人脐静脉内皮细胞的培养观察与鉴定

目的探讨新生儿脐静脉内皮细胞的培养、观察及鉴定方法,建立人血管内皮细胞的培养模型,为体外研究动脉硬化发病机制提供重要的实验手段。方法采用终浓度为0．125%胰蛋白酶+0．01% EDTA或0．1%Ⅱ型胶原酶消化、分离脐静脉内皮细胞进行培养,以0．25%胰蛋白酶进行消化传代,并用光镜、电镜和免疫组化等方法进行内皮细胞的形态观察和鉴定。结果原代培养的内皮细胞在接种后约1h开始贴壁生长,5～7d融合成单层。光镜下内皮细胞呈铺路石状镶嵌排列；免疫组化可见内皮细胞胞浆中人第Ⅷ因子相关抗原呈阳性反应；电镜下可见胞浆巾有W-P小体,证实培养的细胞为内皮细胞。结论用酶灌注消化法消化脐静脉可获得高纯度的内皮细胞,细胞存活率高。本方法为血管内皮细胞的研究提供了实验模型。     

大鼠主动脉内皮细胞原代培养的研究

目的建立简单有效且重复性高的大鼠主动脉血管内皮细胞体外原代培养技术。方法取大鼠主动脉,将主动脉内膜暴露于外,采用不同浓度的Ⅰ型胶原酶以及不同消化时间对组织块进行预消化,然后将大鼠主动脉切成小块按内膜朝下贴在鼠尾胶原包被的培养瓶上进行培养。结果2.0g·L-1型胶原酶消化1h的组织块细胞迁出速度且组织迁出率明显提高(P<0.05),组织贴壁后24h即可见内皮细胞从组织块周围游离出来,呈短梭形或类三角形,d4～5即可进行传代培养,传代后d3～4即可融合成单层,呈典型“铺路石”状排列,经免疫组化S-P法鉴定有第Ⅷ因子相关抗原存在,进一步确认其为内皮细胞。结论经2.0g·L-1Ⅰ型胶原酶消化1h的组织块,组织块迁出率及内皮细胞迁出速度明显提高,从而大大缩短原代培养的时间。     

体外诱导大鼠骨髓间充质干细胞为血管内皮细胞的研究

目的研究骨髓间充质干细胞（mesenchymal stem cells，MSCs）定向分化为血管内皮细胞，为组织工程血管化及细胞移植修复损伤组织提供理想的细胞来源。方法贴壁法分离SD大鼠MSCs进行体外培养和连续传代，采用第四代MSCs，以血管内皮生长因子（vascular endothelial growth factor，VEGF）和碱性成纤维细胞生长因子（basic fibroblast growth facotr，bFGF）进行体外诱导，免疫荧光染色方法鉴定内皮细胞，并通过透射电镜观察内皮细胞W-P小体。结果免疫荧光染色证实实验组细胞阳性表达，透射电镜观察到内皮细胞特有的WP小体。结论MSCs可以在体外定向分化为具有血管内皮细胞特性的细胞。     

白花丹参对过氧化氢致人脐静脉血管内皮细胞损伤的保护作用

目的研究白花丹参对过氧化氢（H2O2）所致人脐静脉血管内皮细胞（HUVEC）损失的保护作用。方法应用酶消化灌注法培养人脐静脉血管内皮细胞,采用形态学观察和Ⅷ因子抗体免疫荧光检测法进行鉴定;取对数生长期的细胞分组进行干预,应用形态学观察法和MTT法检测各组血管内皮细胞的活性。结果H2O2损伤模型组细胞损伤明显,经白花丹参高、中、低各剂量组的干预,受损情况均有较明显地改善,使细胞活性（OD值）增高。结论白花丹参对H2O2所致的HUVEC损伤具有保护作用。     

人脐静脉血管内皮细胞的高效分离与培养

目的:建立人脐静脉内皮细胞(HUVEC)体外分离、培养、鉴定的高效方法.方法:胰酶消化法从脐静脉获得HUVEC,观察其形态并传代培养,检测其摄取Dil标记的乙酰化低密度脂蛋白(Dil-AcLDL)的情况,传3代流式榆测内皮细胞特异性表达.结果:消化分离的细胞呈小圆形且聚集成团;2h细胞开始贴壁,呈条索状;7d细胞增多呈漩涡样生长;此后细胞继续增多接近融合,呈铺路石样改变.流式检测细胞高表达血管内皮细胞特异性标志CD144、vWF、CD31,部分表达CD34,而白细胞共同抗原CD45为阴性表达.镜下发现贴壁细胞原代、第5代摄取Dil-AcLDL,胞浆呈现红色荧光,提示为有活性的内皮细胞.结论:利用胰酶消化法能从人脐静脉获得大量内皮细胞,体外培养后能成功增殖传代.     

酶消化法培养老龄SD大鼠血管平滑肌细胞

目的：采用酶消化法培养老龄SD大鼠血管平滑肌细胞,为血管疾病,特别是为动脉粥样硬化研究提供大量的原代平滑肌细胞。方法：无菌取老龄SD大鼠主动脉,0.2%Ⅱ型胶原酶消化分离细胞,采用自然纯化、差速贴壁纯化平滑肌细胞,免疫组化鉴定平滑肌细胞α肌动蛋白。结果：免疫组化染色显示细胞纯度在95%以上。结论：酶消化法分离获取SD大鼠平滑肌细胞方法简单,易掌握,采用本方法可稳定获得大量的平滑肌细胞供实验使用。     

一种高纯度和高活力的大鼠脑微血管内皮细胞的提取及原代培养方法

目的建立一种纯度和活力较高、简单实用的大鼠脑微血管内皮细胞分离及原代培养方法,为建立体外血脑屏障提供材料。方法采集1-2周SD大鼠大脑皮质,应用Ⅱ型胶原酶和分散酶/胶原酶连续消化法,筛网过滤法及20%BSA和44%Percoll两次梯度离心法获得脑微血管段后,接种于培养瓶中进行原代培养,采用倒置显微镜对所培养的细胞进行形态学观察,以Ⅷ因子相关抗原免疫染色法对其进行鉴定。结果体外培养2 h后,微血管内皮细胞爬出血管段进行贴壁生长,3⁴ d呈典型的铺路卵石样结构,Ⅷ因子相关抗原免疫组化检测内皮细胞表达呈阳性,可见细胞胞质呈棕色,阳性细胞占99%以上。结论该方法能成功地分离并培养出高纯度的大鼠脑微血管内皮细胞,对体外血脑屏障的建立以及脑微血管内皮细胞的生物学特性和功能的深入研究具有重要意义。     

家兔主动脉平滑肌细胞原代培养的研究

目的探索高效快速的家兔血管平滑肌细胞体外原代培养技术,为分子生物学实验提供足量可靠的细胞来源。方法取家兔主动脉中层,用刀片切成约1mm3,采用不同浓度的Ⅰ型胶原酶以及不同消化时间对组织块进行预消化,然后将组织块贴在培养瓶上进行培养。结果1.5g.L-1胶原酶消化6h后组织块细胞迁出速度及组织迁出率明显提高(P<0.05),组织块贴壁后24h即可见血管平滑肌细胞从组织块周围游离出来,呈梭形或星形,d4～5即可进行传代培养,传代后d3～4即可融合成致密单层细胞,呈典型的峰谷交错状,经免疫组化SP法鉴定有α-actin的表达,进一步确认其为血管平滑肌细胞。结论经1.5g.L-1胶原酶消化6h后组织块细胞迁出速度且迁出率明显提高,从而大大缩短原代培养的时间。     

Leptin对HUVECs的氧化应激和增殖作用

目的观察Leptin对脐静脉内皮细胞（HuvEcs）的氧化应激作用及增殖的影响及其有关机制的探讨。方法体外胶原酶法消化分离培养HUVECs进行免疫荧光鉴定;观察Leptin对HUVECs的活性氧簇（ROS）水平的变化的影响;运用MTT比色法测定leptin及抑制剂对HU—VECs增殖的影响,并分别测定增殖率及抑制率。结果胶原酶消化法分离培养的内皮细胞经纯度高;Leptin能显著增加HUVECs内活性氧簇的产量。（与对照组比较,P〈0．01）,上述变化可以被NADPH氧化酶抑制剂-二苯基碘（DPI）和PKC抑制剂（PKCinhibitor）所抑制;Leptin能显著促进HUVECs增殖,与对照组比较,增殖率达36％,运用DPI和PKC抑制剂可以明显抑制leptin对HUVECs的增殖作用．DPI的抑制率达37％,PKC抑制剂的抑制率达43％,（与对照组比较,P均〈0．01）。结论体外消化分离培养的脐静脉内皮细胞纯度较高可以成功运用于实验;Leptin能提高HUVECs内ROS的产量,即leptin可以促进内皮细胞的氧化应激反应;Leptin可以通过PKC途径提高ROS水平促进HUVECs增殖。     

大鼠肾小管上皮细胞分离及其体外原代培养

目的探讨大鼠肾小管上皮细胞的原代培养方法。方法在原有肾小管细胞培养方法的基础上进行改良,采用机械研磨过筛、Ⅰ型胶原酶消化法分离出肾小管节段,在DMEM/F12[含5%胎牛血清(FBS)和胰岛素-转铁蛋白-硒]培养基中培养,并进行ZO-1免疫荧光染色鉴定。结果细胞培养至第3天完全贴壁,为多边鹅卵石样;免疫荧光染色显示90%以上的细胞为ZO-1表达阳性。结论采用机械研磨过筛、Ⅰ型胶原酶消化、DMEM/F12(含5%FBS和胰岛素-转铁蛋白-硒)培养基中培养的方法可以获得纯度达90%以上的肾小管上皮细胞,可满足对肾小管上皮细胞的研究。     

人骨髓来源内皮前体细胞体外培养和鉴定的研究

目的:明确人骨髓单个核细胞中是否存在内皮前体细胞(EPCs),探讨从人骨髓中分离、培养EPCs的方法及体外进行EPCs的鉴定。方法:采集正常人骨髓单个核细胞进行体外培养,应用流式细胞术进行内皮细胞表面标记物检测,应用免疫荧光术对培养细胞进行内皮细胞功能特性检测。结果:人骨髓单个核细胞经体外培养,收获细胞可以表达内皮细胞的特异性抗原,包括CD34、VWF、KDR等,并显示出能摄取乙酰化低密度脂蛋白(ac-LDL),结合荆豆凝集素(UEA-1)等内皮细胞的特性。结论:人骨髓中存在具有内皮细胞潜能的EPCs,其具有内皮细胞的表型特征和功能,有希望作为种子细胞用于冠心病的血管新生治疗。     

Immunological changes in the intestines and skin after senna administration

Abstract

Context: It has been reported that chronic sennoside use is associated with the development of melanosis coli, colonic adenoma, and/or carcinomas.

Objectives: In this study, we investigated the immunological changes in the colon and skin after the administration of senna.

Materials and methods: In this study, we investigated the colon and epidermis of C57/BL6j mice after a single administration of 10?mg/kg of senna [Cassia angustifolia (Caesalpiniaceae); 3, 6, 12, and 24?h after administration] and after repeated once per week administrations (on days 3, 5, 7, 14, and 21 of administration). The LD₅₀ and ED₅₀ of senna used in this experiment were 165?mg/kg and 13?g/kg, respectively.

Results: We demonstrated that the DOPA-positive cells in the colon increased at 12?h after single administration and were further increased from at 5–28?d after repeated administration. We also studied the physiological changes of the small intestine using the charcoal meal test. We found that there was a tendency for peristalsis to be inhibited after repeated senna administration. In the epidermis, we investigated the number of Langerhans cells, because they are important immune cells of the skin. The number of these cells decreased, especially after repeated administration.

Discussion and conclusion: The present findings suggested that it is necessary to pay attention to not only the intestine but also the skin, during long-term senna treatment.     

Stem cell-derived hepatocytes and their use in toxicology

Better prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future.     

子宫内膜腺上皮细胞和基质细胞的分离培养

目的　建立一种获得高产量子宫内膜腺上皮细胞和基质细胞的分离培养方法。方法　通过胶原酶消化法、系列过滤法及贴壁纯化等技术分离纯化培养人子宫内膜腺上皮细胞和基质细胞 ,并通过光镜及免疫细胞化学染色进行鉴定。结果　腺上皮细胞呈蝌蚪形 ,漩涡状生长。角蛋白免疫细胞化学染色阳性 ,纯度约 90 %。基质细胞呈梭形或多角形 ,平行状生长。波形蛋白染色反应阳性 ,纯度达 95 %。每例子宫肌瘤切除标本可获得 (10～2 5 )× 10 6 原代基质细胞和 (4～ 6 )× 10 6 原代腺上皮细胞 ,每例诊断性刮宫获得的原代细胞数约为前者的 1/ 2～2 / 3。结论　改良培养程序可获得高产量的纯化的子宫内膜腺上皮细胞和基质细胞。     

Effects of subtoxic concentrations of TiO(2) and ZnO nanoparticles on human lymphocytes, dendritic cells and exosome production

Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, but the knowledge of their possible interactions with the immune system is very limited. Our aims were to investigate if commercially available TiO₂ and ZnO nanoparticles may affect different human immune cells and their production of exosomes, nano-sized vesicles that have a role in cell to cell communication. We found that the TiO₂ or ZnO nanoparticles at concentrations from 1 to 100 μg/mL did not affect the viability of primary human peripheral blood mononuclear cells (PBMC). In contrast, monocyte-derived dendritic cells (MDDC) reacted with a dose dependent increase in cell death and caspase activity to ZnO but not to TiO₂ nanoparticles. Non-toxic exposure, 10 μg/mL, to TiO₂ and ZnO nanoparticles did not significantly alter the phenotype of MDDC. Interestingly, ZnO but not TiO₂ nanoparticles induced a down regulation of FcγRIII (CD16) expression on NK-cells in the PBMC population, suggesting that subtoxic concentrations of ZnO nanoparticles might have an effect on FcγR-mediated immune responses. The phenotype and size of exosomes produced by PBMC or MDDC exposed to the nanoparticles were similar to that of exosomes harvested from control cultures. TiO₂ or ZnO nanoparticles could not be detected within or associated to exosomes as analyzed with TEM. We conclude that TiO₂ and ZnO nanoparticles differently affect immune cells and that evaluations of nanoparticles should be performed even at subtoxic concentrations on different primary human immune cells when investigating potential effects on immune functions.     

肿瘤干细胞miRNA及miRNA作为肿瘤标志物的研究和临床应用进展

肿瘤干细胞(CSCs)理论为肿瘤的研究开辟了一个新的方向,CSCs学说认为肿瘤细胞具有异质性,肿瘤中存在干细胞样细胞,该群细胞是一种增殖失控、可形成肿瘤的细胞,只占肿瘤细胞很少部分,具有干细胞特性,是形成不同分化程度肿瘤细胞和肿瘤增长、复发及转移的根源。微小核糖核酸(miRNA)是广泛存在的非编码小RNA,调节着人类1/3的基因,越来越多的证据显示miRNA在肿瘤的发生发展中起着重要的作用,作为重要的转录后调控因子,广泛参与肿瘤相关基因调控的生物程序,使不同类型的肿瘤表现出特异的miRNA表达谱。近年来,CSCs的miRNA研究日益成为热点,已经发现多种CSCs中存在特异性表达的miRNA,对CSCs的生物学行为有了更进一步的认识。有研究发现肿瘤患者血浆中表达某些特异的miRNA,这些miRNA可以作为肿瘤的标志物对患者的病情及预后进行预测和判断。本文就近来CSCs中miRNA研究进展及miRNA作为肿瘤标志物研究进展进行综述。     

胎盘间充质干细胞向神经细胞诱导的实验研究

目的间充质干细胞(MSCs)具有自我更新和多向分化能力的特点，尽管其主要来源骨髓间充质干细胞(BMSCs)已被认为是治疗多种疾病的良好细胞来源。但在严重感染或随着年龄的增长骨髓细胞数及增殖、分化能力均明显下降等情况下，就无法顺利获得足量可用于治疗的BMSC，有必要寻找MSC的新来源。方法消化胎盘组织、贴壁培养获得基质细胞；用流式细胞仪检测其细胞表面标志；比较叔丁对甲氧酚(butylated hydroxyanisole，BHA)、复方丹参注射液、β-巯基乙醇(BME)等对培养的基质细胞向神经细胞分化的作用；采用免疫细胞化学的方法对分化的细胞进行鉴定。结果胎盘组织中分离出的基质细胞与骨髓间充质干细胞有相似形态和细胞表面标志，并可诱导表达神经元和星型胶质细胞标志神经元特异性烯醇化酶(neuron specific enolasen，NSE)、神经微丝(neurofilament，NF)和胶质纤维酸性蛋白(glial fibrilament acidic protein，GFAP)。结论胎盘组织中存在能分化为神经元样细胞的间充质干细胞。胎盘间充质干细胞可能是一种可为临床治疗提供丰富新来源的间充质干细胞。     

Differential cytotoxic effects of mono-(2-ethylhexyl) phthalate on blastomere-derived embryonic stem cells and differentiating neurons

Potential applications of embryonic stem (ES) cells are not limited to regenerative medicine but can also include in vitro screening of various toxicants. In this study, we established mouse ES cell lines from isolated blastomeres of two-cell stage embryos and examined their potential use as an in vitro system for the study of developmental toxicity. Two ES cell lines were established from 69 blastomere-derived blastocysts (2.9%). The blastomere-derived ES (bm-ES) cells were treated with mono-(2-ethylhexyl) phthalate (MEHP) in an undifferentiated state or after directed differentiation into early neural cell types. We observed significantly decreased cell viability when undifferentiated bm-ES cells were exposed to a high dose of MEHP (1000 μM). The cytotoxic effects of MEHP were accompanied by increased DNA fragmentation, nuclear condensation, and activation of Caspase-3, which are biochemical and morphological features of apoptosis. Compared to undifferentiated bm-ES cells, considerably lower doses of MEHP (50 and 100 μM) were sufficient to induce cell death in early neurons differentiated from bm-ES cells. At the lower doses, the number of neural cells positive for the active form of Caspase-3 was greater than that for undifferentiated bm-ES cells. Thus, our data indicate that differentiating neurons are more sensitive to MEHP than undifferentiated ES cells, and that undifferentiated ES cells may have more efficient defense systems against cytotoxic stresses. These findings might contribute to the development of a new predictive screening method for assessment of hazards for developmental toxicity.     

对肿瘤干细胞的认识和可调控的意义

恶性肿瘤严重地威胁着人类的健康，是危害人类生命的重要原因之一。尽管有关研究报告很多，遗憾的是其机制尚不清楚，至今尚无确切有效的抗癌、控癌措施。局部复发和远处转移一直是导致最终治疗失败的主要问题。WHO提出的新目标和理念——肿瘤属于可调控的慢性疾病。新的研究发现在肿瘤组织中有一类具有自我更新及分化潜能的细胞亚群，而且是肿瘤复发及转移的根源。这部分细胞被称马肿瘤干细胞。国内外相继从血液系统肿瘤及多种实体瘤中分离、鉴定了肿瘤干细胞。随着肿瘤干细胞学说的提出及确认，人们对肿瘤复发、转移机制和对策有了新的认识和期望，从而为恶性肿瘤治疗後的复发转移的预防提供了一个全新的途径。现综述如下。     

Chronic Opium Treatment Can Differentially Induce Brain and Liver Cells Apoptosis in Diabetic and Non-diabetic Male and Female Rats

It has been shown that some opium derivatives promote cell death via apoptosis. This study was designed to examine the influence of opium addiction on brain and liver cells apoptosis in male and female diabetic and non-diabetic Wistar rats. This experimental study was performed on normal, opium-addicted, diabetic and diabetic opium-addicted male and female rats. Apoptosis was evaluated by TUNEL and DNA fragmentation assays. Results of this study showed that apoptosis in opium-addicted and diabetic opium-addicted brain and liver cells were significantly higher than the both normal and diabetic rats. In addition, we found that apoptosis in brain cells of opium-addicted and diabetic opium-addicted male rats were significantly higher than opium-addicted and diabetic opium-addicted female, whereas apoptosis in liver cells of opium-addicted and diabetic opium-addicted female rats were significantly higher than opium-addicted and diabetic opium-addicted male. Overall, these results indicate that opium probably plays an important role in brain and liver cells apoptosis, therefore, leading neurotoxicity and hepatotoxicity. These findings also in away possibly means that male brain cells are more susceptible than female and interestingly liver of females are more sensitive than males in induction of apoptosis by opium.