运用植块法培养脑微血管内皮细胞

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。     

大鼠脑微血管内皮细胞的分离与原代培养

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。     

人妊娠子宫平滑肌细胞的原代培养Transgelin蛋白的检测

为了建立最佳的人妊娠子宫平滑肌细胞的原代培养方法和初步检测子宫平滑肌细胞中Transgelin蛋白的表达,采用组织块贴壁法和酶消化法进行人妊娠子宫平滑肌原代培养.发现组织贴块培养的细胞呈典型的梭状肌细胞样生长,经过传代纯化,通过免疫细胞化学方法检测平滑肌肌动蛋白(smooth muscle acting,SMA)进行细胞鉴定及检测Trangsgelin(smooth muscle 22 alpha,SM22-α)蛋白,得到SMA、snd2-α荧光免疫细胞化学染色为阳性.结果表明组织贴块法对妊娠子宫平滑肌细胞损伤小,可获得状态良好的纯净的子宫平滑肌细胞,子宫平滑肌细胞中大量表达sm2-α,为进一步研究sm22-α在子宫平滑肌细胞中作用打下基础.     

大鼠肺微血管周细胞的体外培养及鉴定

目的:探讨SD大鼠肺微血管周细胞(rat pulmonarymicrovessel pericytes,RPMPC)的分离、培养与鉴定方法。方法:采用机械剪切、Ⅰ型胶原酶消化法和微孔过滤结合超高速离心法分离大鼠肺微血管片段,用含15%胎牛血清(FBS)的高糖培养基(DMEM)进行培养,倒置显微镜观察原代RPMPC的形态及生长特性。免疫荧光法检测神经元-胶质抗原2(neuron-glial antigen 2,NG2),α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、结蛋白(desmin)和CD31相关抗原的表达,同时应用免疫细胞化学法检测血小板源性生长因子受体β(PDGFR-β)的表达。CCK-8测定周细胞生长曲线。通过周细胞-内皮细胞共培养成管实验检测细胞成管能力。结果:本方法培养获取的RPMPC纯度较高,并能连续传代。细胞48 h后爬出,呈长梭形、三角形等不规则形,8~10 d细胞汇合,呈栅栏或旋涡状生长,无接触性抑制,前期可见有少量内皮细胞伴随生长,单核偶见双核,核呈卵圆形,胞浆丰富。PDGFR-β、α-SMA、NG2、desmin染色阳性,CD31阴性;周细胞与内皮细胞共培养形成管腔结构。结论:通过本方法能够获得纯度较高的肺微血管周细胞,且所获细胞具有周细胞的特性及功能。     

大鼠主动脉内皮细胞的分离培养和鉴定

目的:探索大鼠主动脉原代内皮细胞体外培养方法,为体外研究提供细胞模型。方法:分离大鼠主动脉,直接贴壁于培养皿中,荧光倒置显微镜观察细胞形态,免疫组化Ⅷ因子相关抗原染色鉴定细胞。结果:约24小时组织块边缘有游离的新生细胞长出,7天即融合成片。消化传代后细胞呈短梭形或三角形,单层生长,铺路石状,Ⅷ因子表达阳性,呈指数增殖。冻存后复苏细胞活性均超过90%。结论:用贴壁法成功建立了大鼠血管内皮细胞体外培养方法,冻存细胞存活率高,为体外研究提供了稳定的模型。     

原代SD大鼠视网膜微血管内皮细胞的培养及鉴定

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;添加培养液,接种于Nunc进口培养皿中。通过细胞形态学观察、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48h后,微血管内皮细胞从血管组织碎片爬出,单个细胞形态为多角形或短梭形,细胞间隙较大,细胞核清晰,胞质丰富;96h后细胞融合,呈典型的单层、扁平、铺路石样镶嵌式排列。细胞Ⅷ因子相关抗原免疫细胞化学染色检测,免疫反应阳性细胞率达98%以上。结论连续物理过筛法结合胶原酶消化法能够成功、高效地分离出原代大鼠视网膜微血管内皮细胞,为视网膜血管疾病的体外基础研究提供有效的细胞模型。     

人脐静脉血管平滑肌促内皮细胞组织因子表达的体外实验研究

目的研究血管平滑肌细胞对血管内皮细胞组织因子表达的影响并探讨其临床意义.方法用贴块法培养人脐静脉平滑肌细胞;酶消化法培养人脐静脉内皮细胞;用培养平滑肌细胞条件培养液(SMC-CM)刺激培养的内皮细胞,一步凝固法检测内皮细胞组织因子的活性;Northern blot检测内皮细胞组织因子的mRNA表达;并用酶联免疫吸附试验检测SMC-CM中IL-1α、IL-1β、TNF-α和VEGF的含量.结果 SMC-CM使内皮细胞组织因子活性呈剂量依赖性增强,作用6h增至最高,最高增强约38倍;SMC-CM使内皮细胞组织因子mRNA表达显著增强;SMC-CM中的组织因子诱导剂不耐热,且并非IL-1α、IL-1β、TNF-α和VEGF等已知的组织因子诱导剂.结论血管平滑肌细胞能促进血管内皮细胞组织因子的表达,提示体内增生的平滑肌细胞,如动脉再狭窄新内膜中的平滑肌细胞可能诱导局部血管内皮细胞活化及表达组织因子,在局部血栓形成中起一定作用.     

组织贴块法建立人气道平滑肌细胞体外培养模型的研究

背景：人气道平滑肌已被证实参与气道重塑,气道平滑肌的重塑已成为慢性呼吸道疾病的主要病理改变之一。人气道平滑肌细胞的培养对慢性呼吸道疾病的研究有重要意义。组织贴块法培养人气道平滑肌细胞是原代培养人气道平滑肌细胞的基本方法之一。目的：采用组织贴块法建立人气道平滑肌体外培养模型。方法：采集人气道组织,用组织贴块法进行人气道平滑肌细胞的原代培养,获得的细胞经形态学和免疫细胞化学染色鉴定。结果：培养的细胞呈典型的”谷峰”状生长,胞浆内特异性的平滑肌肌动蛋白阳性表达,符合平滑肌细胞的形态学特征和生物学特性。结论：组织贴块法易操作,结果可信,并可培养出高纯度活性好的人气道平滑肌细胞,成功建立了体外人气道平滑肌细胞增殖模型,提供了研究慢性呼吸道疾病的细胞培养模型。     

组织贴块法建立人气道平滑肌细胞体外培养模型的研究

背景:人气道平滑肌已被证实参与气道重塑,气道平滑肌的重塑已成为慢性呼吸道疾病的主要病理改变之一。人气道平滑肌细胞的培养对慢性呼吸道疾病的研究有重要意义。组织贴块法培养人气道平滑肌细胞是原代培养人气道平滑肌细胞的基本方法之一。目的:采用组织贴块法建立人气道平滑肌体外培养模型。方法:采集人气道组织,用组织贴块法进行人气道平滑肌细胞的原代培养,获得的细胞经形态学和免疫细胞化学染色鉴定。结果:培养的细胞呈典型的"谷峰"状生长,胞浆内特异性的平滑肌肌动蛋白阳性表达,符合平滑肌细胞的形态学特征和生物学特性。结论:组织贴块法易操作,结果可信,并可培养出高纯度活性好的人气道平滑肌细胞,成功建立了体外人气道平滑肌细胞增殖模型,提供了研究慢性呼吸道疾病的细胞培养模型。     

肺微血管内皮细胞的原代培养

目的:建立简单高效获取大鼠肺微血管内皮细胞(pulmonary microvascular endothelial cells,PMVECs)的培养方法.方法:(1)组织块贴壁法培养大鼠PMVECs;(2)光镜下观察细胞的形态;(3)扫描电镜和透射电镜分别观察细胞表面及内部的结构特征;(4)免疫荧光法鉴定PMVECs.结果:(1)获得的PMVECs长满后呈典型的铺路石或鹅卵石状;(2)扫描电镜下可见内皮细胞表面存在微绒毛等特殊结构;(3)透射电镜观察可见细胞浆内存在韦伯潘力氏小体(Webel Palade body,WPB);(4)免疫组化结果显示细胞浆中有Ⅷ因子相关抗原存在.结论:改良的组织块法培养肺微血管内皮细胞是一种简单、快捷,有效的方法.     

机械力对鼠脑微血管内皮细胞膜电流的影响

采用膜片钳技术以全细胞方式在鼠脑微血管内皮细胞中记录到一延迟外向电流,对Ｋ＾＋具有高度特异性,胞外施加２０ｍｍｏｌ／Ｌ的ＴＥＡ－Ｃｌ在明显抑制该电流。实验的保持电位定在－１００ｍＶ,测试电位从－１００ｍＶ至＋９０ｍＶ,每次增加１０ｍＶ,刺激波宽为２１００ｍｓ。该电流具有ＴＥＡ敏感,并有浓度依赖性,其ＩＣ５０约为２．０ｍｍｏｌ／Ｌ,类似延迟整流性钾电流特征（ＩＫｖ）。机械力作用下可引出一外向电流,膜     

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.     

A Novel Brain Neurovascular Unit Model with Neurons,Astrocytes and Microvascular Endothelial Cells of Rat

A novel triple cell neurovascular unit (NVU) model co-culturing with neurons, brain microvascular endothelial cells (BMECs) and astrocytes was established in this study for investigating the cerebral diseases and screening the candidates of therapeutic drug. We have first performed the cell identification and morphological characterization, analyzed the specific protein expression and determined the blood-brain barrier (BBB) function of the co-culture model under normal condition. Then, we further determined the BBB function, inflammation, cell injury and the variation of neuroprotective factor in this model after anoxia-reoxygenation. The results suggest that this model exhibited a better BBB function and significantly increased expression of P-glycoprotein (Pg-P) and ZO-1 compared with BMECs only or co-culture with astrocytes or neurons. After anoxia-reoxygenation, the pathological changes of this model were basically resemblance to the pathological changes of brain cells and BBB in vivo. And nimodipine, an antagonist of calcium, could reverse those changes as well. According to our observations, we deduce that this triple cell co-culture model exhibits the basic structure, function and cell-cell interaction of NVU, which may offer a more proper in vitro system of NVU for the further investigation of cerebral diseases and drug screening.     

Glycosyltransferase Activities in Cultured Endothelial Cells of Bovine Brain Microvascular Origin

Bovine brain microvascular endothelial cells (BMECs) express GM3 (NeuAc) and GM3 (NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b as well as sialosylparagloboside and sialosyllactosaminylparagloboside as the minor species. To investigate the metabolic basis of this ganglioside pattern, the activities of eight glycosyltransferases (GM3-, GD1a-, GD3-, LM1-, GM2 (NeuAc)-, GM2 (NeuGc)-, LacCer-, and GM1-synthases) in cultured BMECs were studied. It was found that BMECs possessed high activities of GM3- and GD1a-synthases, and low activities of GM2-, GM1-, and GD3-synthases. Thus, the present study provides evidence that endothelial cells are capable of synthesizing gangliosides in situ and that the high content of GM3 in BMEC is closely associated with high activities of GM3-synthase and low activities of GM2-, GM1-, and GD3-synthases.     

Synthesis of Apolipoprotein A-1 in Pig Brain Microvascular Endothelial Cells

In an approach toward the identification of hitherto unknown proteins involved in the function of the blood-brain barrier, we constructed a pig brain microvessel-derived cDNA library that is enriched in blood-brain barrier specific sequences by means of subtractive cloning. Sequence analysis of selected clones revealed that one of the cDNAs encoded porcine apolipoprotein (apo) A-1. The identity of apo A-1 mRNA was further confirmed by in vitro translation of RNA from brain microvascular endothelial cells and subsequent immunoprecipitation with an antibody against human apo A-1. We further investigated the expression of apo A-1 mRNA in several tissues and in endothelial cells of the pig. It is shown that cultured brain microvascular endothelial cells provide an in vitro model to study the expression and function of apo A-1 in the microvasculature of the brain.     

脑微血管内皮细胞条件培养液对星形胶质细胞活性的影响

目的:观察脑微血管内皮细胞与星形胶质细胞的相互关系,探讨血脑屏障维持脑内环境稳定的生理学基础.方法:原代培养大鼠脑皮质微血管内皮细胞,传至三代,收集在指数生长期细胞生长48 h后的务件培养液;将条件培养液分别按20%、30%、40%、50%、60%、70%、80%、90%、100%不同浓度作用于星形胶质细胞,MTT法检测不同浓度内皮细胞条件培养液作用于星形胶质细胞24 h、48h后的活性变化.结果:48h时间点的各浓度内皮细胞条件液组与相应的正常对照组相比差异均有显著统计学意义(P<0.01),内皮细胞条件液对星形胶质细胞表现出显著的抑制效应,而24 h的70%、80%、90%、100%浓度组与相应正常对照组相比也有显著统计学意叉的差异(P<0.01),且有浓度依赖性.结论:正常脑微血管内皮细胞条件培养液抑制了正常星形胶质细胞的活性.     

脑微血管内皮细胞氧糖剥夺条件培养液对PC12细胞活性的影响

目的:观察脑微血管内皮细胞氧糖剥夺后条件培养液对PC12细胞的影响.方法:原代培养大鼠脑皮质微血管内皮细胞,传至三代.制备其正常、氧糖剥夺、复糖复氧三种状态条件液,并观察内皮细胞在这三种状态下的形态改变;将这三种条件培养液作用于PC12细胞,分4组:正常对照组(Normal)、正常内皮细胞条件液组(N-CM)、氧糖剥夺内皮细胞条件液组(I-CM)、复糖复氧内皮细胞条件液组(R-CM).每组分别设6h、12 h、24 h、48 h、72 h、96 h、120 h、144 h 8个时间点,MTS/PMS法检测三种脑微血管内皮细胞条件液作用后的不同时间点PC12细胞活性的变化.结果:氧糖剥夺后脑微血管内皮细胞发生核固缩等明显病理改变,复糖复氧后这些改变有所恢复.N-CM组、R-CM组与相应时间点的Normal组PC12细胞活性相比差异均有统计学意义(P＜0.01).正常和复糖复氧内皮细胞条件液显著抑制了PC12细胞的增殖和活性.N-CM组、R-CM组与相应时间点的I-CM组PC12细胞活性相比差异均有统计学意义(P＜0.01).结论:正常脑微血管内皮细胞条件液抑制了PC12细胞的活性,氧糖剥夺后的脑微血管内皮细胞务件液抑制效应消失,复糖复氧后,这种抑制效应也同时恢复.