One Step Quick Detection of Cancer Cell Surface Marker by Integrated NiFe-based Magnetic Biosensing Cell Cultural Chip

RGD peptides has been used to detect cell surface integrin and direct clinical effective therapeutic drug selection. Herein we report that a quick one step detection of cell surface marker that was realized by a specially designed NiF e-based magnetic biosensing cell chip combined with functionalized magnetic nanoparticles. Magnetic nanoparticles with 20-30 nm in diameter were prepared by coprecipitation and modified with RGD-4C, and the resultant RGD-functionalized magnetic nanoparticles were used for targeting cancer cells cultured on the NiF e-based magnetic biosensing chip and distinguish the amount of cell surface receptor-integrin.Cell lines such as Calu3, Hela, A549, CaF br, HEK293 and HUVEC exhibiting different integrin expression were chosen as test samples. Calu3, Hela, HEK293 and HUVEC cells were successfully identified. This approach has advantages in the qualitative screening test. Compared with traditional method, it is fast, sensitive, low cost,easy-operative, and needs very little human intervention. The novel method has great potential in applications such as fast clinical cell surface marker detection, and diagnosis of early cancer, and can be easily extended to other biomedical applications based on molecular recognition.

Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

血管化类器官的构建策略

背景:有效促进类器官内部血管发生是目前类器官培养中的焦点问题,作为一种新近发展的生物培养技术,血管化的类器官在研究活体组织的发育、疾病形成的机制、组织替代疗法以及药物筛选等方面,都有很大的研究和应用价值。目的:对近年来类器官血管化的方法或策略进行归纳总结,分析类器官血管化形成机制以及构建策略,以期为更加深入地研究类器官的发生机制和为临床转化提供可靠的思路。方法:检索PubMed及中国知网数据库收录的相关文献。英文检索词为“organoids,Vascularization,Vascular,Vascular development,vessel”,中文检索词为“血管发生,血管生成,类器官,干细胞,血管化,预血管化”,最终纳入77篇文献进行归纳总结。结果与结论:①类器官血管化形成机制涉及3个关键因素,即种子细胞、细胞因子与细胞外基质。种子细胞为血管化类器官提供了关键的细胞来源,细胞因子为类器官内部的血管发生起了重要的信号引导作用,细胞外基质为血管细胞提供了外在的生长环境,促进类器官血管化的发生。②血管化类器官的构建策略包括细胞自我重组、微血管碎片渗入、宿主体内移植以及微流控芯片等。体外诱导多能干细胞向血管内皮祖细胞分化能顺利与邻近组织整合并具有血管生成的潜力,故可利用多能干细胞的自我重组功能构建血管化类器官。微血管碎片保留了其细胞复杂性、天然结构和表型可塑性,更利于模拟天然微血管从而促进类器官的血管化。宿主体内移植是目前类器官实现完整血液灌流的最佳方法,而微流控芯片则为实现类器官体外血液供应提供了解决方案。③类器官的多种构建策略如多类干细胞共分化、信号分子的精准调控、微血管渗入和活体宿主移植等,一定程度上在类器官中引入血管成分,使得类器官在功能和成熟度上更接近相应组织。然而缺乏血流灌注仍然是一个难题,迄今为止,仅宿主体内移植才能在类器官中实现有效的血流灌注,因此类器官在血管化方面仍面临许多挑战。

Vascular Channel Formation by Osteosarcoma Cells in Vitro: Vasculogenic Mimicry

Objective: To observe whether there is evidence for vascular channel formation by osteosarcoma cellsin vitro and to illustrate mechanism of vasculogenic mimicry in osteosarcoma.Methods: Osteosarcoma cell lines (U-2OS) were tested for their ability to form tubular networks in three-dimensional culture containing type I collagen. The structures of the tubular networks were observed under a phase contrast microscope and an electron microscope.Results: Observation under light microscopy and electron microscopy showed that high aggressive osteosarcoma cells line (U-2OS) formed networks containing channels when grown in three-dimensional culture containing type I collagen in the absence of endothelial cells or fibroblasts.Conclusion: These observations strongly suggest that aggressive osteosarcoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis and have the ability of vasculogenic mimicry. Key words osteosarcoma cells line - vasculogenesis mimicry - angiogenesis - 3-dimensional cultures This study was supported in part by the National Natural Sciences Foundation of China (No. 30271314).

A Study of Rabbit Lens Epithelial Cells Survival and Growth on the Rabbit Capsular Bag in Vitro

Objective: To study the proliferation, migration and metaplasm of residual rabbit lens epithelial cells (LECs) after extracapsular cataract extraction(ECCE)based on the rabbit capsular bag model in vitro. Methods: Sham cataract surgery, including anterior capsulorhexis, nucleus hydroexpression and aspiration of lens fibers, was performed on 20 rabbit lens. The capsular bags were isolated and pinned to sterile non-toxic silicone rings on petri dishes. The capsular bags were incubated with Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and monitored for 3 weeks by phase-contrast microscopy, after which light microscopy was performed on them.Results: After a latent period of 2-3 d, outgrowth was observed across the posterior capsule. Growth proceeded rapidly so that the posterior capsule was totally covered by a confluent monolayer of cell at 6-8 day. Capsular wrinkles became increasingly apparent as time progressed, causing a marked rise in light scatter. An increase in capsular tension also came.Conclusion: This model exhibits many of the in vito characteristics of the lens capsule after extracapsular surgery and may prove useful in further elucidating the cellular mechanisms of posterior capsule opacification and developing strategies for inhibiting cell growth with this system.

干细胞的趋电性迁移及机制

背景:随着对干细胞技术研究的深入,如何使其准确归巢成为临床应用中的一大难题。除药物和趋化因子等信号的诱导外,电场也被广泛应用于指导干细胞的定向迁移,并可增强其迁移速度和定向性。目的:旨在分析总结电场对干细胞迁移特性的影响,同时综述可能的作用机制。方法:通过检索Pub Med和中国知网数据库,收集截至2024年3月的相关文献,英文检索词:“stem cells,direct current electric field,pulsed electric field,migration,electric field device,mechanism”;中文检索词:“干细胞,直流电场,脉冲电场,迁移,电场装置,机制”。排除不能获取全文和与主题无关的文献。结果与结论:根据筛选要求共纳入58篇文献,包括中文文献15篇及英文文献43篇。文献以脂肪间充质干细胞、骨髓间充质干细胞、神经干细胞、表皮干细胞、人胚胎干细胞和人诱导性多功能干细胞为研究对象,在迁移装置中研究电场的不同参数对上述干细胞迁移的影响及其机制。(1)电场作为一种简单、无创且稳定的干预方式在指导干细胞定向迁移的过程中起着积极作用;(2)不同类型的干细胞发生趋电性迁移的方向不同,同时大部分干细胞的迁移速度和定向性是随着电场强度增加而增加的;(3)不同的电场装置在观测干细胞迁移时的侧重点不同,可根据实验目的而择优选择相关装置;(4)不同干细胞趋电性迁移的机制不完全相同,多数干细胞迁移过程中有MAPK通路、ROCK活化以及PI3K功能的参与,同时还有其他蛋白质复合物与信号通路参与调控该过程;(5)除电场参数不同之外,细胞本身的衰老情况和培养环境也会对趋电性迁移的结果产生影响。总而言之,电场作为一种影响干细胞迁移特性的重要信号,与其他新兴材料结合在组织工程应用中展现出了一定的潜力,有望在指导干细胞归巢方面发挥更加重要的作用,促进骨组织再生和修复以及神经系统、自身免疫系统以及肿瘤等疾病的研究取得更大的突破。

SD大鼠乳鼠原代皮质神经元和小胶质细胞同时提取并培养的实验方法

背景:原代皮质神经元和小胶质细胞在探索神经系统疾病细胞疗法中起着至关重要的作用,而目前获得2种细胞的方法大多较繁琐,需分别单独提取。因此找到一种便捷、快速可同时提取2种细胞的方法十分关键。目的:探索一种新型同时提取原代皮质神经元及小胶质细胞的方法。方法:取24 h内新生SD大鼠乳鼠,将大脑取出放入装有DMEM的培养皿中,去除软脑膜后备用。同一脑组织,先提取原代神经元,再将剩余脑组织用于提取小胶质细胞,整个过程在冰上操作。原代皮质神经元提取培养步骤:镊子夹取2.0-3.0 mm厚度大脑皮质组织置于培养皿,用木瓜蛋白酶消化20 min,中止消化后吹打组织呈互相粘连的组织悬液,吸上清细胞悬液,过滤、分装到15 mL离心管中,离心并重悬后将细胞接种于左旋多聚赖氨酸包被的6孔板爬片上放入细胞培养箱,每隔1 d观察神经元形态,第7天进行MAP2和β-Tubulin免疫荧光染色鉴定。小胶质细胞提取培养步骤:夹取上一步取皮质后剩余脑组织,厚度8-10 mm,置于培养皿,用胰酶消化20 min,中止消化后吹打组织呈匀浆,然后将匀浆转移到培养瓶中培养,第14天将培养瓶封口后进行恒温水平摇床振荡2 h,小胶质细胞脱落于上清中;取纯化后的小胶质细胞继续培养3 d进行Iba1免疫荧光染色鉴定。结果与结论:(1)神经元培养24 h后贴壁、胞体变大,部分神经元长出突触;培养到第3,5天时胞体进一步增大,大部分神经元已呈突触形态,部分神经元成团生长;第7天时,神经元突触延长增粗并互相连接成网。经β-Tubulin、MAP2免疫荧光染色鉴定为神经元。(2)小胶质细胞培养第1天换液后可见细胞贴壁生长;第3,5,7天时细胞密度均较小,细胞形态呈高亮椭圆或圆形,但已基本成团块状生长于其他细胞上层;第10天时小胶质细胞密度明显增大;第14天时小胶质细胞呈致密团块状生长于其他细胞上层,此时可进行分离纯化。取分离纯化后细胞再培养至第3天,经Iba1免疫荧光鉴定为小胶质细胞,且纯度大于95%。(3)结果表明,经此法提取并培养获得的原代皮质神经元和小胶质细胞纯度高、形态好、成活率高。

明胶三维微球装载人脐带间充质干细胞修复慢性肌腱病

背景:肌腱组织因缺少血管而修复困难,如何促进肌腱的恢复和提高肌腱损伤后干细胞治疗的效果,一直是临床及科研的研究热点和重点。目的:将人脐带间充质干细胞与明胶微载体支架结合构建组织工程化干细胞,通过体外实验与大鼠体内实验观察明胶微载体培养的人脐带间充质干细胞对肌腱病的治疗效果及作用机制。方法:(1)体外细胞实验:将人脐带间充质干细胞接种于三维明胶微载体后观察细胞活性以及存活情况,以常规培养的人脐带间充质干细胞作为对照;(2)动物体内实验:将成年SD大鼠随机分为正常组、肌腱病组、2D组(肌腱病+常规培养人脐带间充质干细胞)、3D组(肌腱病+明胶微载体三维培养的人脐带间充质干细胞),每组6只,治疗4周后进行动物行为学检测以及跟腱病理组织形态观察。结果与结论:(1)体外细胞实验:接种于明胶微载体的人脐带间充质干细胞存活率高,且随着时间延长细胞增殖速率增加;与对照组相比,三维明胶微载体培养的细胞活性更好;(2)动物体内实验:治疗4周后,与肌腱病组比较,3D组大鼠下肢功能恢复良好及组织病理学评分显著改善,而2D组也可一定程度改善肌腱病损伤,但效果不及3D组;(3)结果表明,三维明胶微载体培养的人脐带间充质干细胞可以促进肌腱损伤组织修复再生,且修复效果优于常规培养人脐带间充质干细胞。

Morphological Characteristics of Smooth Muscle Cells Isolated from the Rat Ductus Deferens

The aim of this study was to establish a method of isolating and culturing smooth muscle cells from the ductus deferens of rats. Smooth muscle cells were prepared from ductus deferens by explanting technique after dissection of adventitia and intimae, and cultured in vitro. The identification of the smooth muscle cells were verified by using anti u-smooth muscle actin （a-SMA） immunohistochemistry studies. The result suggested that the cells are multi-morphous, showing long fusiform or star shapes. The apophysis of cells contacted and coalesced to each other, in some regions the cells overlapped in multilayer, while in the other regions they formed monolayer that fluctuated and showed a ＂peak-valley＂ shape. They presented a positive reaction through immunohistochemistry studies. The purity of the cells was more than 99% through this method. The culturing of smooth muscle cells by explanting technique is simple and stable.

Modulation of the Nogo signaling pathway to overcome amyloid-β-mediated neurite inhibition in human pluripotent stem cell-derived neurites

Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer's disease.The accumulation of amyloid-βpeptides,a key hallmark of Alzheimer's disease,is believed to induce neuritic abnormalities,including reduced growth,extension,and abnormal growth cone morphology,all of which contribute to decreased connectivity.However,the precise cellular and molecular mechanisms governing this response remain unknown.In this study,we used an innovative approach to demonstrate the effect of amyloid-βon neurite dynamics in both two-dimensional and three-dimensional cultu re systems,in order to provide more physiologically relevant culture geometry.We utilized various methodologies,including the addition of exogenous amyloid-βpeptides to the culture medium,growth substrate coating,and the utilization of human-induced pluripotent stem cell technology,to investigate the effect of endogenous amyloid-βsecretion on neurite outgrowth,thus paving the way for potential future applications in personalized medicine.Additionally,we also explore the involvement of the Nogo signaling cascade in amyloid-β-induced neurite inhibition.We demonstrate that inhibition of downstream ROCK and RhoA components of the Nogo signaling pathway,achieved through modulation with Y-27632(a ROCK inhibitor)and Ibuprofen(a Rho A inhibitor),respectively,can restore and even enhance neuronal connectivity in the presence of amyloid-β.In summary,this study not only presents a novel culture approach that offers insights into the biological process of neurite growth and inhibition,but also proposes a specific mechanism for reduced neural connectivity in the presence of amyloid-βpeptides,along with potential intervention points to restore neurite growth.Thereby,we aim to establish a culture system that has the potential to serve as an assay for measuring preclinical,predictive outcomes of drugs and their ability to promote neurite outgrowth,both generally and in a patient-specific manner.

调整细胞培养条件提高间充质干细胞外泌体治疗骨关节炎的潜能

背景:间充质干细胞外泌体有望发展成为治疗骨关节炎的无细胞疗法,而通过调整细胞培养条件,可以进一步提高其治疗活性和临床转化潜能。目的:综述分析细胞培养条件对外泌体活性的影响,分析其临床转化潜力。方法:查阅国内外有关骨关节炎/软骨损伤修复与间充质干细胞来源外泌体的文献。检索词分别为“外泌体,间充质干细胞,骨关节炎,软骨修复”和“exosomes,extracellular vesicles,mesenchymal stem cells,osteoarthritis,cartilage repair”,检索数据库分别为PubMed和中国知网数据库,检索时限为2010-2023年,最后纳入100篇文献进行总结和分析。结果与结论:(1)在间充质干细胞的培养过程中,提供有利于软骨细胞生长的物理环境,或添加软骨保护小分子、成软骨诱导因子和炎症因子等刺激细胞,可以进一步提高间充质干细胞外泌体在维持软骨细胞表型、促进软骨再生和免疫抑制等方面的调控活性;因此,其他具有相似特征的物理或化学因素,也有可能提升间充质干细胞外泌体治疗骨关节炎的疗效。(2)低氧诱导、脉冲电磁场刺激、生物反应器及软骨保护分子(如人甲状旁腺素1-34)刺激等细胞培养方案具有安全、可规模化生产等优点,可用于制备临床用途的、疗效好的间充质干细胞外泌体。(3)间充质干细胞外泌体有望实现骨关节炎的修正治疗,通过调整细胞培养方案提升治疗活性,可进一步提高其临床转化的可行性。

Postnatal development of rat retina:a continuous observation and comparison between the organotypic retinal explant model and in vivo development

The organotypic retinal explant culture has been established for more than a decade and offers a range of unique advantages compared with in vivo experiments and cell cultures.However,the lack of systematic and continuous comparison between in vivo retinal development and the organotypic retinal explant culture makes this model controversial in postnatal retinal development studies.Thus,we aimed to verify the feasibility of using this model for postnatal retinal development studies by comparing it with the in vivo retina.In this study,we showed that postnatal retinal explants undergo normal development,and exhibit a consistent structure and timeline with retinas in vivo.Initially,we used SOX2 and PAX6 immunostaining to identify retinal progenitor cells.We then examined cell proliferation and migration by immunostaining with Ki-67 and doublecortin,respectively.Ki-67-and doublecortin-positive cells decreased in both in vivo and explants during postnatal retinogenesis,and exhibited a high degree of similarity in abundance and distribution between groups.Additionally,we used Ceh-10 homeodomain-containing homolog,glutamate-ammonia ligase(glutamine synthetase),neuronal nuclei,and ionized calcium-binding adapter molecule 1 immunostaining to examine the emergence of bipolar cells,Müller glia,mature neurons,and microglia,respectively.The timing and spatial patterns of the emergence of these cell types were remarkably consistent between in vivo and explant retinas.Our study showed that the organotypic retinal explant culture model had a high degree of consistency with the progression of in vivo early postnatal retina development.The findings confirm the accuracy and credibility of this model and support its use for long-term,systematic,and continuous observation.

黑枸杞花青素联合人脂肪源性血管外膜细胞支持脐血造血干/祖细胞的增殖

背景:黑枸杞花青素(Anthocyanins in Lycium ruthenicum Murr,ALRM)是黑枸杞中重要的活性成分之一,具有抗氧化、免疫调节等功效。人脂肪源性血管外膜细胞(CD146^(+)hAD-PCs)是骨髓间充质干细胞的前体细胞,在体外具有促进造血干/祖细胞增殖与分化的功能。ALRM联合CD146^(+)hAD-PCs对脐血造血干/组细胞的体外支持作用有待于研究。目的:探讨ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞体外扩增的支持作用。方法:CCK-8法检测不同质量浓度ALRM(0,200,400,600,800,1000 mg/L)对CD146^(+)hAD-PCs增殖的影响;流式细胞术检测ALRM对CD146^(+)hAD-PCs细胞周期的影响。共培养实验分为空白组、ALRM组、CD146^(+)hAD-PCs组、ALRM+CD146^(+)hAD-PCs组,分析ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞的体外支持作用。共培养1,2,4周,比较扩增后细胞数量、集落形成单位数量,流式细胞仪检测细胞免疫表型,ELISA检测细胞因子水平。结果与结论:(1)ALRM质量浓度为200 mg/L时,CD146^(+)hAD-PCs活力最高,CD146^(+)hAD-PCs的G_(0)/G_(1)期细胞比例下降,S期、G_(2)/M期细胞比例上升(P<0.01)。(2)脐血CD34^(+)造血干/祖细胞数量变化:在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组高于ALRM组(P均<0.05),在共培养2,4周时ALRM+CD146^(+)hAD-PCs组高于CD146^(+)hAD-PCs组(P均<0.05),ALRM组与空白组随着共培养时间延长细胞数量逐渐减少。(3)集落形成能力及免疫表型分析:在共培养1,2周时ALRM+CD146^(+)hAD-PCs组的集落形成单位数量高于CD146^(+)hAD-PCs组和ALRM组(P均<0.05);在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组CD45^(+)、CD34^(+)CD33^(-)细胞比例高于CD146^(+)hAD-PCs组(P均<0.01)。(4)细胞因子变化:在共培养4周时ALRM+CD146^(+)hAD-PCs组的白细胞介素2水平高于ALRM组、CD146^(+)hAD-PCs组(P<0.05);在共培养2,4周时ALRM+CD146^(+)hAD-PCs组白细胞介素3水平高于CD146^(+)hAD-PCs组(P<0.05);在共培养1周时ALRM+CD146^(+)h AD-PCs组的粒细胞集落刺激因子水平高于CD146^(+)hAD-PCs组,在共培养2周时高于ALRM组、CD146^(+)hAD-PCs组(P<0.01);在共培养1,2,4周时ALRM组、ALRM+CD146^(+)hAD-PCs组的干扰素γ水平低于CD146^(+)hAD-PCs组(P<0.05)。(5)由于空白组无基质细胞,脐血CD34^(+)造血干/祖细胞在共培养1周之后就无法计数,未进行免疫表型、集落分析和细胞因子检测。(6)结果表明:ALRM可以通过促进CD146^(+)hAD-PCs增殖和细胞周期转化进而促进脐血CD34^(+)造血干/祖细胞的体外扩增,在造血干细胞移植研究方面具有重要价值。

淫羊藿苷含药血清促进3种细胞共培养体系中软骨细胞增殖和干细胞成软骨分化

背景:关节软骨损伤修复能力十分有限,组织工程技术为修复受损软骨提供了新的治疗方案,其中软骨细胞、骨髓间充质干细胞和滑膜间充质干细胞之间的相互影响和诱导作用是自体软骨损伤愈合的基础。目的:构建软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞共培养体系用以模拟软骨细胞体内微环境,并探究其最佳细胞接种比例,同时观察淫羊藿苷含药血清对该体系中软骨细胞增殖和干细胞成软骨分化的影响。方法:提取、培养、鉴定大鼠膝关节软骨细胞、骨髓间充质干细胞和滑膜间充质干细胞,按不同细胞接种比例构建软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞非接触共培养体系,共培养72 h后观察软骨细胞增殖活性和表型能力,选择综合效应最佳的共培养体系;用淫羊藿苷溶液(0.25 mg/m L)灌胃新西兰大白兔制备淫羊藿苷含药血清,对照组共培养体系用含体积分数为10%胎牛血清、1%双抗的高糖DMEM培养液培养,实验组共培养体系在此基础之上加入体积分数为10%淫羊藿苷含药血清进行干预,24,48 h后检测两组软骨细胞增殖活性和Ⅱ型胶原表达,14 d后采用免疫荧光染色检测骨髓间充质干细胞、滑膜间充质干细胞成软骨分化情况。结果与结论:(1)3种细胞以不同比例共培养时均可正常贴壁生长,当软骨细胞、骨髓间充质干细胞、滑膜间充质干细胞接种比例为2∶1∶1时,共培养体系中软骨细胞表现出最佳增殖活性和表型能力;(2)与对照组相比,实验组培养24 h后软骨细胞增殖活性和Ⅱ型胶原表达显著升高(P<0.01),48 h后两组仍有差异(P<0.05),培养14 d后两组骨髓间充质干细胞和滑膜间充质干细胞出现明显的软骨分化,部分细胞呈现圆形或椭圆形,胞浆Ⅱ型胶原免疫荧光染色为阳性,实验组荧光强度明显高于对照组(P<0.01);(3)结果表明,以非接触共培养方法可以成功建立软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞共培养体系且细胞比例为2∶1∶1时软骨细胞增殖活性和表型能力最佳;同时淫羊藿苷含药血清具有促进该体系中软骨细胞增殖及骨髓间充质干细胞、滑膜间充质干细胞成软骨分化的作用。

旋转细胞培养系统对老年人表皮干细胞增殖和干性维持的影响

背景:由于人口老龄化和老年疾病的增加,对老年人表皮干细胞的研究和利用变得越来越重要。但由于老年人表皮干细胞的增殖和干性能力较弱,限制了其在生物医学和临床研究的应用。目的:探讨旋转细胞培养系统培养的老年人表皮干细胞的增殖和干性维持能力。方法:采用传统平面培养方法获取儿童(7-12岁)和老年人(60岁以上)表皮干细胞,经细胞形态学和细胞免疫荧光鉴定后,通过流式细胞术、CCK-8、细胞克隆形成实验分析儿童和老年人表皮干细胞增殖能力的差异;采用旋转细胞培养系统和3D TableTrix®微载体培养儿童和老年人表皮干细胞,通过活/死染色、细胞倍增效率、细胞倍增时间以及实时定量聚合酶链反应检测细胞增殖情况以及干性标志物的表达。结果与结论:①在平面培养条件下,儿童组表皮干细胞较老年组具有更强的增殖能力;②与平面培养相比,经旋转细胞培养系统动态培养的老年人表皮干细胞的细胞倍增效率更高(P<0.01),细胞倍增时间更短(P<0.01);③旋转细胞培养系统动态培养促使老年人表皮干细胞自组装形成基于微载体支架的三维细胞球,具有和儿童组表皮干细胞相似的增殖情况;④动态培养后的老年人表皮干细胞在干性标记物基因(ITGA6和ITGB1)、基底细胞标记物基因(K5)和分化标记物基因(K10和K1)表达上可达到和儿童组相近的表达水平。结果表明:旋转细胞培养系统动态培养不仅可以提高老年人表皮干细胞的培养效率,促进细胞增殖和自组装成三维细胞球,而且可维持老年人表皮干细胞一定的干性,未发生完全终末分化。

三种大鼠椎间盘细胞提取、鉴定及在单层和微团培养中的基质表达

背景:目前获得椎间盘原代细胞的方法大多较繁琐,且缺少同时提取纤维环、终板与髓核原代细胞的相关报道,因此找到同时提取3种细胞的方法十分关键。目的:探索一种同时提取并培养大鼠来源3种椎间盘细胞(纤维环、终板与髓核)的方法,对之进行鉴定并探究单层与微团培养对细胞外基质的影响。方法:取材自3周龄雄性SD大鼠椎间盘组织,从其中分离出软骨终板、髓核与纤维环组织。针对髓核及纤维环组织,先使用0.1%链酶蛋白酶E于37℃消化30 min,然后使用0.2%Ⅱ型胶原酶消化4 h来释放其中的细胞;针对终板组织,直接使用0.2%Ⅱ型胶原酶消化4 h来释放其中的细胞。将去除消化酶后的细胞接种于含有培养基的培养皿中,并观察细胞形态;通过实时定量荧光PCR与蛋白印迹实验检测各细胞标记物的表达水平;单层或微团培养大鼠原代纤维环细胞与软骨终板细胞,并采用阿尔新蓝与番红染色检测细胞外基质的表达能力。结果与结论:①3种椎间盘细胞均在培养4 d后开始贴附在皿底,并逐渐展现出增殖的活力;培养至第8天时,3种细胞增殖显著,形态均呈梭形,其中髓核细胞中存在多囊泡的脊索样细胞;②通过实时定量荧光PCR和/或蛋白免疫印迹实验发现原代髓核细胞中K19与Car3表达较高,原代纤维环细胞中Sparc与Bgn表达较高,原代软骨终板细胞中Pth1r与Lars2表达较高;③原代纤维环、软骨终板细胞微团培养后,经阿尔新蓝与番红染色证明这种培养方式相较于单层培养能够提高细胞外基质的表达能力;④结果表明,通过此次实验方法提取并培养获得的原代椎间盘细胞形态较好并具有较高的胞外基质表达水平,这一成果有望为科研人员提供一种新的科研工具,帮助科研人员更好地理解椎间盘生物学。

细胞周期蛋白依赖性激酶7抑制剂THZ1对脑胶质瘤干细胞干性的调控及机制

背景:THZ1是一种细胞周期蛋白依赖性激酶7的抑制剂,可以抑制多种肿瘤细胞的增殖,但THZ1是否可以通过Wnt/β-catenin信号通路影响脑胶质瘤干细胞的干性尚不清楚。目的:探究THZ1对脑胶质瘤细胞U87干性的影响及机制。方法:培养脑胶质瘤细胞U87形成干细胞球并通过Western blot验证干性相关蛋白的表达;采用CCK-8法检测THZ1作用于U87细胞的半数抑制浓度(IC_(50));通过克隆实验、划痕实验、Transwell迁移实验确定THZ1对U87细胞增殖、迁移的影响;分析THZ1处理对U87干细胞成球率及干细胞球体大小的影响;Western blot检测干性相关蛋白CD133、ABCG2、Nanog、OCT4、SOX2和上皮-间充质转化相关蛋白E-cadherin、N-cadherin、Occludin、Snail以及Wnt/β-catenin通路相关蛋白Axin1、β-Catenin、Wnt-5a、GSK3β、Cyclind-1、C-myc的表达变化。结果与结论:①与贴壁细胞相比,U87干细胞干性相关蛋白Nestin、CD133、ABCG2、Nanog、OCT4、SOX2表达明显升高;②与对照组相比,THZ1减弱了U87细胞的增殖和迁移能力;③THZ1抑制U87干细胞成球率及球体大小,下调干性相关蛋白的表达;④THZ1处理后,U87干细胞中N-cadherin、Snail蛋白表达降低,而E-cadherin、Occludin蛋白表达升高;(5)THZ1处理使U87干细胞中Wnt/β-catenin通路相关蛋白Axin1、β-Catenin、Wnt-5a、GSK3β、Cyclind-1、C-myc表达降低。结果表明:THZ1通过下调Wnt/β-catenin信号通路相关分子表达,抑制脑胶质瘤细胞U87的增殖、迁移,抑制U87干细胞成球能力、干性相关蛋白表达和上皮-间充质转化能力。

Effects of V2O5 on Proliferation and Differentiation of Limb Bud Cells of Rat

The micromass culture was used to determine the effects of vanadium pentoxide (V2O5 ) on the proliferation and differentiation of limb bud cells of rat. In the in vitro test, the results showed that V2O5 had obvious inhibiting effects on both proliferation and differentiation of limb bud cells with a dosedependent response, its proliferating and differentiating IC50 being 13.64 and 4.77μmol/L, respectively. In the in vivo/in vitro test, the results showed that V2O5 had no obvious effect on cell proliferation but had obvious inhibiting effect on cell differentiation. These results indicated that V2O5 might have a specific inhibiting effect on the differentiation of limb bud cells.

基质细胞衍生因子1修饰左旋聚乳酸多孔微球促进软骨细胞增殖和组织形成

背景:二维培养条件下的软骨细胞增殖及表型维持受限,多孔微球作为支架材料可提供三维培养环境,以更好地模拟体内生长条件。基质细胞衍生因子1是有强趋化效力的稳态细胞因子,能够促进细胞的黏附与增殖。目的:明确接枝基质细胞衍生因子1左旋聚乳酸多孔微球对软骨细胞生物学特性及软骨组织形成的影响。方法:(1)体外验证不同质量浓度基质细胞衍生因子1对兔软骨细胞增殖、迁移、表型维持的影响。(2)采用复乳法制备左旋聚乳酸多孔微球,利用碳二亚胺法将基质细胞衍生因子1接枝于左旋聚乳酸多孔微球上,通过酶联免疫吸附实验及孵育基质细胞衍生因子1特异荧光抗体验证接枝情况。(3)将兔软骨细胞分别接种于左旋聚乳酸多孔微球、接枝基质细胞衍生因子1左旋聚乳酸多孔微球上,检测细胞增殖与黏附。(4)在裸鼠背部皮下分别植入甲基丙烯酰胺基明胶-软骨细胞复合体(对照组)、左旋聚乳酸多孔微球-甲基丙烯酰胺基明胶-软骨细胞复合体(多孔微球组)、接枝基质细胞衍生因子1左旋聚乳酸多孔微球-甲基丙烯酰胺基明胶-软骨细胞复合体(多孔微球修饰组),8周后取材,分别进行组织学染色与成软骨相关基因qRT-PCR检测。结果与结论:(1)相较于0,1 000 ng/mL基质细胞衍生因子1,500 ng/mL基质细胞衍生因子1可促进软骨细胞的增殖与迁移,提升软骨细胞内Ⅱ型胶原、弹性蛋白、增殖细胞核抗原、Bcl-2 mRNA表达;(2)基质细胞衍生因子1成功接枝于左旋聚乳酸多孔微球上,接枝率为93.75%;(3)相较于左旋聚乳酸多孔微球,接枝基质细胞衍生因子1左旋聚乳酸多孔微球可促进软骨细胞的增殖、黏附;(4)裸鼠皮下植入8周后,相较于对照组、多孔微球组,多孔微球修饰组具有更明显的软骨陷窝结构、更丰富的软骨特异性基质和Ⅱ型胶原沉积,弹性蛋白、Ⅱ型胶原、增殖细胞核抗原、Bcl-2 mRNA表达升高。结果表明:接枝基质细胞衍生因子1左旋聚乳酸多孔微球有利于软骨细胞的黏附、增殖、表型维持以及体内软骨组织形成。

成年小鼠触液神经元体外分离培养及自我更新能力的鉴定

背景:课题组前期成功在体外分离培养乳鼠触液神经元,尚无研究报道有效分离培养高纯度成年小鼠触液神经元的方法,且触液神经元的自我更新能力是否随着年龄发生变化尚无研究。目的:建立一种高纯度成年小鼠触液神经元体外分离培养的方法,并鉴定成年小鼠触液神经元与乳鼠触液神经元在体外的自我更新能力。方法:从成年3月龄小鼠颈髓分离含有触液神经元的原代细胞贴壁培养并利用融合多模态成像基因的慢病毒转染细胞,通过嘌呤霉素筛选得到高纯度成年小鼠触液神经元细胞,在完全培养基中悬浮培养。通过免疫荧光检测成年小鼠触液神经元表达神经干细胞标记物巢蛋白(Nestin)及SOX2情况,观察成年小鼠触液神经元体外成球与传代能力;将同等数量(5×10^(3))的第3代成年小鼠及乳鼠触液神经元在同等条件下分为2组,分别接种在含有完全培养基的超低黏附培养板中,在体积分数5%CO_(2),37℃恒温箱悬浮培养,通过体外成球、CCK8实验、qPCR和Western blot鉴定成年小鼠及乳鼠触液神经元的自我更新能力。结果与结论:①实验成功在成年小鼠体内分离出高纯度触液神经元,在体外表达Nestin及SOX2,能形成神经球并连续传代。②成年小鼠触液神经元体外自我更新能力较乳鼠相比明显减弱,细胞培养到第4天时乳鼠触液神经元已经形成直径约为150μm的神经球,而成年小鼠触液神经元所形成的神经球直径仅为40μm(P<0.0001)。③CCK8增殖实验结果表明,成年小鼠触液神经元的增殖活性在培养后各时间点显著弱于乳鼠(P<0.0001)。④qPCR和Western blot检测发现成年小鼠触液神经元Nestin及SOX2的mRNA(P<0.0001)和蛋白表达量(P<0.01)较乳鼠显著下降。⑤上述结果证实,成年小鼠触液神经元的体外自我更新能力显著弱于乳鼠。