小鼠脊髓微血管周细胞的体外培养及鉴定

苑晓晨，武清斌，李宏伟，荆瀛黎，李炳蔚，刘淑英，修瑞娟

(中国医学科学院 北京协和医学院 微循环研究所, 北京 100005)



小鼠脊髓微血管周细胞的体外培养及鉴定

苑晓晨，武清斌，李宏伟，荆瀛黎，李炳蔚，刘淑英，修瑞娟*

(中国医学科学院 北京协和医学院 微循环研究所, 北京 100005)

目的应用周细胞培养基(PCM)分离筛选小鼠脊髓微血管周细胞(SCMP)，对其生物学功能进行评价。方法10只3周龄C57小鼠，无菌条件下取脊髓去除软脊膜，剪碎成大约1 mm×1 mm×1 mm。两次酶消化后用含20% 牛血清白蛋白的DMEM离心获得微血管。用内皮细胞培养基(ECM)培养，传代2次后改用PCM培养。倒置显微镜观察细胞增殖状况。免疫细胞化学法检测血小板源性生长因子受体β(PDGFRβ)、神经元-胶质抗原2(NG2)、von Willebrand因子(vWF)和胶质纤维酸性蛋白(GFAP)的表达。流式检测CD140b、CD31、CD11b和GFAP的表达。将PCM换为10%胎牛血清(FBS)的DMEM培养基，检测细胞α-SMA表达的变化。通过周细胞-内皮细胞共培养成管实验检测细胞成管能力。结果接种48 h细胞爬出，7～9 d汇合,周细胞和内皮细胞伴随状增殖。改用PCM培养后内皮细胞减少，周细胞呈优势生长。免疫细胞化学表明PDGFRβ和NG2阳性，vWF和GFAP阴性；流式结果表明细胞PDGFRβ的阳性率为95.52%±2.55%，GFAP为0.63%±0.26%，CD31为0.80%±0.26%，CD11b为1.02%±0.35%。10%胎牛血清的DMEM促进细胞分化，α-SMA表达升高。成管实验周细胞与内皮细胞共同形成管腔样结构。结论通过PCM筛选法能够成功获得纯度较高的SCMP，所获得的细胞具备明显的周细胞的形态和功能特征。

周细胞；脊髓；微血管；细胞分离

脊髓微血管周细胞(spinal cord microvascular pericytes, SCMP)是血-脊髓屏障(blood-spinal cord barrier, BSCB)的重要组成部分，参与调节脊髓微循环血流、内皮细胞紧密连接和BSCB的功能。研究表明SCMP的功能变化与多种脊髓疾病相关[1]。既往关于中枢神经系统周细胞的体外研究多来源于脑组织，分离策略通常是使用酶消化法获得微血管片段，在血管片段的基础上分离培养周细胞，但是此策略容易造成杂细胞的污染。而用免疫磁珠分选和流式分选等方法处理的细胞活性差、产量低，而且成本过高。本研究基于本单位分离脑微血管周细胞的经验[2]，使用商品化的周细胞培养基，通过“培养基筛选法”分离小鼠脊髓微血管周细胞，并对所获得的周细胞的功能进行评价。

1 材料与方法

1.1 材料

1.1.1 实验动物和细胞：清洁级3周龄雄性C57BL/6小鼠[中国医学科学院医学实验动物研究所，许可证号SCXK(京)2009- 0007]。所有的实验方案已获得中国医学科院微循环研究实验动物伦理委员会批准。脊髓微血管内皮细胞(spinal cord microvascular endothelial cells, SCMECs)来自本实验室[3]。

1.1.2 主要试剂:胎牛血清(FBS)、改良杜氏伊格尔培养基(Dulbecco’s modified Eagle medium，DMEM)(高糖)、II型胶原蛋白酶、双抗、庆大霉素、谷氨酰胺和1×PBS、0.05% 胰蛋白酶和含10% FBS的DMEM培养基(北京协和医学院细胞中心)；含10% FBS的内皮细胞培养基(endothelial cell medium，ECM)和含2% FBS周细胞培养基(pericytes cell medium，PCM)(Scien Cell公司)；牛血清白蛋白(BSA)(Amresco公司)；DNA酶I(DNase Ⅰ)和纤维连接蛋白Fibronectin(Sigma公司)，胶原蛋白酶/分散酶(Roche公司)； 抗von Willebrand factor(vWF)抗体(Santa Cruz公司)；抗血小板源生长因子受体(PDGFRβ)、神经元-胶质抗原2(neuron-glia antigen 2，NG2)抗体、von Willebrand因子(von Willebrand factor，vWF)抗体、胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)抗体和α-SMA抗体(Abcam公司)； FITC标记和TRITC标记的二抗(北京中衫金桥生物技术有限公司)。APC标记抗小鼠CD45抗体、PE 标记抗小鼠PDGFRβ/CD140b抗体、PE/Cy7标记抗小鼠CD31抗体、FITC标记抗小鼠CD11b抗体及相应的同型对照(Biolegend公司)eFluor® 660标记抗小鼠GFAP抗体及相应的同型对照抗体、流式染色缓冲液(flow cytometry staining buffer，FCS Buffer)、细胞内固定工作液(intracellular fixation buffer，IC Buffer)和透化工作液(permeabilization buffer)(eBioscience公司);基质胶(BD公司);活细胞荧光示踪剂DiI(红色)和DiO(绿色)(Life Technologies公司)。

1.2 方法

1.2.1 细胞分离与培养：小鼠经3%戊巴比妥钠(30 mg/kg)腹腔注射麻醉，断头处死。取出脊髓组织，用滤纸去除软脊膜，剪碎成约1 mm×1 mm×1 mm大小，整个过程在冰上操作。加入DMEM、Ⅱ型胶原蛋白酶(1 g/L)、DNase Ⅰ(15 mg/L)，37 ℃消化1.5 h。1 000×g离心8 min，弃上清液，20% BSA-DMEM重悬；4 ℃ 1 000×g离心20 min。弃上清，加入DMEM、collagenase/dispase(1 g/L)、DNase Ⅰ(6.7 mg/L)，37 ℃消化1 h。加入DMEM，700×g离心6 min。弃上清，加入含20% BSA-DMEM重悬。4 ℃ 1 000×g离心20 min。弃上清后DMEM重悬，悬液内即含微血管片段。 将微血管片段用DMEM洗2次,1000×g离心8 min,700×g离心5 min。弃上清重悬后用细胞滤器过滤。用ECM将微血管片段接种至两个fibronectin(10 mg/L)包被好的35 mm培养皿。72 h后用PBS清洗3次，以去除漂浮的死细胞以及其他杂质，之后继续用ECM培养。每隔3 d换液1次，自原代细胞从微血管片段爬出到细胞完全汇合需7～9 d。待细胞汇合后用胰蛋白酶消化传代2次，均使用ECM进行培养。从第3次传代后，用PCM培养。取第5代细胞用于细胞鉴定实验(图1)。

图1 分离步骤简图Fig 1 Schematic diagram of the steps

1.2.2 细胞鉴定：1)倒置显微镜观察SCMP的形态:用倒置显微镜每隔24 h观察细胞从微血管片段爬出及增殖状况并拍照。2)细胞免疫化学方法鉴定:待细胞增殖汇合至75%左右，对细胞进行固定和透化处理。分别加入一抗(PDGFRβ、NG2、vWF和GFAP)(1∶200)，4 ℃孵育过夜；PBS洗5 min共3次；分别加入荧光标记二抗，室温下孵育1 h，PBS洗5 min共3次；DAPI染色5 min，PBS清洗；封片。荧光显微镜(DP72，Olympus公司)下观察并拍照，用 Image Pro Plus 7.0(IPP)进行定量分析，实验重复3次。3)流式细胞术检测细胞表面蛋白PDGFRβ、CD11b和CD31:消化并收集细胞，用流式染色工作液将细胞密度调整为1×1010个/ L；取100 μL加入流式细胞仪测定管，添加20 μL Fc受体阻断剂，并孵育10 min；添加荧光直接标记的流式抗体和相应的同型对照抗体，4 ℃避光孵育30 min；流式染色工作液清洗2次后，用PBS重悬至100 μL，用细胞筛制成单细胞悬液，行流式细胞仪检测。实验重复5次。 4)流式细胞术检测细胞胞质蛋白GFAP：消化并收集细胞，用FCSB将细胞密度调整为1×1010个/L；取100 μL加入流式细胞仪测定管，添加100 μL细胞内固定工作液，室温避光孵育30 min； 添加2 mL透化液，400×g室温离心5 min后弃上清，用2 mL透化液重悬；400×g室温离心5 min后弃上清；用100 μL透化液重悬，加入相应的抗体或相应的同型对照抗体，室温避光孵育30 min；用FCSB清洗2次，用PBS重悬至100 μL后用细胞筛制成单细胞悬液，行流式细胞仪检测。实验重复5次。

1.2.3 细胞功能评价：1)含10% FBS的DMEM培养基对细胞分化标志蛋白表达的影响：①两组用PCM培养的第5代细胞，其中一组改用含10% FBS的DMEM培养；另外一组做为对照组继续用PCM培养。4 d后用细胞免疫化学方法和Western blot免疫印记法检测α-SMA的表达。②细胞免疫化学方法鉴定：对细胞进行固定和透化处理。分别加入一抗α-SMA抗体(1∶200)，4 ℃孵育过夜；PBS洗5 min共3次；加入荧光标记二抗，室温下孵育1 h，PBS洗5 min共3次；DAPI染色5 min，PBS清洗；封片油封片。荧光显微镜下观察并拍照，用 Image Pro Plus 7.0(IPP)进行定量分析，实验重复3次。③Western blot免疫印记法：将培养至汇合的细胞收集并裂解。以每孔40 μg蛋白上样于10% SDS-PAGE分离后，将蛋白转移至PVDF膜上。用抗小鼠α-SMA抗体检测，以β-actin为内参。实验结果以同一样本α-SMA蛋白与β-actin蛋白表达量的比值表示。实验重复3次。2)基质胶成管实验:①细胞荧光标记:脊髓微血管内皮细胞用红色荧光素DiI标记，SCMP用绿色荧光素DiO标记。将培养皿中的培养基吸出，加入染色培养基(荧光染料与培养基以1∶200比例混匀)，放置培养箱内孵育20 min；将染色培养基吸出，加入正常培养基清洗3次(每次清洗时用正常培养基孵育10 min后再将其吸出)。②以每孔25 μL基质胶包被96孔板，37 ℃ 30 min使胶凝固。将SCMECs和SCMP以10∶1进行混合后进行接种，每孔接种5×103个细胞，ECM最终体积为100 μL。24 h后观察组成管情况。实验重复3次。

1.3 统计学分析

2 结果

2.1 倒置相差光学显微镜下观察细胞形态

在内皮细胞培养基中，微血管片段呈典型的串珠状或分支状(图2A)；48 h后,可见细胞从微血管片段中爬出(图2B)；4 d后，细胞逐渐增多，细胞形态多样(图2C)；7～9 d后细胞汇合(图2D)；细胞传代后(第2代)继续在ECM中培养，可见纺锤状的内皮细胞和多边形的周细胞呈现伴随状增殖(图2E)；传代后改用PCM培养，长梭状内皮细胞逐渐减少，多边形、多突起的周细胞呈现优势增殖(图2F)。

2.2 SCMP的细胞免疫化学和流式细胞术鉴定

对第5代的细胞进行免疫荧光染色鉴定，可见细胞PDGFRβ和NG2双阳性(图3A～C)；vWF(内皮细胞)和GFAP(星形胶质细胞)阴性(图3D,E)，表明得到的细胞中没有内皮细胞和星形胶质细胞的污染。流式细胞仪检测结果显示，PDGFRβ的阳性率为95.52%±2.55%，而GFAP为0.63%±0.26%，CD31(内皮细胞)为0.80%±0.26%，CD11b(小胶质细胞)为1.02%±0.35%(图4)。

2.310%FBS-DMEM培养基促进SCMP的分化

SCMP在PCM中培养2～3 d即可传代。细胞保持典型的多角形形态但是体积较小，且α-SMA低表达(图5A)；换用DMEM培养4 d后，α-SMA的表达明显上调(图5B)，且部分细胞体积增大，Western blot结果(图5C)表明α-SMA的变化情况与细胞免疫化学法检测结果一致。

A.spinal cord capillary fragments attached after 24 hours; B.the cells migrated out from the microvessels; C, D.after 7～9 days, the cells gradually reached confluency; E.passage 2 cultures contained the spindle-shaped endothelial cell and the rhomboid-shaped pericytes; F.by passage 5, the culture showed the typical rhomboid morphology of pericytes; Scale bar=100 μm; similar results were observed in three independent experiments

Passage 5 pericytes were analyzed by immunofluorescence for the pericyte marker PDGFRβ(A), NG2(B), with merge(C); the cells negatively expressed the von Willebrand factor-related antigen (vWF)(D) and glial fibrillary protein (GFAP)(E); Scale bar=100 μm; nuclei were stained blue with DAPI; similar results were observed in three independent experiments

Passage 5 pericytes uniformly expressed the cell surface molecules PGDFRβ; the cell isolates did not contain cell expressing CD31 (endothelial markers), or CD11b (microglial marker), or GFAP (astrocyte marker); specific staining is shown by the red line; isotype matched control staining is shown by the black line; similar results were observed in five independent experiments

The pericytes were grown in pericyte medium(A) or DMEM+10% FBS(B) for 4 days, then analyzed for α-SMA-green by immunofluorescence and Western blot, when switched into DMEM+10% FBS, α-SMA was up-regulated in the pericytes; *P<0.01 compared with control; Scale bar=100 μm; similar results were observed in three independent experiments

2.4 SCMP与SCMECs联合培养共同成管

红色荧光标记的SCMECs和绿色荧光标记的SCMP以10∶1的比例共培养在基质胶上，24 h后可以观察到两种细胞黏合在一起，共同组成管腔样结构 (图6)。

SCMP and SCMECs were labeled with green or red cell tracker respectively and co-cultured in a 1∶10 ratio in Matrigel in ECM for 24 hours; similar results were observed in four independent experiments; The red-labeled endothelial cells were closely invested with the green-labeled pericytes to form vessel-like structures; Scale bar=100 μm; similar results were observed in four independent experiments图6 SCMP与SCMECs联合培养共同成管Fig 6 SCMP align with SCMECs in co-cultures

3 讨论

有文献报道使用匀浆研磨结合酶消化分离微血管[4-6]，通过振荡消化使细胞从微血管片段上脱离。但得到的微血管活力差，细胞存活率低，时间和力度难掌控。而采用机械剪切、两步酶消化结合20% BSA离心获得微血管片段，避免了研磨对微血管的损伤，增加了细胞的生存概率。

通过微血管片段获取周细胞会面临杂细胞的污染。从微血管上脱落的既包含内皮细胞和周细胞，也有小胶质细胞和星形胶质细胞。根据本实验室分离内皮细胞的经验[7]，用ECM培养内皮细胞时存在周细胞的污染，而且周细胞会抑制内皮细胞的增殖[8]，但在ECM中胶质细胞的生长会受到抑制[9- 10]。因此，先用ECM培养微血管，待胶质细胞被抑制后改用PCM来抑制内皮细胞。细胞免疫化学和流式细胞术鉴定的结果表明，该方法的内皮细胞和胶质细胞的污染显著降低。

用6～8周龄的小鼠分离细胞，从微血管内爬出的细胞数量少，活性低。改用3周龄小鼠，则细胞活力强，存活率高。细胞在25 cm2(或更大)培养瓶中不易生长，而在35 mm 培养皿中细胞能够很快形成汇合，生长状态良好。从而证明中枢神经系统微血管单元的细胞不适合大的生长空间[11]。

周细胞是多能干细胞，在体外培养过程中能够快速分化[12]，也有人认为其在传代过程中能够保持一定的稳定[13]。周细胞常用的标志蛋白为PDGFRβ和NG2[14- 15], 但也有研究表明，周细胞能够表达一种与血管平滑肌细胞相同的收缩蛋白α-SMA。体内和新分离的微血管周细胞不表达α-SMA，而体外培养中周细胞会逐渐表达该蛋白，因此α-SMA被认为是一种周细胞分化的标志蛋白[14]。本研究发现，PCM中的周细胞体积小，α-SMA表达低；换用DMEM培养4 d，部分细胞的体积增大、突触增多，且α-SMA表达明显上调，表明细胞具有一定的分化能力。成管实验表明,分离的SCMP能够与内皮细胞共同形成管腔样结构。

综上所述，本研究建立了一种分离原代SCMP的方法。具有操作简单、经济等优点，可以得到纯度高和活力较强的周细胞。

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Isolation and identification of mouse spinal cord microvessel pericytesinvitro

YUAN Xiao-chen, WU Qing-bin, LI Hong-wei, JING Ying-li, LI Bing-wei, LIU Shu-ying, XIU Rui-juan*

(Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China)

ObjectiveTo establish a method for selective culturing mouse spinal cord microvascular pericytes (SCMP) by pericytes cell medium (PCM) with 2% FBS. To observe the differentiation and function of the cultured cells.MethodsAfter decapitation of 10 of 3-week-old C57 mice, the intact spinal cord was removed from the spinal column. Meninges were cleared from the spinal cord. Tissues were cut into small pieces (approx.1 mm×1 mm×1 mm). The capillary fragments were obtained through two-step enzymatic digestion and 20% bovine serum albumin (BSA) centrifugation. The microvessels were incubated initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. The morphology of pericytes was observed by inverted microscope. Platelet-derived growth factor receptor β(PDGFRβ), neuron-glial antigen 2(NG2)， von Willebrand factor(vWF), and glial fibrillary acidic protein (GFAP) were detected by immun-ofluorescence．PDGFRβ,CD31,CD11b and GFAP were detected by flow cytometry. When switching the cells from pericyte medium into DMEM containing 10% FBS, the α-SMA, a marker of pericyte differentiation, was detected by immunofluorescence and Western blot. The matrigel pericyte-endothelial cell co-culture was used to verify the function of cultured cells.ResultsThe cells migrated from the attached capillary fragments after 48 h, and got confluence after 7 to 9 days. Primary cultures consisted of endothelial cells with pericytes. After switching to PCM, the pericytes grew predominantly and showed the typical rhomboid morphology. Immunofluorescence revealed that PDGFRβ and NG2 were positive， vWF and GFAP were negative. Flow cytometry showed that cells were strongly positive for PDGFRβ(95.52%±2.55%), negative for CD31(0.80%±0.26%), CD11b(1.02%±0.35%) or GFAP(0.63%±0.26%). DMEM containing 10% FBS promoted α-SMA expression, demonstrating the cells could still differentiate. In matrigel co-culture experiments, pericytes aligned with endothelial cords, retaining the ability to associate with endothelial cell.ConclusionsThis method may successfully obtain the highly purified SCMP. The cells have morphological and functional characteristics of pericytes and the ability to differentiate.

pericytes; spinal cord; microvessel; cell isolation

2014- 12- 11

：2015- 01- 19

2014年协和青年教师基金&中央高校基本科研业务费专项资金(33320140062)

*通信作者(correspondingauthor)：xiurj@imc.pumc.edu.cn

1001-6325(2015)05-0688-07

研究论文

R744.9; R-332

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