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。     

初断乳大鼠视网膜微血管周细胞的分离培养

探讨、优化初断乳大鼠视网膜微血管周细胞(retinal microvascular pericytes,RMPs)的分离、培养方案。分别从15只初断乳大鼠及15只成年大鼠中剜取眼球,采用眼科显微手术器械分离获取视网膜,经碎化、消化、过滤处理,收集视网膜微血管片段,予接种培养。MTT法测绘RMPs生长曲线,通过倒置显微镜观察RMPs形态,免疫荧光法鉴定周细胞标记物。比较2组之间视网膜分离操作时间、完整性、原代细胞数量、周细胞形态和表面标记物表达。结果显示,初断乳大鼠视网膜均成功分离,其中24眼视网膜呈整片分出,6眼视网膜破裂呈碎片状,单个眼球视网膜分离时间为14.3~45.5 s。视网膜分离操作时间及完整性与成年大鼠差异无统计学意义(P0.05),初断乳大鼠原代RMPs细胞产量较成年大鼠高,细胞增殖能力强,细胞形态及表面标记物与成年大鼠一致。研究结果表明,初断乳大鼠视网膜是一种可用于RMPs培养的良好组织原料,该实验成功建立了初断乳大鼠RMPs分离培养体系。     

α-SMA在培养生长板软骨细胞中的表达

目的探讨α-SMA在原代培养生长板软骨细胞中的表达及传代培养对其表达的影响。方法分离、培养大鼠肋生长板软骨细胞(rat costochondral growth plate chondrocyte,RGC),免疫细胞化学(ICC)和Western blot分别对1-5代RGC中α-SMA的表达进行定性和半定量分析。结果原代培养的RGC不表达α-SMA,随着传代次数的增加,α-SMA阳性染色的细胞比例从原代的0增加到第5代的24.2±4.3%(n=3),Western blot结果与ICC结果一致。结论本研究首次发现原代培养的RGC并不表达α-SMA,随着RGC传代次数的增加,表达α-SMA的细胞比例和表达量不断升高。     

Notch3对促进胰腺星形细胞活化的基因表达及信号通路的影响

目的:分离培养小鼠胰腺星形细胞(PSCs),检测Notch3对促进PSCs活化的基因表达及信号通路的影响.方法:对小鼠PSCs进行分离培养及传代.采用免疫荧光染色检测活化的小鼠PSCs中α-SMA,fibronectin及collagen Ⅰ的表达;细胞分组为空白对照组(MOCK组),阴性对照组(转染Notch3 si...     

黄芪甲苷激活TGF-β1/Smad2信号通路诱导骨髓间充质干细胞向周细胞分化的作用研究

目的:观察黄芪甲苷促进大鼠骨髓间充质干细胞(Mesenchymal stem cell,MSCs)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法:本研究以大鼠骨髓间充质干细胞为研究对象,采用全骨髓培养法,从SD大鼠乳鼠(5d-7d)股骨提取原代细胞,体外传代纯化,P4-P5代用于实验。实验分阴性对照组,大鼠BMSCs 15%胎牛血清(Fetal Bovine Serum,FBS)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法:本研究以大鼠骨髓间培养基培养,不给予药物干预;阳性对照组,给予转化生长因子β1(TGF-β1)5 ng/mL干预3天;黄芪甲苷(Astragaloside,Ast)组,给予黄芪甲苷4μg/mL分别干预1 d、2 d、3 d、5 d、7d;阻断剂组,TGF-β1受体阻断剂sb431542预处理后,再加入黄芪甲苷4μg/mL分别诱导1 d、2 d、3 d。实时定量PCR(RT-PCR)检测神经元-胶质细胞抗原2(Neuron-glial antigen 2,NG2)、α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)信使核糖核酸(mRNA)表达;印迹法(Western blot)检测NG2、α-SMA、Smad2/3、p-Smad2蛋白表达。结果:经黄芪甲苷诱导刺激后,大鼠骨髓间充质干细胞在mRNA和蛋白水平上,NG2、α-SMA的表达量均升高;Ast 3d组,与阴性对照组和阳性对照组相比,NG2、α-SMA蛋白和mRNA的表达量均显著升高(p0.05)。黄芪甲苷刺激细胞后,TGF-β1/Smad2信号传导通路被激活,从第一天开始,p-Smad2蛋白表达量升高,第3天到达顶峰,随后降低;其中第三天,p-Smad2蛋白表达量显著高于Ast 0d组(p0.01)。加入阻断剂后,黄芪甲苷受到sb431542影响,对细胞的作用减弱,NG2、α-SMA蛋白和mRNA表达量均下调;sb431542+Ast 3d组,与Ast 3d组相比,NG2、α-SMA蛋白和mRNA的表达量显著降低(p0.001)。受阻断剂的影响,黄芪甲苷对TGF-β1/Smad2信号传导通路的作用减弱,前三天p-Smad2蛋白的表达量均降低,sb431542+Ast 3d组,与Ast 3d组相比,p-Smad2蛋白的表达量显著降低(p0.001)。结论:黄芪甲苷具有促进大鼠BMSCs向周细胞分化的作用,其机制与激活TGF-β1/Smad2信号传导通路相关。     

移植骨髓间充质干细胞源性神经元样细胞治疗大鼠脊髓损伤

目的:研究骨髓间充质干细胞源性神经元样细胞移植治疗成鼠脊髓损伤的可行性。方法:选取成年SD大鼠32只,两只用以提取骨髓间充质干细胞,其余被分为3组,其中细胞移植组10只,PBS缓冲液组10只,空白对照组10只。骨髓间充质干细胞分离传代培养并诱导成神经元样细胞后用Hoechst33342标记,损伤1周后采取静脉注射移植的方法移植于大鼠脊髓损伤区,移植六周后用免疫荧光方法检测细胞的存活及与宿主脊髓的整合情况。脊髓损伤后的1~6周对各组动物进行BBB评分,用SPSS12.0进行数据分析。结果:细胞移植组动物的BBB评分提高显著,于其他两组差异有统计学意义。细胞移植组免疫荧光显示,移植细胞在体内大量存活并桥接于脊髓损伤区的两端,存活的多数细胞神经元特异性标记物NSE、NF-200、星形胶质细胞特异性标记物GFAP表达呈阳性。结论:移植定向诱导的神经元样细胞有助于大鼠脊髓损伤后的功能恢复。     

儿童腘窝囊肿液中滑膜干细胞的分离及特性鉴定

为了从儿童腘窝囊肿液中分离滑膜间充质干细胞(synovium-derived mesenchymal stem cells,SMSCs),进行体外培养,鉴定其干细胞特性。本实验采用抽取囊肿液、细胞贴壁培养法从腘窝囊肿患儿的囊肿液中分离出滑膜干细胞,进行体外培养、细胞形态学观察、细胞生长曲线绘制及周期的分析,成骨、成脂、成软骨分化能力检测,流式细胞术检测细胞表面标志物,免疫荧光检测干细胞标志物。实验结果显示可采取本方法稳定地从儿童腘窝囊肿液中获取滑膜间充质干细胞,外观梭型状,体外成指数生长,高表达滑膜间充质干细胞表面标记物(CD73,CD90,CD105),表达胚胎间质细胞标记物波形蛋白(vimentin)和成纤维细胞中α-平滑肌肌动蛋白(α-SMA),经定向诱导培养基培养,可分化为骨,软骨和脂肪细胞,我们可以确定所分离出的细胞即为滑膜间充质干细胞(SMSCs)。本研究证明可从儿童腘窝囊肿液中获取滑膜间充质干细胞,是间充质干细胞有价值的来源。     

建立同时分离培养小鼠肝细胞及肝星状细胞的技术

目的:建立一种简便、经济、高产的同步分离培养肝细胞以及肝星状细胞的方法。方法:在参照国内外方法的基础上加以改良,首先采用肝脏原位胶原酶灌注消化的方法,获得总细胞悬液,经多次低速离心分离肝细胞;再用Nycodenz作为分离介质,通过密度梯度离心法从非实质细胞中得到肝星状细胞。通过台盼蓝染色方法鉴定细胞的活力,用倒置相差显微镜、立体显微镜、CK-18、白蛋白免疫荧光细胞化学染色对培养的肝细胞形态以及功能进行检测。使用Desmin、α-SMA免疫荧光细胞化学对肝星状细胞进行鉴定。结果:成功的在体外同步分离、培养肝细胞及肝星状细胞,肝细胞产率为5-6×107/只小鼠,两只小鼠肝星状细胞产率达1×106个。细胞存活率及纯度均可达90%。肝细胞在培养24h后呈不规则铺路石样形态,此为典型的肝细胞形态,其标志分子CK-18以及白蛋白免疫荧光染色阳性。倒置相差显微镜下可见贴壁后的肝星状细胞呈典型的星形细胞形态,且其标志分子Desmin、α-SMA免疫荧光染色阳性。结论:改良的原位灌注以及分离方法可以同时分离并且培养具有高活性和功能的肝细胞和肝星状细胞。     

黄芪甲苷激活TGF-?茁1/Smad2信号通路诱导骨髓间充质干细胞向周细胞分化的作用研究

摘要 目的：观察黄芪甲苷促进大鼠骨髓间充质干细胞(Mesenchymal stem cell,MSCs)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法：本研究以大鼠骨髓间充质干细胞为研究对象,采用全骨髓培养法,从SD大鼠乳鼠（5d-7d）股骨提取原代细胞,体外传代纯化,P4-P5代用于实验。实验分阴性对照组,大鼠BMSCs 15%胎牛血清(Fetal Bovine Serum, FBS)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法：本研究以大鼠骨髓间培养基培养,不给予药物干预;阳性对照组,给予转化生长因子β1(TGF-β1) 5 ng/mL干预3天;黄芪甲苷(Astragaloside,Ast) 组,给予黄芪甲苷4 μg/mL分别干预1 d、2 d、3 d、5 d、7 d;阻断剂组,TGF-β1 受体阻断剂 sb431542 预处理后,再加入黄芪甲苷4 μg/mL分别诱导1 d、2 d、3 d。实时定量PCR( RT-PCR) 检测神经元-胶质细胞抗原2(Neuron-glial antigen 2,NG2)、α-平滑肌肌动蛋白( alpha smooth muscle actin,α-SMA )信使核糖核酸(mRNA)表达;印迹法(Western blot) 检测NG2、α-SMA、Smad2/3、p-Smad2蛋白表达。结果：经黄芪甲苷诱导刺激后,大鼠骨髓间充质干细胞在mRNA和蛋白水平上,NG2、α-SMA 的表达量均升高;Ast 3d组,与阴性对照组和阳性对照组相比,NG2、?-SMA蛋白和mRNA的表达量均显著升高(p<0.05)。黄芪甲苷刺激细胞后,TGF-β1/Smad2信号传导通路被激活,从第一天开始,p-Smad2蛋白表达量升高,第3天到达顶峰,随后降低;其中第三天,p-Smad2蛋白表达量显著高于Ast 0d组(p<0.01) 。加入阻断剂后,黄芪甲苷受到 sb431542影响,对细胞的作用减弱,NG2、α-SMA蛋白和mRNA表达量均下调;sb431542+Ast 3d组,与Ast 3d组相比,NG2、α-SMA 蛋白和mRNA的表达量显著降低(p<0.001)。受阻断剂的影响,黄芪甲苷对TGF-β1/Smad2信号传导通路的作用减弱,前三天p-Smad2蛋白的表达量均降低,sb431542+Ast 3d组,与Ast 3d组相比,p-Smad2蛋白的表达量显著降低(p<0.001)。结论：黄芪甲苷具有促进大鼠BMSCs向周细胞分化的作用,其机制与激活TGF-β1/Smad2信号传导通路相关。     

周细胞的原代培养

目的:探讨Wistar大鼠视网膜毛细血管周细胞(pericyte,PC)的原代培养和鉴定方法。方法:结合视网膜微血管的消化分离,采用含10%胎牛血清的DMEM培养基选择性培养PC,通过活细胞观察原代PC的形态、生长特性以及与血管碎片之间的关系,同时应用免疫细胞化学染色来鉴定PC。结果:选择性培养获得的PC的纯度达到95%以上,并能连续传代。该细胞呈长梭形或星芒状,漩涡或栅栏状生长,无接触性抑制,单核,偶见双核,核卵圆形,细胞浆丰富,α-SMA、PDGFR-β染色阳性。结论:通过对视网膜微血管的消化分离能够获得较为纯净的PC。     

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.     

The impact of pericytes on the blood-brain barrier integrity depends critically on the pericyte differentiation stage

The blood-brain barrier consists of the cerebral microvascular endothelium, pericytes, astrocytes and neurons. In this study we analyzed the differentiation stage dependent influence of primary porcine brain capillary pericytes on the barrier integrity of primary porcine brain capillary endothelial cells. At first, we were able to induce two distinct differentiation stages of the primary pericytes in vitro. TGFβ treated pericytes expressed more α-SMA and actin while desmin, vimentin and nestin expression was decreased when compared to bFGF induced cells. Further analysis of α-SMA revealed that most of the pericytes differentiated with TGFβ expressed functional α-SMA while only few cells expressed functional α-SMA in the presence of bFGF. In addition the permeability factors VEGF, MMP-2 and MMP-9 were higher secreted by the α-SMA positive phenotype indicating a proangiogenic role of this TGFβ induced pericyte differentiation stage. Higher level of VEGF, MMP-2 and MMP-9 were as well detected in the TGFβ pretreated pericyte coculture with endothelial cells when compared to the influence of the bFGF pretreated pericytes. The TEER measurement of the barrier integrity of endothelial cells revealed that bFGF induced α-SMA negative pericytes stabilize the barrier integrity while α-SMA positive pericytes differentiated by TGFβ decrease the barrier integrity. These results together reveal the potential of pericytes to regulate the endothelial barrier integrity in a differentiation stage dependant pathway.     

Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.     

Immunolocalization of smooth muscle-like cells in the quail ovary.

We localized alpha-smooth muscle actin (alpha-SMA) in the quail ovary, using the peroxidase-anti-peroxidase technique. Special attention was paid to the influence of fixation on the immunoreactivity of the antigen. The immunostaining of alpha-SMA largely depended on the nature of the fixative. The antigen could most successfully be localized in ovaries fixed in Carnoy's fluid. We also localized alpha-SMA and desmin in semi-thin glycol methacrylate sections of the pre-ovulatory follicle, using the immunogold-silver staining method. The sections were pretreated with Lugol's iodine or sodium metaperiodate to enhance the immunoreactivity. alpha-SMA was demonstrated in the cells of the chordae, the tunica albuginea, and the theca externa of each follicle. These structures were inter-connected, forming an ovarian suspensory apparatus. The thecal cells of prelampbrush follicles also expressed alpha-SMA. In the wall of the pre-ovulatory follicle, desmin was found in the cells of the chordae and the tunica albuginea, and in a few cells of the theca externa. In the theca interna, desmin, and sometimes alpha-SMA, was observed in cells adjacent to the endothelium of sinusoids, which are probably pericytes. Our results support the hypothesis that in birds the ovarian follicles possess a thecal contractile system, that is presumably involved in the ovulatory process.     

The angiopoietin1–Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8

In mammalian central nervous system (CNS), the integrity of the blood–spinal cord barrier (BSCB), formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement membrane of capillaries and the accessory structures, is important for relatively independent activities of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated and coordinated by a variety of physiological or pathological factors, similar with but not quite the same as its counterpart, the blood–brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1–Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8.     

Contractile proteins in pericytes at the blood-brain and blood-retinal barriers

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (α-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that α-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to α-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.     

Differential fate of multipotent and lineage-restricted neural precursors following transplantation into the adult CNS

Multiple classes of precursor cells have been isolated and characterized from the developing spinal cord including multipotent neuroepithelial (NEP) stem cells and lineage-restricted precursors for neurons (NRPs) and glia (GRPs). We have compared the survival, differentiation and integration of multipotent NEP cells with lineage-restricted NRPs and GRPs using cells isolated from transgenic rats that express the human placental alkaline phosphatase gene. Our results demonstrate that grafted NEP cells survive poorly, with no cells observed 3 days after transplant in the adult hippocampus, striatum and spinal cord, indicating that most CNS regions are not compatible with transplants of multipotent cells derived from fetal CNS. By contrast, at 3 weeks and 5 weeks post-engraftment, lineage-restricted precursors showed selective migration along white-matter tracts and robust survival in all three CNS regions. The grafted precursors expressed the mature neuronal markers NeuN and MAP2, the astrocytic marker GFAP, the oligodendrocytic markers RIP, NG2 and Sox-10, and the synaptic marker synaptophysin. Similar behavior was observed when these precursors were transplanted into the injured spinal cord. Predifferentiated, multipotent NEP cells also survive and integrate, which indicates that lineage-restricted CNS precursors are well suited for transplantation into the adult CNS and provide a promising cellular replacement candidate.     

Blood-neural barrier: its diversity and coordinated cell-to-cell communication

The cerebral microvessels possess barrier characteristics which are tightly sealed excluding many toxic substances and protecting neural tissues. The specialized blood-neural barriers as well as the cerebral microvascular barrier are recognized in the retina, inner ear, spinal cord, and cerebrospinal fluid. Microvascular endothelial cells in the brain closely interact with other components such as astrocytes, pericytes, perivascular microglia and neurons to form functional 'neurovascular unit'. Communication between endothelial cells and other surrounding cells enhances the barrier functions, consequently resulting in maintenance and elaboration of proper brain homeostasis. Furthermore, the disruption of the neurovascular unit is closely involved in cerebrovascular disorders. In this review, we focus on the location and function of these various blood-neural barriers, and the importance of the cell-to-cell communication for development and maintenance of the barrier integrity at the neurovascular unit. We also demonstrate the close relation between the alteration of the blood-neural barriers and cerebrovascular disorders.     

Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.     

Histochemical Localization of Caldesmon in the CNS and Ganglia of the Mouse

The author has recently reported the distribution of the cytoskeleton-associated protein caldesmon in spleen and lymph nodes detected with different antibodies against caldesmon (J Histochem Cytochem 58:183–193, 2010). Here the author reports the distribution of caldesmon in the CNS and ganglia of the mouse using the same antibodies. Western blot analysis of mouse brain and spinal cord showed the preponderance of l-caldesmon and suggested at least two l-caldesmon isoforms in the brain. Immunostaining revealed the predominant reactivity of smooth muscle cells and cells resembling pericytes of many large and small blood vessels, ependymocytes, and secretory cells of the pineal gland and pituitary gland. Neuronal perikarya and neuropil in general displayed no or weak immunoreactivity, but there was stronger labeling of neuronal perikarya in dorsal root and trigeminal ganglia. In the brain, staining of the neuropil was stronger in the molecular layers of the dentate gyrus and cerebellum. Results show that caldesmon is expressed in many different cell types in the CNS and ganglia, consistent with the notion that l-caldesmon is ubiquitously expressed, but it appears most concentrated in smooth muscle cells, pericytes, epithelial cells, secretory cells, and neuronal perikarya in dorsal root and trigeminal ganglia.