鼠视网膜血管内皮细胞体外培养的改良及鉴定

背景视网膜血管内皮细胞（RVECs）的体外培养是进行视网膜血管性疾病研究的基础,人眼球供体的缺乏是限制人血管内皮细胞培养的主要原因,与人高度同源的大鼠血管内皮细胞的培养方法已经建立,但其分离方法对细胞损伤较大,影响细胞的培养效率。目的建立和改良大鼠RVECs的培养方法。方法选取5只SD大鼠眼球,获得视网膜,用组织块培养法,将大鼠视网膜用质量分数0．1％胶原酶消化,并加入质量分数20％胎牛血清、血管内皮生长因子（VEGF）、血管内皮细胞生长添加物（ECGS）,用血管内皮细胞培养基中和后充分吹打,制备细胞及组织悬液,接种于人纤维粘连蛋白（FN）包被的培养瓶中。用Ⅷ因子相关抗体鉴定培养的内皮细胞。结果组织块法消化培养2d可见细胞从组织块游出,培养4d可伸出触角呈梭形,5～6d可融合生长,培养14d后的细胞呈单层生长,培养的细胞Ⅷ因子相关抗体染色阳性。传代培养的细胞接种2h可重新贴壁,恢复融合生长状态。结论视网膜组织块培养并用胶原酶消化可获得活性较强的细胞和碎片,再用高选择性的血管内皮细胞培养基和FN促贴壁进行选择性培养,可获得纯度较高的RVECs。     

牛视网膜微血管内皮细胞的体外分离培养

目的探讨一种方便可靠的牛视网膜微血管内皮细胞(borein retinal microvas cular endothelial cell,BREC)体外培养方法.方法采用机械剪碎牛视网膜血管,用含牛血清、肝素、血管内皮生长因子(Vascularendothelial growthfactor,VEGF)的条件化培养基培养牛视网膜血管内皮细胞,观察细胞生长状况,制作生长曲线.应用Von Willebrand因子抗体免疫鉴定BREC,并通过透射电镜观察BREC的超微结构.结果添加了VEGF的条件化培养基对BREC有很好的选择刺激作用,初期细胞呈鲤鱼样聚集在微血管碎片周围,对数生长期细胞似铺路石样大片单层生长,存在接触抑制.Von Willebrand因子抗体染色阳性,获得BREC纯度在95％以上,能够连续传代.培养的细胞电镜下符合内皮细胞的特征.结论含VEGF培养基的应用可以获得高纯度、具有良好生长特性的BREC,是一种简便、效果稳定可靠的体外培养牛视网膜微血管内皮细胞的方法.     

大鼠视网膜微血管内皮细胞的分离和培养

目的探讨大鼠视网膜毛细血管内皮细胞(rat retinal capillary endothelial cells,RRCEC)的体外分离和选择性培养方法。方法采用单纯视网膜剪碎法获得的视网膜微血管碎片,结合0.25%胶原酶消化,经细胞筛网过滤,原代细胞悬液接种于纤维粘连蛋白(fibronectin,FN)包被的培养皿中,培养液为含20%胎牛血清(FBS)、100×103U·L-1肝素钠、10mg·L-1碱性成纤维细胞生长因子(bFGF)的DMEM液。原代培养5～6d细胞较少时,采用机械除杂法进行细胞分离,而在传代时利用进行选择性消化和贴壁法除杂分离以获得较纯RRCEC。应用Ⅷ因子相关抗体通过免疫组化方法对所培养的RRCEC进行鉴定。结果大鼠视网膜组织经过胶原酶适当消化后,产生大量具有良好生长能力的微血管碎片,贴壁后可见RRCEC自微血管碎片中游出,融合后呈铺路石样单层生长。传代培养时5～6d可融合生长。选择性培养获得的RRCEC纯度高,能连续传代,保持性状不变。对Ⅷ因子相关抗体染色阳性。结论视网膜剪碎、胶原酶、FBS、bFGF、培养皿处理及机械除杂法分离,选择性消化和贴壁等应用可获得较纯的RRCEC,方便简单,具有良好的重复性。     

牛视网膜微血管内皮细胞和周细胞的体外培养

目的 探讨牛视网膜微血管内皮细胞（ bovine retinal endothelial cells, BREC ）和周细胞（bovine retinal pericytes, BRP）的体外选择性培养方法。 方法 结合视网膜微血管的消化分离，采用含10％人血清、100 μg/ml 肝素（Heparin）的Dulbecco改良Eagle培养基(Dulbecco′s modified Eagle′s medium, DMEM)和含20％胎牛血清的DMEM培养基分别选择性培养BREC和BRP。通过荧光显微镜观察乙酰化低密度脂蛋白(acetylated low density lipoprotein, Dil-Ac-LDL)吞噬情况并应用Von Willebrand因子抗体通过免疫组织化学方法鉴定BREC；以及α-平滑肌肌动蛋白抗体免疫组织化学鉴定BRP。 结果 通过选择性培养获得的BREC和BRP的纯度达到98％以上，并能连续传代。BREC早期似小鲤鱼状聚集在微血管碎片周围，呈片状鹅卵石样单层生长，存在接触性抑制；周细胞多散布在距血管碎片稍远处，形状不规则，呈无接触性抑制的生长。BREC可吞噬Dil-Ac-LDL，细胞浆内有荧光表达 ；消化传代后BREC中Von Willebrand因子表达阳性，而α-平滑肌肌动蛋白表达阴性；BRP的α-平滑肌肌动蛋白表达阳性，而Von Willebrand因子表达阴性。 结论 选择性培养基的应用和培养皿的处理，可分别获得较高纯度的BREC和BRP，简单且具有良好的重复性，无需额外步骤来去除BREC中混杂的BRP。 （中华眼底病杂志，2004，20：23-26）     

选择性培养牛视网膜血管内皮细胞和周细胞

目的　建立选择性培养牛视网膜血管内皮细胞和周细胞的简要方法。方法　采用机械除杂法或等密度沉降分离法 ,培养皿用不同的基质成分包埋 ,培养液中加入有利于细胞生长的添加物。结果　原代培养的牛视网膜血管内皮细胞和周细胞纯净度 >95 % ,能连续传代。周细胞在传代后不规则形状及细胞内微丝更明显。视网膜血管内皮细胞融合后呈“铺路石”样改变。视网膜血管内皮细胞对Ⅷ因子相关抗体染色阳性。视网膜周细胞对单克隆抗体α 平滑肌肌动蛋白抗体染色阳性。结论　用明胶包被培养皿 ,在培养液中加入肝素、内皮细胞生长因子及高浓度的胎牛血清可获得较纯的内皮细胞。而周细胞最适合的培养液为DMEM加入 2 0 g·L-1胎牛血清。利用Percoll分离液也可分离培养出较纯的牛视网膜血管内皮细胞和周细胞     

视网膜微血管内皮细胞的体外分离和培养方法新进展

体外分离与选择性培养视网膜微血管内皮细胞的方法有多种,其中新报道的组织块培养结合胶原酶消化法因操作简便、经济,获得的内皮细胞纯度较高、活性良好等优点而被广泛应用.受人眼球供体的限制,研究者通常会选用分离与人高度同源的鼠视网膜微血管内皮细胞(retinal microvascular endothelial cell,RMVEC)进行体外实验.本文就目前常用的视网膜微血管内皮细胞体外培养方法从不同动物种属、添加因子、细胞纯化、体外鉴定等方面进行阐述,同时简要说明众多方法在操作过程中的优缺点,为研究人员选择合适的培养方法提供参考.     

牛视网膜毛细血管周细胞的选择性培养

目的 :选择性分离、培养牛视网膜毛细血管周细胞。方法 :采用有限胶原酶消化和筛网过滤法培养周细胞。结果 :原代培养时 ,周细胞形态不规则 ,呈现出典型的非接触性抑制重叠融合生长 ;传代培养后增长速度加快 ,第 3天进入对数生长期 ,第 10天进入平台期。结论 :获得了较纯净的视网膜毛细血管周细胞 (纯净度 >98% ) ,并能连续传代。     

高浓度胰岛素对视网膜M(u)ller细胞VEGF表达的影响

目的:探讨在高浓度胰岛素条件下,体外培养的兔视网膜Muller细胞的血管内皮细胞生长因子(vascular endothelial growthfactor,VEGF)的变化.方法:在不同浓度胰岛素条件下(4,8,12kU/L),体外培养兔视网膜Muiler细胞,采用免疫细胞化学法、原位杂交法,定性测定Muller细胞分泌VEGF的变化,采用ELISA方法,定量测定Muiler细胞分泌VEGF的变化.结果:高浓度胰岛素能明显增强VEGF的表达(P<0.05).结论:高浓度胰岛素可能通过刺激Miiller细胞编码VEGF基因的转录,进而增强VEGF蛋白的表达,而在糖尿病视网膜病变的新生血管生成中发挥重要作用.     

大鼠视网膜微血管内皮细胞的原代培养及鉴定改进

【摘要】 目的 探讨大鼠视网膜微血管内皮细胞原代培养改进方法,为研究视网膜新生血管疾病的病理机制奠定基础。设计 实验研究。研究对象 大鼠视网膜微血管内皮细胞。方法 采用机械剪除大鼠视网膜血管联合酶消化分离,用含胎牛血清、内皮细胞培养特制添加剂等的条件培养基培养及纯化大鼠视网膜微血管内皮细胞,倒置相差显微镜下观察细胞生长状态。应用因子VIII、血小板内皮细胞黏附分子-1（CD31）免疫鉴定细胞。主要指标 微血管内皮细胞形态,免疫荧光染色。结果 原代培养的大鼠视网膜微血管内皮细胞贴壁后散在分布,呈单层排列,多为梭形,边界清楚,少量呈铺路石样螺旋向外生长,3~5天后融合,呈簇状单层分布。免疫荧光细胞染色显示,因子VIII抗体染色核周出现黄绿色荧光反应,CD31因子抗体染色以胞浆着色为主。细胞纯度较高,荧光染色阳性细胞率为95%。结论 采用机械剪除大鼠视网膜血管、胰蛋白酶和胶原酶消化、含特制添加剂培养基的应用、明胶包被培养瓶等综合法可获得较纯的大鼠视网膜微血管内皮细胞。（眼科,2012,21：261-263）     

新生Long Evans大视网膜小胶质细胞原代培养及鉴定

目的:建立稳定高产的新生Long Evans大鼠视网膜小胶质细胞(retinamicrogliacellRMG)体外培养、分离的方法。方法:取出生3d内的LongEvans新生大鼠视网膜神经上皮层进行混合细胞培养,10～12d后振荡分离,纯化培养得到小胶质细胞;小胶质细胞的特异性标记IB-4及CD11b进行组化及细胞免疫荧光鉴定,扫描电镜观察细胞表面突起。结果:得到纯度较高的小胶质细胞,IB-4组化及CD11b细胞免疫荧光鉴定纯度分别为94.5%和95.8%,扫描电镜观察细胞表面为棘状突起,明显与巨噬细胞的细胞表面区别。结论:本方法可以得到纯度高的视网膜小胶质细胞。     

A simple method for the in vitro culture of human retinal capillary endothelial cells.

A simple method for the culture of human retinal capillary endothelial cells in vitro is described in this report. Through a process of gentle mechanical disruption and sieving, a sufficient yield of retinal microvessels was obtained from one or two human eyes to allow the culture of endothelial cells in abundance. The cells were factor VIII-positive and demonstrated typical vascular endothelial morphology. Pericyte contamination was prevented by using human platelet-poor serum in primary culture and passaging cells with a low concentration of trypsin. Proliferation assays revealed that the cells grew best in Iscove's modified Dulbecco's modified Eagle's medium with 15% fetal calf serum (FCS). There was no difference in induced proliferation when FCS was compared to human platelet-poor serum. The method seemed to be as good as and much simpler than other recently described techniques. The study of human retinal capillary endothelial cells cultured in this way may shed light on the earliest stages of diabetic retinopathy and other diseases of the retinal microvasculature, particularly AIDS-related retinopathy and radiation retinopathy.     

Actin in cultured bovine retinal capillary pericytes: morphological and functional correlation

Actin in cultured bovine retinal capillary pericytes was identified and partially characterized biochemically. The filamentous actin was localized in bovine retinal capillary pericytes using a fluorescent mushroom toxin (nitrobenzoxadiazole-phallacidin) specific for actin. One-dimensional SDS-polyacrylamide-gel electrophoresis of urea-extracted proteins from bovine retinal capillary pericytes revealed a 46,000 MW protein band corresponding to an actin standard, which comprised 7.3% of the total urea-soluble proteins. Actin-activated skeletal muscle myosin Mg2+-ATPase assay, using [gamma-32P]-ATP as substrate, demonstrated functional actin in bovine retinal capillary pericyte extracts after DEAE-cellulose anion-exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.25 and 0.35 M KCl. The presence of functional actin in pericytes indicated the ability to generate contractile force. This contraction-generating ability may allow pericytes to regulate microvessel caliber and to maintain the integrity of the capillary wall. A lack of this function when pericytes are preferentially lost in diabetic retinal microangiopathy could destabilize the microvessel wall and predispose the capillary to further pathologic changes.     

Polyol formation and NADPH-dependent reductases in dog retinal capillary pericytes and endothelial cells

PURPOSE: Dogs fed a diet containing 30% galactose experience retinal vascular changes similar to those in human diabetic retinopathy, with selective pericyte loss as an initial lesion. In the present study the relationship among reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductases, polyol formation, and flux through the polyol pathway in cultured dog retinal capillary cells were investigated. METHODS: Pericytes and endothelial cells were cultured from retina of beagle dogs. NADPH-dependent reductases were characterized by chromatofocusing after gel filtration. Sugars in cultured cells were analyzed by gas chromatography, and flux through the polyol pathway was investigated by 19F nuclear magnetic resonance (NMR) with 3-fluoro-3-deoxy-D-glucose (3FG) as a substrate. The presence of aldose reductase and sorbitol dehydrogenase in these cells was examined by northern blot analysis. RESULTS: Two distinct peaks corresponding to aldose reductase and aldehyde reductase, the latter being dominant, were observed in pericytes by chromatofocusing. Culture in medium containing either 10 mM D-galactose or 30 mM D-glucose resulted in the accumulation of sugar alcohol in pericytes that was markedly reduced by aldose reductase inhibitors. 19F NMR spectra obtained from pericytes cultured for 5 days in medium containing 2 mM 3FG displayed the marked accumulation of 3-fluoro-deoxysorbitol but not 3-fluoro-deoxyfructose. No 3FG metabolism was observed in similarly cultured endothelial cells. With northern blot analysis, aldose reductase was detected in pericytes but not in endothelial cells. Sorbitol dehydrogenase was below the detectable limit in pericytes and endothelial cells. CONCLUSIONS: Aldose, aldehyde, and glyceraldehyde reductases are present in dog retinal capillary pericytes, with aldehyde reductase being the major reductase present. Polyol accumulation easily occurs in pericytes but not in endothelial cells.     

选择性牛眼上皮样小梁细胞的体外培养

目的选择性行牛眼上皮样小梁细胞的体外培养，为进一步研究原发性开角型青光眼提供实验材料。方法利用组织块法进行小梁细胞的原代培养，之后利用机械划除法、酶消化法、反复贴壁法，对牛眼上皮样小梁细胞进行选择性培养和传代。结果成功选择性培养出形态、性状良好的牛眼上皮样小梁细胞。结论选择性牛眼上皮样小梁细胞的体外培养所获细胞形态单一，可以用此种方法进行牛小梁细胞的体外培养并为实验应用做好准备。     

兔视网膜Müller细胞原代培养

目的 建立成年兔视网膜Müller细胞体外原代培养方法.方法 运用组织块培养法.分离出成年兔视网膜神经感觉层,剪成1 mm×1 mm大小组织块,置于含有20%胎牛血清的Dulbecco改良Eagle培养液/F12培养,采用倒置相差显微镜、透射电子显微镜观察及荧光免疫组织化学染色的方法进行培养细胞的鉴定.结果 培养的细胞块3 d后可见部分细胞突起长出,7 d后整个组织块周围放射状长出较多细胞.倒置相差显微镜下,培养细胞呈一端细胞体丰富膨大,另一端有一长突起,细胞核椭圆形,常有2个或2个以上核仁;透射电子显微镜下,细胞胞体大,细胞浆丰富,内含丰富的8～10 nm的微丝.荧光免疫组织化学法显示细胞呈神经胶质纤维酸性蛋白和胞内视黄醛结合蛋白阳性,阳性率达95%以上.结论 运用组织块培养法可培养出兔视网膜Müller细胞.

Abstract：

Objective To develop a method for the primary culture of retinal Müller cells of adult rabbit in vitro. Methods Retina was isolated from adult rabbit, cut into 1 mm × 1 mm pieces, and placed into Dulbecco modified Eagle medium/F12 containing 20 % fetal bovine serum to culture. Cultured cells were identified by inverted phase contrast microscope, transmissim electron microscope and immunohistochemistry staining method. Results Visible cell processes grew out from the retinal tissues after three days culture, and more cells grew radically around the retina after seven days culture. The cultured cells were often inflated at one side and had one long process at another side, and the nuclei were elliptical and there were two or more than two nucleoli under inverted phase contrast microscope. The cytoplasm was rich and contained abundant microfilaments in eight to ten nanometers under transmission electron microscope. Immunohistochemistry assay showed that 95% of the cells were positive for glial can be cultured by the explant culture method.