小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

人早孕胎盘绒毛膜滋养层细胞体外培养模型的建立

目的通过对早孕胎盘绒毛膜滋养层细胞的分离,纯化和培养,寻找一种稳定、简便可获得较高纯度滋养层细胞的培养方法。方法通过胰酶/DNA酶联合消化法对妊娠6-10周绒毛组织进行消化,获得单细胞悬液,比较Per-coll密度梯度离心和淋巴细胞分离液对滋养层细胞的分离纯化效果。含10?S的DMEM/F12培养基培养,并比较是否应用鼠尾胶原对细胞贴壁和生长的影响。通过免疫荧光方法对滋养层细胞进行鉴定。结果经简化Percoll密度梯度离心分离纯化的滋养层细胞纯度高,明显优于淋巴细胞分离液的分离效果(P<0.001);细胞生长表面预先经鼠尾胶原处理后,细胞贴壁良好,分裂生长旺盛。结论利用简化Percoll密度梯度离心法分离细胞,并在应用鼠尾胶原的条件下进行培养,可以获得满意的人绒毛膜滋养层细胞的体外培养模型。     

优思悦对多囊卵巢综合征模型大鼠卵巢功能的影响及其机制研究

摘要 目的：探讨优思悦对硫酸脱氢表雄酮（dehydroepiandrosterone,DHEA）诱导的多囊卵巢综合征模型大鼠体内性激素：睾酮( testosterone,T)、雌二醇( estradiol,E2 )、促黄体生成素( luteinizing hormone,LH)及卵泡刺激素( follicle－stimulating hormone,FSH)及PI3K /AKT信号通路的的影响。方法：将60只雌性SD大鼠随机均分为3组,包括空白组、模型组及治疗组。空白组每日颈部皮下注射0.2 mL大豆油,其余各组每日颈部皮下注射DHEA 60 mg/kg+0.2 mL大豆油,持续注射35 d。造模第21 d时,每日上午制备大鼠阴道涂片,将其放置在显微镜下观察,根据细胞形态判定大鼠的动情周期,无动情周期规律视为造模成功。造模第36 d时,连续4 w对大鼠进行灌胃,每天一次。检测大鼠阴道分泌物的细胞形态、大鼠卵巢中卵泡发育的情况。放射免疫法测定大鼠血清中T、E2、LH、FSH的含量;RT-qPCR法检测大鼠卵巢组织中PI3K /AKT信号通路相关因子（IＲS-1、AKT-2、CSK-3β、GLUT-4 mRNA和PTEN mRNA）的表达变化。结果：空白组大鼠的动情周期有规律性,而模型组大鼠的动情周期失去规律性,且模型组大鼠卵巢中的卵泡呈囊状扩张,血清中T、LH水平明显升高（P<0.05）,同时卵巢组织中的IRS-1、AKT-2、CSK-3β、GLUT-4 mRNA表达减少（P<0.05）,PTEN mRNA表达增多（P<0.05）。与模型组比较,优思悦可改善PCOS模型大鼠动情周期,降低血清T、LH水平（P<0.05）,改善卵泡发育,减少囊状扩张卵泡的形成,上调IRS-1、AKT-2、CSK-3β、GLUT-4 mRNA表达量,下调PTEN mRNA 表达量。结论：优思悦可有效治疗DHEA诱导的PCOS模型大鼠,与卵巢中的PI3K /AKT信号通路相关因子的基因表达相关。     

正常月经周期妇女卵泡液中激素水平的变化

本文测定了24例正常月经妇女在不同时相、不同大小卵泡的卵泡液中雌二醇(E_2)、孕酮(P_0)、雄烯二酮(A)、睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)和催乳素(PRL)的含量,并分析其与外周血中相应激素浓度的关系。测定结果显示:小卵泡的卵泡液中E_2、Po,FSH,LH水平低于大卵泡中水平,而A和T水平则相反。排卵前大卵泡中E_2(9815nmol/L),P_0(3316nmol/L),FSH(1.34IU/L)和LH(3.9lIU/L)达最高值。A(280nmol/L)和T(137nmol/L)却较小卵泡中水平低(相应为692nmol/L和176nmol/L)。PRL水平在大小卵泡中无显著性差异。卵泡液中甾体激素水平高于外周血7—20.000倍,FSH、LH水平为外周血的10—80％,PRL水平为60％—3倍。     

一种高效、快捷、经济的小鼠胰岛细胞分离纯化方法

采用改良的胶原酶P胆总管内逆行灌注膨胀胰腺的分离方法,比较Ficoll-400不连续密度梯度离心法和人工挑取法两种纯化胰岛的方法,用双硫腙(DTZ)对胰岛细胞团进行特异性染色计算胰岛产量及纯度,用台盼蓝染色判定胰岛细胞活性,使用间接免疫荧光法检测体外培养7 d后胰岛的功能.采用人工挑取法平均每只小鼠可获取的胰岛细胞团(245±35.6个)明显高于密度梯度离心法(120±26.3个)(n=5,P<0.05),两种方法分离纯化的胰岛活力均大于98%;但人工挑取法获得胰岛细胞团的纯度(100%)要高于密度梯度离心法(90%);纯化胰岛所用时间,采用人工挑取法(22±4 min)也少于密度梯度离心法(31±3 min).间接免疫荧光染色检测体外培养7 d后的胰岛细胞团,两种方法分离纯化的胰岛细胞团均具有良好的功能.胶原酶P胆总管内灌注消化分离法联合人工挑取纯化胰岛细胞团的方法,是一种高效、快捷、经济的小鼠胰岛细胞团分离纯化方法.     

17beta-雌二醇对谷氨酸诱导的SH-SY5Y细胞增殖和凋亡的影响

目的：探讨17β-雌二醇对谷氨酸诱导的SH-SY5Y细胞损伤的保护作用及可能机制。方法：选取SH-SY5Y细胞传代培养, 分为四组：(1)阴性对照组;(2)氧化损伤组：0.1 mmol·L-1谷氨酸作用24 h;(3)17β- 雌二醇低、高浓度组：加入(1.0× 10-4mmol·L-1和 1.0× 10-3 mmol·L-1)17β- 雌二醇作用24 h之后,加入谷氨酸作用24 h。采用MTT 比色法检测细胞存活率,流式细胞仪检测细胞活 性氧(ROS)水平,Hoechst-PI染色观查细胞凋亡,分光光度计检测上清液中Caspase-3 及Caspase-9 含量。结果：7β- 雌二醇能明显 抑制谷氨酸诱导的细胞活性的下降,减少谷氨酸所致SH-SY5Y 细胞内ROS 的生成,降低细胞凋亡率,减少凋亡因子的活性。结 论：17β-雌二醇对神经细胞损伤具有保护作用,这可能与其抗氧化作用有关。     

一种同时分离培养大鼠肝细胞及肝枯否细胞技术的建立

目的:建立简便、经济的同步分离培养肝细胞及kupffer细胞的方法.方法:采用肝脏原位灌洗结合离体胶原酶灌注消化的方法获得总细胞悬液,差速离心分离肝细胞及肝非实质细胞,经多次低速离心可分离肝细胞,经percoll密度梯度离心以及选择性贴壁法得到纯化的kupffer细胞.台盼蓝染色鉴定细胞活力.使用倒置相差显微镜、HE染色、PAS染色及白蛋白免疫组织化学染色对培养肝细胞的形态及功能进行检测.使用光学显微镜、荧光显微镜及CD68免疫荧光染色鉴定分离的kupffer细胞.结果:体外成功的同步分离培养了肝细胞及kupffer细胞,肝细胞产率为1.37± 0.53× 108/大鼠,kupffer得率为3.45± 0.41×106/g肝脏.细胞存活率及纯度都可达90％.肝细胞培养24h后呈典型肝细胞形态,7天后仍具有糖原合成和白蛋白合成能力.贴壁后的kupffer细胞呈典型的星型或三角形,且其标志分子CD68免疫荧光染色阳性.结论:应用改良的原位灌注方法可以很好的同时分离具有活性及功能的肝细胞和kupffer细胞.     

骨髓间充质干细胞对环磷酰胺诱导卵巢损伤的保护作用

目的:研究骨髓间充质干细胞(MBSc)对注射环磷酰胺(CTX)引起的大鼠卵巢损伤的保护作用。方法:将30只SD大鼠按随机数字表随机分为三组,即对照组,模型组及细胞移植组,分别经尾静脉接受生理盐水,CTX和CTX+MBSc移植。监测大鼠体重,动情周期的变化,用药结束后一周处死大鼠,测定血清雌二醇(E2),卵泡刺激素(FSH),黄体生成素(LH)的变化,观察各组大鼠卵巢形态学及卵泡数量变化,并利用原位细胞凋亡TUNEL法检测3组大鼠卵巢中细胞凋亡指数(AI)。结果:与对照组比较,模型组大鼠体重下降,动情周期延长,卵泡数量下降,E2下降,FSH及LH升高,组织细胞AI增加。与模型组比较,细胞移植组大鼠体重增加,动情周期缩短,卵泡数量增加,尤其是中大卵泡数增加,E2上升,LH下降,FSH接近正常。结论:MBSc移植能通过减少卵巢组织AI而在一定程度上减少环磷酰胺引起的卵巢功能损伤。     

人腔前卵泡分离及培养

采用机械吹打、胶原酶消化、镜下显微解剖及胶原酶消化加镜下显微解剖4种卵泡提取方式并相互对比.将分离得到的腔前卵泡在含FSH0.5IU/mL、1.0IU/mL和2.0IU/mL的培养液中培养,检测其E2分泌量.与机械吹打、胶原酶消化相比,胶原酶消化加镜下显微解剖法不仅提取卵泡多(P＜0.01),而且可以得到原始、初级、次级各级卵泡.但操作时间较长(P＜0.01).得到的腔前卵泡在FSH为0.5IU/mL、1.0IU/mL和2.01IU/mL的培养液中培养,所分泌的E2分别为10.86pg±4.11pg、31.55pg±9.34pg和43.82pg±18.76pg,较之对照组的4.99pg±2.09pg有显著性差异(P＜0.01),E2的分泌量与FSH浓度呈剂量依赖性关系.FSH有促进腔前卵泡分泌E2的作用.     

Percoll密度梯度离心法分离大鼠肺Ⅱ型上皮细胞

目的建立大鼠肺泡Ⅱ型上皮细胞（AT-Ⅱ）的改良体外原代培养法。方法采用循环冲洗、支气管灌洗、胰蛋白酶消化、滤网过滤并用轻比重及重比重PercolL密度梯度离心,分离AT-Ⅱ。原代培养后通过碱性磷酸酶（alkaline phosphatase,AKP）法和电镜鉴定AT-Ⅱ。结果经AKP染色和电镜鉴定所得的AT-Ⅱ纯度为（80．2±6．3）％,每只大鼠可分离出1×10^7AT-Ⅱ。结论采用Percoll密度梯度离心法能分离出较高纯度的AT-Ⅱ。     

微流控芯片技术在细胞生物学研究中的应用进展

20世纪90年代以来,微流控芯片技术得到了快速发展。由于具有小型化、集成化、高通量、低消耗、分析快速等特点,微流控芯片作为一种新型的生物学研究平台,能够提供传统方法不具备的精细和可控制的细胞研究条件,在细胞生物学研究领域中得到了广泛关注。该文主要介绍其在细胞培养、分选、裂解、计数、凋亡检测、迁移、单细胞捕获、细胞间作用等方面的研究进展。     

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell‐based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

Background: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. Methods: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. Results: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r=0.87, p<0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r=0,68, p<0,05) only in fetal bovine serum in the absence of galactose. Conclusion: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

Engineering Cardiac Cell Junctions In Vitro to Study the Intercalated Disc

Abstract

This review article discusses a recent work using engineered cardiac cells to study the function of the intercalated disc putting emphasis on mechanical and electrical coupling.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.     

Mammalian testis: a target of in vivo electroporation

Mammalian spermatogenesis consists of three biologically significant processes: stem cell self-renewal and differentiation, meiosis, and haploid cell morphogenesis. Understanding the molecular mechanisms behind these processes might provide clues to the puzzle of species preservation and evolution, and to treatments for male infertility. However, few useful in vitro systems exist to investigate these processes at present. To elucidate these mechanisms, in vivo electroporation of the testis might be a convenient option. Since DNA solution can be injected into the seminiferous tubule via the rete testis, similar to germ cell transplantation, it is easy to transfect expression vectors into various differentiated germ cells and supporting Sertoli cells with adequate electric shock. Unfortunately, it is difficult to create transgenic animals using this method because of its low efficiency. However, gain- and loss-of-function assays, promoter assays, and tagged-protein behavior assays can be conducted with this technique, as in in vitro culture systems.