脐带间充质干细胞对角膜内皮细胞增殖能力的影响

目的:探讨并优化人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)体外获取及培养增殖的方法并鉴定;观察兔角膜内皮细胞(cornea endothelial cells,CEC)与hUCMSCs共培养后增殖能力及细胞周期的变化.方法:用酶消化法体外分离培养hUCMSCs,流式细胞仪检测hUCMSCs免疫表型:将CEC分别与hUCMSCs、兔角膜基质细胞在Transwell体系中共培养,通过流式细胞术观察内皮细胞增殖能力及细胞周期的变化.结果:采用酶消化法能有效分离纯化hUCMSCs.接种24 h,贴壁细胞形态多为长梭形、多边形或成纤维细胞样形态,大小均一.流式细胞仪分析第3代细胞均表达CD29、CD44、CD105,不表达造血干细胞标记CD34、CD45和HLA-DR.与空白对照组CEC周期比较脐带间充质干细胞组与基质细胞组角膜内皮细胞S期细胞比例增加.G1期细胞比例下降,干细胞组增加幅度高于基质细胞组.干细胞组角膜内皮细胞S期细胞比例比空白对照组的平均增加近17%,增殖能力显著提高.结论:经鉴定用酶消化法体外能成功分离培养出hUCMSCs,将其与CEC共培养能促进CEC增殖.     

脂肪干细胞诱导神经干细胞分化的体外实验研究

目的研究脂肪干细胞（ADSCs）体外诱导神经干细胞（NSCs）分化的作用。方法从新生BALB／c小鼠中分别分离获得ADSCs及NSCs,进行体外培养和传代。构建ADSCs与NSCs共培养体系,以单纯NSCs组为对照,进行ADSCs诱导分化研究。共培养4、8、12d后行神经元特异性神经丝200免疫组化鉴定,统计NSCs分化为神经元的百分率。结果共培养后8—12d,可见大量成熟神经元,大多为多极神经元,少部分为双极神经元或假单极神经元,带有较长的轴突。共培养组NSCs分化为神经元的百分率大约为21％,而单纯NSCs培养组约为4％。共培养组神经元的转化率与单纯NSCs培养组神经元的转化率之间有显著性差异（P〈0．05）。结论ADSCs在体外能够促进NSCs向神经元转化。     

杜仲叶β-谷甾醇对成骨细胞和卵巢颗粒细胞的影响

目的研究杜仲叶β-谷甾醇(β-sitosterol)对骨代谢平衡的调控作用及其机制。方法采用溶剂法、柱层析与重结晶等技术从杜仲叶提取部位Ⅰ中分离得到白色针状晶体B,根据理化性质和光谱分析鉴定化学结构为β-谷甾醇;建立体外大鼠成骨细胞、卵巢颗粒细胞单独培养与混合培养模型,研究杜仲叶β-谷甾醇对骨代谢平衡的调节作用。结果杜仲叶β-谷甾醇可明显促进卵巢颗粒细胞分泌雌二醇E2,有效提高成骨细胞护骨素与破骨细胞分化因子的比值(OPG/ODF),是调节骨代谢平衡的机制之一。结论杜仲叶β-谷甾醇通过提高成骨细胞OPG/ODF比值和刺激卵巢颗粒细胞分化E2,从而促进和加强成骨作用。     

原代培养人肺癌相关成纤维细胞对肺癌细胞放射敏感性影响研究

目的 探讨在体外直接接触共培养条件下人肺癌相关成纤维细胞(CAF)对肺癌细胞系放射敏感性的影响。方法 采用人肺癌组织进行原代培养后获取人肺CAF,采用免疫荧光技术鉴定。CAF与肺癌细胞系A₅₄₉、H₁₂₉₉进行直接接触共培养,采用细胞克隆形成实验观察其对肺癌细胞系放射敏感性的影响。结果 新鲜人肺腺癌经组织块贴壁法原代培养出可稳定传代的CAF,其特异表达α-平滑肌肌动蛋白、波行蛋白和成纤维细胞活化蛋白,而无细胞角质蛋白-18表达。在0 Gy照射时单独组和共培养组A₅₄₉细胞的克隆形成率分别为(20.0±3.9)%和(32.3±5.5)%(P<0.05),H₁₂₉₉细胞的分别为(20.6±3.1)%和(35.2±2.3)%(P<0.05)。单独组和共培养组A₅₄₉细胞的SF₂分别为0.727±0.061和0.782±0.089(P>0.05),H₁₂₉₉细胞分别为0.692±0.065和0.782±0.037(P>0.05);人肺CAF对A₅₄₉、H₁₂₉₉细胞的放射保护增益比分别为1.29和1.25。结论 人肺CAF与肺癌细胞系直接接触共培养后使肺癌细胞放射敏感性降低,原因可能为CAF促进了肿瘤细胞增殖。     

Co-culture of mouse spermatogonial stem cells with sertoli cell as a feeder layer,stimulates the proliferation and spermatogonial stemness profile

Objectives

Sertoli cells effect the fate map of spermatogonial stem cells (SSCs) to self-renew via providing the special microenvironments. Maintenance of proliferation and self-renewal activity of SSCs may be usable as a therapeutic strategy, leads to increase the recovery of male fertility. This research was aimed to evaluate the effect of mouse sertoli cells on spermatogonia stem cells proliferation and the expression pattern of stemness markers.

Inflammation and (secondary) genotoxicity of Ni and NiO nanoparticles

Abstract

Nanoparticle-induced genotoxicity can arise through different mechanisms, and generally, primary and secondary genotoxicity can be distinguished where the secondary is driven by an inflammatory response. It is, however, yet unclear how a secondary genotoxicity can be detected using in vitro methods. The aim of this study was to investigate inflammation and genotoxicity caused by agglomerated nickel (Ni) and nickel oxide (NiO) nanoparticles and, furthermore, to explore the possibility to test secondary (inflammation-driven) genotoxicity in vitro. As a benchmark particle to compare with, we used crystalline silica (quartz). A proteome profiler antibody array was used to screen for changes in release of 105 different cytokines and the results showed an increased secretion of various cytokines including vascular endothelial growth factor (VEGF) following exposure of macrophages (differentiated THP-1 cells). Both Ni and NiO caused DNA damage (comet assay) following exposure of human bronchial epithelial cells (HBEC) and interestingly conditioned media (CM) from exposed macrophages also resulted in DNA damage (2- and 3-fold increase for Ni and NiO, respectively). Similar results were also found when using a co-culture system of macrophages and epithelial cells. In conclusion, this study shows that it is possible to detect a secondary genotoxicity in lung epithelial cells by using in vitro methods based on conditioned media or co-cultures. Further investigation is needed in order to find out what factors that are causing this secondary genotoxicity and whether such effects are caused by numerous nanoparticles.     

In vitro evaluation of (S)-ibuprofen toxicity on joint cells and explants of cartilage and synovial membrane

Intra-articular drug delivery systems (DDSs) are envisaged as interesting alternative to locally release nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen to reduce pain in patients with osteoarthritis. The present study examines the toxicity of (S)-ibuprofen on chondrocytes and synoviocytes isolated from sheep shoulder joint and cultured in monolayers during 72 h, and on joint explants (cartilage and capsule) cultured in mono- or in co-culture for 13 days. (S)-ibuprofen (5 μM up to 1 mM) did not reduce the cell viability and protein content when added on chondrocyte monolayers, while at 1 mM (S)-ibuprofen reduced (by 8%, p = 0.01) the synoviocytes viability compared to untreated cells. During co-culture of joint explants, (S)-ibuprofen at 50 μM significantly reduced by 35% the spontaneous release of glycosaminoglycans (GAGs) from cartilage (p = 0.0065) whereas in monoculture, (S)-ibuprofen was inactive on GAG metabolism. (S)-ibuprofen at 1 mM significantly reduced cell lysis (lactate dehydrogenase leakage) by 74% during monoculture of capsule explants (p = 0.0136) and by 35% during co-culture of explants (p = 0.0013). Our findings demonstrate that the active isomer of ibuprofen at micro- and millimolar levels was not toxic for chondrocytes and synoviocytes and may reduce at 1 mM the cell lysis during culture of joint explants. The limited toxicity of (S)-ibuprofen at low and high concentration in sheep joint shoulder makes this enantiomer a promising drug candidate for the loading of intra-articular DDS.     

骨髓间充质干细胞对大鼠胶质瘤细胞增殖的影响

目的研究骨髓间充质干细胞（BMSC）对大鼠脑胶质瘤C6细胞增殖的影响,并且探讨其相关机制。方法提取SD大鼠BMSC,进行体外培养、扩增。应用MTt比色法检测不同浓度BMSC上清液对C6细胞系增殖的抑制作用。用Transwell小室将BMSC与C6细胞进行双层培养,以HE染色法检测C6细胞形态变化。用划痕实验检测细胞迁移情况。结果MTF结果显示BMSC上清液对C6细胞有抑制作用,120h后1：8、1：4、1：2的BMSC上清稀释液、BMSC上清原液培养组的生长抑率分别为17．1％、26．0％、39．9％、43．1％;与BMSC进行双层培养后的C6细胞其形态发生了明显改变,由圆形或多角形变为长梭形,细胞包体伸出长突起;划痕实验显示在36h时对照组迁移率为100％,划痕完全愈合,而BMSC上清组划痕未见全愈合,迁移率为82％。结论①BMSC上清液能抑制胶质瘤C6细胞的恶性增殖,而且抑制效应呈剂量依赖性;②BMSC对C6的运动和迁移产生了抑制作用,从而降低了其恶性侵袭程度。     

兔髓核细胞诱导大鼠骨髓间充质干细胞分化为髓核细胞的研究

目的 研究体外新西兰大白兔髓核细胞(nucleus pulposus cells)与SD大鼠骨髓间充质干细胞(mes-enchymal stem cells,MSCs)共培养时,兔髓核细胞与大鼠MSCs直接和间接接触对MSCs分化为髓核细胞的影响.方法 DAPI (4‘,6-二咪基-2‘-苯吲哚盐酸)标记原代髓核细胞后,分别与第三代MSCs按接触组和非接触组(Transwell培养系统)共培养.每隔24小时应用免疫荧光观察MSCs的形态学变化,并用RT-PCR方法检测Ⅱ型胶原和可凝集蛋白多糖(Aggrecan)的表达.结果 直接接触培养组中可见分化的MSCs形态和功能接近髓核细胞;非直接接触组的MSCs未见变化.结论 髓核细胞与MSCs的直接接触,是诱导MSCs分化为髓核细胞的重要因素.