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1.
目的建立一种简单高效的小鼠肺微血管内皮细胞原代培养方法。方法选取2~3周ICR小鼠,剪开胸腹腔,取距离肺边缘约1.5mm组织,剪碎成米粒状,置于含有培养液的离心管中,离心洗涤后种瓶进行原代培养。通过细胞形态学观察、细胞Ⅷ因子相关抗原免疫细胞化学染色鉴定所培养的细胞。结果接种24h后,红细胞从贴壁的肺组织块边缘向四周游离;48h后,肺微血管内皮细胞爬出,单个细胞形态为多角形或短梭形,细胞间隙较大,胞核清晰,胞浆丰富;96h后细胞融合,呈典型的单层、铺路石样镶嵌式排列。细胞Ⅷ因子免疫细胞化学染色检测,胞质呈棕红色,表达为阳性,阳性细胞率达98%以上。结论随机组织块法能够成功高效分离培养出原代小鼠肺微血管内皮细胞。 相似文献
2.
目的 探讨大鼠视网膜微血管内皮细胞的体外分离培养方法.方法 选取6~8周龄SD大鼠5只,摘取眼球,挤压球壁后,人工剥离视网膜.将大鼠视网膜剪碎,依次经过200μm、75μm不锈钢筛网;收集滤液,离心,弃上清,予0.1%II型胶原酶消化15~20min;再次将消化液通过45μm尼龙筛网,反复冲洗筛网,收集网上物;添加培养... 相似文献
3.
肺微血管内皮细胞通透性调控的信号转导机制 总被引:4,自引:0,他引:4
肺微血管内皮细胞通透性增加是急性呼吸窘迫综合征等疾病的病理基础,多种信号转导系统参与其通透性调控,如细胞内Ca2 、蛋白激酶C、环磷酸腺苷、丝裂原激活蛋白激酶、小G蛋白.这些信号转导系统的激活和调控机制各异,并且相互关联组成复杂的信号网络.肺微血管内皮细胞中信号转导的相互作用将是研究方向之一. 相似文献
4.
大鼠脑微血管内皮细胞的分离与原代培养 总被引:14,自引:0,他引:14
为了建立大鼠脑微血管内皮细胞体外培养模型,探索纯度较高的大鼠脑微血管内皮细胞分离和原代培养的方法并进行形态学观察。采用2~3周龄的SD大鼠,解剖得到大脑皮质,两次酶消化及牛血清白蛋白或葡聚糖和Percoll梯度离心获得较纯的脑微血管段后,接种于涂布基质的培养皿进行原代培养;培养的细胞采用相差显微镜形态学观察、透射电镜观察及Ⅷ因子相关抗原免疫组化检测鉴定。结果发现,培养12h即可见细胞从贴壁的脑微血管段周围长出,细胞呈短梭形,区域性单层生长,5~7天内皮细胞融合,内皮细胞纯度达90%以上;内皮细胞的贴壁和生长有赖于所涂布的基质,纤连蛋白/Ⅳ型胶原优于鼠尾胶和明胶;Ⅷ因子相关抗原免疫组化检测内皮细胞表达阳性,透射电镜观察可见相邻内皮细胞间存在紧密连接结构。提示该方法能成功进行纯度较高的大鼠脑微血管内皮细胞原代培养,可用于脑微血管内皮的生理、生化及药理学研究,亦可用于构建大鼠血脑屏障模型。 相似文献
5.
本文旨在探讨趋化因子CCL2在成年大鼠原代心肌微血管内皮细胞(cardiac microvascular endothelial cell, CMEC)血管形成中的作用。分离原代大鼠CMEC,用CD31和VIII因子免疫染色鉴定,观察不同时间点基质胶(Matrigel)上CMEC血管形成情况,用real-time RT-PCR和ELISA分别检测在血管形成过程中CCL2的表达和分泌情况。结果显示:(1)大鼠原代CMEC分离成功,该细胞具有形成血管能力,成网数和节点数在Matrigel处理后4 h显著增加;(2) CMEC血管形成过程中CCL2和CCR2的表达量显著上调,并且CCL2的分泌量明显增加;(3) CCL2阻断抗体和CCR2拮抗剂均可显著降低CMEC血管形成能力。上述结果提示,大鼠原代CMEC在血管形成过程中可分泌CCL2,并且CCL2-CCR2信号通路促进了CMEC血管形成。 相似文献
6.
运用植块法培养脑微血管内皮细胞 总被引:1,自引:0,他引:1
探讨简易可行的脑微血管内皮细胞(brain microvascular endothelial cells,BMECs培)养方法,为研究BMECs细胞在脑血管疾病中的重要作用提供技术支持。分离出生后1~7天内的SD乳鼠大脑皮质区,植块法培养BMECs细胞。用倒置显微镜观察BMECs细胞的形态以及从皮质块迁出的过程;MTT比色法检测BMECs细胞的生长曲线;采用免疫组化染色检测VIII因子相关抗原和CD34抗原,以鉴定内皮细胞。结果发现,大脑皮质块植块法培养的大鼠BMECs细胞呈单层贴壁生长,细胞形态以长梭形、多角形三角形、四边形为主,呈典型的“铺路石”样征象,经鉴定为内皮细胞,第三代纯度达95%以上。提示该方法具有经济、简便、要求条件不高,易于纯化的优点,可作为大鼠BMECs细胞体外培养的良好模型。 相似文献
7.
培养的大鼠脑微血管内皮细胞生化特性观察 总被引:3,自引:0,他引:3
用胶原酶消化、差异离心和尼龙网过滤的方法分离鼠脑微血管内皮细胞,建立其体外长期培养方法。经形态学、免疫组化,酶学鉴定,培养细胞为脑微血管内皮细胞,动态观察培养细胞酶含量变化,发现随着细胞培养时间的延长,血管紧张素转换酶Ⅰ(ACE)呈上升趋势,而γ-谷氨酰胺转化酶(γ-GT)和硷性磷酸酶(ALP)则明显下降,实验结果提示,血脑屏障的主要功能酶γ-GT和ALP可作为体外脑微血管内皮细胞的标志酶,但要维持长时间体外表达则需要某种因子的介导。本实验可为体外研究血脑屏障及相关疾病提供帮助。 相似文献
8.
大鼠肺微血管内皮细胞培养及鉴定 总被引:17,自引:0,他引:17
取大鼠周边肺组织进行肺微血管内皮细胞培养。将肺组织切成小块,用含有20%新生牛血清、肝素90μg/ml、L-谷胺酰胺4mmol、青霉素100U/ml和链霉素100μg/ml的RPMI-1640培养基培养。血细胞立即从肺组织周围游出。继而是肺微血管内皮细胞。成纤维细胞及其他细胞72h才游出。培养60h后取出肺组织块,培养瓶中只有微血管内皮细胞和血细胞。后者可通过传代除去。获得的肺微血管内皮细胞具有规律的鹅卵石样形态和对异植物血凝素结合试验及八因子相关抗原免疫荧光染色均阳性。 相似文献
9.
休克淋巴液对大鼠肺微血管内皮细胞的损伤作用 总被引:7,自引:1,他引:6
无菌条件下复制大鼠重症失血性休克模型,引流肠系膜淋巴液或收集门静脉血,同时,引流正常淋巴液、正常门静脉血。以不同处理因素与原代培养的第三代肺微血管内皮细胞(PMVEC)共同孵育,通过光镜、透射电镜、扫描电镜观察细胞形态及超微结构,MTT法检测不同终浓度的休克淋巴液及正常淋巴液对PMVEC增殖的影响;流式细胞仪检测PMVEC周期变化;同时进行细胞核DNA电泳分析。结果表明,休克淋巴液对PMVEC具有损伤作用,表现为细胞收缩、核固缩等,扫描电镜可观察到凋亡小体;随着休克淋巴液终浓度增加,PMVEC的增殖活力逐渐降低,显著低于正常淋巴液组;4%终浓度的休克淋巴液作用PMVEC 4h后,G0-G1期细胞比值增大,S G2-M期细胞比值下降,其他处理因素无明显变化,同时细胞核DNA电泳形成典型的阶梯状电泳图谱(DNA ladder)。结果提示,休克淋巴液可导致PMVEC形态学及超微结构损伤,同时抑制细胞增殖、影响细胞周期、诱导细胞凋亡。 相似文献
10.
鼠脑微血管内皮细胞的分离与长期培养 总被引:15,自引:1,他引:14
采用胶原酶消化、差速离心、尼龙网滤过技术分离和获取鼠脑微血管内皮细胞,接种后4h换液使获得的内皮细胞纯化,体外进行长期培养。细胞在体外生长176天,传至30代,细胞初期成活率为92%,纯度近90%。经形态学、超微结构和免疫组化鉴定,培养细胞为血管内皮细胞。培养至第30代的细胞仍能合成和分泌PGI2、ACE等,ⅧF:Ag阳性表达,染色体为二倍体(2n=42),基本保持着细胞的主要特征。该分离和培养方法的建立,将为研究与脑血管相关疾病提供有用工具。 相似文献
11.
Primary culture of microvascular endothelial cells from bovine retina 总被引:11,自引:0,他引:11
Phillip D. Bowman A. Lorris Betz Gary W. Goldstein 《In vitro cellular & developmental biology. Plant》1982,18(7):626-632
Summary To provide an in vitro system for studying retinal capillary function we have developed methods for isolation and culture
of microvascular endothelial cells from retina. Retinal microvessels were isolated by homogenization of the retina and collection
of the microvessels onto nylon mesh. Treatment of the isolated microvessels with collagenase and dispase followed by Percoll
gradient centrifugation yielded endothelial cells that were largely free of pericytes. A homogeneous population of endothelial
cells that were capable of at least six population doublings was obtained by plating onto a fibronectin coated substrate in
plasma derived serum. The endothelial origin of these cells was confirmed by the presence of Factor VIII antigen, angiotensin
converting enzyme activity, numerous tight junctions, and a cell surface that did not bind platelets. A second cell type,
which did not exhibit these cell markers and which is presumably the intramural pericyte, was obtained when the isolated microvessels
were plated on tissue culture grade plastic in fetal bovine serum.
Supported by Research Grants 5R01-EY03772 and 5R01-ES02380 from the U.S. Public Health Service (G. W. G.) and Established
Investigator Award 31-107 from the American Heart Association (A. L. B.). 相似文献
12.
Solodushko V Fouty B 《American journal of physiology. Lung cellular and molecular physiology》2007,292(3):L671-L677
Endothelial cells perform a number of important functions including release of vasodilators, control of the coagulation cascade, and restriction of solutes and fluid from the extravascular space. Regulation of fluid balance is of particular importance in the microcirculation of the lung where the loss of endothelial barrier function can lead to alveolar flooding and life-threatening hypoxemia. Significant heterogeneity exists between endothelial cells lining the microcirculation and cells from larger pulmonary arteries, however, and these differences may be relevant in restoring barrier function following vascular injury. Using well-defined populations of rat endothelial cells harvested from the pulmonary microcirculation [pulmonary microvascular endothelial cells (PMVEC)] and from larger pulmonary arteries [pulmonary artery endothelial cells (PAEC)], we compared their growth characteristics in low serum conditions. Withdrawal of serum inhibited proliferation and induced G0/G1 arrest in PAEC, whereas PMVEC failed to undergo G0/G1 arrest and continued to proliferate. Consistent with this observation, PMVEC had an increased cdk4 and cdk2 kinase activity with hyperphosphorylated (inactive) retinoblastoma (Rb) relative to PAEC as well as a threefold increase in cyclin D1 protein levels; overexpression of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 induced G0/G1 arrest. While serum withdrawal failed to induce G0/G1 arrest in nonconfluent PMVEC, confluence was associated with hypophosphorylated Rb and growth arrest; loss of confluence led to resumption of growth. These data suggest that nonconfluent PMVEC continue to proliferate independently of growth factors. This proliferative characteristic may be important in restoring confluence (and barrier function) in the pulmonary microcirculation following endothelial injury. 相似文献
13.
Isolation and culture of rat microvascular endothelial cells 总被引:3,自引:0,他引:3
The purpose of this study is to identify the separation techniques that result in pure cultures of rat microvascular endothelial cells (MECs). A multistep process is used to optimize the separation of the cells from rat epididymal fat pads, obtaining as pure a culture as possible within a relatively short processing time. The process initially employs the digestion, filtration, and density gradient separation steps. We further describe the use of an attachment phase that allows the differential adherence of contaminating cell types. Immunomagnetic purification is the final step in the process and is performed using anti-PECAM-1 (CD31) monoclonal antibody-labeled DynaBeads. 相似文献
14.
15.
Pulmonary endothelial cells possess a variety of enzymes on their surface. However, the topographical distribution of these enzymes is not known. In this report we describe a simple technique for the preparation of surface replicas of pulmonary endothelial cells using an unmodified critical point drying apparatus and a high vacuum freeze-etch unit. The technique should be applicable for use with all cells in monolayer culture. Endothelial cells grown on glass slides or coverslips were washed thoroughly, fixed and dehydrated. The monolayers were then critical point dried. Surface replicas were prepared in a Balzer's freeze-etch unit. The advantages of surface replicas are that they can be examined with the resolving power of the transmission electron microscope and can be used to examine whole cells. The potential for the surface replica technique may lie in mapping specific enzymes, receptors and cell surface factors. Therefore, to provide a basis for such studies, we have described the appearance of pulmonary endothelial cells in surface replicas. 相似文献
16.
W F Patton M U Yoon J S Alexander N Chung-Welch H B Hechtman D Shepro 《Journal of cellular physiology》1990,143(1):140-149
Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells. 相似文献
17.
18.
Klinger JR Warburton R Carino GP Murray J Murphy C Napier M Harrington EO 《Experimental cell research》2006,312(4):401-410
Previous studies have described a protective effect of atrial natriuretic peptide (ANP) against agonist-induced permeability in endothelial cells derived from various vascular beds. In the current study, we assessed the effects of the three natriuretic peptides on thrombin-induced barrier dysfunction in rat lung microvascular endothelial cells (LMVEC). Both ANP and brain natriuretic peptide (BNP) attenuated the effect of thrombin on increased endothelial monolayer permeability and significantly enhanced the rate of barrier restoration. C-type natriuretic peptide (CNP) had no effect on the degree of thrombin-induced monolayer permeability, but did enhance the restoration of the endothelial barrier, similar to ANP and BNP. In contrast, the non-guanylyl cyclase-linked natriuretic peptide receptor specific ligand, cyclic-atrial natriuretic factor (c-ANF), delayed the rate of barrier restoration following exposure to thrombin. All three natriuretic peptides promoted cGMP production in the endothelial cells; however, 8-bromo-cGMP alone did not significantly affect thrombin modulation of endothelial barrier function. ANP and BNP, but not CNP or c-ANF, blunted thrombin-induced RhoA GTPase activation. We conclude that ANP and BNP protect against thrombin-induced barrier dysfunction in the pulmonary microcirculation by a cGMP-independent mechanism, possibly by attenuation of RhoA activation. 相似文献
19.
Isolation and culture of pulmonary artery endothelial cells. 总被引:17,自引:0,他引:17
It has become increasingly evident that endothelial cells function as far more than a mechanical barrier between blood and parenchyma. Endothelial cells from one vesicular bed are known to differ structurally from those of another, and it has been suggested that they may differ functionally. Further to test the hypothesis that endothelial cells from one site may differ in terms of function from those of another site, it is necessary to test endothelium from various source after having obtained these cells in pure, well-characterized cultures. To facilitate such studies, we herein describe in detail means for the isolation, culture and characterization of endothelial cells from calf pulmonary artery. These cells may be of major interest in terms of specific metabolic activities as it has become evident that the lungs play a prominent role in determining the hormonal composition of systemic arterial blood. 相似文献
20.
Yabin Wang Kazim Narsinh Li Zhao Dongdong Sun Dongjuan Wang Zheng Zhang Zhongchan Sun Rongqing Zhang Haichang Wang Feng Cao 《Cell biology international》2011,35(2):135-140
Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10?9, 1×10?8, 1×10?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10?8 and 10?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway. 相似文献