitor Cells

PurposeLimbal niche cells (LNCs) play a vital role in the maintenance of limbal epithelial stem/progenitor cells (LESCs). Four methods have been reported to isolate and expand LNCs: digestion by collagenase alone (C-LNC), collagenase following dispase removal of the limbal epithelium (DC-LNC), dissection of dispase-isolated limbal epithelial sheets (D-LNC), and explant cultures of limbal stromal tissues (Ex-LNC). This study aimed to isolate LNCs using those four methods and to compare their capacity to maintain LESCs.MethodsLNCs were isolated from the rat corneal limbus by the following methods: C-LNC, DC-LNC, D-LNC, and Ex-LNC. Quantitative real-time PCR and immunofluorescence staining were used to analyze the expression of embryonic stem cell (ESC) markers. The ability to maintain LESCs was assessed on the basis of colony-forming capacity and the expression of progenitor, proliferation, and differentiation markers in three-dimensional (3D) Matrigel and Transwell systems. Notch signaling of LESCs supported by different LNCs in Transwell inserts was analyzed by quantitative real-time PCR.ResultsDC-LNCs exhibited lower expression of CK12 during isolation and expansion. Among P4-expanded LNCs, DC-LNCs expressed significantly higher levels of Sox2, Oct4, Nanog, and N-cadherin than C-LNCs, D-LNCs, and Ex-LNCs. Compared with other LNCs, DC-LNCs were more effective in maintaining LESCs with higher holoclone-forming efficiency, greater expression of ΔNp63α and Ki67, and lower expression of CK12. DC-LNCs were also more capable of downregulating Notch signaling of LESCs.ConclusionsDC-LNCs were more effective in expressing ESC markers and maintaining LESCs compared to other LNCs. This study identifies an optimal method for the isolation of LNCs in tissue engineering and ocular surface reconstruction.     

The role of the tumor-microenvironment in lung cancer-metastasis and its relationship to potential therapeutic targets

Non-small cell lung cancer (NSCLC) accounts for >80% of lung cancer cases and currently has an overall five-year survival rate of only 15%. Patients presenting with advanced stage NSCLC die within 18-months of diagnosis. Metastatic spread accounts for >70% of these deaths. Thus elucidation of the mechanistic basis of NSCLC-metastasis has potential to impact on patient quality of life and survival.     

不同剂量γ射线照射后胎儿骨髓CD34~ 造血干细胞死亡规律

目的　探讨不同剂量γ射线照射后胎儿骨髓CD34 造血干细胞的死亡规律。方法　以流式细胞术PE -CD34 FITC AnnexinV 7AAD三色荧光法 ,多参数分析了受不同剂量γ射线照射后 ,不同时间点的胎儿骨髓CD34 造血干细胞凋亡、坏死和存活的时效和量效变化特点。结果　受大剂量率γ射线 1次 5Gy和 8Gy照射后 ,胎儿骨髓CD34 造血干细胞的死亡是在照射后为期 1周的连续性死亡 ,既有凋亡又有坏死 ,但是以凋亡为主 ;受大剂量率γ射线 1次 10Gy和 12Gy照射后 ,胎儿骨髓CD34 造血干细胞则在受照后当天即大量坏死 ,在持续 1周内全部死亡。结论　受大剂量率γ射线 1次照射后 ,造血干细胞在 1周内发生了增殖期死亡而连续性空出所占据的龛位     

缺血性脑损伤与神经干细胞的再生

脑缺血可在成年哺乳动物脑内诱发神经再生,这可能有潜在的治疗价值。本文阐述了脑缺血后神经干细胞的增殖、迁移、分化和整合情况及血管微生态的重要作用,分析了侧脑室下区、齿状回和其他部位的神经再生情况以及老年脑神经再生的特点。     

人工耳蜗植入术的应用解剖学研究

目的探讨后鼓室入路人工耳蜗植入术的有关解剖,为人工耳蜗植入手术提供解剖学基础。方法取成人颞骨标本60侧(左、右各30侧),在放大6倍的手术显微镜下进行观察和测量,结果进行统计学处理。结果圆窗龛(窝)的形态大体分为2种类型:近圆形(52%)和近半圆形(48%)。圆窗龛下壁有一窦腔者占86.7%。岬下脚出现率为11.7%,岬小桥出现率为33.3%。颈静脉窝高位,窝顶达圆窗龛上缘者占11.7%,均未影响圆窗龛的形态。外耳道上棘至锥隆起、鼓索隆起、圆窗龛前唇(缘)、面神经管锥曲的距离分别是(18.02±1.21)mm、(15.22±1.32)mm、(20.09±1.20)mm、(14.96±2.03)mm。鼓索隆起至面神经管锥曲、锥隆起的距离分别是(3.33±0.42)mm、(3.79±0.56)mm。锥隆起向后至面神经管的距离平均为(3.58±0.47)mm。结论后鼓室进路即是通过面神经隐窝的进路,面神经隐窝大小各异,鼓索隆起至面神经管锥曲和至锥隆起的距离,可作为面神经隐窝的宽度。人工耳蜗植入术经面神经隐窝入路较理想,距圆窗龛较近,有利于电极插入。该入路靠近面神经管,手术操作时应注意保护面神经。圆窗龛前唇遮盖圆窗膜,手术时必须磨去前唇暴露圆窗膜。     

Premetastatic niches,exosomes and circulating tumor cells: Early mechanisms of tumor dissemination and the relation to surgery

The physiological stress response to surgery promotes wound healing and functional recovery and includes the activation of neural, inflammatory and proangiogenic signaling pathways. Paradoxically, the same pathways also promote metastatic spread and growth of residual cancer. Human and animal studies show that cancer surgery can increase survival, migration and proliferation of residual tumor cells. To secure the survival and growth of disseminated tumor cells, the formation of premetastatic niches in target organs involves a complex interplay between microenvironment, immune system, circulating tumor cells, as well as chemical mediators and exosomes secreted by the primary tumor. This review describes the current understanding of the early mechanisms of dissemination, as well as how surgery may facilitate disease progression.     

Splenectomy reduces lung metastases and tumoral and metastatic niche inflammation

The immune microenvironment plays a crucial role in supporting tumor growth and metastasis. Tumor-associated macrophages (TAMs) and neutrophils (TANs) are essential components of this microenvironment and affect tumor growth and progression in almost all solid neoplasms. Furthermore, TAMs, TANs and tumor-infiltrating dendritic cells (TIDCs) are found to infiltrate specific distant organs to prepare them as a site for metastatic cell seeding, forming the pre-metastatic niche. The spleen was identified as a major reservoir and source of circulating and tumor infiltrating immune cells. However, discrepancies about its role in supporting tumor growth exist. Thus, here we investigated the role of splenectomy in primary tumor and metastatic growth, and in the formation of an inflammatory niche. In a murine 4T1 and E0771 breast and Panc02 pancreatic cancer model, our results show that while splenectomy reduces the number of infiltrating TAMs, TANs and TIDCs within primary tumors, it does not affect its growth. In line, fewer TAMs, TANs and TIDCs accumulate in the metastatic microenvironment after splenectomy. Interestingly though, this affected metastatic growth depending on the metastatic route/site. The number of hematogenous breast cancer lung metastases was reduced after splenectomy but no effect was observed in breast or pancreatic lymph node metastases. Moreover, we observed that the immune composition of the pre-metastatic niche in lungs of breast cancer bearing mice was altered, and that this could cause the reduction of metastases. Altogether, our results highlight that splenectomy affects the immune microenvironment not only of primary tumors but also of pre-metastatic and metastatic sites.     

Molecular and cellular insights into the coxsackie–adenovirus receptor: role in cellular interactions in the stem cell niche

In recent years, progress has been made in characterizing the molecular and cellular elements that are responsible for the regeneration in the damaged brain and highlighting the key role of the stromal-vascular ‘environment’ to orchestrate secondary neurogenesis and repair. Indeed, the ability of the stem cells to self-renew and differentiate is tightly regulated by stromal ependymal cells and endothelial cells expressing molecular cues that constitute the extracellular stem cell ‘niche’. Several soluble growth factors such as EGF, TGFβ, FGF2, SDF-1α and Noggin are important signals for the stem cell niche but little is known about the role of membrane-bound molecules in intercellular communications between the niche and the stem cells. In this mini-review, we highlight the emerging role of a family of adhesion molecules in the control of secondary neurogenesis. The coxsackie–adenovirus receptor (CAR) is a 46 kDa transmembrane protein and a member of the immunoglobulin super family. It is close structurally and evolutionary to other adhesion molecules involved in cell–cell interactions during embryogenesis, broadly expressed in the developing central nervous system but restricted to ependymal cells in the adult brain. This unique location and its newly established signalling properties further support the role of CAR in intercellular communications. Elucidating the other signalling molecules and manipulating the stromal-vascular niche for example by adenovirus gene therapy remain important goals for future clinical applications.     

中医药对骨髓造血生态位调控与构建的研究进展

骨髓造血生态位是造血干、祖细胞赖以生存的场所,不仅关系到机体造血机能的正常运转,而且其稳态失调及异常可诱发多种血液疾病,甚至可被诸如白血病干细胞等恶性细胞病态适配,以致预后严重不良,因此对骨髓生态位的调控与构建具有重要意义。概述了骨髓造血生态位的构成与生理病理基础,从中药活性组分与中药复方的调控展开,对复方药物组成和药效靶点进行图形分析,结合血液病临床,将分散研究加以整合与总结,以期为中医药对血液病的研究和治疗提供新的方向与视角。